Harper’s Illustrated Biochemistry, Twenty-Sixth Edition

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This book was set in Garamond by Pine Tree Composition

The editors were Janet Foltin, Jim Ransom, and Janene Matragrano Oransky.

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Authors

David A. Bender, PhD

Peter A. Mayes, PhD, DSc

Sub-Dean Royal Free and University College Medical

Emeritus Professor of Veterinary Biochemistry, Royal

School, Assistant Faculty Tutor and Tutor to Med-

Veterinary College, University of London

ical Students, Senior Lecturer in Biochemistry, De-

partment of Biochemistry and Molecular Biology,

University College London

Robert K. Murray, MD, PhD

Professor

(Emeritus) of Biochemistry, University of

Toronto

Kathleen M. Botham, PhD, DSc

Reader in Biochemistry, Royal Veterinary College,

University of London

Margaret L. Rand, PhD

Scientist, Research Institute, Hospital for Sick Chil-

Daryl K. Granner, MD

dren, Toronto, and Associate Professor, Depart-

ments of Laboratory Medicine and Pathobiology

Joe C. Davis Professor of Biomedical Science, Director,

and Department of Biochemistry, University of

Vanderbilt Diabetes Center, Professor of Molecular

Toronto

Physiology and Biophysics and of Medicine, Vander-

bilt University, Nashville, Tennessee

Victor W. Rodwell, PhD

Professor of Biochemistry, Purdue University, West

Frederick W. Keeley, PhD

Lafayette, Indiana

Associate Director and Senior Scientist, Research Insti-

tute, Hospital for Sick Children, Toronto, and Pro-

fessor, Department of Biochemistry, University of

P. Anthony Weil, PhD

Toronto

Professor of Molecular Physiology and Biophysics,

Vanderbilt University School of Medicine, Nash-

Peter J. Kennelly, PhD

ville, Tennessee

Professor of Biochemistry, Virginia Polytechnic Insti-

tute and State University, Blacksburg, Virginia

vii

Preface

The authors and publisher are pleased to present the twenty-sixth edition of Harper’s Illustrated Biochemistry. Review

of Physiological Chemistry was first published in 1939 and revised in 1944, and it quickly gained a wide readership. In

1951, the third edition appeared with Harold A. Harper, University of California School of Medicine at San Fran-

cisco, as author. Dr. Harper remained the sole author until the ninth edition and co-authored eight subsequent edi-

tions. Peter Mayes and Victor Rodwell have been authors since the tenth edition, Daryl Granner since the twentieth

edition, and Rob Murray since the twenty-first edition. Because of the increasing complexity of biochemical knowl-

edge, they have added co-authors in recent editions.

Fred Keeley and Margaret Rand have each co-authored one chapter with Rob Murray for this and previous edi-

tions. Peter Kennelly joined as a co-author in the twenty-fifth edition, and in the present edition has co-authored

with Victor Rodwell all of the chapters dealing with the structure and function of proteins and enzymes. The follow-

ing additional co-authors are very warmly welcomed in this edition: Kathleen Botham has co-authored, with Peter

Mayes, the chapters on bioenergetics, biologic oxidation, oxidative phosphorylation, and lipid metabolism. David

Bender has co-authored, also with Peter Mayes, the chapters dealing with carbohydrate metabolism, nutrition, diges-

tion, and vitamins and minerals. P. Anthony Weil has co-authored chapters dealing with various aspects of DNA, of

RNA, and of gene expression with Daryl Granner. We are all very grateful to our co-authors for bringing their ex-

pertise and fresh perspectives to the text.

CHANGES IN THE TWENTY-SIXTH EDITION

A major goal of the authors continues to be to provide both medical and other students of the health sciences with a

book that both describes the basics of biochemistry and is user-friendly and interesting. A second major ongoing

goal is to reflect the most significant advances in biochemistry that are important to medicine. However, a third

major goal of this edition was to achieve a substantial reduction in size, as feedback indicated that many readers pre-

fer shorter texts.

To achieve this goal, all of the chapters were rigorously edited, involving their amalgamation, division, or dele-

tion, and many were reduced to approximately one-half to two-thirds of their previous size. This has been effected

without loss of crucial information but with gain in conciseness and clarity.

Despite the reduction in size, there are many new features in the twenty-sixth edition. These include:

• A new chapter on amino acids and peptides, which emphasizes the manner in which the properties of biologic

peptides derive from the individual amino acids of which they are comprised.

• A new chapter on the primary structure of proteins, which provides coverage of both classic and newly emerging

“proteomic” and “genomic” methods for identifying proteins. A new section on the application of mass spectrometry

to the analysis of protein structure has been added, including comments on the identification of covalent modifica-

tions.

• The chapter on the mechanisms of action of enzymes has been revised to provide a comprehensive description of

the various physical mechanisms by which enzymes carry out their catalytic functions.

• The chapters on integration of metabolism, nutrition, digestion and absorption, and vitamins and minerals have

been completely re-written.

• Among important additions to the various chapters on metabolism are the following: update of the information

on oxidative phosphorylation, including a description of the rotary ATP synthase; new insights into the role of

GTP in gluconeogenesis; additional information on the regulation of acetyl-CoA carboxylase; new information on

receptors involved in lipoprotein metabolism and reverse cholesterol transport; discussion of the role of leptin in

fat storage; and new information on bile acid regulation, including the role of the farnesoid X receptor (FXR).

• The chapter on membrane biochemistry in the previous edition has been split into two, yielding two new chapters

on the structure and function of membranes and intracellular traffic and sorting of proteins.

• Considerable new material has been added on RNA synthesis, protein synthesis, gene regulation, and various as-

pects of molecular genetics.

• Much of the material on individual endocrine glands present in the twenty-fifth edition has been replaced with

new chapters dealing with the diversity of the endocrine system, with molecular mechanisms of hormone action,

and with signal transduction.

PREFACE

• The chapter on plasma proteins, immunoglobulins, and blood coagulation in the previous edition has been split

into two new chapters on plasma proteins and immunoglobulins and on hemostasis and thrombosis.

• New information has been added in appropriate chapters on lipid rafts and caveolae, aquaporins, connexins, dis-

orders due to mutations in genes encoding proteins involved in intracellular membrane transport, absorption of

iron, and conformational diseases and pharmacogenomics.

• A new and final chapter on “The Human Genome Project” (HGP) has been added, which builds on the material

covered in Chapters 35 through 40. Because of the impact of the results of the HGP on the future of biology and

medicine, it appeared appropriate to conclude the text with a summary of its major findings and their implica-

tions for future work.

• As initiated in the previous edition, references to useful Web sites have been included in a brief Appendix at the

end of the text.

ORGANIZATION OF THE BOOK

The text is divided into two introductory chapters (“Biochemistry & Medicine” and “Water & pH”) followed by six

main sections.

Section I deals with the structures and functions of proteins and enzymes, the workhorses of the body. Because

almost all of the reactions in cells are catalyzed by enzymes, it is vital to understand the properties of enzymes before

considering other topics.

Section II explains how various cellular reactions either utilize or release energy, and it traces the pathways by

which carbohydrates and lipids are synthesized and degraded. It also describes the many functions of these two

classes of molecules.

Section III deals with the amino acids and their many fates and also describes certain key features of protein ca-

tabolism.

Section IV describes the structures and functions of the nucleotides and nucleic acids, and covers many major

topics such as DNA replication and repair, RNA synthesis and modification, and protein synthesis. It also discusses

new findings on how genes are regulated and presents the principles of recombinant DNA technology.

Section V deals with aspects of extracellular and intracellular communication. Topics covered include membrane

structure and function, the molecular bases of the actions of hormones, and the key field of signal transduction.

Section VI consists of discussions of eleven special topics: nutrition, digestion, and absorption; vitamins and

minerals; intracellular traffic and sorting of proteins; glycoproteins; the extracellular matrix; muscle and the cy-

toskeleton; plasma proteins and immunoglobulins; hemostasis and thrombosis; red and white blood cells; the me-

tabolism of xenobiotics; and the Human Genome Project.

ACKNOWLEDGMENTS

The authors thank Janet Foltin for her thoroughly professional approach. Her constant interest and input have had a

significant impact on the final structure of this text. We are again immensely grateful to Jim Ransom for his excel-

lent editorial work; it has been a pleasure to work with an individual who constantly offered wise and informed alter-

natives to the sometimes primitive text transmitted by the authors. The superb editorial skills of Janene Matragrano

Oransky and Harriet Lebowitz are warmly acknowledged, as is the excellent artwork of Charissa Baker and her col-

leagues. The authors are very grateful to Kathy Pitcoff for her thoughtful and meticulous work in preparing the

Index. Suggestions from students and colleagues around the world have been most helpful in the formulation of this

edition. We look forward to receiving similar input in the future.

Robert K. Murray, MD, PhD

Daryl K. Granner, MD

Peter A. Mayes, PhD, DSc

Victor W. Rodwell, PhD

Toronto, Ontario

Nashville, Tennessee

London

West Lafayette, Indiana

March 2003

Contents

Authors

vii

Preface

1. Biochemistry & Medicine

Robert K. Murray, MD, PhD

2. Water & pH

Victor W. Rodwell, PhD, & Peter J. Kennelly, PhD

SECTION I. STRUCTURES & FUNCTIONS OF PROTEINS & ENZYMES

3. Amino Acids & Peptides

Victor W. Rodwell, PhD, & Peter J. Kennelly, PhD

4. Proteins: Determination of Primary Structure

Victor W. Rodwell, PhD, & Peter J. Kennelly, PhD

5. Proteins: Higher Orders of Structure

Victor W. Rodwell, PhD, & Peter J. Kennelly, PhD

6. Proteins: Myoglobin & Hemoglobin

Victor W. Rodwell, PhD, & Peter J. Kennelly, PhD

7. Enzymes: Mechanism of Action

Victor W. Rodwell, PhD, & Peter J. Kennelly, PhD

8. Enzymes: Kinetics

Victor W. Rodwell, PhD, & Peter J. Kennelly, PhD

9. Enzymes: Regulation of Activities

Victor W. Rodwell, PhD, & Peter J. Kennelly, PhD

SECTION II. BIOENERGETICS & THE METABOLISM OF CARBOHYDRATES

& LIPIDS

10. Bioenergetics: The Role of ATP

Peter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DSc

11. Biologic Oxidation

Peter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DSc

12. The Respiratory Chain & Oxidative Phosphorylation

Peter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DSc

13. Carbohydrates of Physiologic Significance

Peter A. Mayes, PhD, DSc, & David A. Bender, PhD

102

iii

CONTENTS

14. Lipids of Physiologic Significance

Peter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DSc

111

15. Overview of Metabolism

Peter A. Mayes, PhD, DSc, & David A. Bender, PhD

122

16. The Citric Acid Cycle: The Catabolism of Acetyl-CoA

Peter A. Mayes, PhD, DSc, & David A. Bender, PhD

130

17. Glycolysis & the Oxidation of Pyruvate

Peter A. Mayes, PhD, DSc, & David A. Bender, PhD

136

18. Metabolism of Glycogen

Peter A. Mayes, PhD, DSc, & David A. Bender, PhD

145

19. Gluconeogenesis & Control of the Blood Glucose

Peter A. Mayes, PhD, DSc, & David A. Bender, PhD

153

20. The Pentose Phosphate Pathway & Other Pathways of Hexose Metabolism

Peter A. Mayes, PhD, DSc, & David A. Bender, PhD

163

21. Biosynthesis of Fatty Acids

Peter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DSc

173

22. Oxidation of Fatty Acids: Ketogenesis

Peter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DSc

180

23. Metabolism of Unsaturated Fatty Acids & Eicosanoids

Peter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DSc

190

24. Metabolism of Acylglycerols & Sphingolipids

Peter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DSc

197

25. Lipid Transport & Storage

Peter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DSc

205

26. Cholesterol Synthesis, Transport, & Excretion

Peter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DSc

219

27. Integration of Metabolism—the Provision of Metabolic Fuels

David A. Bender, PhD, & Peter A. Mayes, PhD, DSc

231

SECTION III. METABOLISM OF PROTEINS & AMINO ACIDS

237

28. Biosynthesis of the Nutritionally Nonessential Amino Acids

Victor W. Rodwell, PhD

237

29. Catabolism of Proteins & of Amino Acid Nitrogen

Victor W. Rodwell, PhD

242

CONTENTS

30. Catabolism of the Carbon Skeletons of Amino Acids

Victor W. Rodwell, PhD

249

31. Conversion of Amino Acids to Specialized Products

Victor W. Rodwell, PhD

264

32. Porphyrins & Bile Pigments

Robert K. Murray, MD, PhD

270

SECTION IV. STRUCTURE, FUNCTION, & REPLICATION

OF INFORMATIONAL MACROMOLECULES

286

33. Nucleotides

Victor W. Rodwell, PhD

286

34. Metabolism of Purine & Pyrimidine Nucleotides

Victor W. Rodwell, PhD

293

35. Nucleic Acid Structure & Function

Daryl K. Granner, MD

303

36. DNA Organization, Replication, & Repair

Daryl K. Granner, MD, & P. Anthony Weil, PhD

314

37. RNA Synthesis, Processing, & Modification

Daryl K. Granner, MD, & P. Anthony Weil, PhD

341

38. Protein Synthesis & the Genetic Code

Daryl K. Granner, MD

358

39. Regulation of Gene Expression

Daryl K. Granner, MD, & P. Anthony Weil, PhD

374

40. Molecular Genetics, Recombinant DNA, & Genomic Technology

Daryl K. Granner, MD, & P. Anthony Weil, PhD

396

SECTION V. BIOCHEMISTRY OF EXTRACELLULAR

& INTRACELLULAR COMMUNICATION

415

41. Membranes: Structure & Function

Robert K. Murray, MD, PhD, & Daryl K. Granner, MD

415

42. The Diversity of the Endocrine System

Daryl K. Granner, MD

434

43. Hormone Action & Signal Transduction

Daryl K. Granner, MD

456

Biochemistry & Medicine

Robert K. Murray, MD, PhD

INTRODUCTION

biochemistry is increasingly becoming their common

language.

Biochemistry can be defined as the science concerned

with the chemical basis of life (Gk bios “life”). The cell is

the structural unit of living systems. Thus, biochem-

A Reciprocal Relationship Between

istry can also be described as the science concerned with

Biochemistry & Medicine Has Stimulated

the chemical constituents of living cells and with the reac-

Mutual Advances

tions and processes they undergo. By this definition, bio-

The two major concerns for workers in the health sci-

chemistry encompasses large areas of cell biology, of

molecular biology, and of molecular genetics.

ences—and particularly physicians—are the understand-

ing and maintenance of health and the understanding

and effective treatment of diseases. Biochemistry im-

The Aim of Biochemistry Is to Describe &

pacts enormously on both of these fundamental con-

Explain, in Molecular Terms, All Chemical

cerns of medicine. In fact, the interrelationship of bio-

Processes of Living Cells

chemistry and medicine is a wide, two-way street.

The major objective of biochemistry is the complete

Biochemical studies have illuminated many aspects of

understanding, at the molecular level, of all of the

health and disease, and conversely, the study of various

chemical processes associated with living cells. To

aspects of health and disease has opened up new areas

achieve this objective, biochemists have sought to iso-

of biochemistry. Some examples of this two-way street

late the numerous molecules found in cells, determine

are shown in Figure 1-1. For instance, a knowledge of

their structures, and analyze how they function. Many

protein structure and function was necessary to eluci-

techniques have been used for these purposes; some of

date the single biochemical difference between normal

them are summarized in Table 1-1.

hemoglobin and sickle cell hemoglobin. On the other

hand, analysis of sickle cell hemoglobin has contributed

significantly to our understanding of the structure and

A Knowledge of Biochemistry Is Essential

function of both normal hemoglobin and other pro-

to All Life Sciences

teins. Analogous examples of reciprocal benefit between

The biochemistry of the nucleic acids lies at the heart of

biochemistry and medicine could be cited for the other

genetics; in turn, the use of genetic approaches has been

paired items shown in Figure 1-1. Another example is

critical for elucidating many areas of biochemistry.

the pioneering work of Archibald Garrod, a physician

Physiology, the study of body function, overlaps with

in England during the early 1900s. He studied patients

biochemistry almost completely. Immunology employs

with a number of relatively rare disorders

(alkap-

numerous biochemical techniques, and many immuno-

tonuria, albinism, cystinuria, and pentosuria; these are

logic approaches have found wide use by biochemists.

described in later chapters) and established that these

Pharmacology and pharmacy rest on a sound knowl-

conditions were genetically determined. Garrod desig-

edge of biochemistry and physiology; in particular,

nated these conditions as inborn errors of metabo-

most drugs are metabolized by enzyme-catalyzed reac-

lism. His insights provided a major foundation for the

tions. Poisons act on biochemical reactions or processes;

development of the field of human biochemical genet-

this is the subject matter of toxicology. Biochemical ap-

ics. More recent efforts to understand the basis of the

proaches are being used increasingly to study basic as-

genetic disease known as familial hypercholesterol-

pects of pathology (the study of disease), such as in-

emia, which results in severe atherosclerosis at an early

flammation, cell injury, and cancer. Many workers in

age, have led to dramatic progress in understanding of

microbiology, zoology, and botany employ biochemical

cell receptors and of mechanisms of uptake of choles-

approaches almost exclusively. These relationships are

terol into cells. Studies of oncogenes in cancer cells

not surprising, because life as we know it depends on

have directed attention to the molecular mechanisms

biochemical reactions and processes. In fact, the old

involved in the control of normal cell growth. These

barriers among the life sciences are breaking down, and

and many other examples emphasize how the study of

CHAPTER 1

Table 1-1. The principal methods and

NORMAL BIOCHEMICAL PROCESSES ARE

preparations used in biochemical laboratories.

THE BASIS OF HEALTH

The World Health Organization

(WHO) defines

Methods for Separating and Purifying Biomolecules¹

health as a state of “complete physical, mental and so-

Salt fractionation (eg, precipitation of proteins with ammo-

cial well-being and not merely the absence of disease

nium sulfate)

and infirmity.” From a strictly biochemical viewpoint,

Chromatography: Paper; ion exchange; affinity; thin-layer;

health may be considered that situation in which all of

gas-liquid; high-pressure liquid; gel filtration

the many thousands of intra- and extracellular reactions

Electrophoresis: Paper; high-voltage; agarose; cellulose

that occur in the body are proceeding at rates commen-

acetate; starch gel; polyacrylamide gel; SDS-polyacryl-

surate with the organism’s maximal survival in the

amide gel

physiologic state. However, this is an extremely reduc-

Ultracentrifugation

Methods for Determining Biomolecular Structures

tionist view, and it should be apparent that caring for

Elemental analysis

the health of patients requires not only a wide knowl-

UV, visible, infrared, and NMR spectroscopy

edge of biologic principles but also of psychologic and

Use of acid or alkaline hydrolysis to degrade the biomole-

social principles.

cule under study into its basic constituents

Use of a battery of enzymes of known specificity to de-

Biochemical Research Has Impact on

grade the biomolecule under study (eg, proteases, nucle-

Nutrition & Preventive Medicine

ases, glycosidases)

Mass spectrometry

One major prerequisite for the maintenance of health is

Specific sequencing methods (eg, for proteins and nucleic

that there be optimal dietary intake of a number of

acids)

chemicals; the chief of these are vitamins, certain

X-ray crystallography

amino acids, certain fatty acids, various minerals, and

Preparations for Studying Biochemical Processes

water. Because much of the subject matter of both bio-

Whole animal (includes transgenic animals and animals

chemistry and nutrition is concerned with the study of

with gene knockouts)

various aspects of these chemicals, there is a close rela-

Isolated perfused organ

tionship between these two sciences. Moreover, more

Tissue slice

emphasis is being placed on systematic attempts to

Whole cells

maintain health and forestall disease, ie, on preventive

Homogenate

Isolated cell organelles

medicine. Thus, nutritional approaches to—for exam-

Subfractionation of organelles

ple—the prevention of atherosclerosis and cancer are

Purified metabolites and enzymes

receiving increased emphasis. Understanding nutrition

Isolated genes (including polymerase chain reaction and

depends to a great extent on a knowledge of biochem-

site-directed mutagenesis)

istry.

¹Most of these methods are suitable for analyzing the compo-

nents present in cell homogenates and other biochemical prepa-

Most & Perhaps All Disease Has

rations. The sequential use of several techniques will generally

a Biochemical Basis

permit purification of most biomolecules. The reader is referred

to texts on methods of biochemical research for details.

We believe that most if not all diseases are manifesta-

tions of abnormalities of molecules, chemical reactions,

or biochemical processes. The major factors responsible

disease can open up areas of cell function for basic bio-

for causing diseases in animals and humans are listed in

chemical research.

Table 1-2. All of them affect one or more critical

The relationship between medicine and biochem-

chemical reactions or molecules in the body. Numerous

istry has important implications for the former. As long

examples of the biochemical bases of diseases will be en-

as medical treatment is firmly grounded in a knowledge

countered in this text; the majority of them are due to

of biochemistry and other basic sciences, the practice of

causes 5, 7, and 8. In most of these conditions, bio-

medicine will have a rational basis that can be adapted

chemical studies contribute to both the diagnosis and

to accommodate new knowledge. This contrasts with

treatment. Some major uses of biochemical investiga-

unorthodox health cults and at least some “alternative

tions and of laboratory tests in relation to diseases are

medicine” practices, which are often founded on little

summarized in Table 1-3.

more than myth and wishful thinking and generally

Additional examples of many of these uses are pre-

lack any intellectual basis.

sented in various sections of this text.

BIOCHEMISTRY & MEDICINE

BIOCHEMISTRY

Nucleic

acids

Proteins

Lipids

Carbohydrates

Genetic

Sickle cell

Athero-

Diabetes

diseases

anemia

sclerosis

mellitus

MEDICINE

Figure 1-1. Examples of the two-way street connecting biochemistry and

medicine. Knowledge of the biochemical molecules shown in the top part of the

diagram has clarified our understanding of the diseases shown in the bottom

half—and conversely, analyses of the diseases shown below have cast light on

many areas of biochemistry. Note that sickle cell anemia is a genetic disease and

that both atherosclerosis and diabetes mellitus have genetic components.

Impact of the Human Genome Project

(HGP) on Biochemistry & Medicine

Table 1-3. Some uses of biochemical

Remarkable progress was made in the late 1990s in se-

investigations and laboratory tests in

quencing the human genome. This culminated in July

relation to diseases.

2000, when leaders of the two groups involved in this

effort (the International Human Genome Sequencing

Consortium and Celera Genomics, a private company)

Use

Example

announced that over 90% of the genome had been se-

To reveal the funda-

Demonstration of the na-

quenced. Draft versions of the sequence were published

mental causes and

ture of the genetic de-

mechanisms of diseases

fects in cystic fibrosis.

To suggest rational treat-

A diet low in phenylalanine

Table 1-2. The major causes of diseases. All of

ments of diseases based

for treatment of phenyl-

on (1) above

ketonuria.

the causes listed act by influencing the various

To assist in the diagnosis

Use of the plasma enzyme

biochemical mechanisms in the cell or in the

of specific diseases

creatine kinase MB

body.¹

(CK-MB) in the diagnosis

of myocardial infarction.

1. Physical agents: Mechanical trauma, extremes of temper-

To act as screening tests

Use of measurement of

ature, sudden changes in atmospheric pressure, radia-

for the early diagnosis

blood thyroxine or

tion, electric shock.

of certain diseases

thyroid-stimulating hor-

2. Chemical agents, including drugs: Certain toxic com-

mone (TSH) in the neo-

pounds, therapeutic drugs, etc.

natal diagnosis of con-

3. Biologic agents: Viruses, bacteria, fungi, higher forms of

genital hypothyroidism.

parasites.

To assist in monitoring

Use of the plasma enzyme

4. Oxygen lack: Loss of blood supply, depletion of the

the progress (eg, re-

alanine aminotransferase

oxygen-carrying capacity of the blood, poisoning of

covery, worsening, re-

(ALT) in monitoring the

the oxidative enzymes.

mission, or relapse) of

progress of infectious

5. Genetic disorders: Congenital, molecular.

certain diseases

hepatitis.

6. Immunologic reactions: Anaphylaxis, autoimmune

To assist in assessing

Use of measurement of

disease.

the response of dis-

blood carcinoembryonic

7. Nutritional imbalances: Deficiencies, excesses.

eases to therapy

antigen (CEA) in certain

8. Endocrine imbalances: Hormonal deficiencies, excesses.

patients who have been

treated for cancer of the

¹Adapted, with permission, from Robbins SL, Cotram RS, Kumar V:

colon.

The Pathologic Basis of Disease, 3rd ed. Saunders, 1984.

CHAPTER 1

in early 2001. It is anticipated that the entire sequence

• The judicious use of various biochemical laboratory

will be completed by 2003. The implications of this

tests is an integral component of diagnosis and moni-

work for biochemistry, all of biology, and for medicine

toring of treatment.

are tremendous, and only a few points are mentioned

• A sound knowledge of biochemistry and of other re-

here. Many previously unknown genes have been re-

lated basic disciplines is essential for the rational

vealed; their protein products await characterization.

practice of medical and related health sciences.

New light has been thrown on human evolution, and

procedures for tracking disease genes have been greatly

refined. The results are having major effects on areas

REFERENCES

such as proteomics, bioinformatics, biotechnology, and

Fruton JS: Proteins, Enzymes, Genes: The Interplay of Chemistry and

pharmacogenomics. Reference to the human genome

Biology. Yale Univ Press, 1999. (Provides the historical back-

will be made in various sections of this text. The

ground for much of today’s biochemical research.)

Human Genome Project is discussed in more detail in

Garrod AE: Inborn errors of metabolism. (Croonian Lectures.)

Chapter 54.

Lancet 1908;2:1, 73, 142, 214.

International Human Genome Sequencing Consortium. Initial se-

SUMMARY

quencing and analysis of the human genome. Nature

2001:409;860. (The issue [15 February] consists of articles

• Biochemistry is the science concerned with studying

dedicated to analyses of the human genome.)

the various molecules that occur in living cells and

Kornberg A: Basic research: The lifeline of medicine. FASEB J

organisms and with their chemical reactions. Because

1992;6:3143.

life depends on biochemical reactions, biochemistry

Kornberg A: Centenary of the birth of modern biochemistry.

has become the basic language of all biologic sci-

FASEB J 1997;11:1209.

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McKusick VA: Mendelian Inheritance in Man. Catalogs of Human

Genes and Genetic Disorders, 12th ed. Johns Hopkins Univ

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Press, 1998. [Abbreviated MIM]

of life forms, from relatively simple viruses and bacte-

Online Mendelian Inheritance in Man (OMIM): Center for Med-

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ical Genetics, Johns Hopkins University and National Center

• Biochemistry and medicine are intimately related.

for Biotechnology Information, National Library of Medi-

Health depends on a harmonious balance of bio-

cine, 1997. http://www.ncbi.nlm.nih.gov/omim/

chemical reactions occurring in the body, and disease

(The numbers assigned to the entries in MIM and OMIM will be

cited in selected chapters of this work. Consulting this exten-

reflects abnormalities in biomolecules, biochemical

sive collection of diseases and other relevant entries—specific

reactions, or biochemical processes.

proteins, enzymes, etc—will greatly expand the reader’s

• Advances in biochemical knowledge have illumi-

knowledge and understanding of various topics referred to

nated many areas of medicine. Conversely, the study

and discussed in this text. The online version is updated al-

of diseases has often revealed previously unsuspected

most daily.)

aspects of biochemistry. The determination of the se-

Scriver CR et al (editors): The Metabolic and Molecular Bases of In-

herited Disease, 8th ed. McGraw-Hill, 2001.

quence of the human genome, nearly complete, will

Venter JC et al: The Sequence of the Human Genome. Science

have a great impact on all areas of biology, including

2001;291:1304. (The issue [16 February] contains the Celera

biochemistry, bioinformatics, and biotechnology.

draft version and other articles dedicated to analyses of the

• Biochemical approaches are often fundamental in il-

human genome.)

luminating the causes of diseases and in designing

Williams DL, Marks V: Scientific Foundations of Biochemistry in

appropriate therapies.

Clinical Practice, 2nd ed. Butterworth-Heinemann, 1994.

Water & pH

Victor W. Rodwell, PhD, & Peter J. Kennelly, PhD

BIOMEDICAL IMPORTANCE

oxygen atom pulls electrons away from the hydrogen

nuclei, leaving them with a partial positive charge,

Water is the predominant chemical component of liv-

while its two unshared electron pairs constitute a region

ing organisms. Its unique physical properties, which in-

of local negative charge.

clude the ability to solvate a wide range of organic and

Water, a strong dipole, has a high dielectric con-

inorganic molecules, derive from water’s dipolar struc-

stant. As described quantitatively by Coulomb’s law,

ture and exceptional capacity for forming hydrogen

the strength of interaction F between oppositely

bonds. The manner in which water interacts with a sol-

charged particles is inversely proportionate to the di-

vated biomolecule influences the structure of each. An

electric constant ε of the surrounding medium. The di-

excellent nucleophile, water is a reactant or product in

electric constant for a vacuum is unity; for hexane it is

many metabolic reactions. Water has a slight propensity

1.9; for ethanol it is 24.3; and for water it is 78.5.

to dissociate into hydroxide ions and protons. The

Water therefore greatly decreases the force of attraction

acidity of aqueous solutions is generally reported using

between charged and polar species relative to water-free

the logarithmic pH scale. Bicarbonate and other buffers

environments with lower dielectric constants. Its strong

normally maintain the pH of extracellular fluid be-

dipole and high dielectric constant enable water to dis-

tween 7.35 and 7.45. Suspected disturbances of acid-

solve large quantities of charged compounds such as

base balance are verified by measuring the pH of arter-

salts.

ial blood and the CO₂ content of venous blood. Causes

of acidosis (blood pH < 7.35) include diabetic ketosis

and lactic acidosis. Alkalosis (pH > 7.45) may, for ex-

Water Molecules Form Hydrogen Bonds

ample, follow vomiting of acidic gastric contents. Regu-

An unshielded hydrogen nucleus covalently bound to

lation of water balance depends upon hypothalamic

an electron-withdrawing oxygen or nitrogen atom can

mechanisms that control thirst, on antidiuretic hor-

interact with an unshared electron pair on another oxy-

mone (ADH), on retention or excretion of water by the

gen or nitrogen atom to form a hydrogen bond. Since

kidneys, and on evaporative loss. Nephrogenic diabetes

water molecules contain both of these features, hydro-

insipidus, which involves the inability to concentrate

gen bonding favors the self-association of water mole-

urine or adjust to subtle changes in extracellular fluid

cules into ordered arrays (Figure 2-2). Hydrogen bond-

osmolarity, results from the unresponsiveness of renal

ing profoundly influences the physical properties of

tubular osmoreceptors to ADH.

water and accounts for its exceptionally high viscosity,

surface tension, and boiling point. On average, each

molecule in liquid water associates through hydrogen

WATER IS AN IDEAL BIOLOGIC SOLVENT

bonds with 3.5 others. These bonds are both relatively

weak and transient, with a half-life of about one mi-

Water Molecules Form Dipoles

crosecond. Rupture of a hydrogen bond in liquid water

A water molecule is an irregular, slightly skewed tetra-

requires only about 4.5 kcal/mol, less than 5% of the

hedron with oxygen at its center (Figure 2-1). The two

energy required to rupture a covalent O H bond.

hydrogens and the unshared electrons of the remaining

Hydrogen bonding enables water to dissolve many

two sp³-hybridized orbitals occupy the corners of the

organic biomolecules that contain functional groups

tetrahedron. The 105-degree angle between the hydro-

which can participate in hydrogen bonding. The oxy-

gens differs slightly from the ideal tetrahedral angle,

gen atoms of aldehydes, ketones, and amides provide

109.5 degrees. Ammonia is also tetrahedral, with a 107-

pairs of electrons that can serve as hydrogen acceptors.

degree angle between its hydrogens. Water is a dipole,

Alcohols and amines can serve both as hydrogen accep-

a molecule with electrical charge distributed asymmetri-

tors and as donors of unshielded hydrogen atoms for

cally about its structure. The strongly electronegative

formation of hydrogen bonds (Figure 2-3).

CHAPTER 2

CH₃

105°

CH CH

CH₂

CH₃

Figure 2-1. The water molecule has tetrahedral

R^II

geometry.

R^I

R^III

INTERACTION WITH WATER INFLUENCES

Figure 2-3. Additional polar groups participate in

THE STRUCTURE OF BIOMOLECULES

hydrogen bonding. Shown are hydrogen bonds formed

Covalent & Noncovalent Bonds Stabilize

between an alcohol and water, between two molecules

Biologic Molecules

of ethanol, and between the peptide carbonyl oxygen

and the peptide nitrogen hydrogen of an adjacent

The covalent bond is the strongest force that holds

amino acid.

molecules together (Table 2-1). Noncovalent forces,

while of lesser magnitude, make significant contribu-

tions to the structure, stability, and functional compe-

phosphatidyl serine or phosphatidyl ethanolamine con-

tence of macromolecules in living cells. These forces,

tact water while their hydrophobic fatty acyl side chains

which can be either attractive or repulsive, involve in-

cluster together, excluding water. This pattern maxi-

teractions both within the biomolecule and between it

mizes the opportunities for the formation of energeti-

and the water that forms the principal component of

cally favorable charge-dipole, dipole-dipole, and hydro-

the surrounding environment.

gen bonding interactions between polar groups on the

biomolecule and water. It also minimizes energetically

unfavorable contact between water and hydrophobic

Biomolecules Fold to Position Polar &

groups.

Charged Groups on Their Surfaces

Most biomolecules are amphipathic; that is, they pos-

Hydrophobic Interactions

sess regions rich in charged or polar functional groups

as well as regions with hydrophobic character. Proteins

Hydrophobic interaction refers to the tendency of non-

tend to fold with the R-groups of amino acids with hy-

polar compounds to self-associate in an aqueous envi-

drophobic side chains in the interior. Amino acids with

ronment. This self-association is driven neither by mu-

charged or polar amino acid side chains (eg, arginine,

tual attraction nor by what are sometimes incorrectly

glutamate, serine) generally are present on the surface

referred to as

“hydrophobic bonds.” Self-association

in contact with water. A similar pattern prevails in a

arises from the need to minimize energetically unfavor-

phospholipid bilayer, where the charged head groups of

able interactions between nonpolar groups and water.

Table 2-1. Bond energies for atoms of biologic

H H

significance.

H O

Bond

Energy

Bond

Energy

H H

Type

(kcal/mol)

Type

(kcal/mol)

O—O

O==O

S—S

C—H

C—N

C==S

108

Figure 2-2. Left: Association of two dipolar water

S—H

O—H

110

molecules by a hydrogen bond (dotted line). Right:

C—C

C==C

147

Hydrogen-bonded cluster of four water molecules.

C—O

C==N

147

Note that water can serve simultaneously both as a hy-

N—H

C==O

164

drogen donor and as a hydrogen acceptor.

WATER & pH

While the hydrogens of nonpolar groups such as the

the backbone to water while burying the relatively hy-

methylene groups of hydrocarbons do not form hydro-

drophobic nucleotide bases inside. The extended back-

gen bonds, they do affect the structure of the water that

bone maximizes the distance between negatively

surrounds them. Water molecules adjacent to a hy-

charged backbone phosphates, minimizing unfavorable

drophobic group are restricted in the number of orien-

electrostatic interactions.

tations (degrees of freedom) that permit them to par-

ticipate in the maximum number of energetically

WATER IS AN EXCELLENT NUCLEOPHILE

favorable hydrogen bonds. Maximal formation of mul-

Metabolic reactions often involve the attack by lone

tiple hydrogen bonds can be maintained only by in-

pairs of electrons on electron-rich molecules termed

creasing the order of the adjacent water molecules, with

nucleophiles on electron-poor atoms called elec-

a corresponding decrease in entropy.

trophiles. Nucleophiles and electrophiles do not neces-

It follows from the second law of thermodynamics

sarily possess a formal negative or positive charge.

that the optimal free energy of a hydrocarbon-water

Water, whose two lone pairs of sp³ electrons bear a par-

mixture is a function of both maximal enthalpy (from

tial negative charge, is an excellent nucleophile. Other

hydrogen bonding) and minimum entropy (maximum

nucleophiles of biologic importance include the oxygen

degrees of freedom). Thus, nonpolar molecules tend to

atoms of phosphates, alcohols, and carboxylic acids; the

form droplets with minimal exposed surface area, re-

sulfur of thiols; the nitrogen of amines; and the imid-

ducing the number of water molecules affected. For the

azole ring of histidine. Common electrophiles include

same reason, in the aqueous environment of the living

the carbonyl carbons in amides, esters, aldehydes, and

cell the hydrophobic portions of biopolymers tend to

ketones and the phosphorus atoms of phosphoesters.

be buried inside the structure of the molecule, or within

Nucleophilic attack by water generally results in the

a lipid bilayer, minimizing contact with water.

cleavage of the amide, glycoside, or ester bonds that

hold biopolymers together. This process is termed hy-

Electrostatic Interactions

drolysis. Conversely, when monomer units are joined

together to form biopolymers such as proteins or glyco-

Interactions between charged groups shape biomolecu-

gen, water is a product, as shown below for the forma-

lar structure. Electrostatic interactions between oppo-

tion of a peptide bond between two amino acids.

sitely charged groups within or between biomolecules

are termed salt bridges. Salt bridges are comparable in

strength to hydrogen bonds but act over larger dis-

tances. They thus often facilitate the binding of charged

⁺H₃N

OH + H

molecules and ions to proteins and nucleic acids.

O^-

Alanine

Van der Waals Forces

Van der Waals forces arise from attractions between

Valine

transient dipoles generated by the rapid movement of

electrons on all neutral atoms. Significantly weaker

than hydrogen bonds but potentially extremely numer-

H₂O

ous, van der Waals forces decrease as the sixth power of

the distance separating atoms. Thus, they act over very

short distances, typically 2-4 Å.

⁺H₃N

O^-

Multiple Forces Stabilize Biomolecules

The DNA double helix illustrates the contribution of

multiple forces to the structure of biomolecules. While

each individual DNA strand is held together by cova-

While hydrolysis is a thermodynamically favored re-

lent bonds, the two strands of the helix are held to-

action, the amide and phosphoester bonds of polypep-

gether exclusively by noncovalent interactions. These

tides and oligonucleotides are stable in the aqueous en-

noncovalent interactions include hydrogen bonds be-

vironment of the cell. This seemingly paradoxic

tween nucleotide bases

(Watson-Crick base pairing)

behavior reflects the fact that the thermodynamics gov-

and van der Waals interactions between the stacked

erning the equilibrium of a reaction do not determine

purine and pyrimidine bases. The helix presents the

the rate at which it will take place. In the cell, protein

charged phosphate groups and polar ribose sugars of

catalysts called enzymes are used to accelerate the rate

CHAPTER 2

of hydrolytic reactions when needed. Proteases catalyze

H₇O3+. The proton is nevertheless routinely repre-

the hydrolysis of proteins into their component amino

sented as H⁺, even though it is in fact highly hydrated.

acids, while nucleases catalyze the hydrolysis of the

Since hydronium and hydroxide ions continuously

phosphoester bonds in DNA and RNA. Careful control

recombine to form water molecules, an individual hy-

of the activities of these enzymes is required to ensure

drogen or oxygen cannot be stated to be present as an

that they act only on appropriate target molecules.

ion or as part of a water molecule. At one instant it is

an ion. An instant later it is part of a molecule. Individ-

Many Metabolic Reactions Involve

ual ions or molecules are therefore not considered. We

Group Transfer

refer instead to the probability that at any instant in

time a hydrogen will be present as an ion or as part of a

In group transfer reactions, a group G is transferred

water molecule. Since 1 g of water contains 3.46 × 10²²

from a donor D to an acceptor A, forming an acceptor

molecules, the ionization of water can be described sta-

group complex A-G:

tistically. To state that the probability that a hydrogen

exists as an ion is 0.01 means that a hydrogen atom has

D−G + A = A−G + D

one chance in 100 of being an ion and 99 chances out

of 100 of being part of a water molecule. The actual

The hydrolysis and phosphorolysis of glycogen repre-

probability of a hydrogen atom in pure water existing as

sent group transfer reactions in which glucosyl groups

a hydrogen ion is approximately 1.8 × 10−9. The proba-

are transferred to water or to orthophosphate. The

bility of its being part of a molecule thus is almost

equilibrium constant for the hydrolysis of covalent

unity. Stated another way, for every hydrogen ion and

bonds strongly favors the formation of split products.

hydroxyl ion in pure water there are 1.8 billion or 1.8 ×

The biosynthesis of macromolecules also involves group

10⁹ water molecules. Hydrogen ions and hydroxyl ions

transfer reactions in which the thermodynamically un-

nevertheless contribute significantly to the properties of

favored synthesis of covalent bonds is coupled to fa-

water.

vored reactions so that the overall change in free energy

For dissociation of water,

favors biopolymer synthesis. Given the nucleophilic

character of water and its high concentration in cells,

why are biopolymers such as proteins and DNA rela-

][OH− ]

tively stable? And how can synthesis of biopolymers

[H O

]

occur in an apparently aqueous environment? Central

to both questions are the properties of enzymes. In the

where brackets represent molar concentrations (strictly

absence of enzymic catalysis, even thermodynamically

speaking, molar activities) and K is the dissociation

highly favored reactions do not necessarily take place

constant. Since one mole (mol) of water weighs 18 g,

rapidly. Precise and differential control of enzyme ac-

one liter (L) (1000 g) of water contains 1000 × 18 =

tivity and the sequestration of enzymes in specific or-

55.56 mol. Pure water thus is 55.56 molar. Since the

ganelles determine under what physiologic conditions a

probability that a hydrogen in pure water will exist as a

given biopolymer will be synthesized or degraded.

hydrogen ion is 1.8 × 10−9, the molar concentration of

Newly synthesized polymers are not immediately hy-

H+ ions (or of OH⁻ ions) in pure water is the product

drolyzed, in part because the active sites of biosynthetic

of the probability, 1.8 × 10−9, times the molar concen-

enzymes sequester substrates in an environment from

tration of water, 55.56 mol/L. The result is 1.0 × 10−7

which water can be excluded.

mol/L.

We can now calculate K for water:

Water Molecules Exhibit a Slight but

Important Tendency to Dissociate

−

−7

][OH

]

[10

][10

]

K =

The ability of water to ionize, while slight, is of central

[H O]

[5556]

importance for life. Since water can act both as an acid

−14

−16

0 018×10

=18×10

mol

and as a base, its ionization may be represented as an

intermolecular proton transfer that forms a hydronium

The molar concentration of water, 55.56 mol/L, is

ion (H₃O⁺) and a hydroxide ion (OH⁻):

too great to be significantly affected by dissociation. It

−

H O

therefore is considered to be essentially constant. This

constant may then be incorporated into the dissociation

The transferred proton is actually associated with a

constant K to provide a useful new constant K_w termed

cluster of water molecules. Protons exist in solution not

the ion product for water. The relationship between

only as H₃O⁺, but also as multimers such as H₅O2+ and

Kw and K is shown below:

WATER & pH

−

termediates, whose phosphoryl group contains two dis-

][OH

]

−16

=18×10

mol/L

sociable protons, the first of which is strongly acidic.

[

H O2

]

The following examples illustrate how to calculate

−

the pH of acidic and basic solutions.

(K )[

H O2

]

][OH

]

Example 1: What is the pH of a solution whose hy-

−16

(18 ×10

mol/L)(5556

mol/L)

drogen ion concentration is 3.2 × 10−4 mol/L?

−14

=1 00×10

(mol

)

pH =

−log [H

]

−4

Note that the dimensions of K are moles per liter and

−log (3.2×10

)

those of K_w are moles² per liter². As its name suggests,

−4

−log (3.2)−log (10

)

the ion product K_w is numerically equal to the product

of the molar concentrations of H⁺ and OH⁻:

−0.5+40

=35

−

K_w

=[H+

[

]

Example 2: What is the pH of a solution whose hy-

At 25 °C, K_w = (10−7)², or 10−14 (mol/L)². At tempera-

droxide ion concentration is 4.0 × 10−4 mol/L? We first

tures below 25 °C, K_w is somewhat less than 10−14; and

define a quantity pOH that is equal to −log [OH⁻] and

at temperatures above 25 °C it is somewhat greater than

that may be derived from the definition of K_w:

10−14. Within the stated limitations of the effect of tem-

perature, K_w equals 10^-14 (mol/L)² for all aqueous so-

−

−14

K_w

=[H

][OH

]

=10

lutions, even solutions of acids or bases. We shall use

Kw to calculate the pH of acidic and basic solutions.

Therefore:

−

pH IS THE NEGATIVE LOG OF THE

log [H

]+log [OH

]= log

10−14

HYDROGEN ION CONCENTRATION

The term pH was introduced in 1909 by Sörensen,

who defined pH as the negative log of the hydrogen ion

pH + pOH = 14

concentration:

To solve the problem by this approach:

pH =

− log [H+

]

−

−4

[OH

] =

40×10

This definition, while not rigorous, suffices for many

biochemical purposes. To calculate the pH of a solution:

pOH=

−log [OH−]

−4

1. Calculate hydrogen ion concentration [H⁺].

−log (40×10

)

2. Calculate the base 10 logarithm of [H⁺].

−4

−log (40)−log

(10

)

3. pH is the negative of the value found in step 2.

−0.60+4.0

For example, for pure water at 25°C,

=34

−7

pH =

−log [H

]

−log 10

−(−7) = 7.0

Now:

Low pH values correspond to high concentrations of

pH =14 −pOH = 14

− .

H+ and high pH values correspond to low concentra-

= 10.6

tions of H⁺.

−2

Acids are proton donors and bases are proton ac-

Example 3: What are the pH values of (a) 2.0 × 10

ceptors. Strong acids (eg, HCl or H₂SO₄) completely

mol/L KOH and of (b) 2.0 × 10−6 mol/L KOH? The

dissociate into anions and cations even in strongly acidic

OH⁻ arises from two sources, KOH and water. Since

solutions (low pH). Weak acids dissociate only partially

pH is determined by the total [H⁺] (and pOH by the

in acidic solutions. Similarly, strong bases (eg, KOH or

total [OH⁻]), both sources must be considered. In the

NaOH)—but not weak bases (eg, Ca[OH]₂)—are

first case

(a), the contribution of water to the total

completely dissociated at high pH. Many biochemicals

[OH⁻] is negligible. The same cannot be said for the

are weak acids. Exceptions include phosphorylated in-

second case (b):

CHAPTER 2

below are the expressions for the dissociation constant

Concentration (mol/L)

(K_a ) for two representative weak acids, RCOOH and

(a)

(b)

RNH3+.

Molarity of KOH

2.0 × 10−2

2.0 × 10−6

[OH⁻] from KOH

2.0 × 10−2

2.0 × 10−6

R—COOH =R—COO−+H

[OH⁻] from water

1.0 × 10−7

−

[R—COO

][H

]

Total [OH⁻]

2.00001 × 10−2

2.1 × 10−6

[R—COOH]

R—NH =R—NH +H

Once a decision has been reached about the significance

[R—NH ][H

]

of the contribution by water, pH may be calculated as

above.

—

]

The above examples assume that the strong base

KOH is completely dissociated in solution and that the

Since the numeric values of K_a for weak acids are nega-

concentration of OH⁻ ions was thus equal to that of the

tive exponential numbers, we express K_a as pK_a, where

KOH. This assumption is valid for dilute solutions of

strong bases or acids but not for weak bases or acids.

= − log K

Since weak electrolytes dissociate only slightly in solu-

tion, we must use the dissociation constant to calcu-

Note that pK_a is related to K_a as pH is to [H⁺]. The

late the concentration of [H⁺] (or [OH⁻]) produced by

stronger the acid, the lower its pK_a value.

a given molarity of a weak acid (or base) before calcu-

pK_a is used to express the relative strengths of both

lating total [H⁺] (or total [OH⁻]) and subsequently pH.

acids and bases. For any weak acid, its conjugate is a

strong base. Similarly, the conjugate of a strong base is

a weak acid. The relative strengths of bases are ex-

Functional Groups That Are Weak Acids

pressed in terms of the pK_a of their conjugate acids. For

Have Great Physiologic Significance

polyproteic compounds containing more than one dis-

Many biochemicals possess functional groups that are

sociable proton, a numerical subscript is assigned to

weak acids or bases. Carboxyl groups, amino groups,

each in order of relative acidity. For a dissociation of

and the second phosphate dissociation of phosphate es-

the type

ters are present in proteins and nucleic acids, most

coenzymes, and most intermediary metabolites. Knowl-

R—NH3+

→R—NH₂

edge of the dissociation of weak acids and bases thus is

basic to understanding the influence of intracellular pH

the pK_a is the pH at which the concentration of the

on structure and biologic activity. Charge-based separa-

acid RNH3+ equals that of the base RNH₂.

tions such as electrophoresis and ion exchange chro-

From the above equations that relate K_a to [H⁺] and

matography also are best understood in terms of the

to the concentrations of undissociated acid and its con-

dissociation behavior of functional groups.

jugate base, when

We term the protonated species

(eg, HA or

RNH3+) the acid and the unprotonated species (eg,

−

—

COO

] = [R—COOH]

A− or RNH₂) its conjugate base. Similarly, we may

refer to a base (eg, A⁻ or RNH₂) and its conjugate

or when

acid (eg, HA or RNH3+). Representative weak acids

(left), their conjugate bases (center), and the pK_a values

(right) include the following:

—

] = [R

—

]

−

R—CH —COOH

R—CH

—COO

4−5

then

R—CH

—NH

R—CH

—NH

=9−10

K_a

]

−

H CO

HCO

−

−2

Thus, when the associated (protonated) and dissociated

H PO

HPO

=72

(conjugate base) species are present at equal concentra-

tions, the prevailing hydrogen ion concentration [H⁺]

We express the relative strengths of weak acids and

is numerically equal to the dissociation constant, K_a. If

bases in terms of their dissociation constants. Shown

the logarithms of both sides of the above equation are

WATER & pH

taken and both sides are multiplied by −1, the expres-

Substitute pH and pK_a for −log [H⁺] and −log K_a, re-

sions would be as follows:

spectively; then:

Ka =[H+]

[HA]

pH=pK

−log

−

−log

K_a

−log [H

]

Since −log K_a is defined as pK_a, and −log [H⁺] de-

Inversion of the last term removes the minus sign

fines pH, the equation may be rewritten as

and gives the Henderson-Hasselbalch equation:

−

]

pH=pK

+ log

[HA]

ie, the pK_a of an acid group is the pH at which the pro-

tonated and unprotonated species are present at equal

The Henderson-Hasselbalch equation has great pre-

concentrations. The pK_a for an acid may be determined

dictive value in protonic equilibria. For example,

by adding 0.5 equivalent of alkali per equivalent of

acid. The resulting pH will be the pK_a of the acid.

(1) When an acid is exactly half-neutralized, [A⁻] =

[HA]. Under these conditions,

The Henderson-Hasselbalch Equation

−

]

Describes the Behavior

pH=pK

+ log

+log

[HA]

of Weak Acids & Buffers

The Henderson-Hasselbalch equation is derived below.

Therefore, at half-neutralization, pH = pK_a.

A weak acid, HA, ionizes as follows:

(2) When the ratio [A⁻]/[HA] = 100:1,

HA = H+ +A⁻

−

]

pH=pK

log

The equilibrium constant for this dissociation is

[HA]

log 100 / 1=pK

−

[H+][A

]

[HA]

(3) When the ratio [A⁻]/[HA] = 1:10,

Cross-multiplication gives

pH=pK

+ log 1/ 10 =pK

+(−1)

−

][A

] =

[HA

]

If the equation is evaluated at ratios of [A⁻]/[HA]

ranging from 10³ to 10−3 and the calculated pH values

Divide both sides by [A⁻]:

are plotted, the resulting graph describes the titration

curve for a weak acid (Figure 2-4).

[HA]

]

−

]

Solutions of Weak Acids & Their Salts

Take the log of both sides:

Buffer Changes in pH

Solutions of weak acids or bases and their conjugates



[HA]

log [H

]= log

exhibit buffering, the ability to resist a change in pH



−





^a [A

]

following addition of strong acid or base. Since many

[HA]

metabolic reactions are accompanied by the release or

log

+log

−

uptake of protons, most intracellular reactions are

]

buffered. Oxidative metabolism produces CO2, the an-

hydride of carbonic acid, which if not buffered would

Multiply through by −1:

produce severe acidosis. Maintenance of a constant pH

involves buffering by phosphate, bicarbonate, and pro-

[HA]

−log [H

] =

−log

−

log

teins, which accept or release protons to resist a change

−

]

CHAPTER 2

1.0

to the pK_a. A solution of a weak acid and its conjugate

base buffers most effectively in the pH range pK_a ± 1.0

0.8

pH unit.

Figure 2-4 also illustrates the net charge on one

0.6

molecule of the acid as a function of pH. A fractional

charge of −0.5 does not mean that an individual mole-

0.4

cule bears a fractional charge, but the probability that a

given molecule has a unit negative charge is 0.5. Con-

0.2

sideration of the net charge on macromolecules as a

function of pH provides the basis for separatory tech-

niques such as ion exchange chromatography and elec-

trophoresis.

Figure 2-4. Titration curve for an acid of the type

Acid Strength Depends on

HA. The heavy dot in the center of the curve indicates

Molecular Structure

the pK_a 5.0.

Many acids of biologic interest possess more than one

dissociating group. The presence of adjacent negative

charge hinders the release of a proton from a nearby

in pH. For experiments using tissue extracts or en-

group, raising its pK_a. This is apparent from the pK_a

zymes, constant pH is maintained by the addition of

values for the three dissociating groups of phosphoric

buffers such as MES ([2-N-morpholino]ethanesulfonic

acid and citric acid (Table 2-2). The effect of adjacent

acid, pK_a 6.1), inorganic orthophosphate (pK_a2 7.2),

charge decreases with distance. The second pK_a for suc-

HEPES (N-hydroxyethylpiperazine-N9-2-ethanesulfonic

cinic acid, which has two methylene groups between its

acid, pK_a

6.8), or Tris

(tris[hydroxymethyl] amino-

carboxyl groups, is 5.6, whereas the second pK_a for glu-

methane, pK_a 8.3). The value of pK_a relative to the de-

sired pH is the major determinant of which buffer is se-

lected.

Buffering can be observed by using a pH meter

Table 2-2. Relative strengths of selected acids of

while titrating a weak acid or base (Figure 2-4). We

can also calculate the pH shift that accompanies addi-

biologic significance. Tabulated values are the pK_a

tion of acid or base to a buffered solution. In the exam-

values (−log of the dissociation constant) of

ple, the buffered solution (a weak acid, pK_a

= 5.0, and

selected monoprotic, diprotic, and triprotic acids.

its conjugate base) is initially at one of four pH values.

We will calculate the pH shift that results when 0.1

Monoprotic Acids

meq of KOH is added to 1 meq of each solution:

Formic

3.75

Lactic

3.86

Acetic

4.76

Initial pH

5.00

5.37

5.60

5.86

Ammonium ion

9.25

[A⁻]_initial

0.50

0.70

0.80

0.88

[HA]_initial

0.50

0.30

0.20

0.12

Diprotic Acids

([A⁻]/[HA])_initial

1.00

2.33

4.00

7.33

Carbonic

pK₁

6.37

Addition of 0.1 meq of KOH produces

pK₂

10.25

[A⁻]_final

0.60

0.80

0.90

0.98

Succinic

pK₁

4.21

[HA]_final

0.40

0.20

0.10

0.02

pK₂

5.64

([A⁻]/[HA])_final

1.50

4.00

9.00

49.0

Glutaric

pK₁

4.34

log ([A⁻]/[HA])_final

0.176

0.602

0.95

1.69

pK₂

5.41

Final pH

5.18

5.60

5.95

6.69

Triprotic Acids

∆pH

0.18

0.60

0.95

1.69

Phosphoric

pK₁

2.15

pK₂

6.82

pK₃

12.38

Citric

pK₁

3.08

Notice that the change in pH per milliequivalent of

pK₂

4.74

OH⁻ added depends on the initial pH. The solution re-

pK₃

5.40

sists changes in pH most effectively at pH values close

WATER & pH

taric acid, which has one additional methylene group,

• Macromolecules exchange internal surface hydrogen

is 5.4.

bonds for hydrogen bonds to water. Entropic forces

dictate that macromolecules expose polar regions to

pK_a Values Depend on the Properties

an aqueous interface and bury nonpolar regions.

of the Medium

• Salt bonds, hydrophobic interactions, and van der

Waals forces participate in maintaining molecular

The pK_a of a functional group is also profoundly influ-

structure.

enced by the surrounding medium. The medium may

• pH is the negative log of [H⁺]. A low pH character-

either raise or lower the pK_a depending on whether the

izes an acidic solution, and a high pH denotes a basic

undissociated acid or its conjugate base is the charged

solution.

species. The effect of dielectric constant on pK_a may be

observed by adding ethanol to water. The pK_a of a car-

• The strength of weak acids is expressed by pK_a, the

negative log of the acid dissociation constant. Strong

boxylic acid increases, whereas that of an amine decreases

because ethanol decreases the ability of water to solvate

acids have low pK_a values and weak acids have high

pK_a values.

a charged species. The pK_a values of dissociating groups

in the interiors of proteins thus are profoundly affected

• Buffers resist a change in pH when protons are pro-

by their local environment, including the presence or

duced or consumed. Maximum buffering capacity

absence of water.

occurs ± 1 pH unit on either side of pK_a. Physiologic

buffers include bicarbonate, orthophosphate, and

proteins.

SUMMARY

REFERENCES

• Water forms hydrogen-bonded clusters with itself and

with other proton donors or acceptors. Hydrogen

Segel IM: Biochemical Calculations. Wiley, 1968.

bonds account for the surface tension, viscosity, liquid

Wiggins PM: Role of water in some biological processes. Microbiol

state at room temperature, and solvent power of water.

Rev 1990;54:432.

• Compounds that contain O, N, or S can serve as hy-

drogen bond donors or acceptors.

SECTION I

Structures & Functions

of Proteins & Enzymes

Amino Acids & Peptides

Victor W. Rodwell, PhD, & Peter J. Kennelly, PhD

BIOMEDICAL IMPORTANCE

more than 20 amino acids, its redundancy limits the

available codons to the 20 L-α-amino acids listed in

In addition to providing the monomer units from which

Table 3-1, classified according to the polarity of their R

the long polypeptide chains of proteins are synthesized,

groups. Both one- and three-letter abbreviations for each

the L-α-amino acids and their derivatives participate in

amino acid can be used to represent the amino acids in

cellular functions as diverse as nerve transmission and

peptides (Table 3-1). Some proteins contain additional

the biosynthesis of porphyrins, purines, pyrimidines,

amino acids that arise by modification of an amino acid

and urea. Short polymers of amino acids called peptides

already present in a peptide. Examples include conver-

perform prominent roles in the neuroendocrine system

sion of peptidyl proline and lysine to 4-hydroxyproline

as hormones, hormone-releasing factors, neuromodula-

and 5-hydroxylysine; the conversion of peptidyl gluta-

tors, or neurotransmitters. While proteins contain only

mate to γ-carboxyglutamate; and the methylation,

L-α-amino acids, microorganisms elaborate peptides

formylation, acetylation, prenylation, and phosphoryla-

that contain both D- and L-α-amino acids. Several of

tion of certain aminoacyl residues. These modifications

these peptides are of therapeutic value, including the an-

extend the biologic diversity of proteins by altering their

tibiotics bacitracin and gramicidin A and the antitumor

solubility, stability, and interaction with other proteins.

agent bleomycin. Certain other microbial peptides are

toxic. The cyanobacterial peptides microcystin and

nodularin are lethal in large doses, while small quantities

Only L-

-Amino Acids Occur in Proteins

promote the formation of hepatic tumors. Neither hu-

With the sole exception of glycine, the α-carbon of

mans nor any other higher animals can synthesize 10 of

amino acids is chiral. Although some protein amino

the 20 common L-α-amino acids in amounts adequate

acids are dextrorotatory and some levorotatory, all share

to support infant growth or to maintain health in adults.

the absolute configuration of L-glyceraldehyde and thus

Consequently, the human diet must contain adequate

are L-α-amino acids. Several free L-α-amino acids fulfill

quantities of these nutritionally essential amino acids.

important roles in metabolic processes. Examples in-

clude ornithine, citrulline, and argininosuccinate that

PROPERTIES OF AMINO ACIDS

participate in urea synthesis; tyrosine in formation of

thyroid hormones; and glutamate in neurotransmitter

The Genetic Code Specifies

biosynthesis. D-Amino acids that occur naturally in-

20 L-

-Amino Acids

clude free D-serine and D-aspartate in brain tissue,

Of the over 300 naturally occurring amino acids, 20 con-

D-alanine and D-glutamate in the cell walls of gram-

stitute the monomer units of proteins. While a nonre-

positive bacteria, and D-amino acids in some nonmam-

dundant three-letter genetic code could accommodate

malian peptides and certain antibiotics.

Table 3-1. L- α-Amino acids present in proteins.

Name

Symbol

Structural Formula

pK₁

pK₂

pK₃

With Aliphatic Side Chains

-COOH

-NH3+

R Group

Glycine

Gly [G]

2.4

9.8

H CH COO^-

Alanine

Ala [A]

2.4

9.9

CH₃

CH COO^-

H₃C

CH COO^-

Valine

Val [V]

2.2

9.7

H₃C

NH₃

H₃C

CH₂

COO^-

Leucine

Leu [L]

2.3

9.7

H₃C

NH₃

CH₃

CH₂

Isoleucine

Ile [I]

CH CH COO^-

2.3

9.8

CH₃

With Side Chains Containing Hydroxylic (OH) Groups

Serine

Ser [S]

2.2

9.2

about 13

COO^-

NH₃

Threonine

Thr [T]

2.1

9.1

about 13

COO^-

NH₃

Tyrosine

Tyr [Y]

See below.

With Side Chains Containing Sulfur Atoms

Cysteine

Cys [C]

CH₂

COO^-

1.9

10.8

8.3

NH₃

Methionine

Met [M]

CH₂

COO^-

2.1

9.3

S CH

NH₃

With Side Chains Containing Acidic Groups or Their Amides

Aspartic acid

Asp [D]

2.0

9.9

3.9

^-OOC

CH₂

CH COO

Asparagine

Asn [N]

2.1

8.8

H₂N

CH₂

COO^-

NH₃

^–OOC

CH₂

CH COO^-

Glutamic acid

Glu [E]

2.1

9.5

4.1

H₂N

CH₂

COO^-

Glutamine

Gln [Q]

2.2

9.1

NH₃

(continued)

CHAPTER 3

Table 3-1. L-α-Amino acids present in proteins. (continued)

Name

Symbol

Structural Formula

pK₁

pK₂

pK₃

With Side Chains Containing Basic Groups

-COOH

-NH3+

R Group

Arginine

Arg [R]

1.8

9.0

12.5

CH₂

CH COO^-

NH₂

NH₃

NH₂

CH₂

CH COO

Lysine

Lys [K]

2.2

9.2

10.8

NH₃

CH₂

CH COO^-

Histidine

His [H]

1.8

9.3

6.0

Containing Aromatic Rings

Histidine

His [H]

See above.

CH₂

CH COO^-

Phenylalanine

Phe [F]

2.2

9.2

Tyrosine

Tyr [Y]

2.2

9.1

10.1

CH₂

COO^-

Tryptophan

Trp [W]

2.4

9.4

CH₂

COO^-

Imino Acid

Proline

Pro [P]

2.0

10.6

COO^-

H₂

Amino Acids May Have Positive, Negative,

Molecules that contain an equal number of ioniz-

or Zero Net Charge

able groups of opposite charge and that therefore bear

no net charge are termed zwitterions. Amino acids in

Charged and uncharged forms of the ionizable

blood and most tissues thus should be represented as in

COOH and NH3+ weak acid groups exist in solu-

A, below.

tion in protonic equilibrium:

NH₃

NH₂

R—COOH =R—COO−+H

R—NH

R—

While both RCOOH and RNH3+ are weak acids,

RCOOH is a far stronger acid than RNH3+. At

physiologic pH (pH 7.4), carboxyl groups exist almost

Structure B cannot exist in aqueous solution because at

entirely as RCOO⁻ and amino groups predomi-

any pH low enough to protonate the carboxyl group

nantly as RNH3+. Figure 3-1 illustrates the effect of

the amino group would also be protonated. Similarly,

pH on the charged state of aspartic acid.

at any pH sufficiently high for an uncharged amino

AMINO ACIDS & PEPTIDES

H⁺

O^-

pK₁ = 2.09

pK₂ = 3.86

pK₃ = 9.82

NH₃₊

(α-COOH)

NH₃₊

(β-COOH)

NH₃₊

(— NH₃₊)

NH₂

^-O

In strong acid

Around pH 3;

Around pH 6-8;

In strong alkali

(below pH 1);

net charge = 0

net charge = -1

(above pH 11);

net charge = +1

net charge = -2

Figure 3-1. Protonic equilibria of aspartic acid.

group to predominate, a carboxyl group will be present

At Its Isoelectric pH (pI), an Amino Acid

as RCOO⁻. The uncharged representation B (above)

Bears No Net Charge

is, however, often used for reactions that do not involve

The isoelectric species is the form of a molecule that

protonic equilibria.

has an equal number of positive and negative charges

and thus is electrically neutral. The isoelectric pH, also

pK_a Values Express the Strengths

called the pI, is the pH midway between pK_a values on

of Weak Acids

either side of the isoelectric species. For an amino acid

such as alanine that has only two dissociating groups,

The acid strengths of weak acids are expressed as their

there is no ambiguity. The first pK_a (R COOH) is

pK_a (Table 3-1). The imidazole group of histidine and

2.35 and the second pK_a (RNH3+) is 9.69. The iso-

the guanidino group of arginine exist as resonance hy-

electric pH (pI) of alanine thus is

brids with positive charge distributed between both ni-

trogens (histidine) or all three nitrogens (arginine) (Fig-

ure

3-2). The net charge on an amino acid—the

pK +pK

2.35+9.69

pl=

6.02

algebraic sum of all the positively and negatively

charged groups present—depends upon the pK_a values

of its functional groups and on the pH of the surround-

For polyfunctional acids, pI is also the pH midway be-

ing medium. Altering the charge on amino acids and

tween the pK_a values on either side of the isoionic

their derivatives by varying the pH facilitates the physi-

species. For example, the pI for aspartic acid is

cal separation of amino acids, peptides, and proteins

(see Chapter 4).

pK +pK

2.09+

3.96

pl=

3.02

For lysine, pI is calculated from:

pK +pK

N H

pl=

Similar considerations apply to all polyprotic acids (eg,

proteins), regardless of the number of dissociating

groups present. In the clinical laboratory, knowledge of

the pI guides selection of conditions for electrophoretic

separations. For example, electrophoresis at pH 7.0 will

separate two molecules with pI values of 6.0 and 8.0

C NH₂

because at pH 8.0 the molecule with a pI of 6.0 will

NH₂

have a net positive charge, and that with pI of 8.0 a net

negative charge. Similar considerations apply to under-

Figure 3-2. Resonance hybrids of the protonated

standing chromatographic separations on ionic sup-

forms of the R groups of histidine and arginine.

ports such as DEAE cellulose (see Chapter 4).

CHAPTER 3

pK_a Values Vary With the Environment

THE

-R GROUPS DETERMINE THE

The environment of a dissociable group affects its pK_a.

PROPERTIES OF AMINO ACIDS

The pK_a values of the R groups of free amino acids in

Since glycine, the smallest amino acid, can be accommo-

aqueous solution (Table 3-1) thus provide only an ap-

dated in places inaccessible to other amino acids, it often

proximate guide to the pK_a values of the same amino

occurs where peptides bend sharply. The hydrophobic R

acids when present in proteins. A polar environment

groups of alanine, valine, leucine, and isoleucine and the

favors the charged form (R COO⁻ or RNH3+),

aromatic R groups of phenylalanine, tyrosine, and tryp-

and a nonpolar environment favors the uncharged form

tophan typically occur primarily in the interior of cy-

(R COOH or RNH₂). A nonpolar environment

tosolic proteins. The charged R groups of basic and

thus raises the pK_a of a carboxyl group (making it a

acidic amino acids stabilize specific protein conforma-

weaker acid) but lowers that of an amino group (making

tions via ionic interactions, or salt bonds. These bonds

it a stronger acid). The presence of adjacent charged

also function in “charge relay” systems during enzymatic

groups can reinforce or counteract solvent effects. The

catalysis and electron transport in respiring mitochon-

pK_a of a functional group thus will depend upon its lo-

dria. Histidine plays unique roles in enzymatic catalysis.

cation within a given protein. Variations in pK_a can en-

The pK_a of its imidazole proton permits it to function at

compass whole pH units (Table 3-2). pK_a values that

neutral pH as either a base or an acid catalyst. The pri-

diverge from those listed by as much as three pH units

mary alcohol group of serine and the primary thioalco-

are common at the active sites of enzymes. An extreme

hol (SH) group of cysteine are excellent nucleophiles

example, a buried aspartic acid of thioredoxin, has a

and can function as such during enzymatic catalysis.

pK_a above 9—a shift of over six pH units!

However, the secondary alcohol group of threonine,

while a good nucleophile, does not fulfill an analogous

The Solubility and Melting Points

role in catalysis. The  OH groups of serine, tyrosine,

of Amino Acids Reflect

and threonine also participate in regulation of the activ-

Their Ionic Character

ity of enzymes whose catalytic activity depends on the

phosphorylation state of these residues.

The charged functional groups of amino acids ensure

that they are readily solvated by—and thus soluble in—

polar solvents such as water and ethanol but insoluble

FUNCTIONAL GROUPS DICTATE THE

in nonpolar solvents such as benzene, hexane, or ether.

CHEMICAL REACTIONS OF AMINO ACIDS

Similarly, the high amount of energy required to dis-

Each functional group of an amino acid exhibits all of

rupt the ionic forces that stabilize the crystal lattice

its characteristic chemical reactions. For carboxylic acid

account for the high melting points of amino acids

groups, these reactions include the formation of esters,

(> 200 °C).

amides, and acid anhydrides; for amino groups, acyla-

Amino acids do not absorb visible light and thus are

tion, amidation, and esterification; and for  OH and

colorless. However, tyrosine, phenylalanine, and espe-

 SH groups, oxidation and esterification. The most

cially tryptophan absorb high-wavelength

(250-290

important reaction of amino acids is the formation of a

nm) ultraviolet light. Tryptophan therefore makes the

peptide bond (shaded blue).

major contribution to the ability of most proteins to

absorb light in the region of 280 nm.

⁺H3N

O^-

Table 3-2. Typical range of pK_a values for

ionizable groups in proteins.

Alanyl

Cysteinyl

Valine

Dissociating Group

pK_a Range

α-Carboxyl

3.5-4.0

Amino Acid Sequence Determines

Non-α COOH of Asp or Glu

4.0-4.8

Primary Structure

Imidazole of His

6.5-7.4

SH of Cys

8.5-9.0

The number and order of all of the amino acid residues

OH of Tyr

9.5-10.5

in a polypeptide constitute its primary structure.

α-Amino

8.0-9.0

Amino acids present in peptides are called aminoacyl

ε-Amino of Lys

9.8-10.4

residues and are named by replacing the -ate or -ine suf-

Guanidinium of Arg

~12.0

fixes of free amino acids with -yl (eg, alanyl, aspartyl, ty-

AMINO ACIDS & PEPTIDES

rosyl). Peptides are then named as derivatives of the

carboxyl terminal aminoacyl residue. For example, Lys-

CH₂

Leu-Tyr-Gln is called lysyl-leucyl-tyrosyl-glutamine.

The -ine ending on glutamine indicates that its α-car-

boxyl group is not involved in peptide bond formation.

CH₂

COO^-

Peptide Structures Are Easy to Draw

NH₃₊

Prefixes like tri- or octa- denote peptides with three or

eight residues, respectively, not those with three or

COO^-

eight peptide bonds. By convention, peptides are writ-

ten with the residue that bears the free α-amino group

Figure 3-3. Glutathione (γ-glutamyl-cysteinyl-

at the left. To draw a peptide, use a zigzag to represent

glycine). Note the non-α peptide bond that links

the main chain or backbone. Add the main chain atoms,

Glu to Cys.

which occur in the repeating order: α-nitrogen, α-car-

bon, carbonyl carbon. Now add a hydrogen atom to

each α-carbon and to each peptide nitrogen, and an

releasing hormone (TRH) is cyclized to pyroglutamic

oxygen to the carbonyl carbon. Finally, add the appro-

acid, and the carboxyl group of the carboxyl terminal

priate R groups (shaded) to each α-carbon atom.

prolyl residue is amidated. Peptides elaborated by fungi,

bacteria, and lower animals can contain nonprotein

amino acids. The antibiotics tyrocidin and gramicidin S

Cα

are cyclic polypeptides that contain D-phenylalanine

and ornithine. The heptapeptide opioids dermorphin

H₃C H

and deltophorin in the skin of South American tree

⁺H₃N

COO^-

frogs contain D-tyrosine and D-alanine.

CH₂

Peptides Are Polyelectrolytes

^-OOC

The peptide bond is uncharged at any pH of physiologic

interest. Formation of peptides from amino acids is

Three-letter abbreviations linked by straight lines

therefore accompanied by a net loss of one positive and

represent an unambiguous primary structure. Lines are

one negative charge per peptide bond formed. Peptides

omitted for single-letter abbreviations.

nevertheless are charged at physiologic pH owing to their

Glu - Ala - Lys - Gly - Tyr - Ala

carboxyl and amino terminal groups and, where present,

their acidic or basic R groups. As for amino acids, the net

E A K G Y A

charge on a peptide depends on the pH of its environ-

Where there is uncertainty about the order of a portion

ment and on the pK_a values of its dissociating groups.

of a polypeptide, the questionable residues are enclosed

in brackets and separated by commas.

The Peptide Bond Has Partial

Glu - Lys - (Ala, Gly, Tyr) - His - Ala

Double-Bond Character

Although peptides are written as if a single bond linked

the α-carboxyl and α-nitrogen atoms, this bond in fact

Some Peptides Contain Unusual

exhibits partial double-bond character:

Amino Acids

O^-

In mammals, peptide hormones typically contain only

the α-amino acids of proteins linked by standard pep-

tide bonds. Other peptides may, however, contain non-

protein amino acids, derivatives of the protein amino

acids, or amino acids linked by an atypical peptide

bond. For example, the amino terminal glutamate of

There thus is no freedom of rotation about the bond

glutathione, which participates in protein folding and

that connects the carbonyl carbon and the nitrogen of a

in the metabolism of xenobiotics

(Chapter

53), is

peptide bond. Consequently, all four of the colored

linked to cysteine by a non-α peptide bond (Figure

atoms of Figure 3-4 are coplanar. The imposed semi-

3-3). The amino terminal glutamate of thyrotropin-

rigidity of the peptide bond has important conse-

CHAPTER 3

mixture of free amino acids is then treated with 6-amino-

R′

N-hydroxysuccinimidyl carbamate, which reacts with

their α-amino groups, forming fluorescent derivatives

that are then separated and identified using high-pressure

122°

121° C

liquid chromatography (see Chapter 5). Ninhydrin, also

120°

widely used for detecting amino acids, forms a purple

117°

110°

120°

product with α-amino acids and a yellow adduct with

the imine groups of proline and hydroxyproline.

R′′

SUMMARY

0.36 nm

•

Both D-amino acids and non-α-amino acids occur

in nature, but only L-α-amino acids are present in

Figure 3-4. Dimensions of a fully extended poly-

proteins.

peptide chain. The four atoms of the peptide bond

•

All amino acids possess at least two weakly acidic

(colored blue) are coplanar. The unshaded atoms are

functional groups, RNH3+ and R COOH.

the α-carbon atom, the α-hydrogen atom, and the α-R

Many also possess additional weakly acidic functional

group of the particular amino acid. Free rotation can

groups such as  OH, SH, guanidino, or imid-

occur about the bonds that connect the α-carbon with

azole groups.

the α-nitrogen and with the α-carbonyl carbon (blue

•

The pK_a values of all functional groups of an amino

arrows). The extended polypeptide chain is thus a semi-

acid dictate its net charge at a given pH. pI is the pH

rigid structure with two-thirds of the atoms of the back-

at which an amino acid bears no net charge and thus

bone held in a fixed planar relationship one to another.

does not move in a direct current electrical field.

The distance between adjacent α-carbon atoms is 0.36

•

Of the biochemical reactions of amino acids, the

nm (3.6 Å). The interatomic distances and bond angles,

most important is the formation of peptide bonds.

which are not equivalent, are also shown. (Redrawn and

•

The R groups of amino acids determine their unique

reproduced, with permission, from Pauling L, Corey LP,

biochemical functions. Amino acids are classified as

Branson HR: The structure of proteins: Two hydrogen-

basic, acidic, aromatic, aliphatic, or sulfur-containing

bonded helical configurations of the polypeptide chain.

based on the properties of their R groups.

Proc Natl Acad Sci U S A 1951;37:205.)

•

Peptides are named for the number of amino acid

residues present, and as derivatives of the carboxyl

terminal residue. The primary structure of a peptide

quences for higher orders of protein structure. Encir-

is its amino acid sequence, starting from the amino-

cling arrows (Figure 3 - 4) indicate free rotation about

terminal residue.

the remaining bonds of the polypeptide backbone.

•

The partial double-bond character of the bond that

links the carbonyl carbon and the nitrogen of a pep-

Noncovalent Forces Constrain Peptide

tide renders four atoms of the peptide bond coplanar

Conformations

and restricts the number of possible peptide confor-

Folding of a peptide probably occurs coincident with

mations.

its biosynthesis (see Chapter 38). The physiologically

active conformation reflects the amino acid sequence,

REFERENCES

steric hindrance, and noncovalent interactions (eg, hy-

drogen bonding, hydrophobic interactions) between

Doolittle RF: Reconstructing history with amino acid sequences.

Protein Sci 1992;1:191.

residues. Common conformations include α-helices

and β-pleated sheets (see Chapter 5).

Kreil G: D-Amino acids in animal peptides. Annu Rev Biochem

1997;66:337.

Nokihara K, Gerhardt J: Development of an improved automated

ANALYSIS OF THE AMINO ACID

gas-chromatographic chiral analysis system: application to

CONTENT OF BIOLOGIC MATERIALS

non-natural amino acids and natural protein hydrolysates.

Chirality 2001;13:431.

In order to determine the identity and quantity of each

Sanger F: Sequences, sequences, and sequences. Annu Rev Biochem

amino acid in a sample of biologic material, it is first nec-

1988;57:1.

essary to hydrolyze the peptide bonds that link the amino

Wilson NA et al: Aspartic acid 26 in reduced Escherichia coli thiore-

acids together by treatment with hot HCl. The resulting

doxin has a pK_a greater than 9. Biochemistry 1995;34:8931.

Proteins: Determination of

Primary Structure

Victor W. Rodwell, PhD, & Peter J. Kennelly, PhD

BIOMEDICAL IMPORTANCE

Column Chromatography

Proteins perform multiple critically important roles. An

Column chromatography of proteins employs as the

internal protein network, the cytoskeleton

(Chapter

stationary phase a column containing small spherical

49), maintains cellular shape and physical integrity.

beads of modified cellulose, acrylamide, or silica whose

Actin and myosin filaments form the contractile ma-

surface typically has been coated with chemical func-

chinery of muscle (Chapter 49). Hemoglobin trans-

tional groups. These stationary phase matrices interact

ports oxygen (Chapter 6), while circulating antibodies

with proteins based on their charge, hydrophobicity,

search out foreign invaders (Chapter 50). Enzymes cat-

and ligand-binding properties. A protein mixture is ap-

alyze reactions that generate energy, synthesize and de-

plied to the column and the liquid mobile phase is per-

grade biomolecules, replicate and transcribe genes,

colated through it. Small portions of the mobile phase

process mRNAs, etc (Chapter 7). Receptors enable cells

or eluant are collected as they emerge (Figure 4-1).

to sense and respond to hormones and other environ-

mental cues (Chapters 42 and 43). An important goal

Partition Chromatography

of molecular medicine is the identification of proteins

whose presence, absence, or deficiency is associated

Column chromatographic separations depend on the

with specific physiologic states or diseases. The primary

relative affinity of different proteins for a given station-

sequence of a protein provides both a molecular finger-

ary phase and for the mobile phase. Association be-

print for its identification and information that can be

tween each protein and the matrix is weak and tran-

used to identify and clone the gene or genes that en-

sient. Proteins that interact more strongly with the

code it.

stationary phase are retained longer. The length of time

that a protein is associated with the stationary phase is a

function of the composition of both the stationary and

mobile phases. Optimal separation of the protein of in-

PROTEINS & PEPTIDES MUST BE

terest from other proteins thus can be achieved by care-

PURIFIED PRIOR TO ANALYSIS

ful manipulation of the composition of the two phases.

Highly purified protein is essential for determination of

its amino acid sequence. Cells contain thousands of dif-

Size Exclusion Chromatography

ferent proteins, each in widely varying amounts. The

isolation of a specific protein in quantities sufficient for

Size exclusion—or gel filtration—chromatography sep-

analysis thus presents a formidable challenge that may

arates proteins based on their Stokes radius, the diam-

require multiple successive purification techniques.

eter of the sphere they occupy as they tumble in solu-

Classic approaches exploit differences in relative solu-

tion. The Stokes radius is a function of molecular mass

bility of individual proteins as a function of pH (iso-

and shape. A tumbling elongated protein occupies a

electric precipitation), polarity

(precipitation with

larger volume than a spherical protein of the same mass.

ethanol or acetone), or salt concentration (salting out

Size exclusion chromatography employs porous beads

with ammonium sulfate). Chromatographic separations

(Figure 4-2). The pores are analogous to indentations

partition molecules between two phases, one mobile

in a river bank. As objects move downstream, those that

and the other stationary. For separation of amino acids

enter an indentation are retarded until they drift back

or sugars, the stationary phase, or matrix, may be a

into the main current. Similarly, proteins with Stokes

sheet of filter paper (paper chromatography) or a thin

radii too large to enter the pores (excluded proteins) re-

layer of cellulose, silica, or alumina (thin-layer chro-

main in the flowing mobile phase and emerge before

matography; TLC).

proteins that can enter the pores (included proteins).

CHAPTER 4

Figure 4-1. Components of a simple liquid chromatography apparatus. R: Reser-

voir of mobile phase liquid, delivered either by gravity or using a pump. C: Glass or

plastic column containing stationary phase. F: Fraction collector for collecting por-

tions, called fractions, of the eluant liquid in separate test tubes.

Proteins thus emerge from a gel filtration column in de-

Ion Exchange Chromatography

scending order of their Stokes radii.

In ion exchange chromatography, proteins interact with

the stationary phase by charge-charge interactions. Pro-

Absorption Chromatography

teins with a net positive charge at a given pH adhere to

For absorption chromatography, the protein mixture is

beads with negatively charged functional groups such as

applied to a column under conditions where the pro-

carboxylates or sulfates (cation exchangers). Similarly,

tein of interest associates with the stationary phase so

proteins with a net negative charge adhere to beads with

tightly that its partition coefficient is essentially unity.

positively charged functional groups, typically tertiary or

Nonadhering molecules are first eluted and discarded.

quaternary amines (anion exchangers). Proteins, which

Proteins are then sequentially released by disrupting the

are polyanions, compete against monovalent ions for

forces that stabilize the protein-stationary phase com-

binding to the support—thus the term “ion exchange.”

plex, most often by using a gradient of increasing salt

For example, proteins bind to diethylaminoethyl

concentration. The composition of the mobile phase is

(DEAE) cellulose by replacing the counter-ions (gener-

altered gradually so that molecules are selectively re-

ally Cl⁻ or CH₃COO⁻) that neutralize the protonated

leased in descending order of their affinity for the sta-

amine. Bound proteins are selectively displaced by grad-

tionary phase.

ually raising the concentration of monovalent ions in

PROTEINS: DETERMINATION OF PRIMARY STRUCTURE

Figure 4-2. Size-exclusion chromatography. A: A mixture of large molecules

(diamonds) and small molecules (circles) are applied to the top of a gel filtration

column. B: Upon entering the column, the small molecules enter pores in the sta-

tionary phase matrix from which the large molecules are excluded. C: As the mo-

bile phase flows down the column, the large, excluded molecules flow with it

while the small molecules, which are temporarily sheltered from the flow when in-

side the pores, lag farther and farther behind.

the mobile phase. Proteins elute in inverse order of the

fied by affinity chromatography using immobilized sub-

strength of their interactions with the stationary phase.

strates, products, coenzymes, or inhibitors. In theory,

Since the net charge on a protein is determined by

only proteins that interact with the immobilized ligand

the pH (see Chapter 3), sequential elution of proteins

adhere. Bound proteins are then eluted either by compe-

may be achieved by changing the pH of the mobile

tition with soluble ligand or, less selectively, by disrupt-

phase. Alternatively, a protein can be subjected to con-

ing protein-ligand interactions using urea, guanidine

secutive rounds of ion exchange chromatography, each

hydrochloride, mildly acidic pH, or high salt concentra-

at a different pH, such that proteins that co-elute at one

tions. Stationary phase matrices available commercially

pH elute at different salt concentrations at another pH.

contain ligands such as NAD⁺ or ATP analogs. Among

the most powerful and widely applicable affinity matri-

Hydrophobic Interaction Chromatography

ces are those used for the purification of suitably modi-

fied recombinant proteins. These include a Ni2+ matrix

Hydrophobic interaction chromatography separates

that binds proteins with an attached polyhistidine “tag”

proteins based on their tendency to associate with a sta-

and a glutathione matrix that binds a recombinant pro-

tionary phase matrix coated with hydrophobic groups

tein linked to glutathione S-transferase.

(eg, phenyl Sepharose, octyl Sepharose). Proteins with

exposed hydrophobic surfaces adhere to the matrix via

hydrophobic interactions that are enhanced by a mobile

Peptides Are Purified by Reversed-Phase

phase of high ionic strength. Nonadherent proteins are

High-Pressure Chromatography

first washed away. The polarity of the mobile phase is

The stationary phase matrices used in classic column

then decreased by gradually lowering the salt concentra-

chromatography are spongy materials whose compress-

tion. If the interaction between protein and stationary

ibility limits flow of the mobile phase. High-pressure liq-

phase is particularly strong, ethanol or glycerol may be

uid chromatography (HPLC) employs incompressible

added to the mobile phase to decrease its polarity and

silica or alumina microbeads as the stationary phase and

further weaken hydrophobic interactions.

pressures of up to a few thousand psi. Incompressible

matrices permit both high flow rates and enhanced reso-

Affinity Chromatography

lution. HPLC can resolve complex mixtures of lipids or

Affinity chromatography exploits the high selectivity of

peptides whose properties differ only slightly. Reversed-

most proteins for their ligands. Enzymes may be puri-

phase HPLC exploits a hydrophobic stationary phase of

CHAPTER 4

aliphatic polymers 3-18 carbon atoms in length. Peptide

through the acrylamide matrix determines the rate of

mixtures are eluted using a gradient of a water-miscible

migration. Since large complexes encounter greater re-

organic solvent such as acetonitrile or methanol.

sistance, polypeptides separate based on their relative

molecular mass (M_r). Individual polypeptides trapped

Protein Purity Is Assessed by

in the acrylamide gel are visualized by staining with

dyes such as Coomassie blue (Figure 4-4).

Polyacrylamide Gel Electrophoresis

(PAGE)

Isoelectric Focusing (IEF)

The most widely used method for determining the pu-

rity of a protein is SDS-PAGE—polyacrylamide gel

Ionic buffers called ampholytes and an applied electric

electrophoresis (PAGE) in the presence of the anionic

field are used to generate a pH gradient within a poly-

detergent sodium dodecyl sulfate (SDS). Electrophore-

acrylamide matrix. Applied proteins migrate until they

sis separates charged biomolecules based on the rates at

reach the region of the matrix where the pH matches

which they migrate in an applied electrical field. For

their isoelectric point (pI), the pH at which a peptide’s

SDS-PAGE, acrylamide is polymerized and cross-

net charge is zero. IEF is used in conjunction with SDS-

linked to form a porous matrix. SDS denatures and

PAGE for two-dimensional electrophoresis, which sepa-

binds to proteins at a ratio of one molecule of SDS per

rates polypeptides based on pI in one dimension and

two peptide bonds. When used in conjunction with 2-

based on M_r in the second (Figure 4-5). Two-dimen-

mercaptoethanol or dithiothreitol to reduce and break

sional electrophoresis is particularly well suited for sepa-

disulfide bonds (Figure 4 -3), SDS separates the com-

rating the components of complex mixtures of proteins.

ponent polypeptides of multimeric proteins. The large

number of anionic SDS molecules, each bearing a

SANGER WAS THE FIRST TO DETERMINE

charge of

−1, on each polypeptide overwhelms the

THE SEQUENCE OF A POLYPEPTIDE

charge contributions of the amino acid functional

groups. Since the charge-to-mass ratio of each SDS-

Mature insulin consists of the 21-residue A chain and

polypeptide complex is approximately equal, the physi-

the 30-residue B chain linked by disulfide bonds. Fred-

cal resistance each peptide encounters as it moves

erick Sanger reduced the disulfide bonds (Figure 4-3),

HCOOH

C₂H₅

SO −

Figure 4-4. Use of SDS-PAGE to observe successive

purification of a recombinant protein. The gel was

Figure 4-3. Oxidative cleavage of adjacent polypep-

stained with Coomassie blue. Shown are protein stan-

tide chains linked by disulfide bonds (shaded) by per-

dards (lane S) of the indicated mass, crude cell extract

formic acid (left) or reductive cleavage by β-mercap-

(E), high-speed supernatant liquid (H), and the DEAE-

toethanol (right) forms two peptides that contain

Sepharose fraction (D). The recombinant protein has a

cysteic acid residues or cysteinyl residues, respectively.

mass of about 45 kDa.

PROTEINS: DETERMINATION OF PRIMARY STRUCTURE

pH = 3

pH = 10

IEF

SDS

PAGE

Figure 4-5. Two-dimensional IEF-SDS-PAGE. The

gel was stained with Coomassie blue. A crude bacter-

ial extract was first subjected to isoelectric focusing

(IEF) in a pH 3-10 gradient. The IEF gel was then

placed horizontally on the top of an SDS gel, and the

proteins then further resolved by SDS-PAGE. Notice

the greatly improved resolution of distinct polypep-

tides relative to ordinary SDS-

PAGE gel (Figure 4-4).

separated the A and B chains, and cleaved each chain

Large Polypeptides Are First Cleaved Into

into smaller peptides using trypsin, chymotrypsin, and

Smaller Segments

pepsin. The resulting peptides were then isolated and

While the first 20-30 residues of a peptide can readily

treated with acid to hydrolyze peptide bonds and gener-

be determined by the Edman method, most polypep-

ate peptides with as few as two or three amino acids.

tides contain several hundred amino acids. Conse-

Each peptide was reacted with 1-fluoro-2,4-dinitroben-

quently, most polypeptides must first be cleaved into

zene (Sanger’s reagent), which derivatizes the exposed

smaller peptides prior to Edman sequencing. Cleavage

α-amino group of amino terminal residues. The amino

also may be necessary to circumvent posttranslational

acid content of each peptide was then determined.

modifications that render a protein’s α-amino group

While the ε-amino group of lysine also reacts with

“blocked”, or unreactive with the Edman reagent.

Sanger’s reagent, amino-terminal lysines can be distin-

It usually is necessary to generate several peptides

guished from those at other positions because they react

using more than one method of cleavage. This reflects

with 2 mol of Sanger’s reagent. Working backwards to

both inconsistency in the spacing of chemically or enzy-

larger fragments enabled Sanger to determine the com-

matically susceptible cleavage sites and the need for sets

plete sequence of insulin, an accomplishment for which

of peptides whose sequences overlap so one can infer

he received a Nobel Prize in 1958.

the sequence of the polypeptide from which they derive

(Figure 4-7). Reagents for the chemical or enzymatic

THE EDMAN REACTION ENABLES

cleavage of proteins include cyanogen bromide (CNBr),

PEPTIDES & PROTEINS

trypsin, and Staphylococcus aureus V8 protease (Table

TO BE SEQUENCED

4-1). Following cleavage, the resulting peptides are pu-

rified by reversed-phase HPLC—or occasionally by

Pehr Edman introduced phenylisothiocyanate (Edman’s

SDS-PAGE—and sequenced.

reagent) to selectively label the amino-terminal residue

of a peptide. In contrast to Sanger’s reagent, the

phenylthiohydantoin (PTH) derivative can be removed

MOLECULAR BIOLOGY HAS

under mild conditions to generate a new amino terminal

REVOLUTIONIZED THE DETERMINATION

residue (Figure 4-6). Successive rounds of derivatization

OF PRIMARY STRUCTURE

with Edman’s reagent can therefore be used to sequence

many residues of a single sample of peptide. Edman se-

Knowledge of DNA sequences permits deduction of

quencing has been automated, using a thin film or solid

the primary structures of polypeptides. DNA sequenc-

matrix to immobilize the peptide and HPLC to identify

ing requires only minute amounts of DNA and can

PTH amino acids. Modern gas-phase sequencers can

readily yield the sequence of hundreds of nucleotides.

analyze as little as a few picomoles of peptide.

To clone and sequence the DNA that encodes a partic-

CHAPTER 4

Peptide X Peptide Y

Peptide Z

NH₂

Carboxyl terminal

Amino terminal

portion of

peptide X

peptide Y

R′

Figure 4-7. The overlapping peptide Z is used to de-

Phenylisothiocyanate (Edman reagent)

duce that peptides X and Y are present in the original

and a peptide

protein in the order X → Y, not Y ← X.

sequence can be determined and the genetic code used

to infer the primary structure of the encoded poly-

peptide.

The hybrid approach enhances the speed and effi-

ciency of primary structure analysis and the range of

proteins that can be sequenced. It also circumvents ob-

stacles such as the presence of an amino-terminal block-

ing group or the lack of a key overlap peptide. Only a

few segments of primary structure must be determined

R′

by Edman analysis.

A phenylthiohydantoic acid

DNA sequencing reveals the order in which amino

acids are added to the nascent polypeptide chain as it is

H⁺, nitro-

H₂O

synthesized on the ribosomes. However, it provides no

methane

information about posttranslational modifications such

as proteolytic processing, methylation, glycosylation,

phosphorylation, hydroxylation of proline and lysine,

NH₂

and disulfide bond formation that accompany matura-

+ N

tion. While Edman sequencing can detect the presence

of most posttranslational events, technical limitations

often prevent identification of a specific modification.

A phenylthiohydantoin and a peptide

shorter by one residue

Table 4-1. Methods for cleaving polypeptides.

Figure 4-6. The Edman reaction. Phenylisothio-

cyanate derivatizes the amino-terminal residue of a

Method

Bond Cleaved

peptide as a phenylthiohydantoic acid. Treatment with

CNBr

Met-X

acid in a nonhydroxylic solvent releases a phenylthio-

hydantoin, which is subsequently identified by its chro-

Trypsin

Lys-X and Arg-X

matographic mobility, and a peptide one residue

Chymotrypsin

Hydrophobic amino acid-X

shorter. The process is then repeated.

Endoproteinase Lys-C

Lys-X

Endoproteinase Arg-C

Arg-X

ular protein, some means of identifying the correct

clone—eg, knowledge of a portion of its nucleotide se-

Endoproteinase Asp-N

X-Asp

quence—is essential. A hybrid approach thus has

V8 protease

Glu-X, particularly where X is hydro-

emerged. Edman sequencing is used to provide a partial

phobic

amino acid sequence. Oligonucleotide primers modeled

Hydroxylamine

Asn-Gly

on this partial sequence can then be used to identify

clones or to amplify the appropriate gene by the poly-

o-Iodosobenzene

Trp-X

merase chain reaction (PCR) (see Chapter 40). Once an

Mild acid

Asp-Pro

authentic DNA clone is obtained, its oligonucleotide

PROTEINS: DETERMINATION OF PRIMARY STRUCTURE

MASS SPECTROMETRY DETECTS

COVALENT MODIFICATIONS

Mass spectrometry, which discriminates molecules

based solely on their mass, is ideal for detecting the

phosphate, hydroxyl, and other groups on posttransla-

tionally modified amino acids. Each adds a specific and

readily identified increment of mass to the modified

amino acid (Table 4-2). For analysis by mass spec-

trometry, a sample in a vacuum is vaporized under

conditions where protonation can occur, imparting

positive charge. An electrical field then propels the

cations through a magnetic field which deflects them

Figure 4-8. Basic components of a simple mass

at a right angle to their original direction of flight and

spectrometer. A mixture of molecules is vaporized in an

focuses them onto a detector (Figure 4-8). The mag-

ionized state in the sample chamber S. These mole-

netic force required to deflect the path of each ionic

cules are then accelerated down the flight tube by an

species onto the detector, measured as the current ap-

electrical potential applied to accelerator grid A. An ad-

plied to the electromagnet, is recorded. For ions of

justable electromagnet, E, applies a magnetic field that

identical net charge, this force is proportionate to their

deflects the flight of the individual ions until they strike

mass. In a time-of-flight mass spectrometer, a briefly

the detector, D. The greater the mass of the ion, the

applied electric field accelerates the ions towards a de-

higher the magnetic field required to focus it onto the

tector that records the time at which each ion arrives.

detector.

For molecules of identical charge, the velocity to which

they are accelerated—and hence the time required to

reach the detector—will be inversely proportionate to

their mass.

Conventional mass spectrometers generally are used

phase HPLC column are introduced directly into the

to determine the masses of molecules of 1000 Da or

mass spectrometer for immediate determination of

less, whereas time-of-flight mass spectrometers are

their masses.

suited for determining the large masses of proteins.

Peptides inside the mass spectrometer are broken

The analysis of peptides and proteins by mass spec-

down into smaller units by collisions with neutral he-

tometry initially was hindered by difficulties in

lium atoms (collision-induced dissociation), and the

volatilizing large organic molecules. However, matrix-

masses of the individual fragments are determined.

assisted laser-desorption

(MALDI) and electrospray

Since peptide bonds are much more labile than carbon-

dispersion (eg, nanospray) permit the masses of even

carbon bonds, the most abundant fragments will differ

large polypeptides (> 100,000 Da) to be determined

from one another by units equivalent to one or two

with extraordinary accuracy (± 1 Da). Using electro-

amino acids. Since—with the exception of leucine and

spray dispersion, peptides eluting from a reversed-

isoleucine—the molecular mass of each amino acid is

unique, the sequence of the peptide can be recon-

structed from the masses of its fragments.

Table 4-2. Mass increases resulting from

common posttranslational modifications.

Tandem Mass Spectrometry

Complex peptide mixtures can now be analyzed with-

Modification

Mass Increase (Da)

out prior purification by tandem mass spectrometry,

Phosphorylation

which employs the equivalent of two mass spectrome-

ters linked in series. The first spectrometer separates in-

Hydroxylation

dividual peptides based upon their differences in mass.

Methylation

By adjusting the field strength of the first magnet, a sin-

gle peptide can be directed into the second mass spec-

Acetylation

trometer, where fragments are generated and their

Myristylation

210

masses determined. As the sensitivity and versatility of

Palmitoylation

238

mass spectrometry continue to increase, it is displacing

Edman sequencers for the direct analysis of protein pri-

Glycosylation

162

mary structure.

CHAPTER 4

GENOMICS ENABLES PROTEINS TO BE

in the hemoglobin tetramer undergo change pre- and

postpartum. Many proteins undergo posttranslational

IDENTIFIED FROM SMALL AMOUNTS

modifications during maturation into functionally

OF SEQUENCE DATA

competent forms or as a means of regulating their prop-

Primary structure analysis has been revolutionized by

erties. Knowledge of the human genome therefore rep-

genomics, the application of automated oligonucleotide

resents only the beginning of the task of describing liv-

sequencing and computerized data retrieval and analysis

ing organisms in molecular detail and understanding

to sequence an organism’s entire genetic complement.

the dynamics of processes such as growth, aging, and

The first genome sequenced was that of Haemophilus

disease. As the human body contains thousands of cell

influenzae, in

1995. By mid

2001, the complete

types, each containing thousands of proteins, the pro-

genome sequences for over 50 organisms had been de-

teome—the set of all the proteins expressed by an indi-

termined. These include the human genome and those

vidual cell at a particular time—represents a moving

of several bacterial pathogens; the results and signifi-

target of formidable dimensions.

cance of the Human Genome Project are discussed in

Chapter 54. Where genome sequence is known, the

Two-Dimensional Electrophoresis &

task of determining a protein’s DNA-derived primary

Gene Array Chips Are Used to Survey

sequence is materially simplified. In essence, the second

Protein Expression

half of the hybrid approach has already been com-

pleted. All that remains is to acquire sufficient informa-

One goal of proteomics is the identification of proteins

tion to permit the open reading frame (ORF) that

whose levels of expression correlate with medically sig-

encodes the protein to be retrieved from an Internet-

nificant events. The presumption is that proteins whose

accessible genome database and identified. In some

appearance or disappearance is associated with a specific

cases, a segment of amino acid sequence only four or

physiologic condition or disease will provide insights

five residues in length may be sufficient to identify the

into root causes and mechanisms. Determination of the

correct ORF.

proteomes characteristic of each cell type requires the

Computerized search algorithms assist the identifi-

utmost efficiency in the isolation and identification of

cation of the gene encoding a given protein and clarify

individual proteins. The contemporary approach uti-

uncertainties that arise from Edman sequencing and

lizes robotic automation to speed sample preparation

mass spectrometry. By exploiting computers to solve

and large two-dimensional gels to resolve cellular pro-

complex puzzles, the spectrum of information suitable

teins. Individual polypeptides are then extracted and

for identification of the ORF that encodes a particular

analyzed by Edman sequencing or mass spectroscopy.

polypeptide is greatly expanded. In peptide mass profil-

While only about 1000 proteins can be resolved on a

ing, for example, a peptide digest is introduced into the

single gel, two-dimensional electrophoresis has a major

mass spectrometer and the sizes of the peptides are de-

advantage in that it examines the proteins themselves.

termined. A computer is then used to find an ORF

An alternative and complementary approach employs

whose predicted protein product would, if broken

gene arrays, sometimes called DNA chips, to detect the

down into peptides by the cleavage method selected,

expression of the mRNAs which encode proteins.

produce a set of peptides whose masses match those ob-

While changes in the expression of the mRNA encod-

served by mass spectrometry.

ing a protein do not necessarily reflect comparable

changes in the level of the corresponding protein, gene

arrays are more sensitive probes than two-dimensional

PROTEOMICS & THE PROTEOME

gels and thus can examine more gene products.

The Goal of Proteomics Is to Identify the

Entire Complement of Proteins Elaborated

Bioinformatics Assists Identification

by a Cell Under Diverse Conditions

of Protein Functions

While the sequence of the human genome is known,

The functions of a large proportion of the proteins en-

the picture provided by genomics alone is both static

coded by the human genome are presently unknown.

and incomplete. Proteomics aims to identify the entire

Recent advances in bioinformatics permit researchers to

complement of proteins elaborated by a cell under di-

compare amino acid sequences to discover clues to po-

verse conditions. As genes are switched on and off, pro-

tential properties, physiologic roles, and mechanisms of

teins are synthesized in particular cell types at specific

action of proteins. Algorithms exploit the tendency of

times of growth or differentiation and in response to

nature to employ variations of a structural theme to

external stimuli. Muscle cells express proteins not ex-

perform similar functions in several proteins (eg, the

pressed by neural cells, and the type of subunits present

Rossmann nucleotide binding fold to bind NAD(P)H,

PROTEINS: DETERMINATION OF PRIMARY STRUCTURE

nuclear targeting sequences, and EF hands to bind

• Scientists are now trying to determine the primary

Ca2+). These domains generally are detected in the pri-

sequence and functional role of every protein ex-

mary structure by conservation of particular amino

pressed in a living cell, known as its proteome.

acids at key positions. Insights into the properties and

• A major goal is the identification of proteins whose

physiologic role of a newly discovered protein thus may

appearance or disappearance correlates with physio-

be inferred by comparing its primary structure with

logic phenomena, aging, or specific diseases.

that of known proteins.

REFERENCES

SUMMARY

Deutscher MP (editor): Guide to Protein Purification. Methods En-

• Long amino acid polymers or polypeptides constitute

zymol 1990;182. (Entire volume.)

the basic structural unit of proteins, and the structure

Geveart K, Vandekerckhove J: Protein identification methods in

of a protein provides insight into how it fulfills its

proteomics. Electrophoresis 2000;21:1145.

functions.

Helmuth L: Genome research: map of the human genome 3.0. Sci-

• The Edman reaction enabled amino acid sequence

ence 2001;293:583.

analysis to be automated. Mass spectrometry pro-

Khan J et al: DNA microarray technology: the anticipated impact

on the study of human disease. Biochim Biophys Acta

vides a sensitive and versatile tool for determining

1999;1423:M17.

primary structure and for the identification of post-

McLafferty FW et al: Biomolecule mass spectrometry. Science

translational modifications.

1999;284:1289.

• DNA cloning and molecular biology coupled with

Patnaik SK, Blumenfeld OO: Use of on-line tools and databases for

protein chemistry provide a hybrid approach that

routine sequence analyses. Anal Biochem 2001;289:1.

greatly increases the speed and efficiency for determi-

Schena M et al: Quantitative monitoring of gene expression pat-

nation of primary structures of proteins.

terns with a complementary DNA microarray. Science

1995;270:467.

• Genomics—the analysis of the entire oligonucleotide

sequence of an organism’s complete genetic mater-

Semsarian C, Seidman CE: Molecular medicine in the 21st cen-

tury. Intern Med J 2001;31:53.

ial—has provided further enhancements.

Temple LK et al: Essays on science and society: defining disease in

• Computer algorithms facilitate identification of the

the genomics era. Science 2001;293:807.

open reading frames that encode a given protein by

Wilkins MR et al: High-throughput mass spectrometric discovery

using partial sequences and peptide mass profiling to

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1999;289:645.

Proteins: Higher Orders of Structure

Victor W. Rodwell, PhD, & Peter J. Kennelly, PhD

BIOMEDICAL IMPORTANCE

Globular proteins are compact, are roughly spherical

or ovoid in shape, and have axial ratios (the ratio of

Proteins catalyze metabolic reactions, power cellular

their shortest to longest dimensions) of not over 3.

motion, and form macromolecular rods and cables that

Most enzymes are globular proteins, whose large inter-

provide structural integrity to hair, bones, tendons, and

nal volume provides ample space in which to con-

teeth. In nature, form follows function. The structural

struct cavities of the specific shape, charge, and hy-

variety of human proteins therefore reflects the sophis-

drophobicity or hydrophilicity required to bind

tication and diversity of their biologic roles. Maturation

substrates and promote catalysis. By contrast, many

of a newly synthesized polypeptide into a biologically

structural proteins adopt highly extended conforma-

functional protein requires that it be folded into a spe-

tions. These fibrous proteins possess axial ratios of 10

cific three-dimensional arrangement, or conformation.

or more.

During maturation, posttranslational modifications

Lipoproteins and glycoproteins contain covalently

may add new chemical groups or remove transiently

bound lipid and carbohydrate, respectively. Myoglobin,

needed peptide segments. Genetic or nutritional defi-

hemoglobin, cytochromes, and many other proteins

ciencies that impede protein maturation are deleterious

contain tightly associated metal ions and are termed

to health. Examples of the former include Creutzfeldt-

metalloproteins. With the development and applica-

Jakob disease, scrapie, Alzheimer’s disease, and bovine

tion of techniques for determining the amino acid se-

spongiform encephalopathy (mad cow disease). Scurvy

quences of proteins (Chapter 4), more precise classifica-

represents a nutritional deficiency that impairs protein

tion schemes have emerged based upon similarity, or

maturation.

homology, in amino acid sequence and structure.

However, many early classification terms remain in

common use.

CONFORMATION VERSUS

CONFIGURATION

The terms configuration and conformation are often

confused. Configuration refers to the geometric rela-

PROTEINS ARE CONSTRUCTED USING

tionship between a given set of atoms, for example,

MODULAR PRINCIPLES

those that distinguish L- from D-amino acids. Intercon-

Proteins perform complex physical and catalytic func-

version of configurational alternatives requires breaking

tions by positioning specific chemical groups in a pre-

covalent bonds. Conformation refers to the spatial re-

cise three-dimensional arrangement. The polypeptide

lationship of every atom in a molecule. Interconversion

scaffold containing these groups must adopt a confor-

between conformers occurs without covalent bond rup-

mation that is both functionally efficient and phys-

ture, with retention of configuration, and typically via

ically strong. At first glance, the biosynthesis of

rotation about single bonds.

polypeptides comprised of tens of thousands of indi-

vidual atoms would appear to be extremely challeng-

ing. When one considers that a typical polypeptide

PROTEINS WERE INITIALLY CLASSIFIED

can adopt ≥ 10⁵⁰ distinct conformations, folding into

BY THEIR GROSS CHARACTERISTICS

the conformation appropriate to their biologic func-

Scientists initially approached structure-function rela-

tion would appear to be even more difficult. As de-

tionships in proteins by separating them into classes

scribed in Chapters 3 and 4, synthesis of the polypep-

based upon properties such as solubility, shape, or the

tide backbones of proteins employs a small set of

presence of nonprotein groups. For example, the pro-

common building blocks or modules, the amino acids,

teins that can be extracted from cells using solutions at

joined by a common linkage, the peptide bond. A

physiologic pH and ionic strength are classified as sol-

stepwise modular pathway simplifies the folding and

uble. Extraction of integral membrane proteins re-

processing of newly synthesized polypeptides into ma-

quires dissolution of the membrane with detergents.

ture proteins.

PROTEINS: HIGHER ORDERS OF STRUCTURE

THE FOUR ORDERS OF

PROTEIN STRUCTURE

The modular nature of protein synthesis and folding

are embodied in the concept of orders of protein struc-

ture: primary structure, the sequence of the amino

acids in a polypeptide chain; secondary structure, the

folding of short (3- to 30-residue), contiguous segments

of polypeptide into geometrically ordered units; ter-

tiary structure, the three-dimensional assembly of sec-

ψ₀

ondary structural units to form larger functional units

such as the mature polypeptide and its component do-

mains; and quaternary structure, the number and

types of polypeptide units of oligomeric proteins and

- 90

their spatial arrangement.

SECONDARY STRUCTURE

Peptide Bonds Restrict Possible

- 90

Secondary Conformations

Free rotation is possible about only two of the three co-

valent bonds of the polypeptide backbone: the α-car-

Figure 5-1. Ramachandran plot of the main chain

bon (Cα) to the carbonyl carbon (Co) bond and the

phi (Φ) and psi (Ψ) angles for approximately 1000

Cα to nitrogen bond (Figure 3-4). The partial double-

nonglycine residues in eight proteins whose structures

bond character of the peptide bond that links Co to the

were solved at high resolution. The dots represent al-

α-nitrogen requires that the carbonyl carbon, carbonyl

lowable combinations and the spaces prohibited com-

oxygen, and α-nitrogen remain coplanar, thus prevent-

binations of phi and psi angles. (Reproduced, with per-

ing rotation. The angle about the CαN bond is

mission, from Richardson JS: The anatomy and taxonomy

termed the phi (Φ) angle, and that about the CoCα

of protein structures. Adv Protein Chem 1981;34:167.)

bond the psi (Ψ) angle. For amino acids other than

glycine, most combinations of phi and psi angles are

disallowed because of steric hindrance (Figure 5-1).

occur in nature. Schematic diagrams of proteins repre-

The conformations of proline are even more restricted

sent α helices as cylinders.

due to the absence of free rotation of the NCα bond.

The stability of an α helix arises primarily from hy-

Regions of ordered secondary structure arise when a

drogen bonds formed between the oxygen of the pep-

series of aminoacyl residues adopt similar phi and psi

tide bond carbonyl and the hydrogen atom of the pep-

angles. Extended segments of polypeptide (eg, loops)

tide bond nitrogen of the fourth residue down the

can possess a variety of such angles. The angles that de-

polypeptide chain (Figure 5-4). The ability to form the

fine the two most common types of secondary struc-

maximum number of hydrogen bonds, supplemented

ture, the helix and the sheet, fall within the lower

by van der Waals interactions in the core of this tightly

and upper left-hand quadrants of a Ramachandran

packed structure, provides the thermodynamic driving

plot, respectively (Figure 5-1).

force for the formation of an α helix. Since the peptide

bond nitrogen of proline lacks a hydrogen atom to con-

The Alpha Helix

tribute to a hydrogen bond, proline can only be stably

The polypeptide backbone of an α helix is twisted by

accommodated within the first turn of an α helix.

an equal amount about each α-carbon with a phi angle

When present elsewhere, proline disrupts the confor-

of approximately −57 degrees and a psi angle of approx-

mation of the helix, producing a bend. Because of its

imately − 47 degrees. A complete turn of the helix con-

small size, glycine also often induces bends in α helices.

tains an average of 3.6 aminoacyl residues, and the dis-

Many α helices have predominantly hydrophobic R

tance it rises per turn (its pitch) is 0.54 nm (Figure

groups on one side of the axis of the helix and predomi-

5-2). The R groups of each aminoacyl residue in an α

nantly hydrophilic ones on the other. These amphi-

helix face outward (Figure 5-3). Proteins contain only

pathic helices are well adapted to the formation of in-

L-amino acids, for which a right-handed α helix is by

terfaces between polar and nonpolar regions such as the

far the more stable, and only right-handed α helices

hydrophobic interior of a protein and its aqueous envi-

CHAPTER 5

Figure 5-3. View down the axis of an α helix. The

side chains (R) are on the outside of the helix. The van

der Waals radii of the atoms are larger than shown here;

hence, there is almost no free space inside the helix.

(Slightly modified and reproduced, with permission, from

0.54-nm pitch

Stryer L: Biochemistry, 3rd ed. Freeman, 1995. Copyright

(3.6 residues)

0.15 nm

polypeptide chain proceed in the same direction amino

to carboxyl, or an antiparallel sheet, in which they pro-

ceed in opposite directions (Figure 5-5). Either config-

uration permits the maximum number of hydrogen

bonds between segments, or strands, of the sheet. Most

Figure 5-2. Orientation of the main chain atoms of a

β sheets are not perfectly flat but tend to have a right-

peptide about the axis of an α helix.

handed twist. Clusters of twisted strands of β sheet

form the core of many globular proteins (Figure 5-6).

Schematic diagrams represent β sheets as arrows that

point in the amino to carboxyl terminal direction.

ronment. Clusters of amphipathic helices can create a

channel, or pore, that permits specific polar molecules

to pass through hydrophobic cell membranes.

Loops & Bends

Roughly half of the residues in a “typical” globular pro-

The Beta Sheet

tein reside in α helices and β sheets and half in loops,

The second (hence “beta”) recognizable regular sec-

turns, bends, and other extended conformational fea-

ondary structure in proteins is the β sheet. The amino

tures. Turns and bends refer to short segments of

acid residues of a β sheet, when viewed edge-on, form a

amino acids that join two units of secondary structure,

zigzag or pleated pattern in which the R groups of adja-

such as two adjacent strands of an antiparallel β sheet.

cent residues point in opposite directions. Unlike the

A β turn involves four aminoacyl residues, in which the

compact backbone of the α helix, the peptide backbone

first residue is hydrogen-bonded to the fourth, resulting

of the β sheet is highly extended. But like the α helix,

in a tight 180-degree turn (Figure 5-7). Proline and

β sheets derive much of their stability from hydrogen

glycine often are present in β turns.

bonds between the carbonyl oxygens and amide hydro-

Loops are regions that contain residues beyond the

gens of peptide bonds. However, in contrast to the α

minimum number necessary to connect adjacent re-

helix, these bonds are formed with adjacent segments of

gions of secondary structure. Irregular in conformation,

β sheet (Figure 5-5).

loops nevertheless serve key biologic roles. For many

Interacting β sheets can be arranged either to form a

enzymes, the loops that bridge domains responsible for

parallel β sheet, in which the adjacent segments of the

binding substrates often contain aminoacyl residues

PROTEINS: HIGHER ORDERS OF STRUCTURE

N O

Figure 5-5.

Spacing and bond angles of the hydro-

gen bonds of antiparallel and parallel pleated β sheets.

Arrows indicate the direction of each strand. The hydro-

gen-donating α-nitrogen atoms are shown as blue cir-

cles. Hydrogen bonds are indicated by dotted lines. For

Figure 5-4. Hydrogen bonds (dotted lines) formed

clarity in presentation, R groups and hydrogens are

between H and O atoms stabilize a polypeptide in an

omitted. Top: Antiparallel β sheet. Pairs of hydrogen

α-helical conformation. (Reprinted, with permission,

bonds alternate between being close together and

from Haggis GH et al: Introduction to Molecular Biology.

wide apart and are oriented approximately perpendicu-

Wiley, 1964.)

lar to the polypeptide backbone. Bottom: Parallel β

sheet. The hydrogen bonds are evenly spaced but slant

in alternate directions.

that participate in catalysis. Helix-loop-helix motifs

provide the oligonucleotide-binding portion of DNA-

binding proteins such as repressors and transcription

dered regions assume an ordered conformation upon

factors. Structural motifs such as the helix-loop-helix

motif that are intermediate between secondary and ter-

binding of a ligand. This structural flexibility enables

such regions to act as ligand-controlled switches that af-

tiary structures are often termed supersecondary struc-

tures. Since many loops and bends reside on the surface

fect protein structure and function.

of proteins and are thus exposed to solvent, they consti-

tute readily accessible sites, or epitopes, for recognition

Tertiary & Quaternary Structure

and binding of antibodies.

While loops lack apparent structural regularity, they

The term “tertiary structure” refers to the entire three-

exist in a specific conformation stabilized through hy-

dimensional conformation of a polypeptide. It indicates,

drogen bonding, salt bridges, and hydrophobic interac-

in three-dimensional space, how secondary structural

tions with other portions of the protein. However, not

features—helices, sheets, bends, turns, and loops—

all portions of proteins are necessarily ordered. Proteins

assemble to form domains and how these domains re-

may contain “disordered” regions, often at the extreme

late spatially to one another. A domain is a section of

amino or carboxyl terminal, characterized by high con-

protein structure sufficient to perform a particular

formational flexibility. In many instances, these disor-

chemical or physical task such as binding of a substrate

CHAPTER 5

COOH

C_α

O H

CH₃

CH₂OH

C_α

Figure 5-7. A β-turn that links two segments of an-

tiparallel β sheet. The dotted line indicates the hydro-

gen bond between the first and fourth amino acids of

the four-residue segment Ala-Gly-Asp-Ser.

or other ligand. Other domains may anchor a protein to

a membrane or interact with a regulatory molecule that

modulates its function. A small polypeptide such as

345

triose phosphate isomerase (Figure 5-6) or myoglobin

280

(Chapter 6) may consist of a single domain. By contrast,

330

protein kinases contain two domains. Protein kinases

150

185

350

catalyze the transfer of a phosphoryl group from ATP to

a peptide or protein. The amino terminal portion of the

145

230

377

polypeptide, which is rich in β sheet, binds ATP, while

245

310

320

the carboxyl terminal domain, which is rich in α helix,

110

220

260

binds the peptide or protein substrate (Figure 5-8). The

groups that catalyze phosphoryl transfer reside in a loop

300

258

positioned at the interface of the two domains.

120

In some cases, proteins are assembled from more

205

than one polypeptide, or protomer. Quaternary struc-

170

ture defines the polypeptide composition of a protein

125

and, for an oligomeric protein, the spatial relationships

Figure 5-6. Examples of tertiary structure of pro-

between its subunits or protomers. Monomeric pro-

teins. Top: The enzyme triose phosphate isomerase.

teins consist of a single polypeptide chain. Dimeric

Note the elegant and symmetrical arrangement of al-

proteins contain two polypeptide chains. Homodimers

contain two copies of the same polypeptide chain,

ternating β sheets and α helices. (Courtesy of J Richard-

while in a heterodimer the polypeptides differ. Greek

son.) Bottom: Two-domain structure of the subunit of a

letters (α, β, γ etc) are used to distinguish different sub-

homodimeric enzyme, a bacterial class II HMG-CoA re-

units of a heterooligomeric protein, and subscripts indi-

ductase. As indicated by the numbered residues, the

cate the number of each subunit type. For example, α₄

single polypeptide begins in the large domain, enters

designates a homotetrameric protein, and α₂β₂γ a pro-

the small domain, and ends in the large domain. (Cour-

tein with five subunits of three different types.

tesy of C Lawrence, V Rodwell, and C Stauffacher, Purdue

Since even small proteins contain many thousands

University.)

of atoms, depictions of protein structure that indicate

the position of every atom are generally too complex to

be readily interpreted. Simplified schematic diagrams

thus are used to depict key features of a protein’s ter-

PROTEINS: HIGHER ORDERS OF STRUCTURE

from water. Other significant contributors include hy-

drogen bonds and salt bridges between the carboxylates

of aspartic and glutamic acid and the oppositely

charged side chains of protonated lysyl, argininyl, and

histidyl residues. While individually weak relative to a

typical covalent bond of 80-120 kcal/mol, collectively

these numerous interactions confer a high degree of sta-

bility to the biologically functional conformation of a

protein, just as a Velcro fastener harnesses the cumula-

tive strength of multiple plastic loops and hooks.

Some proteins contain covalent disulfide

(S S)

bonds that link the sulfhydryl groups of cysteinyl

residues. Formation of disulfide bonds involves oxida-

tion of the cysteinyl sulfhydryl groups and requires oxy-

gen. Intrapolypeptide disulfide bonds further enhance

the stability of the folded conformation of a peptide,

while interpolypeptide disulfide bonds stabilize the

quaternary structure of certain oligomeric proteins.

THREE-DIMENSIONAL STRUCTURE

IS DETERMINED BY X-RAY

CRYSTALLOGRAPHY OR BY

NMR SPECTROSCOPY

X-Ray Crystallography

Since the determination of the three-dimensional struc-

ture of myoglobin over 40 years ago, the three-dimen-

sional structures of thousands of proteins have been de-

Figure 5-8.

Domain structure. Protein kinases con-

termined by x-ray crystallography. The key to x-ray

crystallography is the precipitation of a protein under

tain two domains. The upper, amino terminal domain

conditions in which it forms ordered crystals that dif-

binds the phosphoryl donor ATP (light blue). The lower,

fract x-rays. This is generally accomplished by exposing

carboxyl terminal domain is shown binding a synthetic

small drops of the protein solution to various combina-

peptide substrate (dark blue).

tions of pH and precipitating agents such as salts and

organic solutes such as polyethylene glycol. A detailed

three-dimensional structure of a protein can be con-

tiary and quaternary structure. Ribbon diagrams (Fig-

structed from its primary structure using the pattern by

ures

5-6 and 5-8) trace the conformation of the

which it diffracts a beam of monochromatic x-rays.

polypeptide backbone, with cylinders and arrows indi-

While the development of increasingly capable com-

cating regions of α helix and β sheet, respectively. In an

puter-based tools has rendered the analysis of complex

even simpler representation, line segments that link the

x-ray diffraction patterns increasingly facile, a major

α carbons indicate the path of the polypeptide back-

stumbling block remains the requirement of inducing

bone. These schematic diagrams often include the side

highly purified samples of the protein of interest to

chains of selected amino acids that emphasize specific

crystallize. Several lines of evidence, including the abil-

structure-function relationships.

ity of some crystallized enzymes to catalyze chemical re-

actions, indicate that the vast majority of the structures

MULTIPLE FACTORS STABILIZE

determined by crystallography faithfully represent the

structures of proteins in free solution.

TERTIARY & QUATERNARY STRUCTURE

Higher orders of protein structure are stabilized primar-

Nuclear Magnetic Resonance

ily—and often exclusively—by noncovalent interac-

Spectroscopy

tions. Principal among these are hydrophobic interac-

tions that drive most hydrophobic amino acid side

Nuclear magnetic resonance (NMR) spectroscopy, a

chains into the interior of the protein, shielding them

powerful complement to x-ray crystallography, mea-

CHAPTER 5

sures the absorbance of radio frequency electromagnetic

Folding Is Modular

energy by certain atomic nuclei. “NMR-active” isotopes

Protein folding generally occurs via a stepwise process.

of biologically relevant atoms include ¹H, ¹³C, ¹⁵N, and

In the first stage, the newly synthesized polypeptide

³¹P. The frequency, or chemical shift, at which a partic-

emerges from ribosomes, and short segments fold into

ular nucleus absorbs energy is a function of both the

secondary structural units that provide local regions of

functional group within which it resides and the prox-

organized structure. Folding is now reduced to the se-

imity of other NMR-active nuclei. Two-dimensional

lection of an appropriate arrangement of this relatively

NMR spectroscopy permits a three-dimensional repre-

small number of secondary structural elements. In the

sentation of a protein to be constructed by determining

second stage, the forces that drive hydrophobic regions

the proximity of these nuclei to one another. NMR

into the interior of the protein away from solvent drive

spectroscopy analyzes proteins in aqueous solution, ob-

the partially folded polypeptide into a “molten globule”

viating the need to form crystals. It thus is possible to

in which the modules of secondary structure rearrange

observe changes in conformation that accompany lig-

to arrive at the mature conformation of the protein.

and binding or catalysis using NMR spectroscopy.

This process is orderly but not rigid. Considerable flexi-

However, only the spectra of relatively small proteins,

bility exists in the ways and in the order in which ele-

≤ 20 kDa in size, can be analyzed with current tech-

ments of secondary structure can be rearranged. In gen-

nology.

eral, each element of secondary or supersecondary

structure facilitates proper folding by directing the fold-

Molecular Modeling

ing process toward the native conformation and away

An increasingly useful adjunct to the empirical determi-

from unproductive alternatives. For oligomeric pro-

nation of the three-dimensional structure of proteins is

teins, individual protomers tend to fold before they as-

the use of computer technology for molecular model-

sociate with other subunits.

ing. The types of models created take two forms. In the

first, the known three-dimensional structure of a pro-

Auxiliary Proteins Assist Folding

tein is used as a template to build a model of the proba-

ble structure of a homologous protein. In the second,

Under appropriate conditions, many proteins will

computer software is used to manipulate the static

spontaneously refold after being previously denatured

model provided by crystallography to explore how a

(ie, unfolded) by treatment with acid or base,

protein’s conformation might change when ligands are

chaotropic agents, or detergents. However, unlike the

bound or when temperature, pH, or ionic strength is

folding process in vivo, refolding under laboratory con-

altered. Scientists also are examining the library of

ditions is a far slower process. Moreover, some proteins

available protein structures in an attempt to devise

fail to spontaneously refold in vitro, often forming in-

computer programs that can predict the three-dimen-

soluble aggregates, disordered complexes of unfolded

sional conformation of a protein directly from its pri-

or partially folded polypeptides held together by hy-

mary sequence.

drophobic interactions. Aggregates represent unproduc-

tive dead ends in the folding process. Cells employ aux-

PROTEIN FOLDING

iliary proteins to speed the process of folding and to

guide it toward a productive conclusion.

The Native Conformation of a Protein

Is Thermodynamically Favored

Chaperones

The number of distinct combinations of phi and psi

angles specifying potential conformations of even a rel-

Chaperone proteins participate in the folding of over

atively small—15-kDa—polypeptide is unbelievably

half of mammalian proteins. The hsp70 (70-kDa heat

vast. Proteins are guided through this vast labyrinth of

shock protein) family of chaperones binds short se-

possibilities by thermodynamics. Since the biologically

quences of hydrophobic amino acids in newly syn-

relevant—or native—conformation of a protein gener-

thesized polypeptides, shielding them from solvent.

ally is that which is most energetically favored, knowl-

Chaperones prevent aggregation, thus providing an op-

edge of the native conformation is specified in the pri-

portunity for the formation of appropriate secondary

mary sequence. However, if one were to wait for a

structural elements and their subsequent coalescence

polypeptide to find its native conformation by random

into a molten globule. The hsp60 family of chaperones,

exploration of all possible conformations, the process

sometimes called chaperonins, differ in sequence and

would require billions of years to complete. Clearly,

structure from hsp70 and its homologs. Hsp60 acts

protein folding in cells takes place in a more orderly

later in the folding process, often together with an

and guided fashion.

hsp70 chaperone. The central cavity of the donut-

PROTEINS: HIGHER ORDERS OF STRUCTURE

shaped hsp60 chaperone provides a sheltered environ-

sheep, and bovine spongiform encephalopathy (mad

ment in which a polypeptide can fold until all hy-

cow disease) in cattle. Prion diseases may manifest

drophobic regions are buried in its interior, eliminating

themselves as infectious, genetic, or sporadic disorders.

aggregation. Chaperone proteins can also “rescue” pro-

Because no viral or bacterial gene encoding the patho-

teins that have become thermodynamically trapped in a

logic prion protein could be identified, the source and

misfolded dead end by unfolding hydrophobic regions

mechanism of transmission of prion disease long re-

and providing a second chance to fold productively.

mained elusive. Today it is believed that prion diseases

are protein conformation diseases transmitted by alter-

Protein Disulfide Isomerase

ing the conformation, and hence the physical proper-

ties, of proteins endogenous to the host. Human prion-

Disulfide bonds between and within polypeptides stabi-

related protein, PrP, a glycoprotein encoded on the

lize tertiary and quaternary structure. However, disul-

short arm of chromosome 20, normally is monomeric

fide bond formation is nonspecific. Under oxidizing

and rich in α helix. Pathologic prion proteins serve as

conditions, a given cysteine can form a disulfide bond

the templates for the conformational transformation of

with the SH of any accessible cysteinyl residue. By

normal PrP, known as PrPc, into PrPsc. PrPsc is rich in

catalyzing disulfide exchange, the rupture of an SS

β sheet with many hydrophobic aminoacyl side chains

bond and its reformation with a different partner cys-

exposed to solvent. PrPsc molecules therefore associate

teine, protein disulfide isomerase facilitates the forma-

strongly with one other, forming insoluble protease-re-

tion of disulfide bonds that stabilize their native confor-

sistant aggregates. Since one pathologic prion or prion-

mation.

related protein can serve as template for the conforma-

tional transformation of many times its number of PrPc

Proline-cis,trans-Isomerase

molecules, prion diseases can be transmitted by the pro-

tein alone without involvement of DNA or RNA.

All X-Pro peptide bonds—where X represents any

residue—are synthesized in the trans configuration.

However, of the X-Pro bonds of mature proteins, ap-

Alzheimer’s Disease

proximately 6% are cis. The cis configuration is partic-

Refolding or misfolding of another protein endogenous

ularly common in β-turns. Isomerization from trans to

to human brain tissue, β-amyloid, is also a prominent

cis is catalyzed by the enzyme proline-cis,trans-iso-

feature of Alzheimer’s disease. While the root cause of

merase (Figure 5-9).

Alzheimer’s disease remains elusive, the characteristic

senile plaques and neurofibrillary bundles contain ag-

SEVERAL NEUROLOGIC DISEASES

gregates of the protein β-amyloid, a 4.3-kDa polypep-

RESULT FROM ALTERED PROTEIN

tide produced by proteolytic cleavage of a larger protein

known as amyloid precursor protein. In Alzheimer’s

CONFORMATION

disease patients, levels of β-amyloid become elevated,

Prions

and this protein undergoes a conformational transfor-

mation from a soluble α helix-rich state to a state rich

The transmissible spongiform encephalopathies, or

in β sheet and prone to self-aggregation. Apolipopro-

prion diseases, are fatal neurodegenerative diseases

tein E has been implicated as a potential mediator of

characterized by spongiform changes, astrocytic gli-

this conformational transformation.

omas, and neuronal loss resulting from the deposition

of insoluble protein aggregates in neural cells. They in-

clude Creutzfeldt-Jakob disease in humans, scrapie in

COLLAGEN ILLUSTRATES THE ROLE OF

POSTTRANSLATIONAL PROCESSING IN

PROTEIN MATURATION

Protein Maturation Often Involves Making

′

& Breaking Covalent Bonds

α₁

The maturation of proteins into their final structural

R₁

α₁

′

R₁

state often involves the cleavage or formation (or both)

of covalent bonds, a process termed posttranslational

modification. Many polypeptides are initially synthe-

Figure 5-9. Isomerization of the N-α₁ prolyl peptide

sized as larger precursors, called proproteins. The

bond from a cis to a trans configuration relative to the

“extra” polypeptide segments in these proproteins

backbone of the polypeptide.

often serve as leader sequences that target a polypeptide

CHAPTER 5

to a particular organelle or facilitate its passage through

rise per residue nearly twice that of an α helix. The

a membrane. Others ensure that the potentially harm-

R groups of each polypeptide strand of the triple helix

ful activity of a protein such as the proteases trypsin

pack so closely that in order to fit, one must be glycine.

and chymotrypsin remains inhibited until these pro-

Thus, every third amino acid residue in collagen is a

teins reach their final destination. However, once these

glycine residue. Staggering of the three strands provides

transient requirements are fulfilled, the now superflu-

appropriate positioning of the requisite glycines

ous peptide regions are removed by selective proteoly-

throughout the helix. Collagen is also rich in proline

sis. Other covalent modifications may take place that

and hydroxyproline, yielding a repetitive Gly-X-Y pat-

add new chemical functionalities to a protein. The mat-

tern (Figure 5-10) in which Y generally is proline or

uration of collagen illustrates both of these processes.

hydroxyproline.

Collagen triple helices are stabilized by hydrogen

Collagen Is a Fibrous Protein

bonds between residues in different polypeptide chains.

The hydroxyl groups of hydroxyprolyl residues also par-

Collagen is the most abundant of the fibrous proteins

ticipate in interchain hydrogen bonding. Additional

that constitute more than 25% of the protein mass in

stability is provided by covalent cross-links formed be-

the human body. Other prominent fibrous proteins in-

tween modified lysyl residues both within and between

clude keratin and myosin. These proteins represent a

polypeptide chains.

primary source of structural strength for cells (ie, the

cytoskeleton) and tissues. Skin derives its strength and

Collagen Is Synthesized as a

flexibility from a crisscrossed mesh of collagen and ker-

atin fibers, while bones and teeth are buttressed by an

Larger Precursor

underlying network of collagen fibers analogous to the

Collagen is initially synthesized as a larger precursor

steel strands in reinforced concrete. Collagen also is

polypeptide, procollagen. Numerous prolyl and lysyl

present in connective tissues such as ligaments and ten-

residues of procollagen are hydroxylated by prolyl hy-

dons. The high degree of tensile strength required to

droxylase and lysyl hydroxylase, enzymes that require

fulfill these structural roles requires elongated proteins

ascorbic acid (vitamin C). Hydroxyprolyl and hydroxy-

characterized by repetitive amino acid sequences and a

lysyl residues provide additional hydrogen bonding ca-

regular secondary structure.

pability that stabilizes the mature protein. In addition,

glucosyl and galactosyl transferases attach glucosyl or

Collagen Forms a Unique Triple Helix

galactosyl residues to the hydroxyl groups of specific

hydroxylysyl residues.

Tropocollagen consists of three fibers, each containing

The central portion of the precursor polypeptide

about 1000 amino acids, bundled together in a unique

then associates with other molecules to form the char-

conformation, the collagen triple helix (Figure 5-10). A

acteristic triple helix. This process is accompanied by

mature collagen fiber forms an elongated rod with an

the removal of the globular amino terminal and car-

axial ratio of about 200. Three intertwined polypeptide

boxyl terminal extensions of the precursor polypeptide

strands, which twist to the left, wrap around one an-

by selective proteolysis. Certain lysyl residues are modi-

other in a right-handed fashion to form the collagen

fied by lysyl oxidase, a copper-containing protein that

triple helix. The opposing handedness of this superhelix

converts ε-amino groups to aldehydes. The aldehydes

and its component polypeptides makes the collagen

can either undergo an aldol condensation to form a

triple helix highly resistant to unwinding—the same

CC double bond or to form a Schiff base (eneimine)

principle used in the steel cables of suspension bridges.

with the ε-amino group of an unmodified lysyl residue,

A collagen triple helix has 3.3 residues per turn and a

which is subsequently reduced to form a CN single

bond. These covalent bonds cross-link the individual

Amino acid

polypeptides and imbue the fiber with exceptional

- Gly - X - Y - Gly - X - Y - Gly - X - Y -

sequence

strength and rigidity.

2º structure

Nutritional & Genetic Disorders Can Impair

Collagen Maturation

The complex series of events in collagen maturation

Triple helix

provide a model that illustrates the biologic conse-

quences of incomplete polypeptide maturation. The

Figure 5-10. Primary, secondary, and tertiary struc-

best-known defect in collagen biosynthesis is scurvy, a

tures of collagen.

result of a dietary deficiency of vitamin C required by

PROTEINS: HIGHER ORDERS OF STRUCTURE

prolyl and lysyl hydroxylases. The resulting deficit in

sized polypeptide fold into secondary structural

the number of hydroxyproline and hydroxylysine

units. Forces that bury hydrophobic regions from

residues undermines the conformational stability of col-

solvent then drive the partially folded polypeptide

lagen fibers, leading to bleeding gums, swelling joints,

into a “molten globule” in which the modules of sec-

poor wound healing, and ultimately to death. Menkes’

ondary structure are rearranged to give the native

syndrome, characterized by kinky hair and growth re-

conformation of the protein.

tardation, reflects a dietary deficiency of the copper re-

•

Proteins that assist folding include protein disulfide

quired by lysyl oxidase, which catalyzes a key step in

isomerase, proline-cis,trans,-isomerase, and the chap-

formation of the covalent cross-links that strengthen

erones that participate in the folding of over half of

collagen fibers.

mammalian proteins. Chaperones shield newly syn-

Genetic disorders of collagen biosynthesis include

thesized polypeptides from solvent and provide an

several forms of osteogenesis imperfecta, characterized

environment for elements of secondary structure to

by fragile bones. In Ehlers-Dahlos syndrome, a group

emerge and coalesce into molten globules.

of connective tissue disorders that involve impaired in-

•

Techniques for study of higher orders of protein

tegrity of supporting structures, defects in the genes

structure include x-ray crystallography, NMR spec-

that encode α collagen-1, procollagen N-peptidase, or

troscopy, analytical ultracentrifugation, gel filtration,

lysyl hydroxylase result in mobile joints and skin abnor-

and gel electrophoresis.

malities.

•

Silk fibroin and collagen illustrate the close linkage of

protein structure and biologic function. Diseases of

SUMMARY

collagen maturation include Ehlers-Danlos syndrome

•

Proteins may be classified on the basis of the solubil-

and the vitamin C deficiency disease scurvy.

ity, shape, or function or of the presence of a pros-

•

Prions—protein particles that lack nucleic acid—

thetic group such as heme. Proteins perform complex

cause fatal transmissible spongiform encephalopa-

physical and catalytic functions by positioning spe-

thies such as Creutzfeldt-Jakob disease, scrapie, and

cific chemical groups in a precise three-dimensional

bovine spongiform encephalopathy. Prion diseases

arrangement that is both functionally efficient and

involve an altered secondary-tertiary structure of a

physically strong.

naturally occurring protein, PrPc. When PrPc inter-

acts with its pathologic isoform PrPSc, its conforma-

•

The gene-encoded primary structure of a polypeptide

is the sequence of its amino acids. Its secondary

tion is transformed from a predominantly α-helical

structure to the β-sheet structure characteristic of

structure results from folding of polypeptides into

hydrogen-bonded motifs such as the α helix, the

PrPSc.

β-pleated sheet, β bends, and loops. Combinations

of these motifs can form supersecondary motifs.

•

Tertiary structure concerns the relationships between

REFERENCES

secondary structural domains. Quaternary structure

of proteins with two or more polypeptides

Branden C, Tooze J: Introduction to Protein Structure. Garland,

1991.

(oligomeric proteins) is a feature based on the spatial

Burkhard P, Stetefeld J, Strelkov SV: Coiled coils: A highly versa-

relationships between various types of polypeptides.

tile protein folding motif. Trends Cell Biol 2001;11:82.

•

Primary structures are stabilized by covalent peptide

Collinge J: Prion diseases of humans and animals: Their causes and

bonds. Higher orders of structure are stabilized by

molecular basis. Annu Rev Neurosci 2001;24:519.

weak forces—multiple hydrogen bonds, salt (electro-

Frydman J: Folding of newly translated proteins in vivo: The role

static) bonds, and association of hydrophobic R

of molecular chaperones. Annu Rev Biochem 2001;70:603.

groups.

Radord S: Protein folding: Progress made and promises ahead.

•

The phi (Φ) angle of a polypeptide is the angle about

Trends Biochem Sci 2000;25:611.

the C_αN bond; the psi (Ψ) angle is that about the

Schmid FX: Proly isomerase: Enzymatic catalysis of slow protein

folding reactions. Ann Rev Biophys Biomol Struct 1993;22:

Cα-Co bond. Most combinations of phi-psi angles

123.

are disallowed due to steric hindrance. The phi-psi

Segrest MP et al: The amphipathic alpha-helix: A multifunctional

angles that form the α helix and the β sheet fall

structural motif in plasma lipoproteins. Adv Protein Chem

within the lower and upper left-hand quadrants of a

1995;45:1.

Ramachandran plot, respectively.

Soto C: Alzheimer’s and prion disease as disorders of protein con-

•

Protein folding is a poorly understood process.

formation: Implications for the design of novel therapeutic

Broadly speaking, short segments of newly synthe-

approaches. J Mol Med 1999;77:412.

Proteins: Myoglobin & Hemoglobin

Victor W. Rodwell, PhD, & Peter J. Kennelly, PhD

BIOMEDICAL IMPORTANCE

Myoglobin Is Rich in α Helix

The heme proteins myoglobin and hemoglobin main-

Oxygen stored in red muscle myoglobin is released dur-

tain a supply of oxygen essential for oxidative metabo-

ing O₂ deprivation (eg, severe exercise) for use in mus-

lism. Myoglobin, a monomeric protein of red muscle,

cle mitochondria for aerobic synthesis of ATP

(see

stores oxygen as a reserve against oxygen deprivation.

Chapter

12). A

153-aminoacyl residue polypeptide

Hemoglobin, a tetrameric protein of erythrocytes,

(MW 17,000), myoglobin folds into a compact shape

transports O₂ to the tissues and returns CO₂ and pro-

that measures 4.5 × 3.5 × 2.5 nm (Figure 6-2). Unusu-

tons to the lungs. Cyanide and carbon monoxide kill

ally high proportions, about 75%, of the residues are

because they disrupt the physiologic function of the

present in eight right-handed, 7-20 residue α helices.

heme proteins cytochrome oxidase and hemoglobin, re-

Starting at the amino terminal, these are termed helices

spectively. The secondary-tertiary structure of the sub-

A-H. Typical of globular proteins, the surface of myo-

units of hemoglobin resembles myoglobin. However,

globin is polar, while—with only two exceptions—the

the tetrameric structure of hemoglobin permits cooper-

interior contains only nonpolar residues such as Leu,

ative interactions that are central to its function. For ex-

Val, Phe, and Met. The exceptions are His E7 and His

ample, 2,3-bisphosphoglycerate

(BPG) promotes the

F8, the seventh and eighth residues in helices E and F,

efficient release of O₂

by stabilizing the quaternary

which lie close to the heme iron where they function in

structure of deoxyhemoglobin. Hemoglobin and myo-

O₂ binding.

globin illustrate both protein structure-function rela-

tionships and the molecular basis of genetic diseases

such as sickle cell disease and the thalassemias.

Histidines F8 & E7 Perform Unique Roles in

Oxygen Binding

The heme of myoglobin lies in a crevice between helices

HEME & FERROUS IRON CONFER THE

E and F oriented with its polar propionate groups fac-

ABILITY TO STORE & TO TRANSPORT

ing the surface of the globin (Figure 6-2). The remain-

OXYGEN

der resides in the nonpolar interior. The fifth coordina-

tion position of the iron is linked to a ring nitrogen of

Myoglobin and hemoglobin contain heme, a cyclic

the proximal histidine, His F8. The distal histidine,

tetrapyrrole consisting of four molecules of pyrrole

His E7, lies on the side of the heme ring opposite to

linked by α-methylene bridges. This planar network of

His F8.

conjugated double bonds absorbs visible light and col-

ors heme deep red. The substituents at the β-positions

of heme are methyl (M), vinyl (V), and propionate (Pr)

The Iron Moves Toward the Plane of the

groups arranged in the order M, V, M, V, M, Pr, Pr, M

Heme When Oxygen Is Bound

(Figure 6-1). One atom of ferrous iron (Fe2+) resides at

the center of the planar tetrapyrrole. Other proteins

The iron of unoxygenated myoglobin lies 0.03 nm

with metal-containing tetrapyrrole prosthetic groups

(0.3 Å) outside the plane of the heme ring, toward His

include the cytochromes (Fe and Cu) and chlorophyll

F8. The heme therefore “puckers” slightly. When O₂

(Mg) (see Chapter 12). Oxidation and reduction of the

occupies the sixth coordination position, the iron

Fe and Cu atoms of cytochromes is essential to their bi-

moves to within 0.01 nm (0.1 Å) of the plane of the

ologic function as carriers of electrons. By contrast, oxi-

heme ring. Oxygenation of myoglobin thus is accompa-

dation of the Fe2+

of myoglobin or hemoglobin to Fe₃

nied by motion of the iron, of His F8, and of residues

destroys their biologic activity.

linked to His F8.

PROTEINS: MYOGLOBIN & HEMOGLOBIN

Apomyoglobin Provides a Hindered

Environment for Heme Iron

When O₂ binds to myoglobin, the bond between the first

oxygen atom and the Fe2+ is perpendicular to the plane of

the heme ring. The bond linking the first and second

Fe2+

oxygen atoms lies at an angle of 121 degrees to the plane

of the heme, orienting the second oxygen away from the

distal histidine (Figure 6-3, left). Isolated heme binds

^-O

carbon monoxide (CO) 25,000 times more strongly than

oxygen. Since CO is present in small quantities in the at-

mosphere and arises in cells from the catabolism of heme,

why is it that CO does not completely displace O₂ from

heme iron? The accepted explanation is that the apopro-

teins of myoglobin and hemoglobin create a hindered

O^-

environment. While CO can bind to isolated heme in its

Figure 6-1. Heme. The pyrrole rings and methylene

preferred orientation, ie, with all three atoms (Fe, C, and

bridge carbons are coplanar, and the iron atom (Fe2+)

O) perpendicular to the plane of the heme, in myoglobin

resides in almost the same plane. The fifth and sixth co-

and hemoglobin the distal histidine sterically precludes

ordination positions of Fe2+ are directed perpendicular

this orientation. Binding at a less favored angle reduces

the strength of the heme-CO bond to about 200 times

to—and directly above and below—the plane of the

that of the heme-O₂ bond (Figure 6 -3, right) at which

heme ring. Observe the nature of the substituent

level the great excess of O₂ over CO normally present

groups on the β carbons of the pyrrole rings, the cen-

dominates. Nevertheless, about 1% of myoglobin typi-

tral iron atom, and the location of the polar side of the

cally is present combined with carbon monoxide.

heme ring (at about 7 o’clock) that faces the surface of

the myoglobin molecule.

THE OXYGEN DISSOCIATION CURVES

FOR MYOGLOBIN & HEMOGLOBIN SUIT

THEIR PHYSIOLOGIC ROLES

O^-

FG2

CD2

Why is myoglobin unsuitable as an O₂ transport pro-

H24

tein but well suited for O₂ storage? The relationship

HC5

between the concentration, or partial pressure, of O₂

CD1

(PO₂) and the quantity of O₂ bound is expressed as an

CD7

O₂ saturation isotherm

(Figure

6-4). The oxygen-

B14

B16

H16

G15

EF3

EF1

A16

NA1

E20

G19

⁺H₃N

AB1

GH4

Figure 6-2. A model of myoglobin at low resolution.

Only the α-carbon atoms are shown. The α-helical re-

Figure 6-3. Angles for bonding of oxygen and car-

gions are named A through H. (Based on Dickerson RE in:

bon monoxide to the heme iron of myoglobin. The dis-

The Proteins, 2nd ed. Vol 2. Neurath H [editor]. Academic

tal E7 histidine hinders bonding of CO at the preferred

Press, 1964. Reproduced with permission.)

(180 degree) angle to the plane of the heme ring.

CHAPTER 6

100

Hemoglobin Is Tetrameric

Myoglobin

Hemoglobins are tetramers comprised of pairs of two

Oxygenated blood

different polypeptide subunits. Greek letters are used to

leaving the lungs

designate each subunit type. The subunit composition

of the principal hemoglobins are α₂β₂ (HbA; normal

adult hemoglobin), α₂γ₂ (HbF; fetal hemoglobin), α₂S₂

Reduced blood

(HbS; sickle cell hemoglobin), and α₂δ₂

(HbA₂; a

returning from tissues

minor adult hemoglobin). The primary structures of

the β, γ, and δ chains of human hemoglobin are highly

Hemoglobin

conserved.

100

120

140

Myoglobin & the Subunits

Gaseous pressure of oxygen (mm Hg)

of Hemoglobin Share Almost Identical

Secondary and Tertiary Structures

Figure 6-4. Oxygen-binding curves of both hemo-

Despite differences in the kind and number of amino

globin and myoglobin. Arterial oxygen tension is about

acids present, myoglobin and the β polypeptide of he-

100 mm Hg; mixed venous oxygen tension is about 40

moglobin A have almost identical secondary and ter-

mm Hg; capillary (active muscle) oxygen tension is

tiary structures. Similarities include the location of the

about 20 mm Hg; and the minimum oxygen tension re-

heme and the eight helical regions and the presence of

quired for cytochrome oxidase is about 5 mm Hg. Asso-

amino acids with similar properties at comparable loca-

ciation of chains into a tetrameric structure (hemoglo-

tions. Although it possesses seven rather than eight heli-

bin) results in much greater oxygen delivery than

cal regions, the α polypeptide of hemoglobin also

would be possible with single chains. (Modified, with

closely resembles myoglobin.

permission, from Scriver CR et al [editors]: The Molecular

and Metabolic Bases of Inherited Disease, 7th ed.

Oxygenation of Hemoglobin

McGraw-Hill, 1995.)

Triggers Conformational Changes

in the Apoprotein

Hemoglobins bind four molecules of O₂ per tetramer,

binding curve for myoglobin is hyperbolic. Myoglobin

one per heme. A molecule of O₂ binds to a hemoglobin

therefore loads O₂ readily at the PO₂ of the lung capil-

tetramer more readily if other O₂ molecules are already

lary bed (100 mm Hg). However, since myoglobin re-

bound (Figure 6-4). Termed cooperative binding,

leases only a small fraction of its bound O₂ at the PO₂

this phenomenon permits hemoglobin to maximize

values typically encountered in active muscle (20 mm

both the quantity of O₂ loaded at the PO₂ of the lungs

Hg) or other tissues (40 mm Hg), it represents an inef-

and the quantity of O₂ released at the PO₂ of the pe-

fective vehicle for delivery of O₂. However, when

ripheral tissues. Cooperative interactions, an exclusive

strenuous exercise lowers the PO₂ of muscle tissue to

property of multimeric proteins, are critically impor-

about 5 mm Hg, myoglobin releases O₂ for mitochon-

tant to aerobic life.

drial synthesis of ATP, permitting continued muscular

activity.

P₅₀ Expresses the Relative Affinities

of Different Hemoglobins for Oxygen

THE ALLOSTERIC PROPERTIES OF

The quantity P₅₀, a measure of O₂ concentration, is the

HEMOGLOBINS RESULT FROM THEIR

partial pressure of O₂ that half-saturates a given hemo-

QUATERNARY STRUCTURES

globin. Depending on the organism, P₅₀

can vary

The properties of individual hemoglobins are conse-

widely, but in all instances it will exceed the PO₂ of the

quences of their quaternary as well as of their secondary

peripheral tissues. For example, values of P₅₀ for HbA

and tertiary structures. The quaternary structure of he-

and fetal HbF are 26 and 20 mm Hg, respectively. In

moglobin confers striking additional properties, absent

the placenta, this difference enables HbF to extract oxy-

from monomeric myoglobin, which adapts it to its

gen from the HbA in the mother’s blood. However,

unique biologic roles. The allosteric (Gk allos “other,”

HbF is suboptimal postpartum since its high affinity

steros “space”) properties of hemoglobin provide, in ad-

for O₂ dictates that it can deliver less O₂ to the tissues.

dition, a model for understanding other allosteric pro-

The subunit composition of hemoglobin tetramers

teins (see Chapter 11).

undergoes complex changes during development. The

PROTEINS: MYOGLOBIN & HEMOGLOBIN

human fetus initially synthesizes a ζ₂ε₂ tetramer. By the

Histidine F8

end of the first trimester, ζ and γ subunits have been re-

F helix

placed by α and ε subunits, forming HbF (α₂γ₂), the

hemoglobin of late fetal life. While synthesis of β sub-

units begins in the third trimester, β subunits do not

completely replace γ subunits to yield adult HbA (α₂β₂)

Steric

repulsion

until some weeks postpartum (Figure 6-5).

Porphyrin

plane

Oxygenation of Hemoglobin Is

Accompanied by Large

Conformational Changes

+O₂

F helix

The binding of the first O₂ molecule to deoxyHb shifts

the heme iron towards the plane of the heme ring from

a position about 0.6 nm beyond it (Figure 6-6). This

motion is transmitted to the proximal (F8) histidine

and to the residues attached thereto, which in turn

causes the rupture of salt bridges between the carboxyl

terminal residues of all four subunits. As a consequence,

one pair of α/β subunits rotates 15 degrees with respect

to the other, compacting the tetramer (Figure 6-7).

Profound changes in secondary, tertiary, and quater-

nary structure accompany the high-affinity O₂-induced

transition of hemoglobin from the low-affinity T (taut)

Figure 6-6. The iron atom moves into the plane of

state to the R (relaxed) state. These changes signifi-

the heme on oxygenation. Histidine F8 and its associ-

cantly increase the affinity of the remaining unoxy-

ated residues are pulled along with the iron atom.

genated hemes for O₂, as subsequent binding events re-

(Slightly modified and reproduced, with permission,

quire the rupture of fewer salt bridges (Figure 6-8).

from Stryer L: Biochemistry, 4th ed. Freeman, 1995.)

The terms T and R also are used to refer to the low-

affinity and high-affinity conformations of allosteric en-

zymes, respectively.

α chain

γ chain

α₁

β₂

α₁

β₂

(fetal)

Axis

β chain (adult)

α₂

and ζ chains

α₂

β₁

(embryonic)

15°

δ chain

T form

R form

Birth

Figure 6-7. During transition of the T form to the R

form of hemoglobin, one pair of subunits (α₂/β₂) ro-

Gestation (months)

Age (months)

tates through 15 degrees relative to the other pair

Figure 6-5. Developmental pattern of the quater-

(α₁/β₁). The axis of rotation is eccentric, and the α₂/β₂

nary structure of fetal and newborn hemoglobins. (Re-

pair also shifts toward the axis somewhat. In the dia-

produced, with permission, from Ganong WF: Review of

gram, the unshaded α₁/β₁ pair is shown fixed while the

Medical Physiology, 20th ed. McGraw-Hill, 2001.)

colored α₂/β₂ pair both shifts and rotates.

CHAPTER 6

T structure

α₁

α₂

O₂

β₁

β₂

O₂

R structure

Figure 6-8. Transition from the T structure to the R structure. In this model, salt

bridges (thin lines) linking the subunits in the T structure break progressively as oxy-

gen is added, and even those salt bridges that have not yet ruptured are progressively

weakened (wavy lines). The transition from T to R does not take place after a fixed

number of oxygen molecules have been bound but becomes more probable as each

successive oxygen binds. The transition between the two structures is influenced by

protons, carbon dioxide, chloride, and BPG; the higher their concentration, the more

oxygen must be bound to trigger the transition. Fully oxygenated molecules in the T

structure and fully deoxygenated molecules in the R structure are not shown because

they are unstable. (Modified and redrawn, with permission, from Perutz MF: Hemoglobin

structure and respiratory transport. Sci Am [Dec] 1978;239:92.)

After Releasing O₂ at the Tissues,

Hemoglobin Transports CO₂ & Protons

to the Lungs

In addition to transporting O₂ from the lungs to pe-

ripheral tissues, hemoglobin transports CO₂, the by-

product of respiration, and protons from peripheral tis-

Deoxyhemoglobin binds one proton for every two

sues to the lungs. Hemoglobin carries CO₂

O₂ molecules released, contributing significantly to the

carbamates formed with the amino terminal nitrogens

buffering capacity of blood. The somewhat lower pH of

of the polypeptide chains.

peripheral tissues, aided by carbamation, stabilizes the

T state and thus enhances the delivery of O₂. In the

lungs, the process reverses. As O₂ binds to deoxyhemo-

globin, protons are released and combine with bicar-

bonate to form carbonic acid. Dehydration of H₂CO₃,

−

CO2+ HbNH

HbN O

catalyzed by carbonic anhydrase, forms CO₂, which is

exhaled. Binding of oxygen thus drives the exhalation

of CO₂ (Figure 6-9).This reciprocal coupling of proton

Carbamates change the charge on amino terminals

and O₂ binding is termed the Bohr effect. The Bohr

from positive to negative, favoring salt bond formation

effect is dependent upon cooperative interactions be-

between the α and β chains.

tween the hemes of the hemoglobin tetramer. Myo-

Hemoglobin carbamates account for about 15% of

globin, a monomer, exhibits no Bohr effect.

the CO₂ in venous blood. Much of the remaining CO₂

is carried as bicarbonate, which is formed in erythro-

Protons Arise From Rupture of Salt Bonds

cytes by the hydration of CO₂

to carbonic acid

When O₂ Binds

(H₂CO₃), a process catalyzed by carbonic anhydrase. At

the pH of venous blood, H₂CO₃ dissociates into bicar-

Protons responsible for the Bohr effect arise from rup-

bonate and a proton.

ture of salt bridges during the binding of O₂ to T state

PROTEINS: MYOGLOBIN & HEMOGLOBIN

Exhaled

2CO₂ + 2H₂O

CARBONIC

ANHYDRASE

2H₂CO₃

PERIPHERAL

TISSUES

2HCO3- + 2H+

Hb • 4O₂

The hemoglobin tetramer binds one molecule of

4O₂

BPG in the central cavity formed by its four subunits.

–

However, the space between the H helices of the β

2H⁺

+ 2HCO

chains lining the cavity is sufficiently wide to accom-

4O₂

Hb • 2H⁺

modate BPG only when hemoglobin is in the T state.

(buffer)

BPG forms salt bridges with the terminal amino groups

2H₂CO

of both β chains via Val NA1 and with Lys EF6 and

LUNGS

CARBONIC

ANHYDRASE

His H21 (Figure 6-10). BPG therefore stabilizes de-

oxygenated (T state) hemoglobin by forming additional

2CO₂ + 2H₂O

salt bridges that must be broken prior to conversion to

the R state.

Generated by

Residue H21 of the γ subunit of fetal hemoglobin

the Krebs cycle

(HbF) is Ser rather than His. Since Ser cannot form a

Figure 6-9. The Bohr effect. Carbon dioxide gener-

salt bridge, BPG binds more weakly to HbF than to

ated in peripheral tissues combines with water to form

HbA. The lower stabilization afforded to the T state by

carbonic acid, which dissociates into protons and bicar-

BPG accounts for HbF having a higher affinity for O₂

bonate ions. Deoxyhemoglobin acts as a buffer by

than HbA.

binding protons and delivering them to the lungs. In

the lungs, the uptake of oxygen by hemoglobin re-

leases protons that combine with bicarbonate ion,

forming carbonic acid, which when dehydrated by car-

bonic anhydrase becomes carbon dioxide, which then

is exhaled.

His H21

Lys EF6

hemoglobin. Conversion to the oxygenated R state

breaks salt bridges involving β-chain residue His 146.

BPG

Val NA1

The subsequent dissociation of protons from His 146

Val NA1

α-NH₃

drives the conversion of bicarbonate to carbonic acid

(Figure 6-9). Upon the release of O₂, the T structure

and its salt bridges re-form. This conformational

Lys EF6

change increases the pK_a of the β-chain His

146

residues, which bind protons. By facilitating the re-for-

mation of salt bridges, an increase in proton concentra-

tion enhances the release of O₂ from oxygenated (R

His H21

state) hemoglobin. Conversely, an increase in PO₂ pro-

motes proton release.

Figure 6-10. Mode of binding of 2,3-bisphospho-

2,3-Bisphosphoglycerate (BPG) Stabilizes

glycerate to human deoxyhemoglobin. BPG interacts

the T Structure of Hemoglobin

with three positively charged groups on each β chain.

A low PO₂ in peripheral tissues promotes the synthesis

(Based on Arnone A: X-ray diffraction study of binding of

in erythrocytes of 2,3-bisphosphoglycerate (BPG) from

2,3-diphosphoglycerate to human deoxyhemoglobin. Na-

the glycolytic intermediate 1,3-bisphosphoglycerate.

ture 1972;237:146. Reproduced with permission.)

CHAPTER 6

Adaptation to High Altitude

In hemoglobin M, histidine F8 (His F8) has been

replaced by tyrosine. The iron of HbM forms a tight

Physiologic changes that accompany prolonged expo-

ionic complex with the phenolate anion of tyrosine that

sure to high altitude include an increase in the number

stabilizes the Fe3+ form. In α-chain hemoglobin M vari-

of erythrocytes and in their concentrations of hemoglo-

ants, the R-T equilibrium favors the T state. Oxygen

bin and of BPG. Elevated BPG lowers the affinity of

affinity is reduced, and the Bohr effect is absent.

HbA for O₂ (decreases P₅₀), which enhances release of

β-Chain hemoglobin M variants exhibit R-T switching,

O₂ at the tissues.

and the Bohr effect is therefore present.

Mutations (eg, hemoglobin Chesapeake) that favor

NUMEROUS MUTANT HUMAN

the R state increase O₂ affinity. These hemoglobins

HEMOGLOBINS HAVE BEEN IDENTIFIED

therefore fail to deliver adequate O₂ to peripheral tis-

sues. The resulting tissue hypoxia leads to poly-

Mutations in the genes that encode the α or β subunits

cythemia, an increased concentration of erythrocytes.

of hemoglobin potentially can affect its biologic func-

tion. However, almost all of the over 800 known mu-

tant human hemoglobins are both extremely rare and

Hemoglobin S

benign, presenting no clinical abnormalities. When a

In HbS, the nonpolar amino acid valine has replaced

mutation does compromise biologic function, the con-

the polar surface residue Glu6 of the β subunit, gener-

dition is termed a hemoglobinopathy. The URL

ating a hydrophobic “sticky patch” on the surface of

http://globin.cse.psu.edu/

(Globin Gene Server) pro-

the β subunit of both oxyHbS and deoxyHbS. Both

vides information about—and links for—normal and

HbA and HbS contain a complementary sticky patch

mutant hemoglobins.

on their surfaces that is exposed only in the deoxy-

genated, R state. Thus, at low PO₂, deoxyHbS can poly-

Methemoglobin & Hemoglobin M

merize to form long, insoluble fibers. Binding of deoxy-

In methemoglobinemia, the heme iron is ferric rather

HbA terminates fiber polymerization, since HbA lacks

than ferrous. Methemoglobin thus can neither bind nor

the second sticky patch necessary to bind another Hb

transport O₂. Normally, the enzyme methemoglobin

molecule (Figure 6-11). These twisted helical fibers

reductase reduces the Fe3+ of methemoglobin to Fe2+.

distort the erythrocyte into a characteristic sickle shape,

Methemoglobin can arise by oxidation of Fe2+

to Fe₃

rendering it vulnerable to lysis in the interstices of the

as a side effect of agents such as sulfonamides, from

splenic sinusoids. They also cause multiple secondary

hereditary hemoglobin M, or consequent to reduced

clinical effects. A low PO₂ such as that at high altitudes

activity of the enzyme methemoglobin reductase.

exacerbates the tendency to polymerize.

Oxy A

Deoxy A

Oxy S

Deoxy S

Deoxy A Deoxy S

Figure 6-11. Representation of the sticky patch (

) on hemoglobin S and its “receptor” (

)

on deoxyhemoglobin A and deoxyhemoglobin S. The complementary surfaces allow deoxyhe-

moglobin S to polymerize into a fibrous structure, but the presence of deoxyhemoglobin A will

terminate the polymerization by failing to provide sticky patches. (Modified and reproduced, with

permission, from Stryer L: Biochemistry, 4th ed. Freeman, 1995.)

PROTEINS: MYOGLOBIN & HEMOGLOBIN

BIOMEDICAL IMPLICATIONS

different primary structures, myoglobin and the sub-

units of hemoglobin have nearly identical secondary

Myoglobinuria

and tertiary structures.

Following massive crush injury, myoglobin released

•

Heme, an essentially planar, slightly puckered, cyclic

from damaged muscle fibers colors the urine dark red.

tetrapyrrole, has a central Fe2+ linked to all four ni-

Myoglobin can be detected in plasma following a my-

trogen atoms of the heme, to histidine F8, and, in

ocardial infarction, but assay of serum enzymes (see

oxyMb and oxyHb, also to O₂.

Chapter 7) provides a more sensitive index of myocar-

•

The O₂-binding curve for myoglobin is hyperbolic,

dial injury.

but for hemoglobin it is sigmoidal, a consequence of

cooperative interactions in the tetramer. Cooperativ-

ity maximizes the ability of hemoglobin both to load

Anemias

O₂ at the PO₂ of the lungs and to deliver O₂ at the

Anemias, reductions in the number of red blood cells or

P^O2 of the tissues.

of hemoglobin in the blood, can reflect impaired syn-

•

Relative affinities of different hemoglobins for oxy-

thesis of hemoglobin (eg, in iron deficiency; Chapter

gen are expressed as P₅₀, the PO₂ that half-saturates

51) or impaired production of erythrocytes (eg, in folic

them with O₂. Hemoglobins saturate at the partial

acid or vitamin B₁₂ deficiency; Chapter 45). Diagnosis

pressures of their respective respiratory organ, eg, the

of anemias begins with spectroscopic measurement of

lung or placenta.

blood hemoglobin levels.

•

On oxygenation of hemoglobin, the iron, histidine

F8, and linked residues move toward the heme ring.

Thalassemias

Conformational changes that accompany oxygena-

tion include rupture of salt bonds and loosening of

The genetic defects known as thalassemias result from

quaternary structure, facilitating binding of addi-

the partial or total absence of one or more α or β chains

tional O₂.

of hemoglobin. Over 750 different mutations have

•

2,3-Bisphosphoglycerate (BPG) in the central cavity

been identified, but only three are common. Either the

of deoxyHb forms salt bonds with the β subunits

α chain (alpha thalassemias) or β chain (beta thal-

that stabilize deoxyHb. On oxygenation, the central

assemias) can be affected. A superscript indicates

cavity contracts, BPG is extruded, and the quaternary

whether a subunit is completely absent (α⁰ or β⁰) or

structure loosens.

whether its synthesis is reduced (α⁺ or β⁺). Apart from

•

Hemoglobin also functions in CO₂

and proton

marrow transplantation, treatment is symptomatic.

transport from tissues to lungs. Release of O₂ from

Certain mutant hemoglobins are common in many

oxyHb at the tissues is accompanied by uptake of

populations, and a patient may inherit more than one

protons due to lowering of the pK_a of histidine

type. Hemoglobin disorders thus present a complex

residues.

pattern of clinical phenotypes. The use of DNA probes

for their diagnosis is considered in Chapter 40.

•

In sickle cell hemoglobin (HbS), Val replaces the β6

Glu of HbA, creating a “sticky patch” that has a

complement on deoxyHb (but not on oxyHb). De-

Glycosylated Hemoglobin (HbA_1c)

oxyHbS polymerizes at low O₂

concentrations,

When blood glucose enters the erythrocytes it glycosy-

forming fibers that distort erythrocytes into sickle

lates the ε-amino group of lysine residues and the

shapes.

amino terminals of hemoglobin. The fraction of hemo-

•

Alpha and beta thalassemias are anemias that result

globin glycosylated, normally about 5%, is proportion-

from reduced production of α and β subunits of

ate to blood glucose concentration. Since the half-life of

HbA, respectively.

an erythrocyte is typically 60 days, the level of glycosy-

lated hemoglobin (HbA_1c) reflects the mean blood glu-

REFERENCES

cose concentration over the preceding

6-8 weeks.

Measurement of HbA_1c therefore provides valuable in-

Bettati S et al: Allosteric mechanism of haemoglobin: Rupture of

salt-bridges raises the oxygen affinity of the T-structure. J

formation for management of diabetes mellitus.

Mol Biol 1998;281:581.

Bunn HF: Pathogenesis and treatment of sickle cell disease. N Engl

SUMMARY

J Med 1997;337:762.

Faustino P et al: Dominantly transmitted beta-thalassemia arising

• Myoglobin is monomeric; hemoglobin is a tetramer

from the production of several aberrant mRNA species and

of two subunit types (α₂β₂ in HbA). Despite having

one abnormal peptide. Blood 1998;91:685.

Enzymes: Mechanism of Action

Victor W. Rodwell, PhD, & Peter J. Kennelly, PhD

BIOMEDICAL IMPORTANCE

with the ability to simultaneously conduct and inde-

pendently control a broad spectrum of chemical

Enzymes are biologic polymers that catalyze the chemi-

processes.

cal reactions which make life as we know it possible.

The presence and maintenance of a complete and bal-

anced set of enzymes is essential for the breakdown of

ENZYMES ARE CLASSIFIED BY REACTION

nutrients to supply energy and chemical building

TYPE & MECHANISM

blocks; the assembly of those building blocks into pro-

A system of enzyme nomenclature that is comprehen-

teins, DNA, membranes, cells, and tissues; and the har-

sive, consistent, and at the same time easy to use has

nessing of energy to power cell motility and muscle

proved elusive. The common names for most enzymes

contraction. With the exception of a few catalytic RNA

derive from their most distinctive characteristic: their

molecules, or ribozymes, the vast majority of enzymes

ability to catalyze a specific chemical reaction. In gen-

are proteins. Deficiencies in the quantity or catalytic ac-

eral, an enzyme’s name consists of a term that identifies

tivity of key enzymes can result from genetic defects,

the type of reaction catalyzed followed by the suffix

nutritional deficits, or toxins. Defective enzymes can re-

-ase. For example, dehydrogenases remove hydrogen

sult from genetic mutations or infection by viral or bac-

atoms, proteases hydrolyze proteins, and isomerases cat-

terial pathogens (eg, Vibrio cholerae). Medical scientists

alyze rearrangements in configuration. One or more

address imbalances in enzyme activity by using pharma-

modifiers usually precede this name. Unfortunately,

cologic agents to inhibit specific enzymes and are inves-

while many modifiers name the specific substrate in-

tigating gene therapy as a means to remedy deficits in

volved (xanthine oxidase), others identify the source of

enzyme level or function.

the enzyme (pancreatic ribonuclease), specify its mode

of regulation (hormone-sensitive lipase), or name a dis-

ENZYMES ARE EFFECTIVE & HIGHLY

tinguishing characteristic of its mechanism (a cysteine

SPECIFIC CATALYSTS

protease). When it was discovered that multiple forms

of some enzymes existed, alphanumeric designators

The enzymes that catalyze the conversion of one or

were added to distinguish between them (eg, RNA

more compounds (substrates) into one or more differ-

polymerase III; protein kinase Cβ). To address the am-

ent compounds (products) enhance the rates of the

biguity and confusion arising from these inconsistencies

corresponding noncatalyzed reaction by factors of at

in nomenclature and the continuing discovery of new

least 10⁶. Like all catalysts, enzymes are neither con-

enzymes, the International Union of Biochemists (IUB)

sumed nor permanently altered as a consequence of

developed a complex but unambiguous system of en-

their participation in a reaction.

zyme nomenclature. In the IUB system, each enzyme

In addition to being highly efficient, enzymes are

has a unique name and code number that reflect the

also extremely selective catalysts. Unlike most catalysts

type of reaction catalyzed and the substrates involved.

used in synthetic chemistry, enzymes are specific both

Enzymes are grouped into six classes, each with several

for the type of reaction catalyzed and for a single sub-

subclasses. For example, the enzyme commonly called

strate or a small set of closely related substrates. En-

“hexokinase” is designated “ATP:D-hexose-6-phospho-

zymes are also stereospecific catalysts and typically cat-

transferase E.C. 2.7.1.1.” This identifies hexokinase as a

alyze reactions only of specific stereoisomers of a given

member of class 2 (transferases), subclass 7 (transfer of a

compound—for example, D- but not L-sugars, L- but

phosphoryl group), sub-subclass 1 (alcohol is the phos-

not D-amino acids. Since they bind substrates through

phoryl acceptor). Finally, the term “hexose-6” indicates

at least “three points of attachment,” enzymes can even

that the alcohol phosphorylated is that of carbon six of

convert nonchiral substrates to chiral products. Figure

a hexose. Listed below are the six IUB classes of en-

7-1 illustrates why the enzyme-catalyzed reduction of

zymes and the reactions they catalyze.

the nonchiral substrate pyruvate produces L-lactate

rather a racemic mixture of D- and L-lactate. The ex-

1. Oxidoreductases catalyze oxidations and reduc-

quisite specificity of enzyme catalysts imbues living cells

tions.

CHAPTER 7

Prosthetic Groups Are Tightly Integrated

Into an Enzyme’s Structure

Prosthetic groups are distinguished by their tight, stable

incorporation into a protein’s structure by covalent or

noncovalent forces. Examples include pyridoxal phos-

phate, flavin mononucleotide

(FMN), flavin dinu-

cleotide

(FAD), thiamin pyrophosphate, biotin, and

the metal ions of Co, Cu, Mg, Mn, Se, and Zn. Metals

are the most common prosthetic groups. The roughly

Enzyme site

Substrate

one-third of all enzymes that contain tightly bound

metal ions are termed metalloenzymes. Metal ions that

Figure 7-1. Planar representation of the “three-

participate in redox reactions generally are complexed

point attachment” of a substrate to the active site of an

to prosthetic groups such as heme (Chapter 6) or iron-

enzyme. Although atoms 1 and 4 are identical, once

sulfur clusters (Chapter 12). Metals also may facilitate

atoms 2 and 3 are bound to their complementary sites

the binding and orientation of substrates, the formation

on the enzyme, only atom 1 can bind. Once bound to

of covalent bonds with reaction intermediates (Co2+ in

an enzyme, apparently identical atoms thus may be dis-

coenzyme B₁₂ ), or interaction with substrates to render

tinguishable, permitting a stereospecific chemical

them more electrophilic (electron-poor) or nucleo-

change.

philic (electron-rich).

Cofactors Associate Reversibly With

2. Transferases catalyze transfer of groups such as

Enzymes or Substrates

methyl or glycosyl groups from a donor molecule

Cofactors serve functions similar to those of prosthetic

to an acceptor molecule.

groups but bind in a transient, dissociable manner ei-

3. Hydrolases catalyze the hydrolytic cleavage of

ther to the enzyme or to a substrate such as ATP. Un-

C C, C O, CN, P O, and certain other

like the stably associated prosthetic groups, cofactors

bonds, including acid anhydride bonds.

therefore must be present in the medium surrounding

4. Lyases catalyze cleavage of C C, C O, CN,

the enzyme for catalysis to occur. The most common

and other bonds by elimination, leaving double

cofactors also are metal ions. Enzymes that require a

bonds, and also add groups to double bonds.

metal ion cofactor are termed metal-activated enzymes

5. Isomerases catalyze geometric or structural

to distinguish them from the metalloenzymes for

changes within a single molecule.

which metal ions serve as prosthetic groups.

6. Ligases catalyze the joining together of two mole-

cules, coupled to the hydrolysis of a pyrophospho-

Coenzymes Serve as Substrate Shuttles

ryl group in ATP or a similar nucleoside triphos-

phate.

Coenzymes serve as recyclable shuttles—or group

transfer reagents—that transport many substrates from

Despite the many advantages of the IUB system,

their point of generation to their point of utilization.

texts tend to refer to most enzymes by their older and

Association with the coenzyme also stabilizes substrates

shorter, albeit sometimes ambiguous names.

such as hydrogen atoms or hydride ions that are unsta-

ble in the aqueous environment of the cell. Other

chemical moieties transported by coenzymes include

methyl groups (folates), acyl groups (coenzyme A), and

PROSTHETIC GROUPS, COFACTORS,

oligosaccharides (dolichol).

& COENZYMES PLAY IMPORTANT

ROLES IN CATALYSIS

Many Coenzymes, Cofactors, & Prosthetic

Many enzymes contain small nonprotein molecules and

Groups Are Derivatives of B Vitamins

metal ions that participate directly in substrate binding

or catalysis. Termed prosthetic groups, cofactors, and

The water-soluble B vitamins supply important compo-

coenzymes, these extend the repertoire of catalytic ca-

nents of numerous coenzymes. Many coenzymes con-

pabilities beyond those afforded by the limited number

tain, in addition, the adenine, ribose, and phosphoryl

of functional groups present on the aminoacyl side

moieties of AMP or ADP (Figure 7-2). Nicotinamide

chains of peptides.

and riboflavin are components of the redox coenzymes

ENZYMES: MECHANISM OF ACTION

Arg 145

NH₂

CH₂

C O

Tyr 248

H H

HO OH

His 196

O^-

CH₂

Zn²⁺

NH₂

His 69

Glu 72

CH₂

Figure 7-3. Two-dimensional representation of a

O^-

dipeptide substrate, glycyl-tyrosine, bound within the

active site of carboxypeptidase A.

H H

Figure 7-2.

Structure of NAD⁺ and NADP⁺. For

tribute to the extensive size and three-dimensional char-

NAD⁺, R = H. For NADP⁺, R = PO32−.

acter of the active site.

ENZYMES EMPLOY MULTIPLE

NAD and NADP and FMN and FAD, respectively.

MECHANISMS TO FACILITATE

Pantothenic acid is a component of the acyl group car-

CATALYSIS

rier coenzyme A. As its pyrophosphate, thiamin partici-

Four general mechanisms account for the ability of en-

pates in decarboxylation of α-keto acids and folic acid

zymes to achieve dramatic catalytic enhancement of the

and cobamide coenzymes function in one-carbon me-

rates of chemical reactions.

tabolism.

Catalysis by Proximity

CATALYSIS OCCURS AT THE ACTIVE SITE

For molecules to react, they must come within bond-

The extreme substrate specificity and high catalytic effi-

forming distance of one another. The higher their con-

ciency of enzymes reflect the existence of an environ-

centration, the more frequently they will encounter one

ment that is exquisitely tailored to a single reaction.

another and the greater will be the rate of their reaction.

Termed the active site, this environment generally

When an enzyme binds substrate molecules in its active

takes the form of a cleft or pocket. The active sites of

site, it creates a region of high local substrate concentra-

multimeric enzymes often are located at the interface

tion. This environment also orients the substrate mole-

between subunits and recruit residues from more than

cules spatially in a position ideal for them to interact, re-

one monomer. The three-dimensional active site both

sulting in rate enhancements of at least a thousandfold.

shields substrates from solvent and facilitates catalysis.

Substrates bind to the active site at a region comple-

Acid-Base Catalysis

mentary to a portion of the substrate that will not un-

dergo chemical change during the course of the reac-

The ionizable functional groups of aminoacyl side

tion. This simultaneously aligns portions of the

chains and (where present) of prosthetic groups con-

substrate that will undergo change with the chemical

tribute to catalysis by acting as acids or bases. Acid-base

functional groups of peptidyl aminoacyl residues. The

catalysis can be either specific or general. By “specific”

active site also binds and orients cofactors or prosthetic

we mean only protons (H₃O⁺) or OH^- ions. In specific

groups. Many amino acyl residues drawn from diverse

acid or specific base catalysis, the rate of reaction is

portions of the polypeptide chain (Figure 7-3) con-

sensitive to changes in the concentration of protons but

CHAPTER 7

independent of the concentrations of other acids (pro-

enzyme’s active site failed to account for the dynamic

ton donors) or bases (proton acceptors) present in solu-

changes that accompany catalysis. This drawback was

tion or at the active site. Reactions whose rates are re-

addressed by Daniel Koshland’s induced fit model,

sponsive to all the acids or bases present are said to be

which states that when substrates approach and bind to

subject to general acid or general base catalysis.

an enzyme they induce a conformational change, a

change analogous to placing a hand (substrate) into a

Catalysis by Strain

glove (enzyme) (Figure 7-5). A corollary is that the en-

zyme induces reciprocal changes in its substrates, har-

Enzymes that catalyze lytic reactions which involve

nessing the energy of binding to facilitate the transfor-

breaking a covalent bond typically bind their substrates

mation of substrates into products. The induced fit

in a conformation slightly unfavorable for the bond

model has been amply confirmed by biophysical studies

that will undergo cleavage. The resulting strain

of enzyme motion during substrate binding.

stretches or distorts the targeted bond, weakening it

and making it more vulnerable to cleavage.

HIV PROTEASE ILLUSTRATES

ACID-BASE CATALYSIS

Covalent Catalysis

Enzymes of the aspartic protease family, which in-

The process of covalent catalysis involves the formation

cludes the digestive enzyme pepsin, the lysosomal

of a covalent bond between the enzyme and one or more

cathepsins, and the protease produced by the human im-

substrates. The modified enzyme then becomes a reac-

munodeficiency virus (HIV), share a common catalytic

tant. Covalent catalysis introduces a new reaction path-

mechanism. Catalysis involves two conserved aspartyl

way that is energetically more favorable—and therefore

residues which act as acid-base catalysts. In the first stage

faster—than the reaction pathway in homogeneous so-

of the reaction, an aspartate functioning as a general base

lution. The chemical modification of the enzyme is,

(Asp X, Figure 7-6) extracts a proton from a water mole-

however, transient. On completion of the reaction, the

cule, making it more nucleophilic. This resulting nucle-

enzyme returns to its original unmodified state. Its role

ophile then attacks the electrophilic carbonyl carbon of

thus remains catalytic. Covalent catalysis is particularly

the peptide bond targeted for hydrolysis, forming a

common among enzymes that catalyze group transfer

tetrahedral transition state intermediate. A second as-

reactions. Residues on the enzyme that participate in co-

partate (Asp Y, Figure 7-6) then facilitates the decompo-

valent catalysis generally are cysteine or serine and occa-

sition of this tetrahedral intermediate by donating a pro-

sionally histidine. Covalent catalysis often follows a

ton to the amino group produced by rupture of the

“ping-pong” mechanism—one in which the first sub-

peptide bond. Two different active site aspartates thus

strate is bound and its product released prior to the

can act simultaneously as a general base or as a general

binding of the second substrate (Figure 7-4).

acid. This is possible because their immediate environ-

ment favors ionization of one but not the other.

SUBSTRATES INDUCE

CONFORMATIONAL CHANGES

CHYMOTRYPSIN & FRUCTOSE-2,6-

IN ENZYMES

BISPHOSPHATASE ILLUSTRATE

Early in the last century, Emil Fischer compared the

COVALENT CATALYSIS

highly specific fit between enzymes and their substrates

Chymotrypsin

to that of a lock and its key. While the “lock and key

model” accounted for the exquisite specificity of en-

While catalysis by aspartic proteases involves the direct

zyme-substrate interactions, the implied rigidity of the

hydrolytic attack of water on a peptide bond, catalysis

Pyr

Glu

Ala

CHO

CH₂NH₂

CHO

E CHO

E CH₂NH₂

E CHO

Ala

Pyr

Glu

Figure 7-4. Ping-pong mechanism for transamination. ECHO and ECH₂NH₂ represent the enzyme-

pyridoxal phosphate and enzyme-pyridoxamine complexes, respectively. (Ala, alanine; Pyr, pyruvate; KG,

α-ketoglutarate; Glu, glutamate).

ENZYMES: MECHANISM OF ACTION

R′

CH₂

Asp X

Asp Y

R′

CH₂

Asp Y

Asp X

Figure 7-5.

Two-dimensional representation of

Koshland’s induced fit model of the active site of a

R′

lyase. Binding of the substrate AB induces conforma-

tional changes in the enzyme that aligns catalytic

residues which participate in catalysis and strains the

bond between A and B, facilitating its cleavage.

by the serine protease chymotrypsin involves prior for-

mation of a covalent acyl enzyme intermediate. A

CH₂

highly reactive seryl residue, serine 195, participates in

Asp Y

Asp X

a charge-relay network with histidine 57 and aspartate

102. Far apart in primary structure, in the active site

Figure 7-6.

Mechanism for catalysis by an aspartic

these residues are within bond-forming distance of one

protease such as HIV protease. Curved arrows indicate

another. Aligned in the order Asp 102-His 57-Ser 195,

directions of electron movement. 1

Aspartate X acts

they constitute a “charge-relay network” that functions

as a base to activate a water molecule by abstracting a

as a “proton shuttle.”

proton. 2

The activated water molecule attacks the

Binding of substrate initiates proton shifts that in ef-

peptide bond, forming a transient tetrahedral interme-

fect transfer the hydroxyl proton of Ser 195 to Asp 102

diate.

3 Aspartate Y acts as an acid to facilitate break-

(Figure 7-7). The enhanced nucleophilicity of the seryl

down of the tetrahedral intermediate and release of the

oxygen facilitates its attack on the carbonyl carbon of

split products by donating a proton to the newly

the peptide bond of the substrate, forming a covalent

formed amino group. Subsequent shuttling of the pro-

acyl-enzyme intermediate. The hydrogen on Asp 102

ton on Asp X to Asp Y restores the protease to its initial

then shuttles through His 57 to the amino group liber-

state.

ated when the peptide bond is cleaved. The portion of

the original peptide with a free amino group then leaves

the active site and is replaced by a water molecule. The

charge-relay network now activates the water molecule

by withdrawing a proton through His 57 to Asp 102.

The resulting hydroxide ion attacks the acyl-enzyme in-

CHAPTER 7

termediate and a reverse proton shuttle returns a proton

to Ser 195, restoring its original state. While modified

R₁

R₂

during the process of catalysis, chymotrypsin emerges

unchanged on completion of the reaction. Trypsin and

N N H O

elastase employ a similar catalytic mechanism, but the

Ser 195

numbers of the residues in their Ser-His-Asp proton

Asp 102

His 57

shuttles differ.

Fructose-2,6-Bisphosphatase

R₁

R₂

Fructose-2,6-bisphosphatase, a regulatory enzyme of

gluconeogenesis (Chapter 19), catalyzes the hydrolytic

N N H O

release of the phosphate on carbon 2 of fructose 2,6-

Ser 195

bisphosphate. Figure 7-8 illustrates the roles of seven

Asp 102

His 57

active site residues. Catalysis involves a “catalytic triad”

of one Glu and two His residues and a covalent phos-

R₁

NH₂

R₂

phohistidyl intermediate.

N N

CATALYTIC RESIDUES ARE

Ser 195

HIGHLY CONSERVED

Asp 102

His 57

Members of an enzyme family such as the aspartic or

serine proteases employ a similar mechanism to catalyze

R₂

a common reaction type but act on different substrates.

Enzyme families appear to arise through gene duplica-

N N

Ser 195

tion events that create a second copy of the gene which

encodes a particular enzyme. The proteins encoded by

Asp 102

His 57

the two genes can then evolve independently to recog-

nize different substrates—resulting, for example, in

chymotrypsin, which cleaves peptide bonds on the car-

R₂

boxyl terminal side of large hydrophobic amino acids;

and trypsin, which cleaves peptide bonds on the car-

N N H O

boxyl terminal side of basic amino acids. The common

Ser 195

ancestry of enzymes can be inferred from the presence

Asp 102

His 57

of specific amino acids in the same position in each

HOOC

R₂

family member. These residues are said to be conserved

residues. Proteins that share a large number of con-

served residues are said to be homologous to one an-

N N H O

other. Table 7-1 illustrates the primary structural con-

Ser 195

servation of two components of the charge-relay

Asp 102

His 57

network for several serine proteases. Among the most

Figure 7-7. Catalysis by chymotrypsin.

1 The

highly conserved residues are those that participate di-

charge-relay system removes a proton from Ser 195,

rectly in catalysis.

making it a stronger nucleophile. 2 Activated Ser 195

attacks the peptide bond, forming a transient tetrahedral

ISOZYMES ARE DISTINCT ENZYME

intermediate. 3

Release of the amino terminal peptide

FORMS THAT CATALYZE THE

is facilitated by donation of a proton to the newly

SAME REACTION

formed amino group by His 57 of the charge-relay sys-

tem, yielding an acyl-Ser 195 intermediate. 4 His 57 and

Higher organisms often elaborate several physically dis-

Asp 102 collaborate to activate a water molecule, which

tinct versions of a given enzyme, each of which cat-

attacks the acyl-Ser 195, forming a second tetrahedral in-

alyzes the same reaction. Like the members of other

termediate. 5 The charge-relay system donates a pro-

protein families, these protein catalysts or isozymes

ton to Ser 195, facilitating breakdown of tetrahedral in-

arise through gene duplication. Isozymes may exhibit

termediate to release the carboxyl terminal peptide 6 .

subtle differences in properties such as sensitivity to

ENZYMES: MECHANISM OF ACTION

particular regulatory factors (Chapter 9) or substrate

Lys 356

affinity

(eg, hexokinase and glucokinase) that adapt

Arg

352

them to specific tissues or circumstances. Some iso-

zymes may also enhance survival by providing a “back-

up” copy of an essential enzyme.

Arg 307

O H

Glu

+ H

327

THE CATALYTIC ACTIVITY OF ENZYMES

His

392

FACILITATES THEIR DETECTION

Arg 257

His 258

Arg 257

His 258

The minute quantities of enzymes present in cells com-

E • Fru-2,6-P₂

E-P • Fru-6-P

plicate determination of their presence and concentra-

tion. However, the ability to rapidly transform thou-

sands of molecules of a specific substrate into products

Lys 356

imbues each enzyme with the ability to reveal its pres-

Arg

352

ence. Assays of the catalytic activity of enzymes are fre-

quently used in research and clinical laboratories.

Under appropriate conditions (see Chapter 8), the rate

Arg 307

of the catalytic reaction being monitored is proportion-

–

Glu

ate to the amount of enzyme present, which allows its

+ H

327

concentration to be inferred.

His

392

Arg 257

His 258

Arg 257

His 258

Enzyme-Linked Immunoassays

E-P • H₂O

E • P_i

The sensitivity of enzyme assays can also be exploited to

Figure 7-8. Catalysis by fructose-2,6-bisphos-

detect proteins that lack catalytic activity. Enzyme-

phatase. (1) Lys 356 and Arg 257, 307, and 352 stabilize

linked immunoassays (ELISAs) use antibodies cova-

the quadruple negative charge of the substrate by

lently linked to a “reporter enzyme” such as alkaline

phosphatase or horseradish peroxidase, enzymes whose

charge-charge interactions. Glu 327 stabilizes the posi-

products are readily detected. When serum or other

tive charge on His 392. (2) The nucleophile His 392 at-

samples to be tested are placed in a plastic microtiter

tacks the C-2 phosphoryl group and transfers it to His

plate, the proteins adhere to the plastic surface and are

258, forming a phosphoryl-enzyme intermediate. Fruc-

immobilized. Any remaining absorbing areas of the well

tose 6-phosphate leaves the enzyme. (3) Nucleophilic

are then “blocked” by adding a nonantigenic protein

attack by a water molecule, possibly assisted by Glu 327

such as bovine serum albumin. A solution of antibody

acting as a base, forms inorganic phosphate. (4) Inor-

covalently linked to a reporter enzyme is then added.

ganic orthophosphate is released from Arg 257 and Arg

The antibodies adhere to the immobilized antigen and

307. (Reproduced, with permission, from Pilkis SJ et al: 6-

these are themselves immobilized. Excess free antibody

Phosphofructo-2-kinase/fructose-2,6-bisphosphatase: A

molecules are then removed by washing. The presence

metabolic signaling enzyme. Annu Rev Biochem

and quantity of bound antibody are then determined

1995;64:799.)

by adding the substrate for the reporter enzyme.

Table 7-1. Amino acid sequences in the neighborhood of the catalytic sites of several

bovine proteases. Regions shown are those on either side of the catalytic site seryl (S) and

histidyl (H) residues.

Enzyme

Sequence Around Serine S

Sequence Around Histidine H

Trypsin

D S C Q D G S G G P V V C S G K

V V S A A H C Y K S G

Chymotrypsin A

S S C M G D S G G P L V C K K N

V V T A A H G G V T T

Chymotrypsin B

S S C M G D S G G P L V C Q K N

V V T A A H C G V T T

Thrombin

D A C E G D S G G P F V M K S P

V L T A A H C L L Y P

CHAPTER 7

tated by the use of radioactive substrates. An alternative

NAD(P)⁺-Dependent Dehydrogenases Are

strategy is to devise a synthetic substrate whose product

Assayed Spectrophotometrically

absorbs light. For example, p-nitrophenyl phosphate is

The physicochemical properties of the reactants in an

an artificial substrate for certain phosphatases and for

enzyme-catalyzed reaction dictate the options for the

chymotrypsin that does not absorb visible light. How-

assay of enzyme activity. Spectrophotometric assays ex-

ever, following hydrolysis, the resulting p-nitrophen-

ploit the ability of a substrate or product to absorb

ylate anion absorbs light at 419 nm.

light. The reduced coenzymes NADH and NADPH,

Another quite general approach is to employ a “cou-

written as NAD(P)H, absorb light at a wavelength of

pled” assay (Figure 7-10). Typically, a dehydrogenase

340 nm, whereas their oxidized forms NAD(P)⁺ do not

whose substrate is the product of the enzyme of interest

(Figure

7-9). When NAD(P)⁺ is reduced, the ab-

is added in catalytic excess. The rate of appearance or

sorbance at 340 nm therefore increases in proportion

disappearance of NAD(P)H then depends on the rate

to—and at a rate determined by—the quantity of

of the enzyme reaction to which the dehydrogenase has

NAD(P)H produced. Conversely, for a dehydrogenase

been coupled.

that catalyzes the oxidation of NAD(P)H, a decrease in

absorbance at 340 nm will be observed. In each case,

the rate of change in optical density at 340 nm will be

THE ANALYSIS OF CERTAIN ENZYMES

proportionate to the quantity of enzyme present.

AIDS DIAGNOSIS

Of the thousands of different enzymes present in the

Many Enzymes Are Assayed by Coupling

human body, those that fulfill functions indispensable

to a Dehydrogenase

to cell vitality are present throughout the body tissues.

The assay of enzymes whose reactions are not accompa-

Other enzymes or isozymes are expressed only in spe-

nied by a change in absorbance or fluorescence is gener-

cific cell types, during certain periods of development,

ally more difficult. In some instances, the product or re-

or in response to specific physiologic or pathophysio-

maining substrate can be transformed into a more

logic changes. Analysis of the presence and distribution

readily detected compound. In other instances, the re-

of enzymes and isozymes—whose expression is nor-

action product may have to be separated from unre-

mally tissue-, time-, or circumstance-specific—often

acted substrate prior to measurement—a process facili-

aids diagnosis.

1.0

Glucose

ATP, Mg²⁺

0.8

HEXOKINASE

ADP, Mg²⁺

0.6

Glucose 6-phosphate

NADP⁺

0.4

GLUCOSE-6-PHOSPHATE

NADH

DEHYDROGENASE

NADPH + H⁺

0.2

6-Phosphogluconolactone

Figure 7-10. Coupled enzyme assay for hexokinase

NAD⁺

activity. The production of glucose 6-phosphate by

200

250

300

350

400

hexokinase is coupled to the oxidation of this product

by glucose-6-phosphate dehydrogenase in the pres-

Wavelength (nm)

ence of added enzyme and NADP⁺. When an excess of

Figure 7-9. Absorption spectra of NAD⁺ and NADH.

glucose-6-phosphate dehydrogenase is present, the

Densities are for a 44 mg/L solution in a cell with a 1 cm

rate of formation of NADPH, which can be measured at

light path. NADP⁺ and NADPH have spectrums analo-

340 nm, is governed by the rate of formation of glucose

gous to NAD⁺ and NADH, respectively.

6-phosphate by hexokinase.

ENZYMES: MECHANISM OF ACTION

Nonfunctional Plasma Enzymes Aid

heart) and M (for muscle). The subunits can combine

Diagnosis & Prognosis

as shown below to yield catalytically active isozymes of

L-lactate dehydrogenase:

Certain enzymes, proenzymes, and their substrates are

present at all times in the circulation of normal individ-

uals and perform a physiologic function in the blood.

Lactate

Examples of these functional plasma enzymes include

Dehydrogenase

lipoprotein lipase, pseudocholinesterase, and the proen-

Isozyme

Subunits

zymes of blood coagulation and blood clot dissolution

I₁

HHHH

(Chapters 9 and 51). The majority of these enzymes are

I₂

HHHM

synthesized in and secreted by the liver.

I₃

HHMM

Plasma also contains numerous other enzymes that

I₄

HMMM

perform no known physiologic function in blood.

I₅

MMMM

These apparently nonfunctional plasma enzymes arise

from the routine normal destruction of erythrocytes,

leukocytes, and other cells. Tissue damage or necrosis

Distinct genes whose expression is differentially regu-

resulting from injury or disease is generally accompa-

lated in various tissues encode the H and M subunits.

nied by increases in the levels of several nonfunctional

Since heart expresses the H subunit almost exclusively,

plasma enzymes. Table 7-2 lists several enzymes used

isozyme I₁ predominates in this tissue. By contrast,

in diagnostic enzymology.

isozyme I₅ predominates in liver. Small quantities of

lactate dehydrogenase are normally present in plasma.

Isozymes of Lactate Dehydrogenase Are

Following a myocardial infarction or in liver disease,

Used to Detect Myocardial Infarctions

the damaged tissues release characteristic lactate dehy-

L-Lactate dehydrogenase is a tetrameric enzyme whose

drogenase isoforms into the blood. The resulting eleva-

four subunits occur in two isoforms, designated H (for

tion in the levels of the I₁ or I₅ isozymes is detected by

separating the different oligomers of lactate dehydroge-

nase by electrophoresis and assaying their catalytic ac-

tivity (Figure 7-11).

Table 7-2. Principal serum enzymes used in

clinical diagnosis. Many of the enzymes are not

ENZYMES FACILITATE DIAGNOSIS

specific for the disease listed.

OF GENETIC DISEASES

Serum Enzyme

Major Diagnostic Use

While many diseases have long been known to result

from alterations in an individual’s DNA, tools for the

Aminotransferases

detection of genetic mutations have only recently be-

Aspartate aminotransfer-

Myocardial infarction

come widely available. These techniques rely upon the

ase (AST, or SGOT)

catalytic efficiency and specificity of enzyme catalysts.

Alanine aminotransferase

Viral hepatitis

For example, the polymerase chain reaction (PCR) re-

(ALT, or SGPT)

lies upon the ability of enzymes to serve as catalytic am-

Amylase

Acute pancreatitis

plifiers to analyze the DNA present in biologic and

forensic samples. In the PCR technique, a thermostable

Ceruloplasmin

Hepatolenticular degeneration

(Wilson’s disease)

DNA polymerase, directed by appropriate oligonu-

cleotide primers, produces thousands of copies of a

Creatine kinase

Muscle disorders and myocar-

sample of DNA that was present initially at levels too

dial infarction

low for direct detection.

γ-Glutamyl transpeptidase

Various liver diseases

The detection of restriction fragment length poly-

morphisms (RFLPs) facilitates prenatal detection of

Lactate dehydrogenase

Myocardial infarction

hereditary disorders such as sickle cell trait, beta-

(isozymes)

thalassemia, infant phenylketonuria, and Huntington’s

Lipase

Acute pancreatitis

disease. Detection of RFLPs involves cleavage of dou-

ble-stranded DNA by restriction endonucleases, which

Phosphatase, acid

Metastatic carcinoma of the

prostate

can detect subtle alterations in DNA that affect their

recognized sites. Chapter 40 provides further details

Phosphatase, alkaline

Various bone disorders, ob-

concerning the use of PCR and restriction enzymes for

(isozymes)

structive liver diseases

diagnosis.

CHAPTER 7

LACTATE

(Lactate)

SH₂

(Pyruvate)

DEHYDROGENASE

Heart

NAD⁺

NADH + H⁺

Normal

Reduced PMS

Oxidized PMS

Liver

Oxidized NBT

Reduced NBT

(colorless)

(blue formazan)

Figure 7-11. Normal and pathologic patterns of lactate dehydrogenase (LDH) isozymes in human

serum. LDH isozymes of serum were separated by electrophoresis and visualized using the coupled reac-

tion scheme shown on the left. (NBT, nitroblue tetrazolium; PMS, phenazine methylsulfate). At right is

shown the stained electropherogram. Pattern A is serum from a patient with a myocardial infarct; B is nor-

mal serum; and C is serum from a patient with liver disease. Arabic numerals denote specific LDH isozymes.

RECOMBINANT DNA PROVIDES AN

resulting modified protein, termed a fusion protein,

contains a domain tailored to interact with a specific

IMPORTANT TOOL FOR STUDYING

affinity support. One popular approach is to attach an

ENZYMES

oligonucleotide that encodes six consecutive histidine

Recombinant DNA technology has emerged as an im-

residues. The expressed “His tag” protein binds to chro-

portant asset in the study of enzymes. Highly purified

matographic supports that contain an immobilized diva-

samples of enzymes are necessary for the study of their

lent metal ion such as Ni2+. Alternatively, the substrate-

structure and function. The isolation of an individual

binding domain of glutathione S-transferase (GST) can

enzyme, particularly one present in low concentration,

serve as a “GST tag.” Figure 7-12 illustrates the purifi-

from among the thousands of proteins present in a cell

cation of a GST-fusion protein using an affinity support

can be extremely difficult. If the gene for the enzyme of

containing bound glutathione. Fusion proteins also

interest has been cloned, it generally is possible to pro-

often encode a cleavage site for a highly specific protease

duce large quantities of its encoded protein in Esch-

such as thrombin in the region that links the two por-

erichia coli or yeast. However, not all animal proteins

tions of the protein. This permits removal of the added

can be expressed in active form in microbial cells, nor

fusion domain following affinity purification.

do microbes perform certain posttranslational process-

ing tasks. For these reasons, a gene may be expressed in

Site-Directed Mutagenesis Provides

cultured animal cell systems employing the baculovirus

Mechanistic Insights

expression vector to transform cultured insect cells. For

more details concerning recombinant DNA techniques,

Once the ability to express a protein from its cloned

see Chapter 40.

gene has been established, it is possible to employ site-

directed mutagenesis to change specific aminoacyl

residues by altering their codons. Used in combination

Recombinant Fusion Proteins Are Purified

with kinetic analyses and x-ray crystallography, this ap-

by Affinity Chromatography

proach facilitates identification of the specific roles of

Recombinant DNA technology can also be used to cre-

given aminoacyl residues in substrate binding and catal-

ate modified proteins that are readily purified by affinity

ysis. For example, the inference that a particular

chromatography. The gene of interest is linked to an

aminoacyl residue functions as a general acid can be

oligonucleotide sequence that encodes a carboxyl or

tested by replacing it with an aminoacyl residue inca-

amino terminal extension to the encoded protein. The

pable of donating a proton.

ENZYMES: MECHANISM OF ACTION

GST

Enzyme

•

Catalytic mechanisms employed by enzymes include

the introduction of strain, approximation of reac-

tants, acid-base catalysis, and covalent catalysis.

Plasmid encoding GST

Cloned DNA

with thrombin site (T)

encoding enzyme

•

Aminoacyl residues that participate in catalysis are

highly conserved among all classes of a given enzyme

activity.

•

Substrates and enzymes induce mutual conforma-

Ligate together

tional changes in one another that facilitate substrate

recognition and catalysis.

•

The catalytic activity of enzymes reveals their pres-

GST

Enzyme

ence, facilitates their detection, and provides the basis

for enzyme-linked immunoassays.

Transfect cells, add

•

Many enzymes can be assayed spectrophotometri-

inducing agent, then

break cells

cally by coupling them to an NAD(P)⁺-dependent

dehydrogenase.

Apply to glutathione (GSH)

•

Assay of plasma enzymes aids diagnosis and progno-

affinity column

sis. For example, a myocardial infarction elevates

serum levels of lactate dehydrogenase isozyme I₁

Sepharose

GSH

GST

Enzyme

bead

•

Restriction endonucleases facilitate diagnosis of ge-

netic diseases by revealing restriction fragment length

Elute with GSH,

polymorphisms.

treat with thrombin

•

Site-directed mutagenesis, used to change residues

suspected of being important in catalysis or substrate

GSH

GST

Enzyme

binding, provides insights into the mechanisms of

enzyme action.

•

Recombinant fusion proteins such as His-tagged or

Figure 7-12. Use of glutathione S-transferase (GST)

GST fusion enzymes are readily purified by affinity

fusion proteins to purify recombinant proteins. (GSH,

chromatography.

glutathione.)

REFERENCES

SUMMARY

Conyers GB et al: Metal requirements of a diadenosine pyrophos-

phatase from Bartonella bacilliformis. Magnetic resonance and

• Enzymes are highly effective and extremely specific

kinetic studies of the role of Mn2+. Biochemistry

2000;

catalysts.

39:2347.

• Organic and inorganic prosthetic groups, cofactors,

Fersht A: Structure and Mechanism in Protein Science: A Guide to

and coenzymes play important roles in catalysis.

Enzyme Catalysis and Protein Folding. Freeman, 1999.

Coenzymes, many of which are derivatives of B vita-

Suckling CJ: Enzyme Chemistry. Chapman & Hall, 1990.

mins, serve as “shuttles.”

Walsh CT: Enzymatic Reaction Mechanisms. Freeman, 1979.

Enzymes: Kinetics

Victor W. Rodwell, PhD, & Peter J. Kennelly, PhD

BIOMEDICAL IMPORTANCE

A+B → P+Q

(2)

Enzyme kinetics is the field of biochemistry concerned

Unidirectional arrows are also used to describe reac-

with the quantitative measurement of the rates of en-

tions in living cells where the products of reaction (2)

zyme-catalyzed reactions and the systematic study of fac-

are immediately consumed by a subsequent enzyme-

tors that affect these rates. Kinetic analyses permit scien-

catalyzed reaction. The rapid removal of product P or

tists to reconstruct the number and order of the

Q therefore precludes occurrence of the reverse reac-

individual steps by which enzymes transform substrates

tion, rendering equation (2) functionally irreversible

into products. The study of enzyme kinetics also repre-

under physiologic conditions.

sents the principal way to identify potential therapeutic

agents that selectively enhance or inhibit the rates of spe-

cific enzyme-catalyzed processes. Together with site-

CHANGES IN FREE ENERGY DETERMINE

directed mutagenesis and other techniques that probe

THE DIRECTION & EQUILIBRIUM STATE

protein structure, kinetic analysis can also reveal details

OF CHEMICAL REACTIONS

of the catalytic mechanism. A complete, balanced set of

enzyme activities is of fundamental importance for main-

The Gibbs free energy change ∆G (also called either the

taining homeostasis. An understanding of enzyme kinet-

free energy or Gibbs energy) describes both the direc-

ics thus is important for understanding how physiologic

tion in which a chemical reaction will tend to proceed

stresses such as anoxia, metabolic acidosis or alkalosis,

and the concentrations of reactants and products that

toxins, and pharmacologic agents affect that balance.

will be present at equilibrium. ∆G for a chemical reac-

tion equals the sum of the free energies of formation of

the reaction products ∆G_P minus the sum of the free

CHEMICAL REACTIONS ARE DESCRIBED

energies of formation of the substrates ∆G_S. ∆G⁰ de-

USING BALANCED EQUATIONS

notes the change in free energy that accompanies transi-

A balanced chemical equation lists the initial chemical

tion from the standard state, one-molar concentrations

species

(substrates) present and the new chemical

of substrates and products, to equilibrium. A more use-

species (products) formed for a particular chemical re-

ful biochemical term is ∆G0′, which defines ∆G⁰ at a

action, all in their correct proportions or stoichiome-

standard state of 10−7 M protons, pH 7.0 (Chapter 10).

try. For example, balanced equation (1) below describes

If the free energy of the products is lower than that of

the reaction of one molecule each of substrates A and B

the substrates, the signs of ∆G⁰ and ∆G0′ will be nega-

to form one molecule each of products P and Q.

tive, indicating that the reaction as written is favored in

the direction left to right. Such reactions are referred to

A+B → P+ Q

(1)

as spontaneous. The sign and the magnitude of the

free energy change determine how far the reaction will

The double arrows indicate reversibility, an intrinsic

proceed. Equation (3)—

property of all chemical reactions. Thus, for reaction

(1), if A and B can form P and Q, then P and Q can

∆G0=−RT

ln K

(3)

also form A and B. Designation of a particular reactant

as a “substrate” or “product” is therefore somewhat ar-

—illustrates the relationship between the equilibrium

bitrary since the products for a reaction written in one

constant K_eq and ∆G⁰, where R is the gas constant (1.98

direction are the substrates for the reverse reaction. The

cal/mol/°K or 8.31 J/mol/°K) and T is the absolute

term “products” is, however, often used to designate

temperature in degrees Kelvin. K_eq is equal to the prod-

the reactants whose formation is thermodynamically fa-

uct of the concentrations of the reaction products, each

vored. Reactions for which thermodynamic factors

raised to the power of their stoichiometry, divided by

strongly favor formation of the products to which the

the product of the substrates, each raised to the power

arrow points often are represented with a single arrow

of their stoichiometry.

as if they were “irreversible”:

ENZYMES: KINETICS

For the reaction A + B → P + Q—

characteristic changes in free energy, ∆G

F, and ∆GD are

associated with each partial reaction.

[P][Q]

K_eq

(4)

[A][B]

E+R− L^← EL L

∆

(8)

and for reaction (5)

ELRLL

→

E−R+L

∆

(9)

←

(5)

A+A^← P

E+R−L

→←

E−R+L

∆G=

∆G

(8-10)

[P]

K_eq

(6)

[A]2

For the overall reaction (10), ∆G is the sum of ∆G

F and

∆GD. As for any equation of two terms, it is not possi-

—∆G⁰ may be calculated from equation (3) if the con-

ble to infer from ∆G either the sign or the magnitude

centrations of substrates and products present at equi-

of ∆G

F or ∆GD.

librium are known. If ∆G⁰ is a negative number, K_eq

Many reactions involve multiple transition states,

will be greater than unity and the concentration of

each with an associated change in free energy. For these

products at equilibrium will exceed that of substrates. If

reactions, the overall ∆G represents the sum of all of

∆G⁰ is positive, K_eq will be less than unity and the for-

the free energy changes associated with the formation

mation of substrates will be favored.

and decay of all of the transition states. Therefore, it is

Notice that, since ∆G⁰ is a function exclusively of

not possible to infer from the overall G the num-

the initial and final states of the reacting species, it can

ber or type of transition states through which the re-

provide information only about the direction and equi-

action proceeds. Stated another way: overall thermo-

librium state of the reaction. ∆G⁰ is independent of the

dynamics tells us nothing about kinetics.

mechanism of the reaction and therefore provides no

information concerning rates of reactions. Conse-

∆G_F Defines the Activation Energy

quently—and as explained below—although a reaction

may have a large negative ∆G⁰ or ∆G0′, it may never-

Regardless of the sign or magnitude of ∆G, ∆G

F for the

theless take place at a negligible rate.

overwhelming majority of chemical reactions has a pos-

itive sign. The formation of transition state intermedi-

ates therefore requires surmounting of energy barriers.

THE RATES OF REACTIONS

For this reason, ∆G

F is often termed the activation en-

ARE DETERMINED BY THEIR

ergy, E_act, the energy required to surmount a given en-

ACTIVATION ENERGY

ergy barrier. The ease—and hence the frequency—with

which this barrier is overcome is inversely related to

Reactions Proceed via Transition States

Eact. The thermodynamic parameters that determine

how fast a reaction proceeds thus are the ∆G

F values for

The concept of the transition state is fundamental to

formation of the transition states through which the re-

understanding the chemical and thermodynamic basis

action proceeds. For a simple reaction, where means

of catalysis. Equation (7) depicts a displacement reac-

“proportionate to,”

tion in which an entering group E displaces a leaving

group L, attached initially to R.

−E

act

(11)

→

Rate ∝ e RT

E+R−L

E−R+L

←

(7)

Midway through the displacement, the bond between

The activation energy for the reaction proceeding in the

R and L has weakened but has not yet been completely

opposite direction to that drawn is equal to −∆G

severed, and the new bond between E and R is as yet

incompletely formed. This transient intermediate—in

NUMEROUS FACTORS AFFECT

which neither free substrate nor product exists—is

termed the transition state, E R L. Dotted lines

THE REACTION RATE

represent the “partial” bonds that are undergoing for-

The kinetic theory—also called the collision theory—

mation and rupture.

of chemical kinetics states that for two molecules to

Reaction (7) can be thought of as consisting of two

react they must (1) approach within bond-forming dis-

“partial reactions,” the first corresponding to the forma-

tance of one another, or “collide”; and (2) must possess

tion (F) and the second to the subsequent decay (D) of

sufficient kinetic energy to overcome the energy barrier

the transition state intermediate. As for all reactions,

for reaching the transition state. It therefore follows

CHAPTER 8

that anything which increases the frequency or energy of

which can also be written as

collision between substrates will increase the rate of the

reaction in which they participate.

A+B+B→P

(15)

the corresponding rate expression is

Temperature

Rate ∝ [A][B][B]

(16)

Raising the temperature increases the kinetic energy of

molecules. As illustrated in Figure 8-1, the total num-

ber of molecules whose kinetic energy exceeds the en-

ergy barrier E_act (vertical bar) for formation of products

Rate ∝ [A][B]²

(17)

increases from low (A), through intermediate (B), to

high (C) temperatures. Increasing the kinetic energy of

For the general case when n molecules of A react with

molecules also increases their motion and therefore the

m molecules of B,

frequency with which they collide. This combination of

more frequent and more highly energetic and produc-

nA + mB → P

(18)

tive collisions increases the reaction rate.

the rate expression is

Reactant Concentration

]

(19)

The frequency with which molecules collide is directly

proportionate to their concentrations. For two different

Replacing the proportionality constant with an equal

molecules A and B, the frequency with which they col-

sign by introducing a proportionality or rate constant

lide will double if the concentration of either A or B is

k characteristic of the reaction under study gives equa-

doubled. If the concentrations of both A and B are dou-

tions (20) and (21), in which the subscripts 1 and −1

bled, the probability of collision will increase fourfold.

refer to the rate constants for the forward and reverse

For a chemical reaction proceeding at constant tem-

reactions, respectively.

perature that involves one molecule each of A and B,

Rate

[A]

[B]

(20)

A+B→P

(12)

the number of molecules that possess kinetic energy

Rate

−1

[P]

(21)

sufficient to overcome the activation energy barrier will

be a constant. The number of collisions with sufficient

K_eq Is a Ratio of Rate Constants

energy to produce product P therefore will be directly

proportionate to the number of collisions between A

While all chemical reactions are to some extent re-

and B and thus to their molar concentrations, denoted

versible, at equilibrium the overall concentrations of re-

by square brackets.

actants and products remain constant. At equilibrium,

the rate of conversion of substrates to products there-

Rate ∝ [A][B]

(13)

fore equals the rate at which products are converted to

substrates.

Similarly, for the reaction represented by

A +2B→P

(14)

Rate =Rate

−1

(22)

Therefore,

k [A]n[B]

−

k [P

]

(23)

Energy barrier

∞

and

[P]

(24)

−1

[A]

[B]

The ratio of k₁ to k−1 is termed the equilibrium con-

stant, K_eq. The following important properties of a sys-

∞

Kinetic energy

tem at equilibrium must be kept in mind:

(1) The equilibrium constant is a ratio of the reaction

Figure 8-1. The energy barrier for chemical

rate constants (not the reaction rates).

reactions.

ENZYMES: KINETICS

(2) At equilibrium, the reaction rates (not the rate

∆Go =−RT

ln K

(25)

constants) of the forward and back reactions are

equal.

If we include the presence of the enzyme (E) in the cal-

(3) Equilibrium is a dynamic state. Although there is

culation of the equilibrium constant for a reaction,

no net change in the concentration of substrates

or products, individual substrate and product

A+B+Enz^← P+Q+Enz

(26)

molecules are continually being interconverted.

the expression for the equilibrium constant,

(4) The numeric value of the equilibrium constant

Keq can be calculated either from the concentra-

P][Q][Enz]

K_eq

(27)

tions of substrates and products at equilibrium or

A][B][Enz

[

]

from the ratio k₁/k−1.

reduces to one identical to that for the reaction in the

absence of the enzyme:

THE KINETICS OF

ENZYMATIC CATALYSIS

P][Q]

K_eq

(28)

[A][B]

Enzymes Lower the Activation Energy

Barrier for a Reaction

Enzymes therefore have no effect on K_eq.

All enzymes accelerate reaction rates by providing tran-

sition states with a lowered ∆G

F for formation of the

MULTIPLE FACTORS AFFECT THE RATES

transition states. However, they may differ in the way

this is achieved. Where the mechanism or the sequence

OF ENZYME-CATALYZED REACTIONS

of chemical steps at the active site is essentially the same

Temperature

as those for the same reaction proceeding in the absence

of a catalyst, the environment of the active site lowers

Raising the temperature increases the rate of both uncat-

F by stabilizing the transition state intermediates. As

alyzed and enzyme-catalyzed reactions by increasing the

discussed in Chapter 7, stabilization can involve (1)

kinetic energy and the collision frequency of the react-

acid-base groups suitably positioned to transfer protons

ing molecules. However, heat energy can also increase

to or from the developing transition state intermediate,

the kinetic energy of the enzyme to a point that exceeds

(2) suitably positioned charged groups or metal ions

the energy barrier for disrupting the noncovalent inter-

that stabilize developing charges, or (3) the imposition

actions that maintain the enzyme’s three-dimensional

of steric strain on substrates so that their geometry ap-

structure. The polypeptide chain then begins to unfold,

proaches that of the transition state. HIV protease (Fig-

or denature, with an accompanying rapid loss of cat-

ure 7-6) illustrates catalysis by an enzyme that lowers

alytic activity. The temperature range over which an

the activation barrier by stabilizing a transition state in-

enzyme maintains a stable, catalytically competent con-

termediate.

formation depends upon—and typically moderately

Catalysis by enzymes that proceeds via a unique re-

exceeds—the normal temperature of the cells in which

action mechanism typically occurs when the transition

it resides. Enzymes from humans generally exhibit sta-

state intermediate forms a covalent bond with the en-

bility at temperatures up to 45-55 °C. By contrast,

zyme (covalent catalysis). The catalytic mechanism of

enzymes from the thermophilic microorganisms that re-

the serine protease chymotrypsin (Figure 7-7) illus-

side in volcanic hot springs or undersea hydrothermal

trates how an enzyme utilizes covalent catalysis to pro-

vents may be stable up to or above 100 °C.

vide a unique reaction pathway.

The Q₁₀, or temperature coefficient, is the factor

by which the rate of a biologic process increases for a

10 °C increase in temperature. For the temperatures

ENZYMES DO NOT AFFECT K_eq

over which enzymes are stable, the rates of most bio-

Enzymes accelerate reaction rates by lowering the acti-

logic processes typically double for a 10 °C rise in tem-

vation barrier ∆G

F. While they may undergo transient

perature (Q₁₀ = 2). Changes in the rates of enzyme-

modification during the process of catalysis, enzymes

catalyzed reactions that accompany a rise or fall in body

emerge unchanged at the completion of the reaction.

temperature constitute a prominent survival feature for

The presence of an enzyme therefore has no effect on

“cold-blooded” life forms such as lizards or fish, whose

∆G⁰ for the overall reaction, which is a function solely

body temperatures are dictated by the external environ-

of the initial and final states of the reactants. Equation

ment. However, for mammals and other homeothermic

(25) shows the relationship between the equilibrium

organisms, changes in enzyme reaction rates with tem-

constant for a reaction and the standard free energy

perature assume physiologic importance only in cir-

change for that reaction:

cumstances such as fever or hypothermia.

CHAPTER 8

Hydrogen Ion Concentration

the rate of the forward reaction. Assays of enzyme activ-

ity almost always employ a large (10³-10⁷) molar excess

The rate of almost all enzyme-catalyzed reactions ex-

of substrate over enzyme. Under these conditions, v_i is

hibits a significant dependence on hydrogen ion con-

proportionate to the concentration of enzyme. Measur-

centration. Most intracellular enzymes exhibit optimal

ing the initial velocity therefore permits one to estimate

activity at pH values between 5 and 9. The relationship

the quantity of enzyme present in a biologic sample.

of activity to hydrogen ion concentration (Figure 8-2)

reflects the balance between enzyme denaturation at

SUBSTRATE CONCENTRATION AFFECTS

high or low pH and effects on the charged state of the

enzyme, the substrates, or both. For enzymes whose

REACTION RATE

mechanism involves acid-base catalysis, the residues in-

In what follows, enzyme reactions are treated as if they

volved must be in the appropriate state of protonation

had only a single substrate and a single product. While

for the reaction to proceed. The binding and recogni-

most enzymes have more than one substrate, the princi-

tion of substrate molecules with dissociable groups also

ples discussed below apply with equal validity to en-

typically involves the formation of salt bridges with the

zymes with multiple substrates.

enzyme. The most common charged groups are the

For a typical enzyme, as substrate concentration is

negative carboxylate groups and the positively charged

increased, v_i increases until it reaches a maximum value

groups of protonated amines. Gain or loss of critical

Vmax (Figure 8-3). When further increases in substrate

charged groups thus will adversely affect substrate bind-

concentration do not further increase v_i, the enzyme is

ing and thus will retard or abolish catalysis.

said to be “saturated” with substrate. Note that the

shape of the curve that relates activity to substrate con-

ASSAYS OF ENZYME-CATALYZED

centration (Figure 8-3) is hyperbolic. At any given in-

REACTIONS TYPICALLY MEASURE

stant, only substrate molecules that are combined with

the enzyme as an ES complex can be transformed into

THE INITIAL VELOCITY

product. Second, the equilibrium constant for the for-

Most measurements of the rates of enzyme-catalyzed re-

mation of the enzyme-substrate complex is not infi-

actions employ relatively short time periods, conditions

nitely large. Therefore, even when the substrate is pre-

that approximate initial rate conditions. Under these

sent in excess (points A and B of Figure 8-4), only a

conditions, only traces of product accumulate, hence

fraction of the enzyme may be present as an ES com-

the rate of the reverse reaction is negligible. The initial

plex. At points A or B, increasing or decreasing [S]

velocity (v_i ) of the reaction thus is essentially that of

therefore will increase or decrease the number of ES

complexes with a corresponding change in v_i. At point

C (Figure 8-4), essentially all the enzyme is present as

the ES complex. Since no free enzyme remains available

100

for forming ES, further increases in [S] cannot increase

the rate of the reaction. Under these saturating condi-

tions, v_i depends solely on—and thus is limited by—

SH⁺

E ^-

the rapidity with which free enzyme is released to com-

bine with more substrate.

V_max

Low

High

V_max/2

v_i

Figure 8-2. Effect of pH on enzyme activity. Con-

sider, for example, a negatively charged enzyme (EH⁻)

V_max/2

that binds a positively charged substrate (SH⁺). Shown

is the proportion (%) of SH⁺ [\\\] and of EH⁻ [///] as a

K_m

[S]

function of pH. Only in the cross-hatched area do both

the enzyme and the substrate bear an appropriate

Figure 8-3. Effect of substrate concentration on the

charge.

initial velocity of an enzyme-catalyzed reaction.

ENZYMES: KINETICS

= S

= E

Figure 8-4. Representation of an enzyme at low (A), at high (C), and at a substrate concentration

equal to K_m (B). Points A, B, and C correspond to those points in Figure 8-3.

THE MICHAELIS-MENTEN & HILL

equal to [S]. Replacing K_m + [S] with [S] reduces equa-

tion (29) to

EQUATIONS MODEL THE EFFECTS

OF SUBSTRATE CONCENTRATION

max

V [S]

max

[S]

≈

≈V

(31)

The Michaelis-Menten Equation

max

[S]

The Michaelis-Menten equation

(29) illustrates in

mathematical terms the relationship between initial re-

Thus, when [S] greatly exceeds K_m, the reaction velocity

action velocity v_i and substrate concentration

[S],

is maximal (V_max) and unaffected by further increases in

shown graphically in Figure 8-3.

substrate concentration.

(3) When [S] = K_m (point B in Figures 8-3 and

]

8-4).

= max[

(29)

[S]

V [S]

[S]

max

(32)

The Michaelis constant K_m is the substrate concen-

[S]

2[S]

tration at which v_i is half the maximal velocity

(V_max/2) attainable at a particular concentration of

Equation (32) states that when [S] equals K_m, the initial

enzyme. K_m thus has the dimensions of substrate con-

velocity is half-maximal. Equation (32) also reveals that

centration. The dependence of initial reaction velocity

Km is—and may be determined experimentally from—

on [S] and K_m may be illustrated by evaluating the

the substrate concentration at which the initial velocity

Michaelis-Menten equation under three conditions.

is half-maximal.

(1) When [S] is much less than K_m (point A in Fig-

ures 8-3 and 8-4), the term K_m + [S] is essentially equal

A Linear Form of the Michaelis-Menten

to K_m. Replacing K_m + [S] with K_m reduces equation

Equation Is Used to Determine K_m & V_max

(29) to

The direct measurement of the numeric value of V_max

[S]

V



and therefore the calculation of K_m often requires im-

max

(30)

v ≈ V

≈

[S]





practically high concentrations of substrate to achieve

+[S]





saturating conditions. A linear form of the Michaelis-

where ≈ means “approximately equal to.” Since V_max

Menten equation circumvents this difficulty and per-

and K_m are both constants, their ratio is a constant. In

mits V_max and K_m to be extrapolated from initial veloc-

other words, when [S] is considerably below K_m, v_i ∝

ity data obtained at less than saturating concentrations

k[S]. The initial reaction velocity therefore is directly

of substrate. Starting with equation (29),

proportionate to [S].

(2) When [S] is much greater than K_m (point C in

= Vmax[

(29)

Figures 8-3 and 8-4), the term K_m + [S] is essentially

[S]

CHAPTER 8

invert

Stated another way, the smaller the tendency of the en-

zyme and its substrate to dissociate, the greater the affin-

[S]

ity of the enzyme for its substrate. While the Michaelis

(33)

max

[S]

constant K_m often approximates the dissociation con-

stant K_d, this is by no means always the case. For a typi-

factor

cal enzyme-catalyzed reaction,

[S]

(34)

Vmax

[S]

max

[S]

(39)

E+ S

←

→ +

and simplify

−1



the value of [S] that gives v_i = V_max/2 is

= 

(35)









[S]

Vmax

max

−1

[S]=

(40)

Equation (35) is the equation for a straight line, y = ax

+ b, where y = 1/v_i and x = 1/[S]. A plot of 1/v_i as y as a

function of 1/[S] as x therefore gives a straight line

When k−1 » k₂, then

whose y intercept is 1/V_max and whose slope is K_m/V_max.

Such a plot is called a double reciprocal or

+k ≈k

(41)

Lineweaver-Burk plot (Figure 8-5). Setting the y term

−1

of equation (36) equal to zero and solving for x reveals

and

that the x intercept is −1/K_m.

−b

−1

(42)

0= ax+b; therefore, x =

(36)

[S]

≈

≈Kd

−1

Km is thus most easily calculated from the x intercept.

Hence, 1/K_m only approximates 1/K_d under conditions

where the association and dissociation of the ES com-

K_m May Approximate a Binding Constant

plex is rapid relative to the rate-limiting step in cataly-

sis. For the many enzyme-catalyzed reactions for which

The affinity of an enzyme for its substrate is the inverse

k−1 + k2 is not approximately equal to k −1, 1/Km will

of the dissociation constant K_d for dissociation of the

underestimate 1/K_d.

enzyme substrate complex ES.

The Hill Equation Describes the Behavior

(37)

E+ S

←

of Enzymes That Exhibit Cooperative

−1

Binding of Substrate

−1

While most enzymes display the simple saturation ki-

Kd = k

(38)

netics depicted in Figure 8-3 and are adequately de-

scribed by the Michaelis-Menten expression, some en-

zymes bind their substrates in a cooperative fashion

analogous to the binding of oxygen by hemoglobin

(Chapter 6). Cooperative behavior may be encountered

K_m

for multimeric enzymes that bind substrate at multiple

Slope =

V_max

sites. For enzymes that display positive cooperativity in

v_i

binding substrate, the shape of the curve that relates

changes in v_i to changes in [S] is sigmoidal (Figure

8-6). Neither the Michaelis-Menten expression nor its

–

K_m

derived double-reciprocal plots can be used to evaluate

V_max

cooperative saturation kinetics. Enzymologists therefore

employ a graphic representation of the Hill equation

[S]

originally derived to describe the cooperative binding of

O₂

by hemoglobin. Equation (43) represents the Hill

Figure 8-5. Double reciprocal or Lineweaver-Burk

equation arranged in a form that predicts a straight line,

plot of 1/v_i versus 1/[S] used to evaluate K_m and V_max.

where k′ is a complex constant.

ENZYMES: KINETICS

first substrate molecule then enhances the affinity of the

∞

enzyme for binding additional substrate. The greater

the value for n, the higher the degree of cooperativity

and the more sigmoidal will be the plot of v_i versus [S].

A perpendicular dropped from the point where the y

term log v_i/(V_max − v_i) is zero intersects the x axis at a

substrate concentration termed S₅₀, the substrate con-

v_i

centration that results in half-maximal velocity. S₅₀ thus

is analogous to the P₅₀ for oxygen binding to hemoglo-

bin (Chapter 6).

KINETIC ANALYSIS DISTINGUISHES

COMPETITIVE FROM

∞

NONCOMPETITIVE INHIBITION

[S]

Inhibitors of the catalytic activities of enzymes provide

Figure 8-6. Representation of sigmoid substrate

both pharmacologic agents and research tools for study

saturation kinetics.

of the mechanism of enzyme action. Inhibitors can be

classified based upon their site of action on the enzyme,

on whether or not they chemically modify the enzyme,

or on the kinetic parameters they influence. Kinetically,

log

(43)

log[S]−log

k′

we distinguish two classes of inhibitors based upon

max

−v1

whether raising the substrate concentration does or

Equation (43) states that when [S] is low relative to k′,

does not overcome the inhibition.

the initial reaction velocity increases as the nth power

of [S].

Competitive Inhibitors Typically

A graph of log v_i/(V_max − v_i) versus log[S] gives a

Resemble Substrates

straight line (Figure 8-7), where the slope of the line n

The effects of competitive inhibitors can be overcome

is the Hill coefficient, an empirical parameter whose

by raising the concentration of the substrate. Most fre-

value is a function of the number, kind, and strength of

quently, in competitive inhibition the inhibitor, I,

the interactions of the multiple substrate-binding sites

binds to the substrate-binding portion of the active site

on the enzyme. When n = 1, all binding sites behave in-

and blocks access by the substrate. The structures of

dependently, and simple Michaelis-Menten kinetic be-

most classic competitive inhibitors therefore tend to re-

havior is observed. If n is greater than 1, the enzyme is

semble the structures of a substrate and thus are termed

said to exhibit positive cooperativity. Binding of the

substrate analogs. Inhibition of the enzyme succinate

dehydrogenase by malonate illustrates competitive inhi-

bition by a substrate analog. Succinate dehydrogenase

catalyzes the removal of one hydrogen atom from each

of the two methylene carbons of succinate (Figure 8-8).

Both succinate and its structural analog malonate

(−OOC  CH2  COO−) can bind to the active site of

Slope = n

succinate dehydrogenase, forming an ES or an EI com-

plex, respectively. However, since malonate contains

- 1

- 4

S₅₀

– 3

C COO

-2H

C COO^-

Log [S]

^–OOC

^-OOC

C H

SUCCINATE

DEHYDROGENASE

Figure 8-7. A graphic representation of a linear

form of the Hill equation is used to evaluate S₅₀, the

Succinate

Fumarate

substrate concentration that produces half-maximal

velocity, and the degree of cooperativity n.

Figure 8-8. The succinate dehydrogenase reaction.

CHAPTER 8

only one methylene carbon, it cannot undergo dehy-

drogenation. The formation and dissociation of the EI

complex is a dynamic process described by

v_i

→

(44)

EnzI

←

Enz+I

−1

K′_m

for which the equilibrium constant K_i is

K_m

V_max

[S]

K = [En ][] = k

(45)

[EnzI]

−1

Figure 8-9. Lineweaver-Burk plot of competitive in-

hibition. Note the complete relief of inhibition at high

In effect, a competitive inhibitor acts by decreasing

[S] (ie, low 1/[S]).

the number of free enzyme molecules available to

bind substrate, ie, to form ES, and thus eventually

to form product, as described below:

For simple competitive inhibition, the intercept on

E-I

the x axis is

E-S

−1

[I]

(47)





E + P





(46)

A competitive inhibitor and substrate exert reciprocal

Once K_m has been determined in the absence of in-

effects on the concentration of the EI and ES com-

hibitor, K_i can be calculated from equation (47). K_i val-

plexes. Since binding substrate removes free enzyme

ues are used to compare different inhibitors of the same

available to combine with inhibitor, increasing the [S]

enzyme. The lower the value for K_i, the more effective

decreases the concentration of the EI complex and

the inhibitor. For example, the statin drugs that act as

raises the reaction velocity. The extent to which [S]

competitive inhibitors of HMG-CoA reductase (Chap-

must be increased to completely overcome the inhibi-

ter 26) have K_i values several orders of magnitude lower

tion depends upon the concentration of inhibitor pre-

than the K_m for the substrate HMG-CoA.

sent, its affinity for the enzyme K_i, and the K_m of the

enzyme for its substrate.

Simple Noncompetitive Inhibitors Lower

Double Reciprocal Plots Facilitate the

V_max but Do Not Affect K_m

Evaluation of Inhibitors

In noncompetitive inhibition, binding of the inhibitor

Double reciprocal plots distinguish between competi-

does not affect binding of substrate. Formation of both

tive and noncompetitive inhibitors and simplify evalua-

EI and EIS complexes is therefore possible. However,

tion of inhibition constants K_i. v_i is determined at sev-

while the enzyme-inhibitor complex can still bind sub-

eral substrate concentrations both in the presence and

strate, its efficiency at transforming substrate to prod-

in the absence of inhibitor. For classic competitive inhi-

uct, reflected by V_max, is decreased. Noncompetitive

bition, the lines that connect the experimental data

inhibitors bind enzymes at sites distinct from the sub-

points meet at the y axis (Figure 8-9). Since the y inter-

strate-binding site and generally bear little or no struc-

cept is equal to 1/V_max, this pattern indicates that when

tural resemblance to the substrate.

1/[S] approaches 0, v_i is independent of the presence

For simple noncompetitive inhibition, E and EI

of inhibitor. Note, however, that the intercept on the

possess identical affinity for substrate, and the EIS com-

x axis does vary with inhibitor concentration—and that

plex generates product at a negligible rate (Figure 8-10).

since −1/K_m′ is smaller than 1/K_m, K_m′ (the “apparent

More complex noncompetitive inhibition occurs when

Km”) becomes larger in the presence of increasing con-

binding of the inhibitor does affect the apparent affinity

centrations of inhibitor. Thus, a competitive inhibitor

of the enzyme for substrate, causing the lines to inter-

has no effect on V_max but raises K ′_m, the apparent

cept in either the third or fourth quadrants of a double

K_m for the substrate.

reciprocal plot (not shown).

ENZYMES: KINETICS

EAB-EPQ

v_i

A B

V′_max

K_m

V_max

EAB-EPQ

[S]

Figure 8-10. Lineweaver-Burk plot for simple non-

B A

Q P

competitive inhibition.

EA-FP

FB-EQ

Irreversible Inhibitors “Poison” Enzymes

Figure 8-11. Representations of three classes of Bi-

In the above examples, the inhibitors form a dissocia-

Bi reaction mechanisms. Horizontal lines represent the

ble, dynamic complex with the enzyme. Fully active en-

enzyme. Arrows indicate the addition of substrates and

zyme can therefore be recovered simply by removing

departure of products. Top: An ordered Bi-Bi reaction,

the inhibitor from the surrounding medium. However,

characteristic of many NAD(P)H-dependent oxidore-

a variety of other inhibitors act irreversibly by chemi-

ductases. Center: A random Bi-Bi reaction, characteris-

cally modifying the enzyme. These modifications gen-

erally involve making or breaking covalent bonds with

tic of many kinases and some dehydrogenases. Bot-

aminoacyl residues essential for substrate binding, catal-

tom: A ping-pong reaction, characteristic of

ysis, or maintenance of the enzyme’s functional confor-

aminotransferases and serine proteases.

mation. Since these covalent changes are relatively sta-

ble, an enzyme that has been

“poisoned” by an

irreversible inhibitor remains inhibited even after re-

moval of the remaining inhibitor from the surrounding

reactions because the group undergoing transfer is usu-

medium.

ally passed directly, in a single step, from one substrate

to the other. Sequential Bi-Bi reactions can be further

distinguished based on whether the two substrates add

MOST ENZYME-CATALYZED REACTIONS

in a random or in a compulsory order. For random-

INVOLVE TWO OR MORE SUBSTRATES

order reactions, either substrate A or substrate B may

combine first with the enzyme to form an EA or an EB

While many enzymes have a single substrate, many oth-

complex (Figure 8-11, center). For compulsory-order

ers have two—and sometimes more than two—sub-

reactions, A must first combine with E before B can

strates and products. The fundamental principles dis-

combine with the EA complex. One explanation for a

cussed above, while illustrated for single-substrate

compulsory-order mechanism is that the addition of A

enzymes, apply also to multisubstrate enzymes. The

induces a conformational change in the enzyme that

mathematical expressions used to evaluate multisub-

aligns residues which recognize and bind B.

strate reactions are, however, complex. While detailed

kinetic analysis of multisubstrate reactions exceeds the

scope of this chapter, two-substrate, two-product reac-

Ping-Pong Reactions

tions (termed “Bi-Bi” reactions) are considered below.

The term

“ping-pong” applies to mechanisms in

which one or more products are released from the en-

Sequential or Single

zyme before all the substrates have been added. Ping-

Displacement Reactions

pong reactions involve covalent catalysis and a tran-

In sequential reactions, both substrates must combine

sient, modified form of the enzyme

(Figure

7-4).

with the enzyme to form a ternary complex before

Ping-pong Bi-Bi reactions are double displacement re-

catalysis can proceed (Figure 8-11, top). Sequential re-

actions. The group undergoing transfer is first dis-

actions are sometimes referred to as single displacement

placed from substrate A by the enzyme to form product

CHAPTER 8

Increasing

[S₂]

v_i

Figure 8-12. Lineweaver-Burk plot for a two-sub-

strate ping-pong reaction. An increase in concentra-

tion of one substrate (S₁) while that of the other sub-

strate (S₂) is maintained constant changes both the x

S₁

and y intercepts, but not the slope.

P and a modified form of the enzyme (F). The subse-

other combinations of product inhibitor and variable

quent group transfer from F to the second substrate B,

substrate will produce forms of complex noncompeti-

forming product Q and regenerating E, constitutes the

tive inhibition.

second displacement (Figure 8-11, bottom).

Most Bi-Bi Reactions Conform to

SUMMARY

Michaelis-Menten Kinetics

•

The study of enzyme kinetics—the factors that affect

Most Bi-Bi reactions conform to a somewhat more

the rates of enzyme-catalyzed reactions—reveals the

complex form of Michaelis-Menten kinetics in which

individual steps by which enzymes transform sub-

strates into products.

Vmax refers to the reaction rate attained when both sub-

strates are present at saturating levels. Each substrate

•

∆G, the overall change in free energy for a reaction,

has its own characteristic K_m value which corresponds

is independent of reaction mechanism and provides

to the concentration that yields half-maximal velocity

no information concerning rates of reactions.

when the second substrate is present at saturating levels.

•

Enzymes do not affect Keq. Keq, a ratio of reaction

As for single-substrate reactions, double-reciprocal plots

rate constants, may be calculated from the concentra-

can be used to determine V_max and K_m. v_i is measured as

tions of substrates and products at equilibrium or

a function of the concentration of one substrate (the

from the ratio k₁/k−1.

variable substrate) while the concentration of the other

•

Reactions proceed via transition states in which ∆G

substrate (the fixed substrate) is maintained constant. If

is the activation energy. Temperature, hydrogen ion

the lines obtained for several fixed-substrate concentra-

concentration, enzyme concentration, substrate con-

tions are plotted on the same graph, it is possible to dis-

centration, and inhibitors all affect the rates of en-

tinguish between a ping-pong enzyme, which yields

zyme-catalyzed reactions.

parallel lines, and a sequential mechanism, which yields

•

A measurement of the rate of an enzyme-catalyzed

a pattern of intersecting lines (Figure 8-12).

reaction generally employs initial rate conditions, for

Product inhibition studies are used to complement

which the essential absence of product precludes the

kinetic analyses and to distinguish between ordered and

reverse reaction.

random Bi-Bi reactions. For example, in a random-

order Bi-Bi reaction, each product will be a competitive

•

A linear form of the Michaelis-Menten equation sim-

inhibitor regardless of which substrate is designated the

plifies determination of K_m and V_max.

variable substrate. However, for a sequential mecha-

•

A linear form of the Hill equation is used to evaluate

nism (Figure 8-11, bottom), only product Q will give

the cooperative substrate-binding kinetics exhibited

the pattern indicative of competitive inhibition when A

by some multimeric enzymes. The slope n, the Hill

is the variable substrate, while only product P will pro-

coefficient, reflects the number, nature, and strength

duce this pattern with B as the variable substrate. The

of the interactions of the substrate-binding sites. A

Enzymes: Regulation of Activities

Victor W. Rodwell, PhD, & Peter J. Kennelly, PhD

BIOMEDICAL IMPORTANCE

concentration generate corresponding changes in me-

tabolite flux (Figure 9-1). Responses to changes in sub-

The 19th-century physiologist Claude Bernard enunci-

strate level represent an important but passive means for

ated the conceptual basis for metabolic regulation. He

coordinating metabolite flow and maintaining homeo-

observed that living organisms respond in ways that are

stasis in quiescent cells. However, they offer limited

both quantitatively and temporally appropriate to per-

scope for responding to changes in environmental vari-

mit them to survive the multiple challenges posed by

ables. The mechanisms that regulate enzyme activity in

changes in their external and internal environments.

an active manner in response to internal and external

Walter Cannon subsequently coined the term “homeo-

signals are discussed below.

stasis” to describe the ability of animals to maintain a

constant intracellular environment despite changes in

their external environment. We now know that organ-

Metabolite Flow Tends

isms respond to changes in their external and internal

to Be Unidirectional

environment by balanced, coordinated changes in the

Despite the existence of short-term oscillations in

rates of specific metabolic reactions. Many human dis-

metabolite concentrations and enzyme levels, living

eases, including cancer, diabetes, cystic fibrosis, and

cells exist in a dynamic steady state in which the mean

Alzheimer’s disease, are characterized by regulatory dys-

concentrations of metabolic intermediates remain rela-

functions triggered by pathogenic agents or genetic mu-

tively constant over time (Figure 9-2). While all chemi-

tations. For example, many oncogenic viruses elaborate

cal reactions are to some extent reversible, in living cells

protein-tyrosine kinases that modify the regulatory

the reaction products serve as substrates for—and are

events which control patterns of gene expression, con-

removed by—other enzyme-catalyzed reactions. Many

tributing to the initiation and progression of cancer. The

nominally reversible reactions thus occur unidirection-

toxin from Vibrio cholerae, the causative agent of cholera,

ally. This succession of coupled metabolic reactions is

disables sensor-response pathways in intestinal epithelial

accompanied by an overall change in free energy that

cells by ADP-ribosylating the GTP-binding proteins

favors unidirectional metabolite flow (Chapter 10). The

(G-proteins) that link cell surface receptors to adenylyl

unidirectional flow of metabolites through a pathway

cyclase. The consequent activation of the cyclase triggers

with a large overall negative change in free energy is

the flow of water into the intestines, resulting in massive

analogous to the flow of water through a pipe in which

diarrhea and dehydration. Yersinia pestis, the causative

one end is lower than the other. Bends or kinks in the

agent of plague, elaborates a protein-tyrosine phos-

pipe simulate individual enzyme-catalyzed steps with a

phatase that hydrolyzes phosphoryl groups on key cy-

small negative or positive change in free energy. Flow of

toskeletal proteins. Knowledge of factors that control the

water through the pipe nevertheless remains unidirec-

rates of enzyme-catalyzed reactions thus is essential to an

tional due to the overall change in height, which corre-

understanding of the molecular basis of disease. This

sponds to the overall change in free energy in a pathway

chapter outlines the patterns by which metabolic

(Figure 9-3).

processes are controlled and provides illustrative exam-

ples. Subsequent chapters provide additional examples.

COMPARTMENTATION ENSURES

REGULATION OF METABOLITE FLOW

METABOLIC EFFICIENCY

CAN BE ACTIVE OR PASSIVE

& SIMPLIFIES REGULATION

Enzymes that operate at their maximal rate cannot re-

In eukaryotes, anabolic and catabolic pathways that in-

spond to an increase in substrate concentration, and

terconvert common products may take place in specific

can respond only to a precipitous decrease in substrate

subcellular compartments. For example, many of the

concentration. For most enzymes, therefore, the aver-

enzymes that degrade proteins and polysaccharides re-

age intracellular concentration of their substrate tends

side inside organelles called lysosomes. Similarly, fatty

to be close to the K_m value, so that changes in substrate

acid biosynthesis occurs in the cytosol, whereas fatty

ENZYMES: REGULATION OF ACTIVITIES

∆V_B

∆V_A

K_m

∆S

[ S ]

Figure 9-1. Differential response of the rate of an

enzyme-catalyzed reaction, ∆V, to the same incremen-

Figure 9-3. Hydrostatic analogy for a pathway with

tal change in substrate concentration at a substrate

a rate-limiting step (A) and a step with a ∆G value near

concentration of K_m (∆V_A) or far above K_m (∆V_B).

zero (B).

acid oxidation takes place within mitochondria (Chap-

generation from those of NADPH that participate in

ters 21 and 22). Segregation of certain metabolic path-

the reductive steps in many biosynthetic pathways.

ways within specialized cell types can provide further

physical compartmentation. Alternatively, possession of

Controlling an Enzyme That Catalyzes

one or more unique intermediates can permit apparently

a Rate-Limiting Reaction Regulates

opposing pathways to coexist even in the absence of

an Entire Metabolic Pathway

physical barriers. For example, despite many shared in-

termediates and enzymes, both glycolysis and gluconeo-

While the flux of metabolites through metabolic path-

genesis are favored energetically. This cannot be true if

ways involves catalysis by numerous enzymes, active

all the reactions were the same. If one pathway was fa-

control of homeostasis is achieved by regulation of only

vored energetically, the other would be accompanied by

a small number of enzymes. The ideal enzyme for regu-

a change in free energy G equal in magnitude but op-

latory intervention is one whose quantity or catalytic ef-

posite in sign. Simultaneous spontaneity of both path-

ficiency dictates that the reaction it catalyzes is slow rel-

ways results from substitution of one or more reactions

ative to all others in the pathway. Decreasing the

by different reactions favored thermodynamically in the

catalytic efficiency or the quantity of the catalyst for the

opposite direction. The glycolytic enzyme phospho-

“bottleneck” or rate-limiting reaction immediately re-

fructokinase

(Chapter 17) is replaced by the gluco-

duces metabolite flux through the entire pathway. Con-

neogenic enzyme fructose-1,6-bisphosphatase (Chapter

versely, an increase in either its quantity or catalytic ef-

19). The ability of enzymes to discriminate between the

ficiency enhances flux through the pathway as a whole.

structurally similar coenzymes NAD⁺ and NADP⁺ also

For example, acetyl-CoA carboxylase catalyzes the syn-

results in a form of compartmentation, since it segre-

thesis of malonyl-CoA, the first committed reaction of

gates the electrons of NADH that are destined for ATP

fatty acid biosynthesis (Chapter 21). When synthesis of

malonyl-CoA is inhibited, subsequent reactions of fatty

acid synthesis cease due to lack of substrates. Enzymes

that catalyze rate-limiting steps serve as natural “gover-

Large

nors” of metabolic flux. Thus, they constitute efficient

molecules

targets for regulatory intervention by drugs. For exam-

ple, inhibition by “statin” drugs of HMG-CoA reduc-

tase, which catalyzes the rate-limiting reaction of cho-

Small

Nutrients

Wastes

molecules

lesterogenesis, curtails synthesis of cholesterol.

REGULATION OF ENZYME QUANTITY

Small

molecules

The catalytic capacity of the rate-limiting reaction in a

metabolic pathway is the product of the concentration

Figure 9-2. An idealized cell in steady state. Note

of enzyme molecules and their intrinsic catalytic effi-

that metabolite flow is unidirectional.

ciency. It therefore follows that catalytic capacity can be

CHAPTER 9

influenced both by changing the quantity of enzyme

Enzyme levels in mammalian tissues respond to a

present and by altering its intrinsic catalytic efficiency.

wide range of physiologic, hormonal, or dietary factors.

For example, glucocorticoids increase the concentration

of tyrosine aminotransferase by stimulating k_s, and

Control of Enzyme Synthesis

glucagon—despite its antagonistic physiologic effects—

increases k_s fourfold to fivefold. Regulation of liver

Enzymes whose concentrations remain essentially con-

arginase can involve changes either in k_s or in k_deg. After

stant over time are termed constitutive enzymes. By

a protein-rich meal, liver arginase levels rise and argi-

contrast, the concentrations of many other enzymes de-

nine synthesis decreases (Chapter 29). Arginase levels

pend upon the presence of inducers, typically sub-

also rise in starvation, but here arginase degradation de-

strates or structurally related compounds, that initiate

creases, whereas k_s remains unchanged. Similarly, injec-

their synthesis. Escherichia coli grown on glucose will,

tion of glucocorticoids and ingestion of tryptophan

for example, only catabolize lactose after addition of a

both elevate levels of tryptophan oxygenase. While the

β-galactoside, an inducer that initiates synthesis of a

hormone raises k_s for oxygenase synthesis, tryptophan

β-galactosidase and a galactoside permease (Figure 39-3).

specifically lowers k_deg by stabilizing the oxygenase

Inducible enzymes of humans include tryptophan pyr-

against proteolytic digestion.

rolase, threonine dehydrase, tyrosine-α-ketoglutarate

aminotransferase, enzymes of the urea cycle, HMG-CoA

reductase, and cytochrome P450. Conversely, an excess

MULTIPLE OPTIONS ARE AVAILABLE FOR

of a metabolite may curtail synthesis of its cognate

enzyme via repression. Both induction and repression

REGULATING CATALYTIC ACTIVITY

involve cis elements, specific DNA sequences located up-

In humans, the induction of protein synthesis is a com-

stream of regulated genes, and trans-acting regulatory

plex multistep process that typically requires hours to

proteins. The molecular mechanisms of induction and

produce significant changes in overall enzyme level. By

repression are discussed in Chapter 39.

contrast, changes in intrinsic catalytic efficiency ef-

fected by binding of dissociable ligands (allosteric reg-

ulation) or by covalent modification achieve regula-

Control of Enzyme Degradation

tion of enzymic activity within seconds. Changes in

protein level serve long-term adaptive requirements,

The absolute quantity of an enzyme reflects the net bal-

whereas changes in catalytic efficiency are best suited

ance between enzyme synthesis and enzyme degrada-

for rapid and transient alterations in metabolite flux.

tion, where k_s and k_deg represent the rate constants for

the overall processes of synthesis and degradation, re-

spectively. Changes in both the k_s and k_deg of specific

enzymes occur in human subjects.

ALLOSTERIC EFFECTORS REGULATE

CERTAIN ENZYMES

Enzyme

Feedback inhibition refers to inhibition of an enzyme

k_s

k_deg

in a biosynthetic pathway by an end product of that

pathway. For example, for the biosynthesis of D from A

Amino acids

catalyzed by enzymes Enz₁ through Enz₃,

Protein turnover represents the net result of en-

Enz₁

Enz

zyme synthesis and degradation. By measuring the rates

→

of incorporation of ¹⁵N-labeled amino acids into pro-

tein and the rates of loss of ¹⁵N from protein, Schoen-

heimer deduced that body proteins are in a state of “dy-

high concentrations of D inhibit conversion of A to B.

namic equilibrium” in which they are continuously

Inhibition results not from the “backing up” of inter-

synthesized and degraded. Mammalian proteins are de-

mediates but from the ability of D to bind to and in-

graded both by ATP and ubiquitin-dependent path-

hibit Enz₁. Typically, D binds at an allosteric site spa-

ways and by ATP-independent pathways (Chapter 29).

tially distinct from the catalytic site of the target

Susceptibility to proteolytic degradation can be influ-

enzyme. Feedback inhibitors thus are allosteric effectors

enced by the presence of ligands such as substrates,

and typically bear little or no structural similarity to the

coenzymes, or metal ions that alter protein conforma-

substrates of the enzymes they inhibit. In this example,

tion. Intracellular ligands thus can influence the rates at

the feedback inhibitor D acts as a negative allosteric

which specific enzymes are degraded.

effector of Enz₁.

ENZYMES: REGULATION OF ACTIVITIES

In a branched biosynthetic pathway, the initial reac-

tions participate in the synthesis of several products.

Figure 9-4 shows a hypothetical branched biosynthetic

pathway in which curved arrows lead from feedback in-

hibitors to the enzymes whose activity they inhibit. The

S₁

S₂

S₃

S₄

sequences S₃ → A, S₄ → B, S₄ → C, and S₃ → → D

each represent linear reaction sequences that are feed-

S₅

back-inhibited by their end products. The pathways of

nucleotide biosynthesis (Chapter 34) provide specific

examples.

The kinetics of feedback inhibition may be competi-

Figure 9-5. Multiple feedback inhibition in a

tive, noncompetitive, partially competitive, or mixed.

branched biosynthetic pathway. Superimposed on sim-

Feedback inhibitors, which frequently are the small

ple feedback loops (dashed, curved arrows) are multi-

molecule building blocks of macromolecules (eg, amino

ple feedback loops (solid, curved arrows) that regulate

acids for proteins, nucleotides for nucleic acids), typi-

enzymes common to biosynthesis of several end prod-

cally inhibit the first committed step in a particular

ucts.

biosynthetic sequence. A much-studied example is inhi-

bition of bacterial aspartate transcarbamoylase by CTP

(see below and Chapter 34).

phosphate (CTP). Following treatment with mercuri-

Multiple feedback loops can provide additional fine

als, ATCase loses its sensitivity to inhibition by CTP

control. For example, as shown in Figure 9-5, the pres-

but retains its full activity for synthesis of carbamoyl as-

ence of excess product B decreases the requirement for

partate. This suggests that CTP is bound at a different

substrate S₂. However, S₂ is also required for synthesis

(allosteric) site from either substrate. ATCase consists

of A, C, and D. Excess B should therefore also curtail

of multiple catalytic and regulatory subunits. Each cat-

synthesis of all four end products. To circumvent this

alytic subunit contains four aspartate (substrate) sites

potential difficulty, each end product typically only

and each regulatory subunit at least two CTP (regula-

partially inhibits catalytic activity. The effect of an ex-

tory) sites (Chapter 34).

cess of two or more end products may be strictly addi-

tive or, alternatively, may be greater than their individ-

ual effect (cooperative feedback inhibition).

Allosteric & Catalytic Sites Are

Spatially Distinct

The lack of structural similarity between a feedback in-

Aspartate Transcarbamoylase Is a Model

hibitor and the substrate for the enzyme whose activity

Allosteric Enzyme

it regulates suggests that these effectors are not isosteric

Aspartate transcarbamoylase (ATCase), the catalyst for

with a substrate but allosteric

(“occupy another

the first reaction unique to pyrimidine biosynthesis

space”). Jacques Monod therefore proposed the exis-

(Figure

34-7), is feedback-inhibited by cytidine tri-

tence of allosteric sites that are physically distinct from

the catalytic site. Allosteric enzymes thus are those

whose activity at the active site may be modulated by

the presence of effectors at an allosteric site. This hy-

pothesis has been confirmed by many lines of evidence,

including x-ray crystallography and site-directed muta-

genesis, demonstrating the existence of spatially distinct

S₁

S₂

S₃

S₄

active and allosteric sites on a variety of enzymes.

S₅

Allosteric Effects May Be on K_m or on V_max

To refer to the kinetics of allosteric inhibition as “com-

Figure 9-4. Sites of feedback inhibition in a

petitive” or

“noncompetitive” with substrate carries

branched biosynthetic pathway. S₁-S₅ are intermedi-

misleading mechanistic implications. We refer instead

ates in the biosynthesis of end products A-D. Straight

to two classes of regulated enzymes: K-series and V-se-

arrows represent enzymes catalyzing the indicated con-

ries enzymes. For K-series allosteric enzymes, the sub-

versions. Curved arrows represent feedback loops and

strate saturation kinetics are competitive in the sense

indicate sites of feedback inhibition by specific end

that K_m is raised without an effect on V_max. For V-series

products.

allosteric enzymes, the allosteric inhibitor lowers V_max

CHAPTER 9

without affecting the K_m. Alterations in K_m or V_max

REGULATORY COVALENT

probably result from conformational changes at the cat-

MODIFICATIONS CAN BE

alytic site induced by binding of the allosteric effector

REVERSIBLE OR IRREVERSIBLE

at the allosteric site. For a K-series allosteric enzyme,

this conformational change may weaken the bonds be-

In mammalian cells, the two most common forms of

tween substrate and substrate-binding residues. For a

covalent modification are partial proteolysis and

V-series allosteric enzyme, the primary effect may be to

phosphorylation. Because cells lack the ability to re-

alter the orientation or charge of catalytic residues, low-

unite the two portions of a protein produced by hydrol-

ering V_max. Intermediate effects on K_m and V_max, how-

ysis of a peptide bond, proteolysis constitutes an irre-

ever, may be observed consequent to these conforma-

versible modification. By contrast, phosphorylation is a

tional changes.

reversible modification process. The phosphorylation of

proteins on seryl, threonyl, or tyrosyl residues, catalyzed

FEEDBACK REGULATION

by protein kinases, is thermodynamically spontaneous.

Equally spontaneous is the hydrolytic removal of these

IS NOT SYNONYMOUS WITH

phosphoryl groups by enzymes called protein phos-

FEEDBACK INHIBITION

phatases.

In both mammalian and bacterial cells, end products

“feed back” and control their own synthesis, in many

PROTEASES MAY BE SECRETED AS

instances by feedback inhibition of an early biosyn-

CATALYTICALLY INACTIVE PROENZYMES

thetic enzyme. We must, however, distinguish between

feedback regulation, a phenomenologic term devoid

Certain proteins are synthesized and secreted as inactive

of mechanistic implications, and feedback inhibition,

precursor proteins known as proproteins. The propro-

a mechanism for regulation of enzyme activity. For ex-

teins of enzymes are termed proenzymes or zymogens.

ample, while dietary cholesterol decreases hepatic syn-

Selective proteolysis converts a proprotein by one or

thesis of cholesterol, this feedback regulation does not

more successive proteolytic “clips” to a form that ex-

involve feedback inhibition. HMG-CoA reductase, the

hibits the characteristic activity of the mature protein,

rate-limiting enzyme of cholesterologenesis, is affected,

eg, its enzymatic activity. Proteins synthesized as pro-

but cholesterol does not feedback-inhibit its activity.

proteins include the hormone insulin (proprotein =

Regulation in response to dietary cholesterol involves

proinsulin), the digestive enzymes pepsin, trypsin, and

curtailment by cholesterol or a cholesterol metabolite of

chymotrypsin (proproteins = pepsinogen, trypsinogen,

the expression of the gene that encodes HMG-CoA re-

and chymotrypsinogen, respectively), several factors of

ductase (enzyme repression) (Chapter 26).

the blood clotting and blood clot dissolution cascades

(see Chapter 51), and the connective tissue protein col-

lagen (proprotein = procollagen).

MANY HORMONES ACT THROUGH

ALLOSTERIC SECOND MESSENGERS

Proenzymes Facilitate Rapid

Nerve impulses—and binding of hormones to cell sur-

Mobilization of an Activity in Response

face receptors—elicit changes in the rate of enzyme-

to Physiologic Demand

catalyzed reactions within target cells by inducing the re-

lease or synthesis of specialized allosteric effectors called

The synthesis and secretion of proteases as catalytically

second messengers. The primary or “first” messenger is

inactive proenzymes protects the tissue of origin (eg,

the hormone molecule or nerve impulse. Second mes-

the pancreas) from autodigestion, such as can occur in

sengers include 3′,5′-cAMP, synthesized from ATP by

pancreatitis. Certain physiologic processes such as di-

the enzyme adenylyl cyclase in response to the hormone

gestion are intermittent but fairly regular and pre-

epinephrine, and Ca²⁺, which is stored inside the endo-

dictable. Others such as blood clot formation, clot dis-

plasmic reticulum of most cells. Membrane depolariza-

solution, and tissue repair are brought “on line” only in

tion resulting from a nerve impulse opens a membrane

response to pressing physiologic or pathophysiologic

channel that releases calcium ion into the cytoplasm,

need. The processes of blood clot formation and dis-

where it binds to and activates enzymes involved in the

solution clearly must be temporally coordinated to

regulation of contraction and the mobilization of stored

achieve homeostasis. Enzymes needed intermittently

glucose from glycogen. Glucose then supplies the in-

but rapidly often are secreted in an initially inactive

creased energy demands of muscle contraction. Other

form since the secretion process or new synthesis of the

second messengers include 3′,5′-cGMP and polyphos-

required proteins might be insufficiently rapid for re-

phoinositols, produced by the hydrolysis of inositol

sponse to a pressing pathophysiologic demand such as

phospholipids by hormone-regulated phospholipases.

the loss of blood.

ENZYMES: REGULATION OF ACTIVITIES

13 14 15

146

149

245

Pro-CT

13 14 15

146

149

245

π-CT

14-15

147-148

146

149

245

α-CT

Figure 9-6. Selective proteolysis and associated conformational changes form the

active site of chymotrypsin, which includes the Asp102-His57-Ser195 catalytic triad.

Successive proteolysis forms prochymotrypsin (pro-CT), π-chymotrypsin (π-CT), and ul-

timately α-chymotrypsin (α-CT), an active protease whose three peptides remain asso-

ciated by covalent inter-chain disulfide bonds.

Activation of Prochymotrypsin

catalyzing transfer of the terminal phosphoryl group of

Requires Selective Proteolysis

ATP to the hydroxyl groups of seryl, threonyl, or tyro-

syl residues, forming O-phosphoseryl, O-phosphothre-

Selective proteolysis involves one or more highly spe-

onyl, or O-phosphotyrosyl residues, respectively (Figure

cific proteolytic clips that may or may not be accompa-

9-7). Some protein kinases target the side chains of his-

nied by separation of the resulting peptides. Most im-

tidyl, lysyl, arginyl, and aspartyl residues. The unmodi-

portantly, selective proteolysis often results in

fied form of the protein can be regenerated by hy-

conformational changes that “create” the catalytic site

drolytic removal of phosphoryl groups, catalyzed by

of an enzyme. Note that while His 57 and Asp 102 re-

protein phosphatases.

side on the B peptide of α-chymotrypsin, Ser 195 re-

A typical mammalian cell possesses over 1000 phos-

sides on the C peptide (Figure 9-6). The conforma-

phorylated proteins and several hundred protein kinases

tional changes that accompany selective proteolysis of

and protein phosphatases that catalyze their intercon-

prochymotrypsin (chymotrypsinogen) align the three

version. The ease of interconversion of enzymes be-

residues of the charge-relay network, creating the cat-

tween their phospho- and dephospho- forms in part

alytic site. Note also that contact and catalytic residues

can be located on different peptide chains but still be

within bond-forming distance of bound substrate.

ATP

ADP

REVERSIBLE COVALENT MODIFICATION

Mg2+

REGULATES KEY MAMMALIAN ENZYMES

KINASE

Mammalian proteins are the targets of a wide range of

Enz Ser

O PO₃

covalent modification processes. Modifications such as

PHOSPHATASE

glycosylation, hydroxylation, and fatty acid acylation

introduce new structural features into newly synthe-

Mg2+

sized proteins that tend to persist for the lifetime of the

P_i

H₂O

protein. Among the covalent modifications that regu-

late protein function (eg, methylation, adenylylation),

Figure 9-7. Covalent modification of a regulated en-

the most common by far is phosphorylation-dephos-

zyme by phosphorylation-dephosphorylation of a seryl

phorylation. Protein kinases phosphorylate proteins by

residue.

CHAPTER 9

accounts for the frequency of phosphorylation-dephos-

Table 9-1. Examples of mammalian enzymes

phorylation as a mechanism for regulatory control.

whose catalytic activity is altered by covalent

Phosphorylation-dephosphorylation permits the func-

phosphorylation-dephosphorylation.

tional properties of the affected enzyme to be altered

only for as long as it serves a specific need. Once the

Activity State¹

need has passed, the enzyme can be converted back to

its original form, poised to respond to the next stimula-

Enzyme

Low

High

tory event. A second factor underlying the widespread

Acetyl-CoA carboxylase

use of protein phosphorylation-dephosphorylation lies

Glycogen synthase

in the chemical properties of the phosphoryl group it-

Pyruvate dehydrogenase

self. In order to alter an enzyme’s functional properties,

HMG-CoA reductase

any modification of its chemical structure must influ-

Glycogen phosphorylase

ence the protein’s three-dimensional configuration.

Citrate lyase

The high charge density of protein-bound phosphoryl

Phosphorylase b kinase

groups—generally −2 at physiologic pH—and their

HMG-CoA reductase kinase

propensity to form salt bridges with arginyl residues

¹E, dephosphoenzyme; EP, phosphoenzyme.

make them potent agents for modifying protein struc-

ture and function. Phosphorylation generally targets

amino acids distant from the catalytic site itself. Conse-

quent conformational changes then influence an en-

phosphorylation at different sites, or between phosphory-

zyme’s intrinsic catalytic efficiency or other properties.

lation sites and allosteric sites provides the basis for

In this sense, the sites of phosphorylation and other co-

regulatory networks that integrate multiple environ-

valent modifications can be considered another form of

mental input signals to evoke an appropriate coordi-

allosteric site. However, in this case the “allosteric li-

nated cellular response. In these sophisticated regula-

gand” binds covalently to the protein.

tory networks, individual enzymes respond to different

environmental signals. For example, if an enzyme can

be phosphorylated at a single site by more than one

PROTEIN PHOSPHORYLATION

protein kinase, it can be converted from a catalytically

IS EXTREMELY VERSATILE

efficient to an inefficient (inactive) form, or vice versa,

Protein phosphorylation-dephosphorylation is a highly

in response to any one of several signals. If the protein

versatile and selective process. Not all proteins are sub-

kinase is activated in response to a signal different from

ject to phosphorylation, and of the many hydroxyl

the signal that activates the protein phosphatase, the

groups on a protein’s surface, only one or a small subset

phosphoprotein becomes a decision node. The func-

are targeted. While the most common enzyme function

tional output, generally catalytic activity, reflects the

affected is the protein’s catalytic efficiency, phosphory-

phosphorylation state. This state or degree of phos-

lation can also alter the affinity for substrates, location

phorylation is determined by the relative activities of

within the cell, or responsiveness to regulation by al-

the protein kinase and protein phosphatase, a reflection

losteric ligands. Phosphorylation can increase an en-

of the presence and relative strength of the environ-

zyme’s catalytic efficiency, converting it to its active

mental signals that act through each. The ability of

form in one protein, while phosphorylation of another

many protein kinases and protein phosphatases to tar-

converts it into an intrinsically inefficient, or inactive,

get more than one protein provides a means for an en-

form (Table 9-1).

vironmental signal to coordinately regulate multiple

Many proteins can be phosphorylated at multiple

metabolic processes. For example, the enzymes 3-hy-

sites or are subject to regulation both by phosphoryla-

droxy-3-methylglutaryl-CoA reductase and acetyl-CoA

tion-dephosphorylation and by the binding of allosteric

carboxylase—the rate-controlling enzymes for choles-

ligands. Phosphorylation-dephosphorylation at any one

terol and fatty acid biosynthesis, respectively—are

site can be catalyzed by multiple protein kinases or pro-

phosphorylated and inactivated by the AMP-activated

tein phosphatases. Many protein kinases and most pro-

protein kinase. When this protein kinase is activated ei-

tein phosphatases act on more than one protein and are

ther through phosphorylation by yet another protein

themselves interconverted between active and inactive

kinase or in response to the binding of its allosteric acti-

forms by the binding of second messengers or by cova-

vator 5′-AMP, the two major pathways responsible for

lent modification by phosphorylation-dephosphoryla-

the synthesis of lipids from acetyl-CoA both are inhib-

tion.

ited. Interconvertible enzymes and the enzymes respon-

The interplay between protein kinases and protein

sible for their interconversion do not act as mere on

phosphatases, between the functional consequences of

and off switches working independently of one another.

ENZYMES: REGULATION OF ACTIVITIES

They form the building blocks of biomolecular com-

active site. Secretion as an inactive proenzyme facili-

puters that maintain homeostasis in cells that carry out

tates rapid mobilization of activity in response to in-

a complex array of metabolic processes that must be

jury or physiologic need and may protect the tissue

regulated in response to a broad spectrum of environ-

of origin (eg, autodigestion by proteases).

mental factors.

• Binding of metabolites and second messengers to

sites distinct from the catalytic site of enzymes trig-

Covalent Modification Regulates

gers conformational changes that alter V_max or the

Metabolite Flow

Km.

• Phosphorylation by protein kinases of specific seryl,

Regulation of enzyme activity by phosphorylation-

threonyl, or tyrosyl residues—and subsequent de-

dephosphorylation has analogies to regulation by feed-

phosphorylation by protein phosphatases—regulates

back inhibition. Both provide for short-term, readily

the activity of many human enzymes. The protein ki-

reversible regulation of metabolite flow in response to

nases and phosphatases that participate in regulatory

specific physiologic signals. Both act without altering

cascades which respond to hormonal or second mes-

gene expression. Both act on early enzymes of a pro-

senger signals constitute a “bio-organic computer”

tracted, often biosynthetic metabolic sequence, and

that can process and integrate complex environmen-

both act at allosteric rather than catalytic sites. Feed-

tal information to produce an appropriate and com-

back inhibition, however, involves a single protein and

prehensive cellular response.

lacks hormonal and neural features. By contrast, regula-

tion of mammalian enzymes by phosphorylation-

dephosphorylation involves several proteins and ATP

REFERENCES

and is under direct neural and hormonal control.

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SUMMARY

Graves DJ, Martin BL, Wang JH: Co- and Post-translational Modi-

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• Homeostasis involves maintaining a relatively con-

Oxford Univ Press, 1994.

stant intracellular and intra-organ environment de-

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spite wide fluctuations in the external environment

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via appropriate changes in the rates of biochemical

mol Struct 1993;22:199.

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Marks F (editor): Protein Phosphorylation. VCH Publishers, 1996.

• The substrates for most enzymes are usually present

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Inherited Disease, 8th ed. McGraw-Hill, 2000.

• Active control of metabolite flux involves changes in

Sitaramayya A (editor): Introduction to Cellular Signal Transduction.

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SECTION II

Bioenergetics & the Metabolism

of Carbohydrates & Lipids

Bioenergetics:The Role of ATP

Peter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DSc

BIOMEDICAL IMPORTANCE

the system to another or may be transformed into an-

other form of energy. In living systems, chemical en-

Bioenergetics, or biochemical thermodynamics, is the

ergy may be transformed into heat or into electrical, ra-

study of the energy changes accompanying biochemical

diant, or mechanical energy.

reactions. Biologic systems are essentially isothermic

The second law of thermodynamics states that the

and use chemical energy to power living processes.

total entropy of a system must increase if a process

How an animal obtains suitable fuel from its food to

is to occur spontaneously. Entropy is the extent of

provide this energy is basic to the understanding of nor-

disorder or randomness of the system and becomes

mal nutrition and metabolism. Death from starvation

maximum as equilibrium is approached. Under condi-

occurs when available energy reserves are depleted, and

tions of constant temperature and pressure, the rela-

certain forms of malnutrition are associated with energy

tionship between the free energy change (∆G) of a re-

imbalance (marasmus). Thyroid hormones control the

acting system and the change in entropy

(∆S) is

rate of energy release (metabolic rate), and disease re-

expressed by the following equation, which combines

sults when they malfunction. Excess storage of surplus

the two laws of thermodynamics:

energy causes obesity, one of the most common dis-

eases of Western society.

∆G = ∆H− T∆S

FREE ENERGY IS THE USEFUL ENERGY

where ∆H is the change in enthalpy (heat) and T is the

absolute temperature.

IN A SYSTEM

In biochemical reactions, because ∆H is approxi-

Gibbs change in free energy (∆G) is that portion of the

mately equal to ∆E, the total change in internal energy

total energy change in a system that is available for

of the reaction, the above relationship may be expressed

doing work—ie, the useful energy, also known as the

in the following way:

chemical potential.

∆G = ∆E − T∆S

Biologic Systems Conform to the General

∆G is negative, the reaction proceeds sponta-

Laws of Thermodynamics

neously with loss of free energy; ie, it is exergonic. If,

The first law of thermodynamics states that the total

in addition, ∆G is of great magnitude, the reaction goes

energy of a system, including its surroundings, re-

virtually to completion and is essentially irreversible.

mains constant. It implies that within the total system,

On the other hand, if ∆G is positive, the reaction pro-

energy is neither lost nor gained during any change.

ceeds only if free energy can be gained; ie, it is ender-

However, energy may be transferred from one part of

gonic. If, in addition, the magnitude of ∆G is great, the

BIOENERGETICS: THE ROLE OF ATP

system is stable, with little or no tendency for a reaction

occurs with release of free energy. It is coupled to an-

to occur. If ∆G is zero, the system is at equilibrium and

other reaction, in which free energy is required to con-

no net change takes place.

vert metabolite C to metabolite D. The terms exer-

When the reactants are present in concentrations of

gonic and endergonic rather than the normal chemical

1.0 mol/L, ∆G⁰ is the standard free energy change. For

terms “exothermic” and “endothermic” are used to in-

biochemical reactions, a standard state is defined as

dicate that a process is accompanied by loss or gain, re-

having a pH of 7.0. The standard free energy change at

spectively, of free energy in any form, not necessarily as

this standard state is denoted by ∆G0′.

heat. In practice, an endergonic process cannot exist in-

The standard free energy change can be calculated

dependently but must be a component of a coupled ex-

from the equilibrium constant K_eq.

ergonic-endergonic system where the overall net change

is exergonic. The exergonic reactions are termed catab-

olism (generally, the breakdown or oxidation of fuel

0′ = −RT ln K′

∆G

molecules), whereas the synthetic reactions that build

up substances are termed anabolism. The combined

where R is the gas constant and T is the absolute tem-

catabolic and anabolic processes constitute metabo-

perature (Chapter 8). It is important to note that the

lism.

actual ∆G may be larger or smaller than ∆G0′ depend-

If the reaction shown in Figure 10-1 is to go from

ing on the concentrations of the various reactants, in-

left to right, then the overall process must be accompa-

cluding the solvent, various ions, and proteins.

nied by loss of free energy as heat. One possible mecha-

In a biochemical system, an enzyme only speeds up

nism of coupling could be envisaged if a common oblig-

the attainment of equilibrium; it never alters the final

atory intermediate (I) took part in both reactions, ie,

concentrations of the reactants at equilibrium.

A+C→I→B+D

ENDERGONIC PROCESSES PROCEED BY

Some exergonic and endergonic reactions in biologic

COUPLING TO EXERGONIC PROCESSES

systems are coupled in this way. This type of system has

The vital processes—eg, synthetic reactions, muscular

a built-in mechanism for biologic control of the rate of

contraction, nerve impulse conduction, and active

oxidative processes since the common obligatory inter-

transport—obtain energy by chemical linkage, or cou-

mediate allows the rate of utilization of the product of

pling, to oxidative reactions. In its simplest form, this

the synthetic path (D) to determine by mass action the

type of coupling may be represented as shown in Figure

rate at which A is oxidized. Indeed, these relationships

10-1. The conversion of metabolite A to metabolite B

supply a basis for the concept of respiratory control,

the process that prevents an organism from burning out

of control. An extension of the coupling concept is pro-

vided by dehydrogenation reactions, which are coupled

to hydrogenations by an intermediate carrier (Figure

10-2).

An alternative method of coupling an exergonic to

an endergonic process is to synthesize a compound of

high-energy potential in the exergonic reaction and to

incorporate this new compound into the endergonic re-

action, thus effecting a transference of free energy from

the exergonic to the endergonic pathway (Figure 10-3).

The biologic advantage of this mechanism is that the

compound of high potential energy,

∼ E, unlike I

∆G = ∆H− T∆S

Figure 10-1. Coupling of an exergonic to an ender-

Figure 10-2. Coupling of dehydrogenation and hy-

gonic reaction.

drogenation reactions by an intermediate carrier.

CHAPTER 10

Figure 10-4. Adenosine triphosphate (ATP) shown

as the magnesium complex. ADP forms a similar com-

plex with Mg2+.

Figure 10-3. Transfer of free energy from an exer-

gonic to an endergonic reaction via a high-energy in-

The Intermediate Value for the Free

termediate compound (∼ E ).

Energy of Hydrolysis of ATP Has Important

Bioenergetic Significance

The standard free energy of hydrolysis of a number of

in the previous system, need not be structurally related

biochemically important phosphates is shown in Table

to A, B, C, or D, allowing E to serve as a transducer of

10-1. An estimate of the comparative tendency of each

energy from a wide range of exergonic reactions to an

of the phosphate groups to transfer to a suitable accep-

equally wide range of endergonic reactions or processes,

tor may be obtained from the ∆G0′ of hydrolysis at

such as biosyntheses, muscular contraction, nervous ex-

37 °C. The value for the hydrolysis of the terminal

citation, and active transport. In the living cell, the

principal high-energy intermediate or carrier com-

pound (designated ∼ E in Figure 10-3) is adenosine

triphosphate (ATP).

Table 10-1. Standard free energy of hydrolysis

of some organophosphates of biochemical

HIGH-ENERGY PHOSPHATES PLAY A

importance.^1,2

CENTRAL ROLE IN ENERGY CAPTURE

AND TRANSFER

G⁰

Compound

kJ/mol

kcal/mol

In order to maintain living processes, all organisms

must obtain supplies of free energy from their environ-

Phosphoenolpyruvate

−61.9

−14.8

ment. Autotrophic organisms utilize simple exergonic

Carbamoyl phosphate

−51.4

−12.3

processes; eg, the energy of sunlight (green plants), the

1,3-Bisphosphoglycerate

−49.3

−11.8

reaction Fe2+ → Fe3+ (some bacteria). On the other

(to 3-phosphoglycerate)

hand, heterotrophic organisms obtain free energy by

Creatine phosphate

−43.1

−10.3

coupling their metabolism to the breakdown of com-

ATP → ADP + P_i

−30.5

−7.3

plex organic molecules in their environment. In all

ADP → AMP + P_i

−27.6

−6.6

these organisms, ATP plays a central role in the trans-

Pyrophosphate

−27.6

−6.6

ference of free energy from the exergonic to the ender-

Glucose 1-phosphate

−20.9

−5.0

gonic processes

(Figure

10-3). ATP is a nucleoside

Fructose 6-phosphate

−15.9

−3.8

triphosphate containing adenine, ribose, and three

AMP

−14.2

−3.4

phosphate groups. In its reactions in the cell, it func-

Glucose 6-phosphate

−13.8

−3.3

tions as the Mg2+ complex (Figure 10-4).

Glycerol 3-phosphate

−9.2

−2.2

The importance of phosphates in intermediary me-

¹P_i, inorganic orthophosphate.

tabolism became evident with the discovery of the role

²Values for ATP and most others taken from Krebs and Kornberg

of ATP, adenosine diphosphate (ADP), and inorganic

(1957). They differ between investigators depending on the pre-

phosphate (P_i) in glycolysis (Chapter 17).

cise conditions under which the measurements are made.

BIOENERGETICS: THE ROLE OF ATP

phosphate of ATP divides the list into two groups.

Low-energy phosphates, exemplified by the ester

phosphates found in the intermediates of glycolysis,

have ∆G0′ values smaller than that of ATP, while in

high-energy phosphates the value is higher than that

of ATP. The components of this latter group, including

ATP, are usually anhydrides (eg, the 1-phosphate of

1,3-bisphosphoglycerate), enolphosphates

(eg, phos-

phoenolpyruvate), and phosphoguanidines (eg, creatine

phosphate, arginine phosphate). The intermediate posi-

tion of ATP allows it to play an important role in en-

ergy transfer. The high free energy change on hydrolysis

of ATP is due to relief of charge repulsion of adjacent

negatively charged oxygen atoms and to stabilization of

the reaction products, especially phosphate, as reso-

nance hybrids. Other “high-energy compounds” are

thiol esters involving coenzyme A (eg, acetyl-CoA), acyl

carrier protein, amino acid esters involved in protein

synthesis, S-adenosylmethionine

(active methionine),

UDPGlc (uridine diphosphate glucose), and PRPP

(5-phosphoribosyl-1-pyrophosphate).

High-Energy Phosphates Are

Designated by ~ P

Figure 10-5. Structure of ATP, ADP, and AMP show-

ing the position and the number of high-energy phos-

The symbol ∼ P indicates that the group attached to

phates (∼ P ).

the bond, on transfer to an appropriate acceptor, results

in transfer of the larger quantity of free energy. For this

reason, the term group transfer potential is preferred

by some to “high-energy bond.” Thus, ATP contains

two high-energy phosphate groups and ADP contains

one, whereas the phosphate in AMP (adenosine mono-

phosphate) is of the low-energy type, since it is a nor-

mal ester link (Figure 10-5).

HIGH-ENERGY PHOSPHATES ACT AS THE

“ENERGY CURRENCY” OF THE CELL

ATP is able to act as a donor of high-energy phosphate

to form those compounds below it in Table 10-1. Like-

wise, with the necessary enzymes, ADP can accept

high-energy phosphate to form ATP from those com-

pounds above ATP in the table. In effect, an ATP/

ADP cycle connects those processes that generate ∼ P

to those processes that utilize ∼ P

(Figure 10-6), con-

tinuously consuming and regenerating ATP. This oc-

curs at a very rapid rate, since the total ATP/ADP pool

is extremely small and sufficient to maintain an active

tissue for only a few seconds.

There are three major sources of ∼ P taking part in

energy conservation or energy capture:

(1) Oxidative phosphorylation: The greatest quan-

Figure 10-6.

Role of ATP/ADP cycle in transfer of

titative source of ∼ P in aerobic organisms. Free energy

high-energy phosphate.

CHAPTER 10

(1) Glucose+P_i → Glucose 6- phosphate+ H₂O

( ∆G0′ = +13.8 k/ mol)

To take place, the reaction must be coupled with an-

other—more exergonic—reaction such as the hydroly-

sis of the terminal phosphate of ATP.

(2) ATP → ADP+P_i (∆G0′ = −30.5 kJ / mol)

Figure 10-7. Transfer of high-energy phosphate be-

tween ATP and creatine.

When (1) and (2) are coupled in a reaction catalyzed by

hexokinase, phosphorylation of glucose readily pro-

ceeds in a highly exergonic reaction that under physio-

logic conditions is irreversible. Many “activation” reac-

comes from respiratory chain oxidation using molecular

tions follow this pattern.

O₂ within mitochondria (Chapter 11).

(2) Glycolysis: A net formation of two ∼ P results

from the formation of lactate from one molecule of glu-

Adenylyl Kinase (Myokinase)

cose, generated in two reactions catalyzed by phospho-

Interconverts Adenine Nucleotides

glycerate kinase and pyruvate kinase, respectively (Fig-

This enzyme is present in most cells. It catalyzes the fol-

ure 17-2).

lowing reaction:

(3) The citric acid cycle: One ∼ P is generated di-

rectly in the cycle at the succinyl thiokinase step (Figure

16-3).

Phosphagens act as storage forms of high-energy

phosphate and include creatine phosphate, occurring in

vertebrate skeletal muscle, heart, spermatozoa, and

This allows:

brain; and arginine phosphate, occurring in inverte-

(1) High-energy phosphate in ADP to be used in

brate muscle. When ATP is rapidly being utilized as a

the synthesis of ATP.

source of energy for muscular contraction, phosphagens

(2) AMP, formed as a consequence of several acti-

permit its concentrations to be maintained, but when

vating reactions involving ATP, to be recovered by

the ATP/ADP ratio is high, their concentration can in-

rephosphorylation to ADP.

crease to act as a store of high-energy phosphate (Figure

(3) AMP to increase in concentration when ATP

10-7).

becomes depleted and act as a metabolic (allosteric) sig-

When ATP acts as a phosphate donor to form those

nal to increase the rate of catabolic reactions, which in

compounds of lower free energy of hydrolysis (Table

turn lead to the generation of more ATP (Chapter 19).

10-1), the phosphate group is invariably converted to

one of low energy, eg,

When ATP Forms AMP, Inorganic

Pyrophosphate (PP_i) Is Produced

This occurs, for example, in the activation of long-

chain fatty acids (Chapter 22):

ATP Allows the Coupling of

Thermodynamically Unfavorable

Reactions to Favorable Ones

This reaction is accompanied by loss of free energy

as heat, which ensures that the activation reaction will

The phosphorylation of glucose to glucose

6-phos-

go to the right; and is further aided by the hydrolytic

phate, the first reaction of glycolysis (Figure 17-2), is

splitting of PP_i, catalyzed by inorganic pyrophospha-

highly endergonic and cannot proceed under physio-

tase, a reaction that itself has a large ∆G0′ of −27.6 kJ/

logic conditions.

BIOENERGETICS: THE ROLE OF ATP

Thus, adenylyl kinase is a specialized monophosphate

kinase.

SUMMARY

• Biologic systems use chemical energy to power the

living processes.

• Exergonic reactions take place spontaneously with

loss of free energy (∆G is negative). Endergonic reac-

tions require the gain of free energy (∆G is positive)

and only occur when coupled to exergonic reactions.

• ATP acts as the “energy currency” of the cell, trans-

Phosphate cycles and interchange of

Figure 10-8.

ferring free energy derived from substances of higher

adenine nucleotides.

energy potential to those of lower energy potential.

REFERENCES

mol. Note that activations via the pyrophosphate path-

way result in the loss of two ∼ P rather than one ∼ P as

de Meis L: The concept of energy-rich phosphate compounds:

Water, transport ATPases, and entropy energy. Arch Bio-

occurs when ADP and P_i are formed.

chem Biophys 1993;306:287.

Ernster L (editor): Bioenergetics. Elsevier, 1984.

Harold FM: The Vital Force: A Study of Bioenergetics. Freeman,

1986.

Klotz IM: Introduction to Biomolecular Energetics. Academic Press,

1986.

A combination of the above reactions makes it pos-

Krebs HA, Kornberg HL: Energy Transformations in Living Matter.

Springer, 1957.

sible for phosphate to be recycled and the adenine nu-

cleotides to interchange (Figure 10-8).

Other Nucleoside Triphosphates

Participate in the Transfer of

High-Energy Phosphate

By means of the enzyme nucleoside diphosphate ki-

nase, UTP, GTP, and CTP can be synthesized from

their diphosphates, eg,

All of these triphosphates take part in phosphoryla-

tions in the cell. Similarly, specific nucleoside mono-

phosphate kinases catalyze the formation of nucleoside

diphosphates from the corresponding monophosphates.

Biologic Oxidation

Peter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DSc

BIOMEDICAL IMPORTANCE

groups: oxidases, dehydrogenases, hydroperoxidases,

and oxygenases.

Chemically, oxidation is defined as the removal of elec-

trons and reduction as the gain of electrons. Thus, oxi-

dation is always accompanied by reduction of an elec-

OXIDASES USE OXYGEN AS A

tron acceptor. This principle of oxidation-reduction

HYDROGEN ACCEPTOR

applies equally to biochemical systems and is an impor-

tant concept underlying understanding of the nature of

Oxidases catalyze the removal of hydrogen from a sub-

biologic oxidation. Note that many biologic oxidations

strate using oxygen as a hydrogen acceptor.* They form

can take place without the participation of molecular

water or hydrogen peroxide as a reaction product (Fig-

oxygen, eg, dehydrogenations. The life of higher ani-

ure 11-1).

mals is absolutely dependent upon a supply of oxygen

for respiration, the process by which cells derive energy

Some Oxidases Contain Copper

in the form of ATP from the controlled reaction of hy-

drogen with oxygen to form water. In addition, molec-

Cytochrome oxidase is a hemoprotein widely distrib-

ular oxygen is incorporated into a variety of substrates

uted in many tissues, having the typical heme pros-

by enzymes designated as oxygenases; many drugs, pol-

thetic group present in myoglobin, hemoglobin, and

lutants, and chemical carcinogens (xenobiotics) are me-

other cytochromes (Chapter 6). It is the terminal com-

tabolized by enzymes of this class, known as the cy-

ponent of the chain of respiratory carriers found in mi-

tochrome P450 system. Administration of oxygen can

tochondria and transfers electrons resulting from the

be lifesaving in the treatment of patients with respira-

oxidation of substrate molecules by dehydrogenases to

tory or circulatory failure.

their final acceptor, oxygen. The enzyme is poisoned by

carbon monoxide, cyanide, and hydrogen sulfide. It has

. It is now known that

also been termed cytochrome a₃

FREE ENERGY CHANGES CAN

cytochromes a and a₃ are combined in a single protein,

and the complex is known as cytochrome aa₃. It con-

BE EXPRESSED IN TERMS

tains two molecules of heme, each having one Fe atom

OF REDOX POTENTIAL

that oscillates between Fe3+ and Fe2+ during oxidation

In reactions involving oxidation and reduction, the free

and reduction. Furthermore, two atoms of Cu are pre-

energy change is proportionate to the tendency of reac-

sent, each associated with a heme unit.

tants to donate or accept electrons. Thus, in addition to

expressing free energy change in terms of ∆G0′ (Chapter

Other Oxidases Are Flavoproteins

10), it is possible, in an analogous manner, to express it

numerically as an oxidation-reduction or redox po-

Flavoprotein enzymes contain flavin mononucleotide

tential (E′₀). The redox potential of a system (E₀) is

(FMN) or flavin adenine dinucleotide (FAD) as pros-

usually compared with the potential of the hydrogen

thetic groups. FMN and FAD are formed in the body

electrode (0.0 volts at pH 0.0). However, for biologic

from the vitamin riboflavin (Chapter 45). FMN and

systems, the redox potential (E′₀)is normally expressed

FAD are usually tightly—but not covalently—bound to

at pH 7.0, at which pH the electrode potential of the

their respective apoenzyme proteins. Metalloflavopro-

hydrogen electrode is −0.42 volts. The redox potentials

teins contain one or more metals as essential cofactors.

of some redox systems of special interest in mammalian

Examples of flavoprotein enzymes include L-amino

biochemistry are shown in Table 11-1. The relative po-

acid oxidase, an FMN-linked enzyme found in kidney

sitions of redox systems in the table allows prediction of

with general specificity for the oxidative deamination of

the direction of flow of electrons from one redox couple

to another.

Enzymes involved in oxidation and reduction are

* The term “oxidase” is sometimes used collectively to denote all

called oxidoreductases and are classified into four

enzymes that catalyze reactions involving molecular oxygen.

BIOLOGIC OXIDATION

Table 11-1. Some redox potentials of special

versible, these properties enable reducing equivalents to

interest in mammalian oxidation systems.

be freely transferred within the cell. This type of reac-

tion, which enables one substrate to be oxidized at the

expense of another, is particularly useful in enabling ox-

System

E₀ Volts

idative processes to occur in the absence of oxygen,

H+/H₂

−0.42

such as during the anaerobic phase of glycolysis (Figure

NAD⁺/NADH

−0.32

17-2).

Lipoate; ox/red

−0.29

(2) As components in the respiratory chain of elec-

Acetoacetate/3-hydroxybutyrate

−0.27

tron transport from substrate to oxygen (Figure 12-3).

Pyruvate/lactate

−0.19

Oxaloacetate/malate

−0.17

Fumarate/succinate

+0.03

Many Dehydrogenases Depend

Cytochrome b; Fe3+/Fe2+

+0.08

on Nicotinamide Coenzymes

Ubiquinone; ox/red

+0.10

Cytochrome c₁; Fe3+/Fe2+

+0.22

These dehydrogenases use nicotinamide adenine di-

Cytochrome a; Fe3+/Fe2+

+0.29

nucleotide (NAD⁺) or nicotinamide adenine dinu-

Oxygen/water

+0.82

cleotide phosphate

(NADP⁺)—or both—and are

formed in the body from the vitamin niacin (Chapter

45). The coenzymes are reduced by the specific sub-

strate of the dehydrogenase and reoxidized by a suitable

the naturally occurring L-amino acids; xanthine oxi-

electron acceptor (Figure 11-4).They may freely and

dase, which contains molybdenum and plays an impor-

reversibly dissociate from their respective apoenzymes.

tant role in the conversion of purine bases to uric acid

Generally, NAD-linked dehydrogenases catalyze ox-

(Chapter 34), and is of particular significance in uri-

idoreduction reactions in the oxidative pathways of me-

cotelic animals (Chapter 29); and aldehyde dehydro-

tabolism, particularly in glycolysis, in the citric acid

genase, an FAD-linked enzyme present in mammalian

cycle, and in the respiratory chain of mitochondria.

livers, which contains molybdenum and nonheme iron

NADP-linked dehydrogenases are found characteristi-

and acts upon aldehydes and N-heterocyclic substrates.

cally in reductive syntheses, as in the extramitochon-

The mechanisms of oxidation and reduction of these

drial pathway of fatty acid synthesis and steroid synthe-

enzymes are complex. Evidence suggests a two-step re-

sis—and also in the pentose phosphate pathway.

action as shown in Figure 11-2.

Other Dehydrogenases Depend

DEHYDROGENASES CANNOT USE

on Riboflavin

OXYGEN AS A HYDROGEN ACCEPTOR

The flavin groups associated with these dehydrogenases

There are a large number of enzymes in this class. They

are similar to FMN and FAD occurring in oxidases.

perform two main functions:

They are generally more tightly bound to their apoen-

zymes than are the nicotinamide coenzymes. Most of

(1) Transfer of hydrogen from one substrate to an-

the riboflavin-linked dehydrogenases are concerned

other in a coupled oxidation-reduction reaction (Figure

with electron transport in (or to) the respiratory chain

11-3). These dehydrogenases are specific for their sub-

(Chapter 12). NADH dehydrogenase acts as a carrier

strates but often utilize common coenzymes or hydro-

of electrons between NADH and the components of

gen carriers, eg, NAD⁺. Since the reactions are re-

higher redox potential (Figure 12-3). Other dehydro-

genases such as succinate dehydrogenase, acyl-CoA

dehydrogenase, and mitochondrial glycerol-3-phos-

phate dehydrogenase transfer reducing equivalents di-

AH₂

¹/2O₂

AH₂

O₂

rectly from the substrate to the respiratory chain (Fig-

(Red)

ure

12-4). Another role of the flavin-dependent

OXIDASE

dehydrogenases is in the dehydrogenation (by dihy-

drolipoyl dehydrogenase) of reduced lipoate, an inter-

H₂O

H₂O₂

mediate in the oxidative decarboxylation of pyruvate

(Ox)

and α-ketoglutarate

(Figures

12-4 and 17-5). The

electron-transferring flavoprotein is an intermediary

Figure 11-1. Oxidation of a metabolite catalyzed by

carrier of electrons between acyl-CoA dehydrogenase

an oxidase (A) forming H₂O, (B) forming H₂O₂.

and the respiratory chain (Figure 12-4).

CHAPTER 11

H₃C

O H₃C

H₃C

(H⁺+ e^-)

Figure 11-2. Oxidoreduction of isoalloxazine ring in flavin nucleotides via a semi-

quinone (free radical) intermediate (center).

Cytochromes May Also Be Regarded

Peroxidases Reduce Peroxides Using

as Dehydrogenases

Various Electron Acceptors

The cytochromes are iron-containing hemoproteins in

Peroxidases are found in milk and in leukocytes,

which the iron atom oscillates between Fe3+ and Fe2+

platelets, and other tissues involved in eicosanoid me-

during oxidation and reduction. Except for cytochrome

tabolism (Chapter 23). The prosthetic group is proto-

oxidase (previously described), they are classified as de-

heme. In the reaction catalyzed by peroxidase, hydro-

hydrogenases. In the respiratory chain, they are in-

gen peroxide is reduced at the expense of several

volved as carriers of electrons from flavoproteins on the

substances that will act as electron acceptors, such as

one hand to cytochrome oxidase on the other (Figure

ascorbate, quinones, and cytochrome c. The reaction

12-4). Several identifiable cytochromes occur in the

catalyzed by peroxidase is complex, but the overall reac-

respiratory chain, ie, cytochromes b, c₁, c, a, and a₃ (cy-

tion is as follows:

tochrome oxidase). Cytochromes are also found in

other locations, eg, the endoplasmic reticulum

(cy-

PEROXIDASE

tochromes P450 and b₅), and in plant cells, bacteria,

H₂O₂ + AH₂

2H₂O + A

and yeasts.

In erythrocytes and other tissues, the enzyme glu-

tathione peroxidase, containing selenium as a pros-

HYDROPEROXIDASES USE HYDROGEN

thetic group, catalyzes the destruction of H₂O₂ and

PEROXIDE OR AN ORGANIC PEROXIDE

lipid hydroperoxides by reduced glutathione, protecting

AS SUBSTRATE

membrane lipids and hemoglobin against oxidation by

Two type of enzymes found both in animals and plants

peroxides (Chapter 20).

fall into this category: peroxidases and catalase.

Hydroperoxidases protect the body against harmful

Catalase Uses Hydrogen Peroxide as

peroxides. Accumulation of peroxides can lead to gen-

Electron Donor & Electron Acceptor

eration of free radicals, which in turn can disrupt mem-

branes and perhaps cause cancer and atherosclerosis.

Catalase is a hemoprotein containing four heme groups.

(See Chapters 14 and 45.)

In addition to possessing peroxidase activity, it is able

to use one molecule of H₂O₂ as a substrate electron

donor and another molecule of H₂O₂ as an oxidant or

electron acceptor.

AH2

Carrier

BH₂

(Red)

(Ox)

(Red)

CATALASE

2H₂O₂

2H₂O + O₂

Carrier-H₂

(Ox)

(Red)

(Ox)

Under most conditions in vivo, the peroxidase activity

DEHYDROGENASE

SPECIFIC FOR A

SPECIFIC FOR B

of catalase seems to be favored. Catalase is found in

blood, bone marrow, mucous membranes, kidney, and

Figure 11-3. Oxidation of a metabolite catalyzed by

liver. Its function is assumed to be the destruction of

coupled dehydrogenases.

hydrogen peroxide formed by the action of oxidases.

BIOLOGIC OXIDATION

Figure 11-4. Mechanism of oxidation

DEHYDROGENASE

CONH₂

SPECIFIC FOR A

and reduction of nicotinamide coen-

zymes. There is stereospecificity about

AH₂

A Form

position 4 of nicotinamide when it is re-

duced by a substrate AH₂. One of the hy-

drogen atoms is removed from the sub-

CONH₂

A + H⁺

strate as a hydrogen nucleus with two

electrons (hydride ion, H⁻) and is trans-

ferred to the 4 position, where it may be

attached in either the A or the B position

AH₂

CONH₂

according to the specificity determined

by the particular dehydrogenase catalyz-

DEHYDROGENASE

SPECIFIC FOR B

ing the reaction. The remaining hydro-

B Form

gen of the hydrogen pair removed from

the substrate remains free as a hydro-

NAD⁺ + AH₂

NADH + H+ + A

gen ion.

Peroxisomes are found in many tissues, including liver.

Monooxygenases (Mixed-Function

They are rich in oxidases and in catalase, Thus, the en-

Oxidases, Hydroxylases) Incorporate

zymes that produce H₂O₂ are grouped with the enzyme

Only One Atom of Molecular Oxygen

that destroys it. However, mitochondrial and microso-

Into the Substrate

mal electron transport systems as well as xanthine oxi-

dase must be considered as additional sources of H₂O₂.

The other oxygen atom is reduced to water, an addi-

tional electron donor or cosubstrate (Z) being necessary

for this purpose.

OXYGENASES CATALYZE THE DIRECT

TRANSFER & INCORPORATION

A—H+O +ZH →A—OH+H O+Z

OF OXYGEN INTO A SUBSTRATE

MOLECULE

Cytochromes P450 Are Monooxygenases

Oxygenases are concerned with the synthesis or degra-

Important for the Detoxification of Many

dation of many different types of metabolites. They cat-

Drugs & for the Hydroxylation of Steroids

alyze the incorporation of oxygen into a substrate mole-

cule in two steps: (1) oxygen is bound to the enzyme at

Cytochromes P450 are an important superfamily of

the active site, and (2) the bound oxygen is reduced or

heme-containing monooxgenases, and more than 1000

transferred to the substrate. Oxygenases may be divided

such enzymes are known. Both NADH and NADPH

into two subgroups, as follows.

donate reducing equivalents for the reduction of these

cytochromes (Figure 11-5), which in turn are oxidized

by substrates in a series of enzymatic reactions collectively

Dioxygenases Incorporate Both Atoms

known as the hydroxylase cycle (Figure 11-6). In liver

of Molecular Oxygen Into the Substrate

microsomes, cytochromes P450 are found together with

The basic reaction is shown below:

cytochrome b₅ and have an important role in detoxifica-

tion. Benzpyrene, aminopyrine, aniline, morphine, and

A + O →AO

benzphetamine are hydroxylated, increasing their solubil-

ity and aiding their excretion. Many drugs such as phe-

Examples include the liver enzymes, homogentisate

nobarbital have the ability to induce the formation of mi-

dioxygenase

(oxidase) and

3-hydroxyanthranilate

crosomal enzymes and of cytochromes P450.

dioxygenase (oxidase), that contain iron; and L-trypto-

Mitochondrial cytochrome P450 systems are found

phan dioxygenase

(tryptophan pyrrolase)

(Chapter

in steroidogenic tissues such as adrenal cortex, testis,

30), that utilizes heme.

ovary, and placenta and are concerned with the biosyn-

CHAPTER 11

CN^-

NADH

Flavoprotein₂

Cyt b₅

Stearyl-CoA desaturase

Amine oxidase, etc

Flavoprotein₃

NADPH

Flavoprotein₁

Cyt P450

Hydroxylation

Lipid peroxidation

Heme oxygenase

Figure 11-5. Electron transport chain in microsomes. Cyanide (CN⁻) inhibits the

indicated step.

thesis of steroid hormones from cholesterol (hydroxyla-

ing rise to free radical chain reactions (Chapter 14).

tion at C₂₂ and C₂₀ in side-chain cleavage and at the

The ease with which superoxide can be formed from

11β and 18 positions). In addition, renal systems cat-

oxygen in tissues and the occurrence of superoxide dis-

alyzing 1α- and 24-hydroxylations of 25-hydroxychole-

mutase, the enzyme responsible for its removal in all

calciferol in vitamin D metabolism—and cholesterol

aerobic organisms (although not in obligate anaerobes)

7α-hydroxylase and sterol 27-hydroxylase involved in

indicate that the potential toxicity of oxygen is due to

bile acid biosynthesis in the liver (Chapter 26)—are

its conversion to superoxide.

P450 enzymes.

Superoxide is formed when reduced flavins—pre-

sent, for example, in xanthine oxidase—are reoxidized

univalently by molecular oxygen.

SUPEROXIDE DISMUTASE PROTECTS

AEROBIC ORGANISMS AGAINST

⋅

Enz − Flavin − H +O

→Enz − Flavin − H+O

OXYGEN TOXICITY

Transfer of a single electron to O₂ generates the poten-

Superoxide can reduce oxidized cytochrome c

tially damaging superoxide anion free radical (O2−⋅ ),

the destructive effects of which are amplified by its giv-

⋅

O +Cyt c (Fe

)→

O +Cyt c (Fe

)

Substrate A-H

P450-A-H

Fe3+

e^-

P450-A-H

P450

NADPH-CYT P450 REDUCTASE

Fe3+

NADP⁺

FADH₂

2Fe₂S23+

O₂

e^-

NADPH + H

FAD

2Fe₂S

2H⁺

P450-A-H

Fe2+ O₂

H₂O

P450-A-H

Fe2+

O₂

A-OH

Figure 11-6. Cytochrome P450 hydroxylase cycle in microsomes. The system shown is typical

of steroid hydroxylases of the adrenal cortex. Liver microsomal cytochrome P450 hydroxylase does

not require the iron-sulfur protein Fe₂S₂. Carbon monoxide (CO) inhibits the indicated step.

BIOLOGIC OXIDATION

or be removed by superoxide dismutase.

REFERENCES

SUPEROXIDE

Babcock GT, Wikstrom M: Oxygen activation and the conserva-

DISMUTASE

tion of energy in cell respiration. Nature 1992;356:301.

O2− + O2− + 2H⁺

H₂O₂ + O₂

Coon MJ et al: Cytochrome P450: Progress and predictions.

FASEB J 1992;6:669.

In this reaction, superoxide acts as both oxidant and

Ernster L (editor): Bioenergetics. Elsevier, 1984.

reductant. Thus, superoxide dismutase protects aerobic

Mammaerts GP, Van Veldhoven PP: Role of peroxisomes in mam-

organisms against the potential deleterious effects of su-

malian metabolism. Cell Biochem Funct 1992;10:141.

peroxide. The enzyme occurs in all major aerobic tis-

Nicholls DG: Cytochromes and Cell Respiration. Carolina Biological

sues in the mitochondria and the cytosol. Although ex-

Supply Company, 1984.

posure of animals to an atmosphere of 100% oxygen

Raha S, Robinson BH: Mitochondria, oxygen free radicals, disease

causes an adaptive increase in superoxide dismutase,

and aging. Trends Biochem Sci 2000;25:502.

particularly in the lungs, prolonged exposure leads to

Tyler DD: The Mitochondrion in Health and Disease. VCH Pub-

lung damage and death. Antioxidants, eg, α-tocopherol

lishers, 1992.

(vitamin E), act as scavengers of free radicals and reduce

Tyler DD, Sutton CM: Respiratory enzyme systems in mitochon-

the toxicity of oxygen (Chapter 45).

drial membranes. In: Membrane Structure and Function, vol

5. Bittar EE (editor). Wiley, 1984.

Yang CS, Brady JF, Hong JY: Dietary effects on cytochromes

SUMMARY

P450, xenobiotic metabolism, and toxicity. FASEB J 1992;

6:737.

• In biologic systems, as in chemical systems, oxidation

(loss of electrons) is always accompanied by reduc-

tion of an electron acceptor.

• Oxidoreductases have a variety of functions in me-

tabolism; oxidases and dehydrogenases play major

roles in respiration; hydroperoxidases protect the

body against damage by free radicals; and oxygenases

mediate the hydroxylation of drugs and steroids.

• Tissues are protected from oxygen toxicity caused by

the superoxide free radical by the specific enzyme su-

peroxide dismutase.

The Respiratory Chain &

Oxidative Phosphorylation

Peter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DSc

BIOMEDICAL IMPORTANCE

trapping the liberated free energy as high-energy phos-

phate, and the enzymes of β-oxidation and of the citric

Aerobic organisms are able to capture a far greater pro-

acid cycle (Chapters 22 and 16) that produce most of

portion of the available free energy of respiratory sub-

the reducing equivalents.

strates than anaerobic organisms. Most of this takes

place inside mitochondria, which have been termed the

“powerhouses” of the cell. Respiration is coupled to the

Components of the Respiratory Chain

generation of the high-energy intermediate, ATP, by

Are Arranged in Order of Increasing

oxidative phosphorylation, and the chemiosmotic

Redox Potential

theory offers insight into how this is accomplished. A

Hydrogen and electrons flow through the respiratory

number of drugs (eg, amobarbital) and poisons (eg,

chain (Figure 12-3) through a redox span of 1.1 V

cyanide, carbon monoxide) inhibit oxidative phos-

from NAD⁺/NADH to O₂/2H₂O (Table 11-1). The

phorylation, usually with fatal consequences. Several in-

respiratory chain consists of a number of redox carriers

herited defects of mitochondria involving components

that proceed from the NAD-linked dehydrogenase sys-

of the respiratory chain and oxidative phosphorylation

tems, through flavoproteins and cytochromes, to mole-

have been reported. Patients present with myopathy

cular oxygen. Not all substrates are linked to the respi-

and encephalopathy and often have lactic acidosis.

ratory chain through NAD-specific dehydrogenases;

some, because their redox potentials are more positive

(eg, fumarate/succinate; Table

11-1), are linked di-

SPECIFIC ENZYMES ACT AS MARKERS

rectly to flavoprotein dehydrogenases, which in turn are

OF COMPARTMENTS SEPARATED BY

linked to the cytochromes of the respiratory chain (Fig-

THE MITOCHONDRIAL MEMBRANES

ure 12-4).

Ubiquinone or Q (coenzyme Q) (Figure 12-5)

Mitochondria have an outer membrane that is perme-

links the flavoproteins to cytochrome b, the member of

able to most metabolites, an inner membrane that is

the cytochrome chain of lowest redox potential. Q ex-

selectively permeable, and a matrix within

(Figure

ists in the oxidized quinone or reduced quinol form

12-1). The outer membrane is characterized by the

under aerobic or anaerobic conditions, respectively.

presence of various enzymes, including acyl-CoA syn-

The structure of Q is very similar to that of vitamin K

thetase and glycerolphosphate acyltransferase. Adenylyl

and vitamin E (Chapter 45) and of plastoquinone,

kinase and creatine kinase are found in the intermem-

found in chloroplasts. Q acts as a mobile component of

brane space. The phospholipid cardiolipin is concen-

the respiratory chain that collects reducing equivalents

trated in the inner membrane together with the en-

from the more fixed flavoprotein complexes and passes

zymes of the respiratory chain.

them on to the cytochromes.

An additional component is the iron-sulfur protein

(FeS; nonheme iron) (Figure 12-6). It is associated

THE RESPIRATORY CHAIN COLLECTS

with the flavoproteins (metalloflavoproteins) and with

& OXIDIZES REDUCING EQUIVALENTS

cytochrome b. The sulfur and iron are thought to take

Most of the energy liberated during the oxidation of

part in the oxidoreduction mechanism between flavin

carbohydrate, fatty acids, and amino acids is made

and Q, which involves only a single e⁻ change, the iron

available within mitochondria as reducing equivalents

atom undergoing oxidoreduction between Fe2+ and

(H or electrons) (Figure 12-2). Mitochondria con-

Fe3+.

tain the respiratory chain, which collects and trans-

Pyruvate and α-ketoglutarate dehydrogenase have

ports reducing equivalents directing them to their final

complex systems involving lipoate and FAD prior to

reaction with oxygen to form water, the machinery for

the passage of electrons to NAD, while electron trans-

THE RESPIRATORY CHAIN & OXIDATIVE PHOSPHORYLATION

Electrons flow from Q through the series of cyto-

chromes in order of increasing redox potential to mole-

cular oxygen (Figure 12-4). The terminal cytochrome

aa₃ (cytochrome oxidase), responsible for the final com-

Phosphorylating

bination of reducing equivalents with molecular oxy-

complexes

gen, has a very high affinity for oxygen, allowing the

respiratory chain to function at maximum rate until the

tissue has become depleted of O₂. Since this is an irre-

MATRIX

versible reaction (the only one in the chain), it gives di-

rection to the movement of reducing equivalents and to

the production of ATP, to which it is coupled.

Functionally and structurally, the components of

Cristae

the respiratory chain are present in the inner mitochon-

drial membrane as four protein-lipid respiratory chain

complexes that span the membrane. Cytochrome c is

the only soluble cytochrome and, together with Q,

INNER

seems to be a more mobile component of the respira-

MEMBRANE

tory chain connecting the fixed complexes

(Figures

12-7 and 12-8).

OUTER

MEMBRANE

THE RESPIRATORY CHAIN PROVIDES

MOST OF THE ENERGY CAPTURED

DURING CATABOLISM

ADP captures, in the form of high-energy phosphate, a

Figure 12-1. Structure of the mitochondrial mem-

significant proportion of the free energy released by

catabolic processes. The resulting ATP has been called

branes. Note that the inner membrane contains many

the energy “currency” of the cell because it passes on

folds, or cristae.

this free energy to drive those processes requiring en-

ergy (Figure 10-6).

fers from other dehydrogenases, eg, L(+)-3-hydroxyacyl-

There is a net direct capture of two high-energy

CoA dehydrogenase, couple directly with NAD.

phosphate groups in the glycolytic reactions

(Table

The reduced NADH of the respiratory chain is in

17-1), equivalent to approximately 103.2 kJ/mol of

turn oxidized by a metalloflavoprotein enzyme—NADH

glucose. (In vivo, ∆G for the synthesis of ATP from

dehydrogenase. This enzyme contains FeS and FMN,

ADP has been calculated as approximately 51.6 kJ/mol.

is tightly bound to the respiratory chain, and passes re-

(It is greater than ∆G0′ for the hydrolysis of ATP as

ducing equivalents on to Q.

given in Table 10-1, which is obtained under standard

FOOD

ATP

Fat

Fatty acids

β-Oxidation

Glycerol

O₂

Citric

Carbohydrate

Glucose, etc

Acetyl - CoA

acid

H₂O

cycle

Respiratory chain

Protein

Amino acids

MITOCHONDRION

ADP

Extramitochondrial sources of

reducing equivalents

Figure 12-2. Role of the respiratory chain of mitochondria in the conversion of food energy to ATP. Oxidation

of the major foodstuffs leads to the generation of reducing equivalents (2H) that are collected by the respiratory

chain for oxidation and coupled generation of ATP.

CHAPTER 12

AH₂

NAD⁺

FpH₂

2Fe3+

H₂O

Substrate

Flavoprotein

Cytochromes

Figure 12-3. Transport of reducing

NADH

2Fe2+

/2 O₂

equivalents through the respiratory

H⁺

2H⁺

chain.

concentrations of 1.0 mol/L.) Since 1 mol of glucose

dent that the respiratory chain is responsible for a large

yields approximately 2870 kJ on complete combustion,

proportion of total ATP formation.

the energy captured by phosphorylation in glycolysis is

small. Two more high-energy phosphates per mole of

Respiratory Control Ensures

glucose are captured in the citric acid cycle during the

a Constant Supply of ATP

conversion of succinyl CoA to succinate. All of these

phosphorylations occur at the substrate level. When

The rate of respiration of mitochondria can be con-

substrates are oxidized via an NAD-linked dehydrogen-

trolled by the availability of ADP. This is because oxi-

ase and the respiratory chain, approximately 3 mol of

dation and phosphorylation are tightly coupled; ie, oxi-

inorganic phosphate are incorporated into 3 mol of

dation cannot proceed via the respiratory chain without

ADP to form 3 mol of ATP per half mol of O₂ con-

concomitant phosphorylation of ADP. Table

12-1

sumed; ie, the P:O ratio = 3 (Figure 12-7). On the

shows the five conditions controlling the rate of respira-

other hand, when a substrate is oxidized via a flavopro-

tion in mitochondria. Most cells in the resting state are

tein-linked dehydrogenase, only

2 mol of ATP are

in state 4, and respiration is controlled by the availabil-

formed; ie, P:O = 2. These reactions are known as ox-

ity of ADP. When work is performed, ATP is con-

idative phosphorylation at the respiratory chain

verted to ADP, allowing more respiration to occur,

level. Such dehydrogenations plus phosphorylations at

which in turn replenishes the store of ATP. Under cer-

the substrate level can now account for 68% of the free

tain conditions, the concentration of inorganic phos-

energy resulting from the combustion of glucose, cap-

phate can also affect the rate of functioning of the respi-

tured in the form of high-energy phosphate. It is evi-

ratory chain. As respiration increases (as in exercise),

Succinate

Choline

Proline

3-Hydroxyacyl-CoA

3-Hydroxybutyrate

Glutamate

Malate

(FAD)

Isocitrate

FeS

Pyruvate

Lipoate

NAD

(FMN)

Cyt b

Cyt c₁

Cyt c

Cyt aa₃

O₂

(FAD)

FeS

α-Ketoglutarate

FeS

(FAD)

ETF

FeS

(FAD)

FeS: Iron-sulfur protein

(FAD)

ETF: Electron-transferring flavoprotein

Fp: Flavoprotein

Q: Ubiquinone

Cyt: Cytochrome

Glycerol 3-phosphate

Acyl-CoA

Sarcosine

Dimethylglycine

Figure 12-4. Components of the respiratory chain in mitochondria, showing the collecting points for reduc-

ing equivalents from important substrates. FeS occurs in the sequences on the O₂ side of Fp or Cyt b.

THE RESPIRATORY CHAIN & OXIDATIVE PHOSPHORYLATION

(H⁺ + e^- )

CH₃O

CH₃

CH₃O

[CH₂CH CCH₂]_nH

•O

Fully oxidized or

Semiquinone form

Reduced or quinol form

quinone form

(free radical)

(hydroquinone)

Figure 12-5. Structure of ubiquinone (Q). n = Number of isoprenoid units, which is

10 in higher animals, ie, Q₁₀.

the cell approaches state 3 or state 5 when either the ca-

MANY POISONS INHIBIT THE

pacity of the respiratory chain becomes saturated or the

RESPIRATORY CHAIN

P^O2 decreases below the Km for cytochrome a3. There is

also the possibility that the ADP/ATP transporter (Fig-

Much information about the respiratory chain has been

ure 12-9), which facilitates entry of cytosolic ADP into

obtained by the use of inhibitors, and, conversely, this

and ATP out of the mitochondrion, becomes rate-

has provided knowledge about the mechanism of action

limiting.

of several poisons (Figure 12-7). They may be classified

Thus, the manner in which biologic oxidative

as inhibitors of the respiratory chain, inhibitors of ox-

processes allow the free energy resulting from the oxida-

idative phosphorylation, and uncouplers of oxidative

tion of foodstuffs to become available and to be cap-

phosphorylation.

tured is stepwise, efficient (approximately 68%), and

Barbiturates such as amobarbital inhibit NAD-

controlled—rather than explosive, inefficient, and un-

linked dehydrogenases by blocking the transfer from

controlled, as in many nonbiologic processes. The re-

FeS to Q. At sufficient dosage, they are fatal in vivo.

maining free energy that is not captured as high-energy

Antimycin A and dimercaprol inhibit the respiratory

phosphate is liberated as heat. This need not be consid-

chain between cytochrome b and cytochrome c. The

ered “wasted,” since it ensures that the respiratory sys-

classic poisons H₂S, carbon monoxide, and cyanide

tem as a whole is sufficiently exergonic to be removed

inhibit cytochrome oxidase and can therefore totally ar-

from equilibrium, allowing continuous unidirectional

rest respiration. Malonate is a competitive inhibitor of

flow and constant provision of ATP. It also contributes

succinate dehydrogenase.

to maintenance of body temperature.

Atractyloside inhibits oxidative phosphorylation by

inhibiting the transporter of ADP into and ATP out of

the mitochondrion (Figure 12-10).

The action of uncouplers is to dissociate oxidation

in the respiratory chain from phosphorylation. These

Cys

compounds are toxic in vivo, causing respiration to be-

come uncontrolled, since the rate is no longer limited

by the concentration of ADP or P_i. The uncoupler that

has been used most frequently is 2,4-dinitrophenol,

Pr Cys

but other compounds act in a similar manner. The an-

tibiotic oligomycin completely blocks oxidation and

phosphorylation by acting on a step in phosphorylation

(Figures 12-7 and 12-8).

THE CHEMIOSMOTIC THEORY EXPLAINS

Cys

THE MECHANISM OF OXIDATIVE

Cys

PHOSPHORYLATION

Mitchell’s chemiosmotic theory postulates that the

energy from oxidation of components in the respiratory

Figure 12-6. Iron-sulfur-protein complex (Fe₄S₄). S ,

chain is coupled to the translocation of hydrogen ions

acid-labile sulfur; Pr, apoprotein; Cys, cysteine residue.

(protons, H⁺) from the inside to the outside of the

Some iron-sulfur proteins contain two iron atoms and

inner mitochondrial membrane. The electrochemical

two sulfur atoms (Fe₂S₂).

potential difference resulting from the asymmetric dis-

CHAPTER 12

Malonate

Complex II

FAD

Succinate

FeS

Carboxin

TTFA

H₂S

BAL

–

CN-

Antimycin A

Complex IV

Complex I

Complex III

Cyt a

Cyt a₃

NADH

FMN, FeS

Cyt b, FeS, Cyt c₁

Cyt c

O₂

–

Piericidin A

Uncouplers

–

Amobarbital

Uncouplers

Rotenone

Oligomycin

–

Oligomycin

ADP + P_i

ATP

ADP + P_i

ATP

ADP + P_i

ATP

Figure 12-7. Proposed sites of inhibition ( − ) of the respiratory chain by specific drugs, chemicals, and antibi-

otics. The sites that appear to support phosphorylation are indicated. BAL, dimercaprol. TTFA, an Fe-chelating

agent. Complex I, NADH:ubiquinone oxidoreductase; complex II, succinate:ubiquinone oxidoreductase; complex

III, ubiquinol:ferricytochrome c oxidoreductase; complex IV, ferrocytochrome c:oxygen oxidoreductase. Other ab-

breviations as in Figure 12-4.

tribution of the hydrogen ions is used to drive the

units are attached to a membrane protein complex

mechanism responsible for the formation of ATP (Fig-

known as F₀, which also consists of several protein sub-

ure 12-8).

units. F₀ spans the membrane and forms the proton

channel. The flow of protons through F₀ causes it to ro-

complex

tate, driving the production of ATP in the F₁

The Respiratory Chain Is a Proton Pump

(Figure 12-9). Estimates suggest that for each NADH

oxidized, complex I translocates four protons and com-

Each of the respiratory chain complexes I, III, and IV

plexes III and IV translocate 6 between them. As four

(Figures 12-7 and 12-8) acts as a proton pump. The

protons are taken into the mitochondrion for each ATP

inner membrane is impermeable to ions in general but

exported, the P:O ratio would not necessarily be a com-

particularly to protons, which accumulate outside the

plete integer, ie, 3, but possibly 2.5. However, for sim-

membrane, creating an electrochemical potential dif-

plicity, a value of 3 for the oxidation of NADH + H⁺

ference across the membrane (∆µH+).This consists of a

and 2 for the oxidation of FADH₂ will continue to be

chemical potential (difference in pH) and an electrical

used throughout this text.

potential.

Experimental Findings Support

A Membrane-Located ATP Synthase

the Chemiosmotic Theory

Functions as a Rotary Motor to Form ATP

(1) Addition of protons

(acid) to the external

The electrochemical potential difference is used to drive

medium of intact mitochondria leads to the generation

a membrane-located ATP synthase which in the pres-

of ATP.

ence of P_i + ADP forms ATP (Figure 12-8). Scattered

(2) Oxidative phosphorylation does not occur in solu-

over the surface of the inner membrane are the phos-

ble systems where there is no possibility of a vectorial

phorylating complexes, ATP synthase, responsible for

ATP synthase. A closed membrane must be present in

the production of ATP (Figure 12-1). These consist of

order to achieve oxidative phosphorylation (Figure 12-8).

several protein subunits, collectively known as F₁,

(3) The respiratory chain contains components or-

which project into the matrix and which contain the

ganized in a sided manner (transverse asymmetry) as re-

phosphorylation mechanism (Figure 12-8). These sub-

quired by the chemiosmotic theory.

THE RESPIRATORY CHAIN & OXIDATIVE PHOSPHORYLATION

H⁺

Oligomycin

F₀

Phospholipid

bilayer

F₁

H⁺

ATP

SYNTHASE

NADH

+ H⁺

ADP + P_i

ATP + H₂O

NAD⁺

Mitochondrial

Respiratory

III

inner (coupling)

(electron

H⁺

membrane

transport)

chain

¹/2

O₂

H₂O

H⁺

INSIDE

pH gradient (∆pH)

Electrical

potential

OUTSIDE

(∆Ψ)

Figure 12-8. Principles of the chemiosmotic theory of oxidative phosphorylation. The main proton circuit

is created by the coupling of oxidation in the respiratory chain to proton translocation from the inside to the

outside of the membrane, driven by the respiratory chain complexes I, III, and IV, each of which acts as a pro-

ton pump. Q, ubiquinone; C, cytochrome c; F₁, F₀, protein subunits which utilize energy from the proton gra-

dient to promote phosphorylation. Uncoupling agents such as dinitrophenol allow leakage of H⁺ across the

membrane, thus collapsing the electrochemical proton gradient. Oligomycin specifically blocks conduction

of H⁺ through F₀.

The Chemiosmotic Theory Can Account

for Respiratory Control and the Action

of Uncouplers

The electrochemical potential difference across the mem-

brane, once established as a result of proton transloca-

Table 12-1. States of respiratory control.

tion, inhibits further transport of reducing equivalents

through the respiratory chain unless discharged by back-

translocation of protons across the membrane through

Conditions Limiting the Rate of Respiration

the vectorial ATP synthase. This in turn depends on

State 1

Availability of ADP and substrate

availability of ADP and P_i.

State 2

Availability of substrate only

Uncouplers

(eg, dinitrophenol) are amphipathic

State 3

The capacity of the respiratory chain itself, when

(Chapter 14) and increase the permeability of the lipoid

all substrates and components are present in

inner mitochondrial membrane to protons

(Figure

saturating amounts

12-8), thus reducing the electrochemical potential and

State 4

Availability of ADP only

short-circuiting the ATP synthase. In this way, oxida-

State 5

Availability of oxygen only

tion can proceed without phosphorylation.

CHAPTER 12

ing electrical and osmotic equilibrium. The inner

bilipoid mitochondrial membrane is freely permeable

ATP

to uncharged small molecules, such as oxygen, water,

CO₂, and NH₃, and to monocarboxylic acids, such as

ADP

ATP

3-hydroxybutyric, acetoacetic, and acetic. Long-chain

fatty acids are transported into mitochondria via the

carnitine system (Figure 22-1), and there is also a spe-

cial carrier for pyruvate involving a symport that utilizes

the H⁺ gradient from outside to inside the mitochon-

drion (Figure 12-10). However, dicarboxylate and tri-

H⁺

Inside

Inner

OUTSIDE

mitochondrial

INSIDE

membrane

Mitochondrial

N-Ethylmaleimide

inner

membrane

OH^-

Outside

H₂PO4-

N-Ethylmaleimide

Hydroxycinnamate

Pyruvate^-

H⁺

Figure 12-9. Mechanism of ATP production by ATP

HPO₄

synthase. The enzyme complex consists of an F₀ sub-

complex which is a disk of “C” protein subunits. At-

Malate2-

tached is a γ-subunit in the form of a “bent axle.” Pro-

tons passing through the disk of “C” units cause it and

Malate2-

the attached γ-subunit to rotate. The γ-subunit fits in-

Citrate3-

side the F₁ subcomplex of three α- and three β-sub-

+ H⁺

units, which are fixed to the membrane and do not ro-

Malate2-

tate. ADP and P_i are taken up sequentially by the

β-subunits to form ATP, which is expelled as the rotat-

α-Ketoglutarate2-

ing γ-subunit squeezes each β-subunit in turn. Thus,

three ATP molecules are generated per revolution. For

ADP3-

clarity, not all the subunits that have been identified are

shown—eg, the “axle” also contains an ε-subunit.

ATP4-

Atractyloside

Figure 12-10. Transporter systems in the inner mi-

tochondrial membrane. 1 , phosphate transporter;

THE RELATIVE IMPERMEABILITY

2 , pyruvate symport; 3 , dicarboxylate transporter;

OF THE INNER MITOCHONDRIAL

4 , tricarboxylate transporter; 5 , α-ketoglutarate trans-

MEMBRANE NECESSITATES

porter; 6 , adenine nucleotide transporter. N-Ethyl-

EXCHANGE TRANSPORTERS

maleimide, hydroxycinnamate, and atractyloside inhibit

Exchange diffusion systems are present in the mem-

( − ) the indicated systems. Also present (but not

brane for exchange of anions against OH⁻ ions and

shown) are transporter systems for glutamate/aspar-

cations against H⁺ ions. Such systems are necessary for

tate (Figure 12-13), glutamine, ornithine, neutral amino

uptake and output of ionized metabolites while preserv-

acids, and carnitine (Figure 22-1).

THE RESPIRATORY CHAIN & OXIDATIVE PHOSPHORYLATION

carboxylate anions and amino acids require specific

Ionophores Permit Specific Cations

transporter or carrier systems to facilitate their passage

to Penetrate Membranes

across the membrane. Monocarboxylic acids penetrate

Ionophores are lipophilic molecules that complex spe-

more readily in their undissociated and more lipid-solu-

cific cations and facilitate their transport through bio-

ble form.

logic membranes, eg, valinomycin (K⁺). The classic

The transport of di- and tricarboxylate anions is

uncouplers such as dinitrophenol are, in fact, proton

closely linked to that of inorganic phosphate, which

ionophores.

penetrates readily as the H₂PO4− ion in exchange for

OH⁻. The net uptake of malate by the dicarboxylate

transporter requires inorganic phosphate for exchange

A Proton-Translocating Transhydrogenase

in the opposite direction. The net uptake of citrate,

Is a Source of Intramitochondrial NADPH

isocitrate, or cis-aconitate by the tricarboxylate trans-

Energy-linked transhydrogenase, a protein in the inner

porter requires malate in exchange. α-Ketoglutarate

mitochondrial membrane, couples the passage of pro-

transport also requires an exchange with malate. The

tons down the electrochemical gradient from outside to

adenine nucleotide transporter allows the exchange of

inside the mitochondrion with the transfer of H from

ATP and ADP but not AMP. It is vital in allowing

intramitochondrial NADH to NADPH for intramito-

ATP exit from mitochondria to the sites of extramito-

chondrial enzymes such as glutamate dehydrogenase

chondrial utilization and in allowing the return of ADP

and hydroxylases involved in steroid synthesis.

for ATP production within the mitochondrion (Figure

12-11). Na⁺ can be exchanged for H⁺, driven by the

Oxidation of Extramitochondrial NADH

proton gradient. It is believed that active uptake of Ca2+

Is Mediated by Substrate Shuttles

by mitochondria occurs with a net charge transfer of 1

(Ca⁺ uniport), possibly through a Ca2+/H⁺ antiport.

NADH cannot penetrate the mitochondrial mem-

Calcium release from mitochondria is facilitated by ex-

brane, but it is produced continuously in the cytosol by

change with Na⁺.

3-phosphoglyceraldehyde dehydrogenase, an enzyme in

the glycolysis sequence (Figure 17-2). However, under

aerobic conditions, extramitochondrial NADH does not

accumulate and is presumed to be oxidized by the respi-

ratory chain in mitochondria. The transfer of reducing

equivalents through the mitochondrial membrane re-

Inner

OUTSIDE

mitochondrial

INSIDE

quires substrate pairs, linked by suitable dehydrogen-

membrane

ases on each side of the mitochondrial membrane. The

F₁

mechanism of transfer using the glycerophosphate

ATP SYNTHASE

shuttle is shown in Figure 12-12). Since the mitochon-

drial enzyme is linked to the respiratory chain via a

3H⁺

flavoprotein rather than NAD, only 2 mol rather than

3 mol of ATP are formed per atom of oxygen con-

sumed. Although this shuttle is present in some tissues

(eg, brain, white muscle), in others (eg, heart muscle) it

ATP4-

is deficient. It is therefore believed that the malate

shuttle system (Figure

12-13) is of more universal

utility. The complexity of this system is due to the im-

ADP3-

permeability of the mitochondrial membrane to oxalo-

P_i

acetate, which must react with glutamate and transami-

nate to aspartate and α-ketoglutarate before transport

H⁺

through the mitochondrial membrane and reconstitu-

tion to oxaloacetate in the cytosol.

Figure 12-11. Combination of phosphate trans-

porter ( 1 ) with the adenine nucleotide transporter ( 2 )

Ion Transport in Mitochondria

in ATP synthesis. The H⁺/P_i symport shown is equiva-

Is Energy-Linked

lent to the P_i/OH⁻ antiport shown in Figure 12-10. Four

protons are taken into the mitochondrion for each ATP

Mitochondria maintain or accumulate cations such as

exported. However, one less proton would be taken in

K+, Na⁺, Ca2+, and Mg2+, and P_i. It is assumed that a

when ATP is used inside the mitochondrion.

primary proton pump drives cation exchange.

100

CHAPTER 12

OUTER

INNER

MEMBRANE

CYTOSOL

MITOCHONDRION

NAD⁺

Glycerol 3-phosphate

FAD

GLYCEROL-3-PHOSPHATE

DEHYDROGENASE

(CYTOSOLIC)

(MITOCHONDRIAL)

NADH + H⁺

Dihydroxyacetone

FADH₂

phosphate

Respiratory chain

Figure 12-12. Glycerophosphate shuttle for transfer of reducing equivalents from the cytosol into the

mitochondrion.

The Creatine Phosphate Shuttle

ported into the cytosol via protein pores in the outer

Facilitates Transport of High-Energy

mitochondrial membrane, becoming available for gen-

eration of extramitochondrial ATP.

Phosphate From Mitochondria

This shuttle (Figure 12-14) augments the functions of

CLINICAL ASPECTS

creatine phosphate as an energy buffer by acting as a

dynamic system for transfer of high-energy phosphate

The condition known as fatal infantile mitochondrial

from mitochondria in active tissues such as heart and

myopathy and renal dysfunction involves severe dim-

skeletal muscle. An isoenzyme of creatine kinase (CK_m)

inution or absence of most oxidoreductases of the respi-

is found in the mitochondrial intermembrane space,

ratory chain. MELAS (mitochondrial encephalopathy,

catalyzing the transfer of high-energy phosphate to cre-

lactic acidosis, and stroke) is an inherited condition due

atine from ATP emerging from the adenine nucleotide

to NADH:ubiquinone oxidoreductase (complex I) or

transporter. In turn, the creatine phosphate is trans-

cytochrome oxidase deficiency. It is caused by a muta-

INNER

CYTOSOL

MEMBRANE

MITOCHONDRION

NAD

Malate

NAD⁺

MALATE DEHYDROGENASE

NADH

Oxaloacetate

α-KG

Oxaloacetate

NADH

+ H⁺

TRANSAMINASE

Glutamate

Asp

Glutamate

H⁺

Figure 12-13. Malate shuttle for transfer of reducing equivalents from the cytosol into the mitochondrion.

1 Ketoglutarate transporter; 2 , glutamate/aspartate transporter (note the proton symport with glutamate).

THE RESPIRATORY CHAIN & OXIDATIVE PHOSPHORYLATION

101

Energy-requiring

processes

(eg, muscle contraction)

ADP

CK_a

ATP

ADP

Creatine

Creatine-P

CK_c

CK_g

ATP

ADP

Glycolysis

ito

Cytosol

Figure 12-14. The creatine phosphate shuttle of

heart and skeletal muscle. The shuttle allows rapid

CK_m

transport of high-energy phosphate from the mito-

chondrial matrix into the cytosol. CK_a, creatine kinase

Inter-membrane

concerned with large requirements for ATP, eg, mus-

ADP

space

cular contraction; CK_c, creatine kinase for maintaining

Adenine

equilibrium between creatine and creatine phosphate

nucleotide

and ATP/ADP; CK_g, creatine kinase coupling glycolysis

transporter

to creatine phosphate synthesis; CK_m, mitochondrial

creatine kinase mediating creatine phosphate produc-

Oxidative

phosphorylation

tion from ATP formed in oxidative phosphorylation; P,

pore protein in outer mitochondrial membrane.

Matrix

tion in mitochondrial DNA and may be involved in

•

Because the inner mitochondrial membrane is imper-

Alzheimer’s disease and diabetes mellitus. A number of

meable to protons and other ions, special exchange

drugs and poisons act by inhibition of oxidative phos-

transporters span the membrane to allow passage of

phorylation (see above).

ions such as OH^-, Pi−, ATP4−, ADP3−, and metabo-

lites, without discharging the electrochemical gradi-

ent across the membrane.

SUMMARY

•

Many well-known poisons such as cyanide arrest res-

piration by inhibition of the respiratory chain.

• Virtually all energy released from the oxidation of

carbohydrate, fat, and protein is made available in

REFERENCES

mitochondria as reducing equivalents (H or e⁻).

These are funneled into the respiratory chain, where

Balaban RS: Regulation of oxidative phosphorylation in the mam-

they are passed down a redox gradient of carriers to

malian cell. Am J Physiol 1990;258:C377.

their final reaction with oxygen to form water.

Hinkle PC et al: Mechanistic stoichiometry of mitochondrial ox-

idative phosphorylation. Biochemistry 1991;30:3576.

• The redox carriers are grouped into respiratory chain

Mitchell P: Keilin’s respiratory chain concept and its chemiosmotic

complexes in the inner mitochondrial membrane.

consequences. Science 1979;206:1148.

These use the energy released in the redox gradient to

Smeitink J et al: The genetics and pathology of oxidative phosphor-

pump protons to the outside of the membrane, creat-

ylation. Nat Rev Genet 2001;2:342.

ing an electrochemical potential across the membrane.

Tyler DD: The Mitochondrion in Health and Disease. VCH Pub-

• Spanning the membrane are ATP synthase com-

lishers, 1992.

plexes that use the potential energy of the proton gra-

Wallace DC: Mitochondrial DNA in aging and disease. Sci Am

dient to synthesize ATP from ADP and P_i. In this

1997;277(2):22.

way, oxidation is closely coupled to phosphorylation

Yoshida M et al: ATP synthase—a marvellous rotary engine of the

to meet the energy needs of the cell.

cell. Nat Rev Mol Cell Biol 2001;2:669.

Carbohydrates of

Physiologic Significance

Peter A. Mayes, PhD, DSc, & David A. Bender, PhD

BIOMEDICAL IMPORTANCE

(4) Polysaccharides are condensation products of

more than ten monosaccharide units; examples are the

Carbohydrates are widely distributed in plants and ani-

starches and dextrins, which may be linear or branched

mals; they have important structural and metabolic

polymers. Polysaccharides are sometimes classified as

roles. In plants, glucose is synthesized from carbon

hexosans or pentosans, depending upon the identity of

dioxide and water by photosynthesis and stored as

the constituent monosaccharides.

starch or used to synthesize cellulose of the plant frame-

work. Animals can synthesize carbohydrate from lipid

glycerol and amino acids, but most animal carbohy-

BIOMEDICALLY, GLUCOSE IS THE MOST

drate is derived ultimately from plants. Glucose is the

IMPORTANT MONOSACCHARIDE

most important carbohydrate; most dietary carbohy-

drate is absorbed into the bloodstream as glucose, and

The Structure of Glucose Can Be

other sugars are converted into glucose in the liver.

Represented in Three Ways

Glucose is the major metabolic fuel of mammals (ex-

The straight-chain structural formula

(aldohexose;

cept ruminants) and a universal fuel of the fetus. It is

the precursor for synthesis of all the other carbohy-

Figure 13-1A) can account for some of the properties

of glucose, but a cyclic structure is favored on thermo-

drates in the body, including glycogen for storage; ri-

bose and deoxyribose in nucleic acids; and galactose

dynamic grounds and accounts for the remainder of its

chemical properties. For most purposes, the structural

in lactose of milk, in glycolipids, and in combination

with protein in glycoproteins and proteoglycans. Dis-

formula is represented as a simple ring in perspective as

proposed by Haworth (Figure 13-1B). In this represen-

eases associated with carbohydrate metabolism include

diabetes mellitus, galactosemia, glycogen storage

tation, the molecule is viewed from the side and above

the plane of the ring. By convention, the bonds nearest

diseases, and lactose intolerance.

to the viewer are bold and thickened. The six-mem-

bered ring containing one oxygen atom is in the form

CARBOHYDRATES ARE ALDEHYDE

of a chair (Figure 13-1C).

OR KETONE DERIVATIVES OF

POLYHYDRIC ALCOHOLS

Sugars Exhibit Various Forms of Isomerism

(1) Monosaccharides are those carbohydrates that

cannot be hydrolyzed into simpler carbohydrates: They

Glucose, with four asymmetric carbon atoms, can form

may be classified as trioses, tetroses, pentoses, hex-

16 isomers. The more important types of isomerism

oses, or heptoses, depending upon the number of car-

found with glucose are as follows.

bon atoms; and as aldoses or ketoses depending upon

(1) D and L isomerism: The designation of a sugar

whether they have an aldehyde or ketone group. Exam-

isomer as the D form or of its mirror image as the L form

ples are listed in Table 13-1.

(2) Disaccharides are condensation products of two

monosaccharide units. Examples are maltose and su-

Table 13-1. Classification of important sugars.

crose.

(3) Oligosaccharides are condensation products of

two to ten monosaccharides; maltotriose* is an exam-

Aldoses

Ketoses

ple.

Trioses (C₃H₆O₃)

Glycerose

Dihydroxyacetone

Tetroses (C₄H₈O₄)

Erythrose

Erythrulose

Pentoses (C₅H₁₀O₅)

Ribose

Ribulose

*Note that this is not a true triose but a trisaccharide containing

Hexoses (C₆H₁₂O₆)

Glucose

Fructose

three α-glucose residues.

102

CARBOHYDRATES OF PHYSIOLOGIC SIGNIFICANCE

103

¹C H

C OH

C H

Pyran

Furan

C OH

HOCH₂

2OH

HOCH₂

HCOH

HOCH₂

H H

HO OH H OH

H OH

α-D-Glucopyranose

α-D-Glucofuranose

Figure 13-3. Pyranose and furanose forms of glu-

HOCH₂

cose.

Figure 13-1.

D-Glucose. A: straight chain form.

B: α-D-glucose; Haworth projection. C: α-D-glucose;

chair form.

HOCH₂

¹C H

H H

²C H

³CH₂OH

CH₂OH

HO H HO

L-Glycerose

D-Glycerose

COH

(L-glyceraldehyde)

(D-glyceraldehyde)

OH H

H₂

α-D-Fructopyranose

β-D-Fructopyranose

¹C H

HOCH₂

²C H

³C

HO C

⁴C H

H H HO

⁵C H

COH

OH H

⁶CH₂OH

CH₂OH

L-Glucose

D-Glucose

α-D-Fructofuranose

β-D-Fructofuranose

Figure 13-2. D- and L-isomerism of glycerose and

Figure 13-4. Pyranose and furanose forms of fruc-

glucose.

tose.

104

CHAPTER 13

HOCH₂

HO H

H H

H OH H OH

OH H

HO OH

Figure 13-5. Epimerization of

α-D-Galactose

α-D-Glucose

α-D-Mannose

glucose.

is determined by its spatial relationship to the parent

(3) Alpha and beta anomers: The ring structure of

compound of the carbohydrates, the three-carbon

an aldose is a hemiacetal, since it is formed by combina-

sugar glycerose (glyceraldehyde). The L and D forms of

tion of an aldehyde and an alcohol group. Similarly, the

this sugar, and of glucose, are shown in Figure 13-2.

ring structure of a ketose is a hemiketal. Crystalline glu-

The orientation of the H and  OH groups around

cose is α-D-glucopyranose. The cyclic structure is re-

the carbon atom adjacent to the terminal primary alco-

tained in solution, but isomerism occurs about position

hol carbon (carbon 5 in glucose) determines whether

1, the carbonyl or anomeric carbon atom, to give a

the sugar belongs to the D or L series. When the OH

mixture of α-glucopyranose (38%) and β-glucopyra-

group on this carbon is on the right (as seen in Figure

nose (62%). Less than 0.3% is represented by α and β

13-2), the sugar is the D-isomer; when it is on the

anomers of glucofuranose.

left, it is the L-isomer. Most of the monosaccharides

(4) Epimers: Isomers differing as a result of varia-

occurring in mammals are D sugars, and the enzymes

tions in configuration of the  OH and H on car-

responsible for their metabolism are specific for this

bon atoms 2, 3, and 4 of glucose are known as epimers.

configuration. In solution, glucose is dextrorotatory—

Biologically, the most important epimers of glucose are

hence the alternative name dextrose, often used in

mannose and galactose, formed by epimerization at car-

clinical practice.

bons 2 and 4, respectively (Figure 13-5).

The presence of asymmetric carbon atoms also con-

(5) Aldose-ketose isomerism: Fructose has the

fers optical activity on the compound. When a beam

same molecular formula as glucose but differs in its

of plane-polarized light is passed through a solution of

structural formula, since there is a potential keto group

an optical isomer, it will be rotated either to the right,

in position 2, the anomeric carbon of fructose (Figures

dextrorotatory (+); or to the left, levorotatory (−). The

13-4 and 13-7), whereas there is a potential aldehyde

direction of rotation is independent of the stereochem-

group in position 1, the anomeric carbon of glucose

istry of the sugar, so it may be designated D(−), D(+),

(Figures 13-2 and 13-6).

L(−), or L(+). For example, the naturally occurring form

of fructose is the D(−) isomer.

Many Monosaccharides Are

(2) Pyranose and furanose ring structures: The

Physiologically Important

stable ring structures of monosaccharides are similar to

the ring structures of either pyran

(a six-membered

Derivatives of trioses, tetroses, and pentoses and of a

ring) or furan (a five-membered ring) (Figures 13-3

seven-carbon sugar (sedoheptulose) are formed as meta-

and 13-4). For glucose in solution, more than 99% is

bolic intermediates in glycolysis and the pentose phos-

in the pyranose form.

phate pathway. Pentoses are important in nucleotides,

CHO

H C OH

HO C

H C OH

CHO

HO C

H C OH

HO C

CHO

H C OH

HO C

C OH

H C OH

HO C

H C OH

C OH

H C OH

CH₂OH

D-Glycerose

(D-glyceraldehyde) D-Erythrose

D-Lyxose

D-Xylose

D-Arabinose

D-Ribose

D-Galactose

D-Mannose

D-Glucose

Figure 13-6. Examples of aldoses of physiologic significance.

CARBOHYDRATES OF PHYSIOLOGIC SIGNIFICANCE

105

Table 13-2. Pentoses of physiologic importance.

Sugar

Where Found

Biochemical Importance

Clinical Significance

D-Ribose

Nucleic acids.

Structural elements of nucleic acids and

coenzymes, eg, ATP, NAD, NADP, flavo-

proteins. Ribose phosphates are inter-

mediates in pentose phosphate pathway.

D-Ribulose

Formed in metabolic processes.

Ribulose phosphate is an intermediate in

pentose phosphate pathway.

D-Arabinose

Gum arabic. Plum and cherry gums.

Constituent of glycoproteins.

D-Xylose

Wood gums, proteoglycans,

Constituent of glycoproteins.

glycosaminoglycans.

D-Lyxose

Heart muscle.

A constituent of a lyxoflavin isolated from

human heart muscle.

L-Xylulose

Intermediate in uronic acid pathway.

Found in urine in essential

pentosuria.

nucleic acids, and several coenzymes

(Table

13-2).

Sugars Form Glycosides With Other

Glucose, galactose, fructose, and mannose are physio-

Compounds & With Each Other

logically the most important hexoses (Table 13-3). The

biochemically important aldoses are shown in Figure

Glycosides are formed by condensation between the hy-

13-6, and important ketoses in Figure 13-7.

droxyl group of the anomeric carbon of a monosaccha-

In addition, carboxylic acid derivatives of glucose are

ride, or monosaccharide residue, and a second compound

important, including D-glucuronate

(for glucuronide

that may—or may not (in the case of an aglycone)—be

formation and in glycosaminoglycans) and its meta-

another monosaccharide. If the second group is a hy-

bolic derivative, L-iduronate

(in glycosaminoglycans)

droxyl, the O-glycosidic bond is an acetal link because it

(Figure 13-8) and L-gulonate (an intermediate in the

results from a reaction between a hemiacetal group

uronic acid pathway; see Figure 20-4).

(formed from an aldehyde and an OH group) and an-

Table 13-3. Hexoses of physiologic importance.

Sugar

Source

Importance

Clinical Significance

D-Glucose

Fruit juices. Hydrolysis of starch, cane

The “sugar” of the body. The sugar carried

Present in the urine (glycosuria)

sugar, maltose, and lactose.

by the blood, and the principal one used

in diabetes mellitus owing to

by the tissues.

raised blood glucose (hyper-

glycemia).

D-Fructose

Fruit juices. Honey. Hydrolysis of

Can be changed to glucose in the liver

Hereditary fructose intolerance

cane sugar and of inulin (from the

and so used in the body.

leads to fructose accumulation

Jerusalem artichoke).

and hypoglycemia.

D-Galactose

Hydrolysis of lactose.

Can be changed to glucose in the liver

Failure to metabolize leads

and metabolized. Synthesized in the

to galactosemia and cataract.

mammary gland to make the lactose of

milk. A constituent of glycolipids and

glycoproteins.

D-Mannose

Hydrolysis of plant mannans and

A constituent of many glycoproteins.

gums.

106

CHAPTER 13

CH₂OH

C O

CH₂OH

C H

C O

CH₂OH

H C OH

CH₂OH

Dihydroxyacetone

D-Xylulose

D-Ribulose

D-Fructose

D-Sedoheptulose

Figure 13-7. Examples of ketoses of physiologic significance.

other OH group. If the hemiacetal portion is glucose,

COO^-

the resulting compound is a glucoside; if galactose, a

galactoside; and so on. If the second group is an amine,

COO^-

an N-glycosidic bond is formed, eg, between adenine and

ribose in nucleotides such as ATP (Figure 10-4).

HO OH H

Glycosides are widely distributed in nature; the agly-

cone may be methanol, glycerol, a sterol, a phenol, or a

base such as adenine. The glycosides that are important

in medicine because of their action on the heart (car-

Figure 13-8.

α-D-Glucuronate (left) and

diac glycosides) all contain steroids as the aglycone.

β-L-iduronate (right).

These include derivatives of digitalis and strophanthus

such as ouabain, an inhibitor of the Na⁺-K⁺ ATPase of

cell membranes. Other glycosides include antibiotics

such as streptomycin.

HOCH₂

Deoxy Sugars Lack an Oxygen Atom

Deoxy sugars are those in which a hydroxyl group has

been replaced by hydrogen. An example is deoxyribose

H H

(Figure 13-9) in DNA. The deoxy sugar L-fucose (Figure

13-15) occurs in glycoproteins; 2-deoxyglucose is used

OH H

experimentally as an inhibitor of glucose metabolism.

Figure 13-9.

2-Deoxy-D-ribofuranose (β form).

Amino Sugars (Hexosamines) Are

Components of Glycoproteins,

Gangliosides, & Glycosaminoglycans

HOCH₂

The amino sugars include D-glucosamine, a constituent

of hyaluronic acid

(Figure

13-10), D-galactosamine

H H

(chondrosamine), a constituent of chondroitin; and

D-mannosamine. Several antibiotics (eg, erythromycin)

contain amino sugars believed to be important for their

antibiotic activity.

MALTOSE, SUCROSE, & LACTOSE ARE

Figure 13-10. Glucosamine (2-amino-D-glucopyra-

IMPORTANT DISACCHARIDES

nose) (α form). Galactosamine is 2-amino-D-galactopy-

The physiologically important disaccharides are mal-

ranose. Both glucosamine and galactosamine occur as

tose, sucrose, and lactose (Table 13-4; Figure 13-11).

N-acetyl derivatives in more complex carbohydrates,

Hydrolysis of sucrose yields a mixture of glucose and

eg, glycoproteins.

CARBOHYDRATES OF PHYSIOLOGIC SIGNIFICANCE

107

Table 13-4. Disaccharides.

Sugar

Source

Clinical Significance

Maltose

Digestion by amylase or hydrolysis of starch.

Germinating cereals and malt.

Lactose

Milk. May occur in urine during pregnancy.

In lactase deficiency, malabsorption leads to diarrhea and flatulence.

Sucrose

Cane and beet sugar. Sorghum. Pineapple.

In sucrase deficiency, malabsorption leads to diarrhea and flatulence.

Carrot roots.

Trehalose¹

Fungi and yeasts. The major sugar of insect

hemolymph.

¹O-α-D-Glucopyranosyl-(1 → 1)-α-D-glucopyranoside.

fructose which is called

“invert sugar” because the

toes, legumes, and other vegetables. The two main con-

strongly levorotatory fructose changes (inverts) the pre-

stituents are amylose

(15-20%), which has a non-

vious dextrorotatory action of sucrose.

branching helical structure (Figure 13-12); and amy-

lopectin (80-85%), which consists of branched chains

composed of 24-30 glucose residues united by 1 → 4

POLYSACCHARIDES SERVE STORAGE

linkages in the chains and by 1 → 6 linkages at the

& STRUCTURAL FUNCTIONS

branch points.

Polysaccharides include the following physiologically

Glycogen (Figure 13-13) is the storage polysaccha-

important carbohydrates.

ride in animals. It is a more highly branched structure

Starch is a homopolymer of glucose forming an α-

than amylopectin, with chains of 12-14 α-D-glucopyra-

glucosidic chain, called a glucosan or glucan. It is the

nose residues (in α[1 → 4]-glucosidic linkage), with

most abundant dietary carbohydrate in cereals, pota-

branching by means of α(1 → 6)-glucosidic bonds.

Maltose

Lactose

HOCH₂

H H

HO H

HO OH H

H OH H

O-α-D-Glucopyranosyl-(1 → 4)-α-D-glucopyranose

O-β-D-Galactopyranosyl-(1 → 4)-β-D-glucopyranose

Sucrose

Figure 13-11. Structures of important disaccharides. The α and β

HOCH₂

refer to the configuration at the anomeric carbon atom (asterisk). When

the anomeric carbon of the second residue takes part in the formation

H H

of the glycosidic bond, as in sucrose, the residue becomes a glycoside

known as a furanoside or pyranoside. As the disaccharide no longer has

HO OH H

H HO

COH

an anomeric carbon with a free potential aldehyde or ketone group, it

H₂

no longer exhibits reducing properties. The configuration of the

OH H

β-fructofuranose residue in sucrose results from turning the β-fructofu-

ranose molecule depicted in Figure 13-4 through 180 degrees and in-

O-α-D-Glucopyranosyl-(1 → 2)-β-D-fructofuranoside verting it.

CARBOHYDRATES OF PHYSIOLOGIC SIGNIFICANCE

109

Chitin

Inulin is a polysaccharide of fructose (and hence a fruc-

tosan) found in tubers and roots of dahlias, artichokes,

HOCH₂

and dandelions. It is readily soluble in water and is used

to determine the glomerular filtration rate. Dextrins are

H H

intermediates in the hydrolysis of starch. Cellulose is

the chief constituent of the framework of plants. It is in-

OH H H

soluble and consists of β-D-glucopyranose units linked

by β(1 → 4) bonds to form long, straight chains

H HN

CO CH₃

HN CO CH₃

strengthened by cross-linked hydrogen bonds. Cellulose

cannot be digested by mammals because of the absence

N-Acetylglucosamine

of an enzyme that hydrolyzes the β linkage. It is an im-

portant source of “bulk” in the diet. Microorganisms in

the gut of ruminants and other herbivores can hydrolyze

Hyaluronic acid

the β linkage and ferment the products to short-chain

HOCH₂

fatty acids as a major energy source. There is limited

bacterial metabolism of cellulose in the human colon.

COO

H H

Chitin is a structural polysaccharide in the exoskeleton

of crustaceans and insects and also in mushrooms. It

H H

HO O

H H

consists of N-acetyl-D-glucosamine units joined by

β (1 → 4)-glycosidic linkages (Figure 13-14).

OH H H

Glycosaminoglycans

(mucopolysaccharides) are

H HN CO CH₃

complex carbohydrates characterized by their content

of amino sugars and uronic acids. When these chains

are attached to a protein molecule, the result is a pro-

β-Glucuronic acid

N-Acetylglucosamine

teoglycan. Proteoglycans provide the ground or pack-

ing substance of connective tissues. Their property of

holding large quantities of water and occupying space,

Chondroitin 4-sulfate

(Note: There is also a 6-sulfate)

thus cushioning or lubricating other structures, is due

to the large number of OH groups and negative

HOCH₂

charges on the molecules, which, by repulsion, keep the

carbohydrate chains apart. Examples are hyaluronic

COO

^- SO₃O H

acid, chondroitin sulfate, and heparin

(Figure

13-14).

H H

H O

H H

Glycoproteins (mucoproteins) occur in many dif-

ferent situations in fluids and tissues, including the cell

OH H H

membranes (Chapters 41 and 47). They are proteins

H HN CO CH₃

β-Glucuronic acid

N-Acetylgalactosamine sulfate

Table 13-5. Carbohydrates found in

glycoproteins.

Heparin

Hexoses

Mannose (Man)

COSO

Galactose (Gal)

Acetyl hexosamines

N-Acetylglucosamine (GlcNAc)

H H

COO

N-Acetylgalactosamine (GalNAc)

OH H

H H

Pentoses

Arabinose (Ara)

Xylose (Xyl)

–

NH SO₃

OSO

Methyl pentose

L-Fucose (Fuc; see Figure 13-15)

Sialic acids

N-Acyl derivatives of neuraminic acid,

Sulfated glucosamine

Sulfated iduronic acid

eg, N-acetylneuraminic acid (NeuAc; see

Figure 13-16), the predominant sialic

Figure 13-14. Structure of some complex polysac-

acid.

charides and glycosaminoglycans.

110

CHAPTER 13

outside both the external and internal (cytoplasmic)

surfaces. Carbohydrate chains are only attached to the

CH₃

amino terminal portion outside the external surface

(Chapter 41).

HO H

SUMMARY

•

Carbohydrates are major constituents of animal food

Figure 13-15. β-L-Fucose (6-deoxy-β-L-galactose).

and animal tissues. They are characterized by the

type and number of monosaccharide residues in their

molecules.

•

Glucose is the most important carbohydrate in mam-

malian biochemistry because nearly all carbohydrate

containing branched or unbranched oligosaccharide

in food is converted to glucose for metabolism.

chains (see Table 13-5). The sialic acids are N- or

O-acyl derivatives of neuraminic acid (Figure 13-16).

•

Sugars have large numbers of stereoisomers because

Neuraminic acid is a nine-carbon sugar derived from

they contain several asymmetric carbon atoms.

mannosamine (an epimer of glucosamine) and pyru-

•

The monosaccharides include glucose, the “blood

vate. Sialic acids are constituents of both glycoproteins

sugar”; and ribose, an important constituent of nu-

and gangliosides (Chapters 14 and 47).

cleotides and nucleic acids.

•

The disaccharides include maltose (glucosyl glucose),

CARBOHYDRATES OCCUR IN CELL

an intermediate in the digestion of starch; sucrose

MEMBRANES & IN LIPOPROTEINS

(glucosyl fructose), important as a dietary constituent

containing fructose; and lactose (galactosyl glucose),

In addition to the lipid of cell membranes (see Chapters

in milk.

14 and 41), approximately 5% is carbohydrate in glyco-

•

Starch and glycogen are storage polymers of glucose

proteins and glycolipids. Carbohydrates are also present

in plants and animals, respectively. Starch is the

in apo B of lipoproteins. Their presence on the outer

major source of energy in the diet.

surface of the plasma membrane (the glycocalyx) has

•

Complex carbohydrates contain other sugar deriva-

been shown with the use of plant lectins, protein agglu-

tives such as amino sugars, uronic acids, and sialic

tinins that bind with specific glycosyl residues. For

acids. They include proteoglycans and glycosamino-

example, concanavalin A binds α-glucosyl and α-man-

glycans, associated with structural elements of the tis-

nosyl residues. Glycophorin is a major integral mem-

sues; and glycoproteins, proteins containing attached

brane glycoprotein of human erythrocytes and spans

oligosaccharide chains. They are found in many situ-

the lipid membrane, having free polypeptide portions

ations including the cell membrane.

REFERENCES

Binkley RW: Modern Carbohydrate Chemistry. Marcel Dekker,

1988.

CHOH

COO^—

Collins PM (editor): Carbohydrates. Chapman & Hall, 1988.

CHOH

El-Khadem HS: Carbohydrate Chemistry: Monosaccharides and

CH₂OH

Their Oligomers. Academic Press, 1988.

Lehman J (editor) (translated by Haines A.): Carbohydrates: Struc-

ture and Biology. Thieme, 1998.

Lindahl U, Höök M: Glycosaminoglycans and their binding to bio-

logical macromolecules. Annu Rev Biochem 1978;47:385.

Melendes-Hevia E, Waddell TG, Shelton ED: Optimization of

Figure 13-16.

Structure of N-acetylneuraminic acid,

molecular design in the evolution of metabolism: the glyco-

a sialic acid (Ac = CH₃ CO ).

gen molecule. Biochem J 1993;295:477.

Lipids of Physiologic Significance

Peter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DSc

BIOMEDICAL IMPORTANCE

Other complex lipids: Lipids such as sul-

folipids and aminolipids. Lipoproteins may

The lipids are a heterogeneous group of compounds,

also be placed in this category.

including fats, oils, steroids, waxes, and related com-

3. Precursor and derived lipids: These include fatty

pounds, which are related more by their physical than

acids, glycerol, steroids, other alcohols, fatty alde-

by their chemical properties. They have the common

hydes, and ketone bodies (Chapter 22), hydrocar-

property of being (1) relatively insoluble in water and

bons, lipid-soluble vitamins, and hormones.

(2) soluble in nonpolar solvents such as ether and

chloroform. They are important dietary constituents

Because they are uncharged, acylglycerols

(glyc-

not only because of their high energy value but also be-

erides), cholesterol, and cholesteryl esters are termed

cause of the fat-soluble vitamins and the essential fatty

neutral lipids.

acids contained in the fat of natural foods. Fat is stored

in adipose tissue, where it also serves as a thermal insu-

lator in the subcutaneous tissues and around certain or-

FATTY ACIDS ARE ALIPHATIC

gans. Nonpolar lipids act as electrical insulators, al-

CARBOXYLIC ACIDS

lowing rapid propagation of depolarization waves along

Fatty acids occur mainly as esters in natural fats and oils

myelinated nerves. Combinations of lipid and protein

but do occur in the unesterified form as free fatty

(lipoproteins) are important cellular constituents, oc-

acids, a transport form found in the plasma. Fatty acids

curring both in the cell membrane and in the mito-

that occur in natural fats are usually straight-chain de-

chondria, and serving also as the means of transporting

rivatives containing an even number of carbon atoms.

lipids in the blood. Knowledge of lipid biochemistry is

The chain may be saturated (containing no double

necessary in understanding many important biomedical

bonds) or unsaturated (containing one or more double

areas, eg, obesity, diabetes mellitus, atherosclerosis,

bonds).

and the role of various polyunsaturated fatty acids in

nutrition and health.

Fatty Acids Are Named After

LIPIDS ARE CLASSIFIED AS SIMPLE

Corresponding Hydrocarbons

OR COMPLEX

The most frequently used systematic nomenclature

1. Simple lipids: Esters of fatty acids with various al-

names the fatty acid after the hydrocarbon with the

cohols.

same number and arrangement of carbon atoms, with

Fats: Esters of fatty acids with glycerol. Oils

-oic being substituted for the final -e (Genevan sys-

are fats in the liquid state.

tem). Thus, saturated acids end in -anoic, eg, octanoic

b. Waxes: Esters of fatty acids with higher mole-

acid, and unsaturated acids with double bonds end in

cular weight monohydric alcohols.

-enoic, eg, octadecenoic acid (oleic acid).

2. Complex lipids: Esters of fatty acids containing

Carbon atoms are numbered from the carboxyl car-

groups in addition to an alcohol and a fatty acid.

bon (carbon No. 1). The carbon atoms adjacent to the

Phospholipids: Lipids containing, in addition

carboxyl carbon (Nos. 2, 3, and 4) are also known as

to fatty acids and an alcohol, a phosphoric

the α, β, and γ carbons, respectively, and the terminal

acid residue. They frequently have nitrogen-

methyl carbon is known as the ω or n-carbon.

containing bases and other substituents, eg, in

Various conventions use ∆ for indicating the num-

glycerophospholipids the alcohol is glycerol

ber and position of the double bonds (Figure 14-1); eg,

and in sphingophospholipids the alcohol is

∆⁹ indicates a double bond between carbons 9 and 10

sphingosine.

of the fatty acid; ω9 indicates a double bond on the

b. Glycolipids

(glycosphingolipids): Lipids

ninth carbon counting from the ω- carbon. In animals,

containing a fatty acid, sphingosine, and car-

additional double bonds are introduced only between

bohydrate.

the existing double bond (eg, ω9, ω6, or ω3) and the

111

112

CHAPTER 14

18:1;9 or ∆⁹ 18:1

Unsaturated Fatty Acids Contain One

or More Double Bonds

CH₃(CH₂)₇CH

CH(CH₂)₇COOH

(Table 14-2)

Fatty acids may be further subdivided as follows:

ω9,C18:1 or n-9, 18:1

(1) Monounsaturated (monoethenoid, monoenoic)

CH₃CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH

CH(CH₂)₇COOH

acids, containing one double bond.

(2) Polyunsaturated

(polyethenoid, polyenoic)

Figure 14-1. Oleic acid. n − 9 (n minus 9) is equiva-

acids, containing two or more double bonds.

lent to ω9.

(3) Eicosanoids: These compounds, derived from

eicosa-

(20-carbon) polyenoic fatty acids, com-

prise the prostanoids, leukotrienes (LTs), and

carboxyl carbon, leading to three series of fatty acids

lipoxins

(LXs). Prostanoids include prosta-

known as the ω9, ω6, and ω3 families, respectively.

glandins

(PGs), prostacyclins

(PGIs), and

thromboxanes (TXs).

Saturated Fatty Acids Contain

Prostaglandins exist in virtually every mammalian

tissue, acting as local hormones; they have important

No Double Bonds

physiologic and pharmacologic activities. They are syn-

Saturated fatty acids may be envisaged as based on

thesized in vivo by cyclization of the center of the car-

acetic acid (CH₃  COOH) as the first member of the

bon chain of 20-carbon (eicosanoic) polyunsaturated

series in which  CH₂  is progressively added be-

fatty acids (eg, arachidonic acid) to form a cyclopentane

tween the terminal CH₃  and  COOH groups. Ex-

ring (Figure 14-2). A related series of compounds, the

amples are shown in Table 14-1. Other higher mem-

thromboxanes, have the cyclopentane ring interrupted

bers of the series are known to occur, particularly in

with an oxygen atom (oxane ring) (Figure 14-3). Three

waxes. A few branched-chain fatty acids have also been

different eicosanoic fatty acids give rise to three groups

isolated from both plant and animal sources.

of eicosanoids characterized by the number of double

bonds in the side chains, eg, PG₁, PG₂, PG₃. Different

substituent groups attached to the rings give rise to se-

ries of prostaglandins and thromboxanes, labeled A, B,

Table 14-1. Saturated fatty acids.

etc—eg, the “E” type of prostaglandin (as in PGE₂) has

a keto group in position 9, whereas the “F” type has a

Common

Number of

hydroxyl group in this position. The leukotrienes and

Name

C Atoms

lipoxins are a third group of eicosanoid derivatives

Acetic

Major end product of carbohy-

formed via the lipoxygenase pathway (Figure 14-4).

drate fermentation by rumen

They are characterized by the presence of three or four

organisms¹

conjugated double bonds, respectively. Leukotrienes

cause bronchoconstriction as well as being potent

Propionic

An end product of carbohydrate

fermentation by rumen

proinflammatory agents and play a part in asthma.

organisms¹

Butyric

In certain fats in small amounts

Most Naturally Occurring Unsaturated

(especially butter). An end product

Valeric

Fatty Acids Have cis Double Bonds

of carbohydrate fermentation by

Caproic

rumen organisms¹

The carbon chains of saturated fatty acids form a zigzag

pattern when extended, as at low temperatures. At

Lauric

Spermaceti, cinnamon, palm ker-

higher temperatures, some bonds rotate, causing chain

nel, coconut oils, laurels, butter

shortening, which explains why biomembranes become

Myristic

Nutmeg, palm kernel, coconut oils,

thinner with increases in temperature. A type of geo-

myrtles, butter

metric isomerism occurs in unsaturated fatty acids, de-

Palmitic

Common in all animal and plant

pending on the orientation of atoms or groups around

fats

the axes of double bonds, which do not allow rotation.

Stearic

If the acyl chains are on the same side of the bond, it

¹Also formed in the cecum of herbivores and to a lesser extent in

is cis-, as in oleic acid; if on opposite sides, it is trans-, as

the colon of humans.

in elaidic acid, the trans isomer of oleic acid

(Fig-

LIPIDS OF PHYSIOLOGIC SIGNIFICANCE

113

Table 14-2. Unsaturated fatty acids of physiologic and nutritional significance.

Number of C

Atoms and Number

and Position of

Common

Double Bonds

Family

Name

Systematic Name

Occurrence

Monoenoic acids (one double bond)

16:1;9

ω7

Palmitoleic

cis-9-Hexadecenoic

In nearly all fats.

18:1;9

ω9

Oleic

cis-9-Octadecenoic

Possibly the most common fatty acid in

natural fats.

18:1;9

ω9

Elaidic

trans-9-Octadecenoic

Hydrogenated and ruminant fats.

Dienoic acids (two double bonds)

18:2;9,12

ω6

Linoleic

all-cis-9,12-Octadecadienoic

Corn, peanut, cottonseed, soybean,

and many plant oils.

Trienoic acids (three double bonds)

18:3;6,9,12

ω6

γ-Linolenic

all-cis-6,9,12-Octadecatrienoic

Some plants, eg, oil of evening prim-

rose, borage oil; minor fatty acid in

animals.

18:3;9,12,15

ω3

α-Linolenic

all-cis-9,12,15-Octadecatrienoic

Frequently found with linoleic acid but

particularly in linseed oil.

Tetraenoic acids (four double bonds)

20:4;5,8,11,14

ω6

Arachidonic

all-cis-5,8,11,14-Eicosatetraenoic

Found in animal fats and in peanut oil;

important component of phospho-

lipids in animals.

Pentaenoic acids (five double bonds)

20:5;5,8,11,14,17

ω3

Timnodonic

all-cis-5,8,11,14,17-Eicosapentaenoic

Important component of fish oils, eg,

cod liver, mackerel, menhaden, salmon

oils.

Hexaenoic acids (six double bonds)

22:6;4,7,10,13,16,19

ω3

Cervonic

all-cis-4,7,10,13,16,19-Docosahexaenoic

Fish oils, phospholipids in brain.

ure 14-5). Naturally occurring unsaturated long-chain

U shape. This has profound significance on molecular

fatty acids are nearly all of the cis configuration, the

packing in membranes and on the positions occupied

molecules being

“bent”

120 degrees at the double

by fatty acids in more complex molecules such as phos-

bond. Thus, oleic acid has an L shape, whereas elaidic

pholipids. Trans double bonds alter these spatial rela-

acid remains “straight.” Increase in the number of cis

tionships. Trans fatty acids are present in certain foods,

double bonds in a fatty acid leads to a variety of possi-

arising as a by-product of the saturation of fatty acids

ble spatial configurations of the molecule—eg, arachi-

during hydrogenation, or “hardening,” of natural oils

donic acid, with four cis double bonds, has “kinks” or a

in the manufacture of margarine. An additional small

COO—

Figure 14-2. Prostaglandin E₂ (PGE₂).

Figure 14-3. Thromboxane A₂ (TXA₂).

114

CHAPTER 14

more unsaturated than storage lipids. Lipids in tissues

that are subject to cooling, eg, in hibernators or in the

COO-

extremities of animals, are more unsaturated.

TRIACYLGLYCEROLS (TRIGLYCERIDES)*

ARE THE MAIN STORAGE FORMS OF

Figure 14-4. Leukotriene A₄ (LTA₄).

FATTY ACIDS

The triacylglycerols (Figure 14-6) are esters of the tri-

hydric alcohol glycerol and fatty acids. Mono- and di-

contribution comes from the ingestion of ruminant fat

acylglycerols wherein one or two fatty acids are esteri-

that contains trans fatty acids arising from the action of

fied with glycerol are also found in the tissues. These

microorganisms in the rumen.

are of particular significance in the synthesis and hy-

drolysis of triacylglycerols.

Physical and Physiologic Properties

of Fatty Acids Reflect Chain Length

Carbons 1 & 3 of Glycerol Are

and Degree of Unsaturation

Not Identical

The melting points of even-numbered-carbon fatty

To number the carbon atoms of glycerol unambigu-

acids increase with chain length and decrease according

ously, the -sn-

(stereochemical numbering) system is

to unsaturation. A triacylglycerol containing three satu-

used. It is important to realize that carbons 1 and 3 of

rated fatty acids of 12 carbons or more is solid at body

glycerol are not identical when viewed in three dimen-

temperature, whereas if the fatty acid residues are 18:2,

sions (shown as a projection formula in Figure 14-7).

it is liquid to below 0 °C. In practice, natural acylglyc-

Enzymes readily distinguish between them and are

erols contain a mixture of fatty acids tailored to suit

nearly always specific for one or the other carbon; eg,

their functional roles. The membrane lipids, which

glycerol is always phosphorylated on sn-3 by glycerol

must be fluid at all environmental temperatures, are

kinase to give glycerol 3-phosphate and not glycerol

1-phosphate.

CH₃

PHOSPHOLIPIDS ARE THE MAIN LIPID

CONSTITUENTS OF MEMBRANES

Phospholipids may be regarded as derivatives of phos-

Trans form

(elaidic acid)

phatidic acid (Figure 14-8), in which the phosphate is

esterified with the  OH of a suitable alcohol. Phos-

phatidic acid is important as an intermediate in the syn-

thesis of triacylglycerols as well as phosphoglycerols but

120

is not found in any great quantity in tissues.

Cis form

(oleic acid)

Phosphatidylcholines (Lecithins)

Occur in Cell Membranes

110

Phosphoacylglycerols containing choline (Figure 14-8)

are the most abundant phospholipids of the cell mem-

* According to the standardized terminology of the International

Union of Pure and Applied Chemistry (IUPAC) and the Interna-

tional Union of Biochemistry (IUB), the monoglycerides, diglyc-

COO-

erides, and triglycerides should be designated monoacylglycerols,

diacylglycerols, and triacylglycerols, respectively. However, the

Figure 14-5.

Geometric isomerism of ∆⁹, 18:1 fatty

older terminology is still widely used, particularly in clinical medi-

acids (oleic and elaidic acids).

cine.

LIPIDS OF PHYSIOLOGIC SIGNIFICANCE

115

¹CH₂

O C

R₁

¹CH₂

O C

R₁

R₂

²CH

R₂

²CH

³CH₂

O C

R₂

³CH₂

O-

Figure 14-6. Triacylglycerol.

Phosphatidic acid

brane and represent a large proportion of the body’s

store of choline. Choline is important in nervous trans-

CH₃

mission, as acetylcholine, and as a store of labile methyl

O CH₂

CH₂

CH₃

groups. Dipalmitoyl lecithin is a very effective surface-

CH₃

active agent and a major constituent of the surfactant

preventing adherence, due to surface tension, of the

Choline

inner surfaces of the lungs. Its absence from the lungs

of premature infants causes respiratory distress syn-

O CH₂

CH₂NH₃

drome. Most phospholipids have a saturated acyl radi-

cal in the sn-1 position but an unsaturated radical in the

Ethanolamine

sn-2 position of glycerol.

Phosphatidylethanolamine (cephalin) and phos-

NH₃₊

phatidylserine

(found in most tissues) differ from

phosphatidylcholine only in that ethanolamine or ser-

O CH₂

CH COO-

ine, respectively, replaces choline (Figure 14-8).

Serine

Phosphatidylinositol Is a Precursor

of Second Messengers

O H

The inositol is present in phosphatidylinositol as the

stereoisomer, myoinositol (Figure 14-8). Phosphati-

dylinositol

4,5-bisphosphate is an important con-

stituent of cell membrane phospholipids; upon stimula-

tion by a suitable hormone agonist, it is cleaved into

diacylglycerol and inositol trisphosphate, both of

which act as internal signals or second messengers.

Myoinositol

O-

Cardiolipin Is a Major Lipid

of Mitochondrial Membranes

CH₂

Phosphatidic acid is a precursor of phosphatidylglyc-

R₃

erol which, in turn, gives rise to cardiolipin (Figure

CH₂

R₄

CH₂

14-8).

Phosphatidylglycerol

H₂C

O C

R₁

Figure 14-8.

Phosphatidic acid and its derivatives.

The O⁻ shown shaded in phosphatidic acid is substi-

R₂

tuted by the substituents shown to form in (A) 3-phos-

²C

phatidylcholine, (B) 3-phosphatidylethanolamine,

H₂

³C

O C

R₃

and (E) cardiolipin (diphosphatidylglycerol).

Figure 14-7. Triacyl-sn-glycerol.

116

CHAPTER 14

Lysophospholipids Are Intermediates in

¹CH₂

O CH CH

R₁

the Metabolism of Phosphoglycerols

R₂

C O

²CH

These are phosphoacylglycerols containing only one

CH₂

O P O CH₂

CH₂

NH₃

acyl radical, eg, lysophosphatidylcholine

(lysoleci-

thin), important in the metabolism and interconver-

O-

Ethanolamine

sion of phospholipids (Figure 14-9).It is also found in

oxidized lipoproteins and has been implicated in some

Figure 14-10. Plasmalogen.

of their effects in promoting atherosclerosis.

Plasmalogens Occur in Brain & Muscle

sphingolipid of brain and other nervous tissue, found in

These compounds constitute as much as 10% of the

relatively low amounts elsewhere. It contains a number

phospholipids of brain and muscle. Structurally, the

of characteristic C₂₄

fatty acids, eg, cerebronic acid.

plasmalogens resemble phosphatidylethanolamine but

Galactosylceramide (Figure 14-12) can be converted to

possess an ether link on the sn-1 carbon instead of the

sulfogalactosylceramide

(sulfatide), present in high

ester link found in acylglycerols. Typically, the alkyl

amounts in myelin. Glucosylceramide is the predomi-

radical is an unsaturated alcohol (Figure 14-10). In

nant simple glycosphingolipid of extraneural tissues,

some instances, choline, serine, or inositol may be sub-

also occurring in the brain in small amounts. Ganglio-

stituted for ethanolamine.

sides are complex glycosphingolipids derived from glu-

cosylceramide that contain in addition one or more

Sphingomyelins Are Found

molecules of a sialic acid. Neuraminic acid (NeuAc;

in the Nervous System

see Chapter 13) is the principal sialic acid found in

human tissues. Gangliosides are also present in nervous

Sphingomyelins are found in large quantities in brain

tissues in high concentration. They appear to have re-

and nerve tissue. On hydrolysis, the sphingomyelins

ceptor and other functions. The simplest ganglioside

yield a fatty acid, phosphoric acid, choline, and a com-

found in tissues is G_M3, which contains ceramide, one

plex amino alcohol, sphingosine (Figure 14-11). No

molecule of glucose, one molecule of galactose, and one

glycerol is present. The combination of sphingosine

molecule of NeuAc. In the shorthand nomenclature

plus fatty acid is known as ceramide, a structure also

used, G represents ganglioside; M is a monosialo-

found in the glycosphingolipids (see below).

containing species; and the subscript 3 is a number as-

signed on the basis of chromatographic migration. G_M1

GLYCOLIPIDS (GLYCOSPHINGOLIPIDS)

(Figure 14-13), a more complex ganglioside derived

ARE IMPORTANT IN NERVE TISSUES

from G_M3, is of considerable biologic interest, as it is

& IN THE CELL MEMBRANE

known to be the receptor in human intestine for

cholera toxin. Other gangliosides can contain anywhere

Glycolipids are widely distributed in every tissue of the

from one to five molecules of sialic acid, giving rise to

body, particularly in nervous tissue such as brain. They

di-, trisialogangliosides, etc.

occur particularly in the outer leaflet of the plasma

membrane, where they contribute to cell surface car-

bohydrates.

The major glycolipids found in animal tissues are

Ceramide

glycosphingolipids. They contain ceramide and one or

Sphingosine

more sugars. Galactosylceramide is a major glyco-

(CH₂)₁₂

CH CH CH

C R

CH₂

Fatty acid

CH₂

O C

Phosphoric acid

O P

O-

³CH₂

O P O CH₂

CH₂

CH₃

O CH₂

CH₂

N(CH₃)₃

O-

Choline

Figure 14-9. Lysophosphatidylcholine (lysolecithin).

Figure 14-11. A sphingomyelin.

LIPIDS OF PHYSIOLOGIC SIGNIFICANCE

117

Ceramide

Sphingosine

CH₃

(CH

2 )12

CH CH

CH(OH)

(CH₂ )₂₁

CH₃

Fatty acid

2OH

(eg, cerebronic acid)

Galactose

CH₂

OR H

Figure 14-12. Structure of galactosylcer-

amide (galactocerebroside, R = H), and sul-

fogalactosylceramide (a sulfatide, R = SO42−).

STEROIDS PLAY MANY

groups and no carbonyl or carboxyl groups, it is a

sterol, and the name terminates in -ol.

PHYSIOLOGICALLY IMPORTANT ROLES

Cholesterol is probably the best known steroid because

Because of Asymmetry in the Steroid

of its association with atherosclerosis. However, bio-

Molecule, Many Stereoisomers

chemically it is also of significance because it is the pre-

Are Possible

cursor of a large number of equally important steroids

that include the bile acids, adrenocortical hormones,

Each of the six-carbon rings of the steroid nucleus is ca-

sex hormones, D vitamins, cardiac glycosides, sitos-

pable of existing in the three-dimensional conformation

terols of the plant kingdom, and some alkaloids.

either of a “chair” or a “boat” (Figure 14-15). In natu-

All of the steroids have a similar cyclic nucleus re-

rally occurring steroids, virtually all the rings are in the

sembling phenanthrene (rings A, B, and C) to which a

“chair” form, which is the more stable conformation.

cyclopentane ring (D) is attached. The carbon positions

With respect to each other, the rings can be either cis or

on the steroid nucleus are numbered as shown in Figure

trans (Figure 14-16). The junction between the A and

14-14. It is important to realize that in structural for-

B rings can be cis or trans in naturally occurring

mulas of steroids, a simple hexagonal ring denotes a

steroids. That between B and C is trans, as is usually the

completely saturated six-carbon ring with all valences

C/D junction. Bonds attaching substituent groups

satisfied by hydrogen bonds unless shown otherwise; ie,

above the plane of the rings (β bonds) are shown with

it is not a benzene ring. All double bonds are shown as

bold solid lines, whereas those bonds attaching groups

such. Methyl side chains are shown as single bonds un-

below (α bonds) are indicated with broken lines. The A

attached at the farther (methyl) end. These occur typi-

ring of a

5α steroid is always trans to the B ring,

cally at positions 10 and 13 (constituting C atoms 19

whereas it is cis in a 5β steroid. The methyl groups at-

and 18). A side chain at position 17 is usual (as in cho-

tached to C₁₀ and C₁₃ are invariably in the β configura-

lesterol). If the compound has one or more hydroxyl

tion.

Ceramide Glucose Galactose N-Acetylgalactosamine

(Acyl-

sphingo-

NeuAc

Galactose

sine)

Cer Glc Gal GalNAc Gal

NeuAc

Figure 14-13. G_M1 ganglioside, a monosialoganglio-

side, the receptor in human intestine for cholera toxin.

Figure 14-14. The steroid nucleus.

118

CHAPTER 14

chain alcohol dolichol (Figure 14-20), which takes

part in glycoprotein synthesis by transferring carbohy-

drate residues to asparagine residues of the polypeptide

(Chapter 47). Plant-derived isoprenoid compounds in-

“Chair” form

“Boat” form

clude rubber, camphor, the fat-soluble vitamins A, D,

E, and K, and β-carotene (provitamin A).

Figure 14-15. Conformations of stereoisomers of

the steroid nucleus.

LIPID PEROXIDATION IS A SOURCE

OF FREE RADICALS

Cholesterol Is a Significant Constituent

Peroxidation

(auto-oxidation) of lipids exposed to

of Many Tissues

oxygen is responsible not only for deterioration of foods

Cholesterol (Figure 14-17) is widely distributed in all

(rancidity) but also for damage to tissues in vivo,

cells of the body but particularly in nervous tissue. It is

where it may be a cause of cancer, inflammatory dis-

a major constituent of the plasma membrane and of

eases, atherosclerosis, and aging. The deleterious effects

plasma lipoproteins. It is often found as cholesteryl

are considered to be caused by free radicals (ROO^•,

ester, where the hydroxyl group on position 3 is esteri-

RO^•, OH^•) produced during peroxide formation from

fied with a long-chain fatty acid. It occurs in animals

fatty acids containing methylene-interrupted double

but not in plants.

bonds, ie, those found in the naturally occurring

polyunsaturated fatty acids (Figure 14-21). Lipid per-

Ergosterol Is a Precursor of Vitamin D

oxidation is a chain reaction providing a continuous

supply of free radicals that initiate further peroxidation.

Ergosterol occurs in plants and yeast and is important

The whole process can be depicted as follows:

as a precursor of vitamin D (Figure 14-18). When irra-

(1) Initiation:

diated with ultraviolet light, it acquires antirachitic

properties consequent to the opening of ring B.

(n)+

(n–1)+

ROOH+Metal

→ROO•+Metal

Polyprenoids Share the Same Parent

X•+RH → R•+ XH

Compound as Cholesterol

Although not steroids, these compounds are related be-

(2) Propagation:

cause they are synthesized, like cholesterol

(Figure

26-2), from five-carbon isoprene units (Figure 14-19).

They include ubiquinone (Chapter 12), a member of

R•+ O → ROO^•

the respiratory chain in mitochondria, and the long-

ROO•+ RH → ROOH+ R^•, etc

¹⁰ H

Figure 14-16. Generalized steroid nucleus, showing (A) an all-trans configuration be-

tween adjacent rings and (B) a cis configuration between rings A and B.

LIPIDS OF PHYSIOLOGIC SIGNIFICANCE

119

CH₃

CH C CH CH

Figure 14-19. Isoprene unit.

Peroxidation is also catalyzed in vivo by heme com-

Figure 14-17. Cholesterol, 3-hydroxy-5,6-

pounds and by lipoxygenases found in platelets and

cholestene.

leukocytes. Other products of auto-oxidation or en-

zymic oxidation of physiologic significance include

oxysterols (formed from cholesterol) and isoprostanes

(3) Termination:

(prostanoids).

ROO•+ROO•→ROOR+O

AMPHIPATHIC LIPIDS SELF-ORIENT

AT OIL:WATER INTERFACES

ROO•+R•→ROOR

They Form Membranes, Micelles,

R•+ R• → RR

Liposomes, & Emulsions

Since the molecular precursor for the initiation

In general, lipids are insoluble in water since they

process is generally the hydroperoxide product ROOH,

contain a predominance of nonpolar

(hydrocarbon)

lipid peroxidation is a chain reaction with potentially

groups. However, fatty acids, phospholipids, sphin-

devastating effects. To control and reduce lipid peroxi-

golipids, bile salts, and, to a lesser extent, cholesterol

dation, both humans in their activities and nature in-

contain polar groups. Therefore, part of the molecule is

voke the use of antioxidants. Propyl gallate, butylated

hydrophobic, or water-insoluble; and part is hydro-

hydroxyanisole (BHA), and butylated hydroxytoluene

philic, or water-soluble. Such molecules are described

(BHT) are antioxidants used as food additives. Natu-

as amphipathic (Figure 14-22). They become oriented

rally occurring antioxidants include vitamin E (tocoph-

at oil:water interfaces with the polar group in the water

erol), which is lipid-soluble, and urate and vitamin C,

phase and the nonpolar group in the oil phase. A bi-

which are water-soluble. Beta-carotene is an antioxidant

layer of such amphipathic lipids has been regarded as a

at low PO₂. Antioxidants fall into two classes: (1) pre-

basic structure in biologic membranes (Chapter 41).

ventive antioxidants, which reduce the rate of chain ini-

When a critical concentration of these lipids is present

tiation; and (2) chain-breaking antioxidants, which in-

in an aqueous medium, they form micelles. Aggrega-

terfere with chain propagation. Preventive antioxidants

tions of bile salts into micelles and liposomes and the

include catalase and other peroxidases that react with

formation of mixed micelles with the products of fat di-

ROOH and chelators of metal ions such as EDTA

gestion are important in facilitating absorption of lipids

(ethylenediaminetetraacetate) and DTPA (diethylene-

from the intestine. Liposomes may be formed by soni-

triaminepentaacetate). In vivo, the principal chain-

cating an amphipathic lipid in an aqueous medium.

breaking antioxidants are superoxide dismutase, which

They consist of spheres of lipid bilayers that enclose

acts in the aqueous phase to trap superoxide free radi-

part of the aqueous medium. They are of potential clin-

cals (O2•); perhaps urate; and vitamin E, which acts in

ical use—particularly when combined with tissue-

the lipid phase to trap ROO^• radicals (Figure 45-6).

specific antibodies—as carriers of drugs in the circula-

tion, targeted to specific organs, eg, in cancer therapy.

In addition, they are being used for gene transfer into

vascular cells and as carriers for topical and transdermal

CH₂OH

Figure 14-18. Ergosterol.

Figure 14-20. Dolichol—a C₉₅ alcohol.

LIPIDS OF PHYSIOLOGIC SIGNIFICANCE

121

delivery of drugs and cosmetics. Emulsions are much

are amphipathic lipids and have important roles—as

larger particles, formed usually by nonpolar lipids in an

major constituents of membranes and the outer layer

aqueous medium. These are stabilized by emulsifying

of lipoproteins, as surfactant in the lung, as precur-

agents such as amphipathic lipids (eg, lecithin), which

sors of second messengers, and as constituents of ner-

form a surface layer separating the main bulk of the

vous tissue.

nonpolar material from the aqueous phase

(Figure

• Glycolipids are also important constituents of ner-

14-22).

vous tissue such as brain and the outer leaflet of the

cell membrane, where they contribute to the carbo-

SUMMARY

hydrates on the cell surface.

• Cholesterol, an amphipathic lipid, is an important

• Lipids have the common property of being relatively

component of membranes. It is the parent molecule

insoluble in water (hydrophobic) but soluble in non-

from which all other steroids in the body, including

polar solvents. Amphipathic lipids also contain one

major hormones such as the adrenocortical and sex

or more polar groups, making them suitable as con-

hormones, D vitamins, and bile acids, are synthe-

stituents of membranes at lipid:water interfaces.

sized.

• The lipids of major physiologic significance are fatty

• Peroxidation of lipids containing polyunsaturated

acids and their esters, together with cholesterol and

fatty acids leads to generation of free radicals that

other steroids.

may damage tissues and cause disease.

• Long-chain fatty acids may be saturated, monounsat-

urated, or polyunsaturated, according to the number

of double bonds present. Their fluidity decreases

REFERENCES

with chain length and increases according to degree

of unsaturation.

Benzie IFF: Lipid peroxidation: a review of causes, consequences,

• Eicosanoids are formed from 20-carbon polyunsatu-

measurement and dietary influences. Int J Food Sci Nutr

1996;47:233.

rated fatty acids and make up an important group of

Christie WW: Lipid Analysis, 2nd ed. Pergamon Press, 1982.

physiologically and pharmacologically active com-

pounds known as prostaglandins, thromboxanes,

Cullis PR, Fenske DB, Hope MJ: Physical properties and func-

tional roles of lipids in membranes. In: Biochemistry of Lipids,

leukotrienes, and lipoxins.

Lipoproteins and Membranes. Vance DE, Vance JE (editors).

• The esters of glycerol are quantitatively the most sig-

Elsevier, 1996.

nificant lipids, represented by triacylglycerol (“fat”),

Gunstone FD, Harwood JL, Padley FB: The Lipid Handbook.

a major constituent of lipoproteins and the storage

Chapman & Hall, 1986.

form of lipid in adipose tissue. Phosphoacylglycerols

Gurr MI, Harwood JL: Lipid Biochemistry: An Introduction, 4th ed.

Chapman & Hall, 1991.

Overview of Metabolism

Peter A. Mayes, PhD, DSc, & David A. Bender, PhD

BIOMEDICAL IMPORTANCE

Carbohydrate Metabolism Is Centered

on the Provision & Fate of Glucose

The fate of dietary components after digestion and ab-

(Figure 15-2)

sorption constitutes metabolism—the metabolic path-

ways taken by individual molecules, their interrelation-

Glucose is metabolized to pyruvate by the pathway of

ships, and the mechanisms that regulate the flow of

glycolysis, which can occur anaerobically (in the ab-

metabolites through the pathways. Metabolic pathways

sence of oxygen), when the end product is lactate. Aero-

fall into three categories: (1) Anabolic pathways are

bic tissues metabolize pyruvate to acetyl-CoA, which

those involved in the synthesis of compounds. Protein

can enter the citric acid cycle for complete oxidation

synthesis is such a pathway, as is the synthesis of fuel

to CO₂ and H₂O, linked to the formation of ATP

reserves of triacylglycerol and glycogen. Anabolic path-

in the process of oxidative phosphorylation (Figure

ways are endergonic. (2) Catabolic pathways are in-

16-2). Glucose is the major fuel of most tissues.

volved in the breakdown of larger molecules, com-

monly involving oxidative reactions; they are exergonic,

producing reducing equivalents and, mainly via the res-

piratory chain, ATP. (3) Amphibolic pathways occur

Carbohydrate

Protein

Fat

at the “crossroads” of metabolism, acting as links be-

tween the anabolic and catabolic pathways, eg, the cit-

ric acid cycle.

Digestion and absorption

A knowledge of normal metabolism is essential for

an understanding of abnormalities underlying disease.

Normal metabolism includes adaptation to periods of

Simple sugars

Fatty acids

Amino acids

starvation, exercise, pregnancy, and lactation. Abnor-

(mainly glucose)

+ glycerol

mal metabolism may result from nutritional deficiency,

enzyme deficiency, abnormal secretion of hormones, or

the actions of drugs and toxins. An important example

Catabolism

of a metabolic disease is diabetes mellitus.

Acetyl-CoA

PATHWAYS THAT PROCESS THE MAJOR

PRODUCTS OF DIGESTION

The nature of the diet sets the basic pattern of metabo-

Citric

lism. There is a need to process the products of diges-

acid

ATP

cycle

tion of dietary carbohydrate, lipid, and protein. These

are mainly glucose, fatty acids and glycerol, and amino

acids, respectively. In ruminants (and to a lesser extent

in other herbivores), dietary cellulose is fermented by

2CO₂

symbiotic microorganisms to short-chain fatty acids

(acetic, propionic, butyric), and metabolism in these

Figure 15-1.

Outline of the pathways for the catab-

animals is adapted to use these fatty acids as major sub-

olism of dietary carbohydrate, protein, and fat. All the

strates. All the products of digestion are metabolized to

pathways lead to the production of acetyl-CoA, which is

a common product, acetyl-CoA, which is then oxi-

oxidized in the citric acid cycle, ultimately yielding ATP

dized by the citric acid cycle (Figure 15-1).

in the process of oxidative phosphorylation.

122

OVERVIEW OF METABOLISM

123

Diet

cursor of fatty acids and cholesterol (and hence of all

steroids synthesized in the body). Gluconeogenesis is

the process of forming glucose from noncarbohydrate

Glucose

Glycogen

precursors, eg, lactate, amino acids, and glycerol.

Glucose

Lipid Metabolism Is Concerned Mainly

3CO₂

phosphates

With Fatty Acids & Cholesterol

(Figure 15-3)

Pentose phosphate

pathway

The source of long-chain fatty acids is either dietary

lipid or de novo synthesis from acetyl-CoA derived from

Triose

carbohydrate. Fatty acids may be oxidized to acetyl-

Ribose

RNA

phosphates

CoA (β-oxidation) or esterified with glycerol, forming

phosphate

DNA

triacylglycerol (fat) as the body’s main fuel reserve.

Acetyl-CoA formed by β-oxidation may undergo

several fates:

(1) As with acetyl-CoA arising from glycolysis, it is

Pyruvate

Lactate

oxidized to CO₂ + H₂O via the citric acid cycle.

Acylglycerols

(fat)

CO₂

Triacylglycerol

Steroids

Acetyl-CoA

Fatty

(fat)

acids

Cholesterol

Fatty acids

Diet

Citric

acid

cycle

Cholesterol

Carbohydrate

2CO₂

Acetyl-CoA

Amino acids

Cholesterologenesis

Figure 15-2. Overview of carbohydrate metabolism

Ketogenesis

showing the major pathways and end products. Gluco-

neogenesis is not shown.

Ketone

bodies

Citric

Glucose and its metabolites also take part in other

acid

cycle

processes. Examples:

(1) Conversion to the storage

polymer glycogen in skeletal muscle and liver. (2) The

pentose phosphate pathway, an alternative to part of

the pathway of glycolysis, is a source of reducing equiv-

alents (NADPH) for biosynthesis and the source of ri-

2CO₂

bose for nucleotide and nucleic acid synthesis.

(3) Triose phosphate gives rise to the glycerol moiety

Figure 15-3. Overview of fatty acid metabolism

of triacylglycerols.

(4) Pyruvate and intermediates of

showing the major pathways and end products. Ketone

the citric acid cycle provide the carbon skeletons for

bodies comprise the substances acetoacetate, 3-hy-

the synthesis of amino acids; and acetyl-CoA, the pre-

droxybutyrate, and acetone.

124

CHAPTER 15

(2) It is the precursor for synthesis of cholesterol and

METABOLIC PATHWAYS MAY BE

other steroids.

STUDIED AT DIFFERENT LEVELS

(3) In the liver, it forms ketone bodies (acetone, ace-

OF ORGANIZATION

toacetate, and 3-hydroxybutyrate) that are impor-

tant fuels in prolonged starvation.

In addition to studies in the whole organism, the loca-

tion and integration of metabolic pathways is revealed

by studies at several levels of organization. At the tissue

and organ level, the nature of the substrates entering

Much of Amino Acid Metabolism

and metabolites leaving tissues and organs is defined. At

Involves Transamination

the subcellular level, each cell organelle (eg, the mito-

(Figure 15-4)

chondrion) or compartment (eg, the cytosol) has spe-

cific roles that form part of a subcellular pattern of

The amino acids are required for protein synthesis.

metabolic pathways.

Some must be supplied in the diet (the essential amino

acids) since they cannot be synthesized in the body.

The remainder are nonessential amino acids that are

At the Tissue and Organ Level, the Blood

supplied in the diet but can be formed from metabolic

Circulation Integrates Metabolism

intermediates by transamination, using the amino ni-

trogen from other amino acids. After deamination,

Amino acids resulting from the digestion of dietary

amino nitrogen is excreted as urea, and the carbon

protein and glucose resulting from the digestion of car-

skeletons that remain after transamination (1) are oxi-

bohydrate are absorbed and directed to the liver via the

dized to CO₂ via the citric acid cycle, (2) form glucose

hepatic portal vein. The liver has the role of regulating

(gluconeogenesis), or (3) form ketone bodies.

the blood concentration of most water-soluble metabo-

Several amino acids are also the precursors of other

lites

(Figure

15-5). In the case of glucose, this is

compounds, eg, purines, pyrimidines, hormones such

achieved by taking up glucose in excess of immediate

as epinephrine and thyroxine, and neurotransmitters.

requirements and converting it to glycogen (glycogene-

Diet protein

Nonprotein

Tissue protein

Amino acids

nitrogen derivatives

T R A N S A M I N A T I O N

Carbohydrate

Ketone bodies

(glucose)

Amino nitrogen in

Acetyl-CoA

glutamate

DEAMINATION

Citric

NH₃

acid

cycle

Urea

2CO₂

Figure 15-4. Overview of amino acid metabolism showing the major pathways and end products.

OVERVIEW OF METABOLISM

125

Plasma proteins

LIVER

Urea

Protein

Amino acids

CO₂

Glucose

Amino

acids

Glycogen

Protein

Lactate

Urea

Amino

acids

Alanine,

etc

ERYTHROCYTES

Glucose

CO₂

phosphate

Glucose

KIDNEY

Urine

Glycogen

Diet

Carbohydrate

Glucose

BLOOD PLASMA

Protein

MUSCLE

Amino acids

SMALL INTESTINE

Figure 15-5. Transport and fate of major carbohydrate and amino acid substrates and metabolites. Note that

there is little free glucose in muscle, since it is rapidly phosphorylated upon entry.

sis) or to fat (lipogenesis). Between meals, the liver

mucosa. Here they are packaged with protein and se-

acts to maintain the blood glucose concentration from

creted into the lymphatic system and thence into the

glycogen (glycogenolysis) and, together with the kid-

blood stream as chylomicrons, the largest of the plasma

ney, by converting noncarbohydrate metabolites such

lipoproteins. Chylomicrons also contain other lipid-

as lactate, glycerol, and amino acids to glucose (gluco-

soluble nutrients, eg, vitamins. Unlike glucose and

neogenesis). Maintenance of an adequate concentra-

amino acids, chylomicron triacylglycerol is not taken up

tion of blood glucose is vital for those tissues in which it

directly by the liver. It is first metabolized by tissues that

is the major fuel (the brain) or the only fuel (the eryth-

have lipoprotein lipase, which hydrolyzes the triacyl-

rocytes). The liver also synthesizes the major plasma

glycerol, releasing fatty acids that are incorporated into

proteins (eg, albumin) and deaminates amino acids

tissue lipids or oxidized as fuel. The other major source

that are in excess of requirements, forming urea, which

of long-chain fatty acid is synthesis (lipogenesis) from

is transported to the kidney and excreted.

carbohydrate, mainly in adipose tissue and the liver.

Skeletal muscle utilizes glucose as a fuel, forming

Adipose tissue triacylglycerol is the main fuel reserve

both lactate and CO₂. It stores glycogen as a fuel for its

of the body. On hydrolysis (lipolysis) free fatty acids are

use in muscular contraction and synthesizes muscle

released into the circulation. These are taken up by most

protein from plasma amino acids. Muscle accounts for

tissues (but not brain or erythrocytes) and esterified to

approximately 50% of body mass and consequently

acylglycerols or oxidized as a fuel. In the liver, triacyl-

represents a considerable store of protein that can be

glycerol arising from lipogenesis, free fatty acids, and

drawn upon to supply amino acids for gluconeogenesis

chylomicron remnants (see Figures 25-3 and 25-4) is se-

in starvation.

creted into the circulation as very low density lipopro-

Lipids in the diet (Figure 15-6) are mainly triacyl-

tein (VLDL). This triacylglycerol undergoes a fate simi-

glycerol and are hydrolyzed to monoacylglycerols and

lar to that of chylomicrons. Partial oxidation of fatty

fatty acids in the gut, then reesterified in the intestinal

acids in the liver leads to ketone body production (keto-

126

CHAPTER 15

FFA

CO₂

Glucose

Fatty

acids

Ketone

bodies

BLOOD

PLASMA

CO₂

LIVER

LPL

Fatty

acids

Lipoprotein

Fatty

Glucose

acids

LPL

MUSCLE

Diet

MG +

ADIPOSE

fatty acids

TISSUE

SMALL INTESTINE

Figure 15-6. Transport and fate of major lipid substrates and metabolites. (FFA, free fatty acids; LPL, lipopro-

tein lipase; MG, monoacylglycerol; TG, triacylglycerol; VLDL, very low density lipoprotein.)

genesis). Ketone bodies are transported to extrahepatic

Glycolysis, the pentose phosphate pathway, and fatty

tissues, where they act as a fuel source in starvation.

acid synthesis are all found in the cytosol. In gluconeo-

genesis, substrates such as lactate and pyruvate, which

are formed in the cytosol, enter the mitochondrion to

At the Subcellular Level, Glycolysis Occurs

yield oxaloacetate before formation of glucose.

in the Cytosol & the Citric Acid Cycle

The membranes of the endoplasmic reticulum con-

in the Mitochondria

tain the enzyme system for acylglycerol synthesis, and

Compartmentation of pathways in separate subcellular

the ribosomes are responsible for protein synthesis.

compartments or organelles permits integration and

regulation of metabolism. Not all pathways are of equal

THE FLUX OF METABOLITES IN

importance in all cells. Figure 15-7 depicts the subcel-

METABOLIC PATHWAYS MUST BE

lular compartmentation of metabolic pathways in a he-

REGULATED IN A CONCERTED MANNER

patic parenchymal cell.

The central role of the mitochondrion is immedi-

Regulation of the overall flux through a pathway is im-

ately apparent, since it acts as the focus of carbohydrate,

portant to ensure an appropriate supply, when re-

lipid, and amino acid metabolism. It contains the en-

quired, of the products of that pathway. Regulation is

zymes of the citric acid cycle, β-oxidation of fatty acids,

achieved by control of one or more key reactions in

and ketogenesis, as well as the respiratory chain and

the pathway, catalyzed by “regulatory enzymes.” The

ATP synthase.

physicochemical factors that control the rate of an

128

CHAPTER 15

enzyme-catalyzed reaction, eg, substrate concentration,

In vivo, under “steady-state” conditions, there is a net

are of primary importance in the control of the overall

flux from left to right because there is a continuous sup-

rate of a metabolic pathway (Chapter 9).

ply of A and removal of D. In practice, there are invari-

ably one or more nonequilibrium reactions in a meta-

“Nonequilibrium” Reactions Are

bolic pathway, where the reactants are present in

Potential Control Points

concentrations that are far from equilibrium. In at-

tempting to reach equilibrium, large losses of free en-

In a reaction at equilibrium, the forward and reverse re-

ergy occur as heat, making this type of reaction essen-

actions occur at equal rates, and there is therefore no

tially irreversible, eg,

net flux in either direction:

Heat

A↔B↔C↔D

A↔B→C↔D

Inactive

Enz₁

Ca²⁺/calmodulin

cAMP

Cell

membrane

Active

Enz₁

Enz₂

Negative allosteric

Positive allosteric

feed-back

feed-forward

inhibition

activation

–

Ribosomal synthesis

of new enzyme protein

Nuclear production

of mRNA

Induction

Repression

Figure 15-8. Mechanisms of control of an enzyme-catalyzed reaction. Circled

numbers indicate possible sites of action of hormones. 1 , Alteration of mem-

brane permeability; 2 , conversion of an inactive to an active enzyme, usually in-

volving phosphorylation/dephosphorylation reactions; 3 , alteration of the rate

of translation of mRNA at the ribosomal level; 4 , induction of new mRNA forma-

tion; and 5 , repression of mRNA formation. 1 and 2 are rapid, whereas 3 - 5

are slower ways of regulating enzyme activity.

OVERVIEW OF METABOLISM

129

Such a pathway has both flow and direction. The

activity of existing enzyme molecules, or slowly, by al-

enzymes catalyzing nonequilibrium reactions are usu-

tering the rate of enzyme synthesis.

ally present in low concentrations and are subject to a

variety of regulatory mechanisms. However, many of

SUMMARY

the reactions in metabolic pathways cannot be classified

as equilibrium or nonequilibrium but fall somewhere

•

The products of digestion provide the tissues with

between the two extremes.

the building blocks for the biosynthesis of complex

molecules and also with the fuel to power the living

The Flux-Generating Reaction

processes.

Is the First Reaction in a Pathway

•

Nearly all products of digestion of carbohydrate, fat,

That Is Saturated With Substrate

and protein are metabolized to a common metabo-

lite, acetyl-CoA, before final oxidation to CO₂ in the

It may be identified as a nonequilibrium reaction in

citric acid cycle.

which the K_m of the enzyme is considerably lower than

•

Acetyl-CoA is also used as the precursor for biosyn-

the normal substrate concentration. The first reaction

in glycolysis, catalyzed by hexokinase (Figure 17-2), is

thesis of long-chain fatty acids; steroids, including

cholesterol; and ketone bodies.

such a flux-generating step because its K_m for glucose of

0.05 mmol/L is well below the normal blood glucose

•

Glucose provides carbon skeletons for the glycerol

concentration of 5 mmol/L.

moiety of fat and of several nonessential amino acids.

•

Water-soluble products of digestion are transported

ALLOSTERIC & HORMONAL

directly to the liver via the hepatic portal vein. The

liver regulates the blood concentrations of glucose

MECHANISMS ARE IMPORTANT

and amino acids.

IN THE METABOLIC CONTROL OF

•

Pathways are compartmentalized within the cell.

ENZYME-CATALYZED REACTIONS

Glycolysis, glycogenesis, glycogenolysis, the pentose

A hypothetical metabolic pathway is shown in Figure

phosphate pathway, and lipogenesis occur in the cy-

15-8, in which reactions A ↔ B and C ↔ D are equi-

tosol. The mitochondrion contains the enzymes of

librium reactions and B → C is a nonequilibrium reac-

the citric acid cycle, β-oxidation of fatty acids, and of

tion. The flux through such a pathway can be regulated

oxidative phosphorylation. The endoplasmic reticu-

by the availability of substrate A. This depends on its

lum also contains the enzymes for many other

supply from the blood, which in turn depends on either

processes, including protein synthesis, glycerolipid

food intake or key reactions that maintain and release

formation, and drug metabolism.

substrates from tissue reserves to the blood, eg, the

•

Metabolic pathways are regulated by rapid mecha-

glycogen phosphorylase in liver (Figure 18-1) and hor-

nisms affecting the activity of existing enzymes, eg,

mone-sensitive lipase in adipose tissue (Figure 25-7).

allosteric and covalent modification

(often in re-

The flux also depends on the transport of substrate A

sponse to hormone action); and slow mechanisms af-

across the cell membrane. Flux is also determined by

fecting the synthesis of enzymes.

the removal of the end product D and the availability

of cosubstrate or cofactors represented by X and Y. En-

zymes catalyzing nonequilibrium reactions are often al-

REFERENCES

losteric proteins subject to the rapid actions of “feed-

Cohen P: Control of Enzyme Activity, 2nd ed. Chapman & Hall,

back” or “feed-forward” control by allosteric modifiers

1983.

in immediate response to the needs of the cell (Chap-

Fell D: Understanding the Control of Metabolism. Portland Press,

ter 9). Frequently, the product of a biosynthetic path-

1997.

way will inhibit the enzyme catalyzing the first reaction

Frayn KN: Metabolic Regulation—A Human Perspective. Portland

in the pathway. Other control mechanisms depend on

Press, 1996.

the action of hormones responding to the needs of the

Newsholme EA, Crabtree B: Flux-generating and regulatory steps

body as a whole; they may act rapidly, by altering the

in metabolic control. Trends Biochem Sci 1981;6:53.

The Citric Acid Cycle:

The Catabolism of Acetyl-CoA

Peter A. Mayes, PhD, DSc, & David A. Bender, PhD

BIOMEDICAL IMPORTANCE

cated in the mitochondrial matrix, either free or at-

tached to the inner mitochondrial membrane, where

The citric acid cycle (Krebs cycle, tricarboxylic acid

the enzymes of the respiratory chain are also found.

cycle) is a series of reactions in mitochondria that oxi-

dize acetyl residues (as acetyl-CoA) and reduce coen-

REACTIONS OF THE CITRIC ACID

zymes that upon reoxidation are linked to the forma-

tion of ATP.

CYCLE LIBERATE REDUCING

The citric acid cycle is the final common pathway

EQUIVALENTS & CO₂

for the aerobic oxidation of carbohydrate, lipid, and

(Figure 16-3)*

protein because glucose, fatty acids, and most amino

The initial reaction between acetyl-CoA and oxaloac-

acids are metabolized to acetyl-CoA or intermediates of

the cycle. It also has a central role in gluconeogenesis,

etate to form citrate is catalyzed by citrate synthase

which forms a carbon-carbon bond between the methyl

lipogenesis, and interconversion of amino acids. Many

of these processes occur in most tissues, but the liver is

carbon of acetyl-CoA and the carbonyl carbon of ox-

aloacetate. The thioester bond of the resultant citryl-

the only tissue in which all occur to a significant extent.

The repercussions are therefore profound when, for ex-

CoA is hydrolyzed, releasing citrate and CoASH—an

exergonic reaction.

ample, large numbers of hepatic cells are damaged as in

acute hepatitis or replaced by connective tissue (as in

Citrate is isomerized to isocitrate by the enzyme

aconitase (aconitate hydratase); the reaction occurs in

cirrhosis). Very few, if any, genetic abnormalities of

citric acid cycle enzymes have been reported; such ab-

two steps: dehydration to cis-aconitate, some of which

remains bound to the enzyme; and rehydration to isoci-

normalities would be incompatible with life or normal

development.

trate. Although citrate is a symmetric molecule, aconi-

tase reacts with citrate asymmetrically, so that the two

carbon atoms that are lost in subsequent reactions of

THE CITRIC ACID CYCLE PROVIDES

the cycle are not those that were added from acetyl-

SUBSTRATE FOR THE

CoA. This asymmetric behavior is due to channeling—

transfer of the product of citrate synthase directly onto

RESPIRATORY CHAIN

the active site of aconitase without entering free solu-

The cycle starts with reaction between the acetyl moiety

tion. This provides integration of citric acid cycle activ-

of acetyl-CoA and the four-carbon dicarboxylic acid ox-

ity and the provision of citrate in the cytosol as a source

aloacetate, forming a six-carbon tricarboxylic acid, cit-

of acetyl-CoA for fatty acid synthesis. The poison fluo-

rate. In the subsequent reactions, two molecules of CO₂

roacetate is toxic because fluoroacetyl-CoA condenses

are released and oxaloacetate is regenerated

(Figure

with oxaloacetate to form fluorocitrate, which inhibits

16-1). Only a small quantity of oxaloacetate is needed

aconitase, causing citrate to accumulate.

for the oxidation of a large quantity of acetyl-CoA; ox-

Isocitrate undergoes dehydrogenation catalyzed by

aloacetate may be considered to play a catalytic role.

isocitrate dehydrogenase to form, initially, oxalosucci-

The citric acid cycle is an integral part of the process

nate, which remains enzyme-bound and undergoes de-

by which much of the free energy liberated during the

carboxylation to α-ketoglutarate. The decarboxylation

oxidation of fuels is made available. During oxidation

of acetyl-CoA, coenzymes are reduced and subsequently

*From Circular No. 200 of the Committee of Editors of Biochemi-

reoxidized in the respiratory chain, linked to the forma-

cal Journals Recommendations (1975): “According to standard

tion of ATP (oxidative phosphorylation; see Figure

biochemical convention, the ending ate in, eg, palmitate, denotes

16-2 and also Chapter 12). This process is aerobic, re-

any mixture of free acid and the ionized form(s) (according to pH)

quiring oxygen as the final oxidant of the reduced

in which the cations are not specified.” The same convention is

coenzymes. The enzymes of the citric acid cycle are lo-

adopted in this text for all carboxylic acids.

130

THE CITRIC ACID CYCLE: THE CATABOLISM OF ACETYL-CoA

131

Acetyl-CoA

Carbohydrate

Protein

Lipids

(C₂)

CoA

Acetyl-CoA

(C₂)

H₂O

Citrate

Oxaloacetate

Citrate

Oxaloacetate

(C₆)

(C₄)

(C₆)

(C₄)

H₂O

Citric acid

cycle

Cis-aconitate

H₂O

Malate

(C₄)

Isocitrate

H₂O

CO₂

(C₆)

CO₂

Fumarate

Figure 16-1. Citric acid cycle, illustrating the cat-

(C4)

α-Ketoglutarate

alytic role of oxaloacetate.

(C₅)

NAD

CO₂

Succinate

Succinyl-CoA

(C₄)

requires Mg2+ or Mn2+ ions. There are three isoenzymes

(C₄)

of isocitrate dehydrogenase. One, which uses NAD⁺, is

H₂O

found only in mitochondria. The other two use NADP

and are found in mitochondria and the cytosol. Respi-

ratory chain-linked oxidation of isocitrate proceeds al-

most completely through the NAD⁺-dependent en-

zyme.

Oxidative

Cyt b

α-Ketoglutarate undergoes oxidative decarboxyla-

phosphorylation

tion in a reaction catalyzed by a multi-enzyme complex

Cyt c

similar to that involved in the oxidative decarboxylation

of pyruvate (Figure 17-5). The

-ketoglutarate dehy-

drogenase complex requires the same cofactors as the

Cyt aa₃

pyruvate dehydrogenase complex—thiamin diphos-

1/2

O₂

phate, lipoate, NAD⁺, FAD, and CoA—and results in

the formation of succinyl-CoA. The equilibrium of this

–

Anaerobiosis

reaction is so much in favor of succinyl-CoA formation

(hypoxia, anoxia)

that it must be considered physiologically unidirec-

H₂O

Respiratory chain

tional. As in the case of pyruvate oxidation (Chapter

Flavoprotein

17), arsenite inhibits the reaction, causing the substrate,

-ketoglutarate, to accumulate.

Cyt

Cytochrome

Succinyl-CoA is converted to succinate by the en-

zyme succinate thiokinase

(succinyl-CoA synthe-

High-energy phosphate

tase). This is the only example in the citric acid cycle of

Figure 16-2. The citric acid cycle: the major catabo-

substrate-level phosphorylation. Tissues in which glu-

lic pathway for acetyl-CoA in aerobic organisms. Acetyl-

coneogenesis occurs (the liver and kidney) contain two

CoA, the product of carbohydrate, protein, and lipid ca-

isoenzymes of succinate thiokinase, one specific for

tabolism, is taken into the cycle, together with H₂O, and

GDP and the other for ADP. The GTP formed is

oxidized to CO₂ with the release of reducing equivalents

used for the decarboxylation of oxaloacetate to phos-

(2H). Subsequent oxidation of 2H in the respiratory

phoenolpyruvate in gluconeogenesis and provides a

chain leads to coupled phosphorylation of ADP to ATP.

regulatory link between citric acid cycle activity and

the withdrawal of oxaloacetate for gluconeogenesis.

For one turn of the cycle, 11~ P are generated via ox-

Nongluconeogenic tissues have only the isoenzyme that

idative phosphorylation and one ~ P arises at substrate

uses ADP.

level from the conversion of succinyl-CoA to succinate.

CH CO

CoA

Acetyl-CoA

CITRATE SYNTHASE

MALATE

DEHYDROGENASE

C COO^-

CoA SH

CH₂

*OO-

CH₂

COO^-

NADH + H⁺

Oxaloacetate

H₂O

COO^-

NAD⁺

CH₂

COO^-

CH₂

Citrate

L-Malate

ACONITASE

FUMARASE

Fe2+

H₂O

Fluoroacetate

CH₂

H₂O

COO^-

CH COO^-

OOC*

Cis -aconitate

Fumarate

FADH₂

SUCCINATE

ACONITASE

Fe2+

DEHYDROGENASE

FAD

Malonate

CH₂

*OO-

CH₂

COO^-

CH₂

HO CH COO^-

Succinate

Isocitrate

ATP

NAD⁺

Mg2+

CoA SH

NADH + H⁺

ADP + P

ISOCITRATE

SUCCINATE

DEHYDROGENASE

THIOKINASE

COO^-

CH₂

COO

CH₂

Arsenite

NADH + H

CH COO^-

S CoA

CO₂

Succinyl-CoA

NAD

–

COO^-

CH₂

COO

α-KETOGLUTARATE

Oxalosuccinate

DEHYDROGENASE COMPLEX

CH₂

ISOCITRATE

CoA SH

Mn2+

DEHYDROGENASE

COO

CO₂

α-Ketoglutarate

Figure 16-3. Reactions of the citric acid (Krebs) cycle. Oxidation of NADH and FADH₂ in the respiratory chain

leads to the generation of ATP via oxidative phosphorylation. In order to follow the passage of acetyl-CoA through

the cycle, the two carbon atoms of the acetyl radical are shown labeled on the carboxyl carbon (designated by as-

terisk) and on the methyl carbon (using the designation ^•). Although two carbon atoms are lost as CO₂ in one revo-

lution of the cycle, these atoms are not derived from the acetyl-CoA that has immediately entered the cycle but

from that portion of the citrate molecule that was derived from oxaloacetate. However, on completion of a single

turn of the cycle, the oxaloacetate that is regenerated is now labeled, which leads to labeled CO₂ being evolved

during the second turn of the cycle. Because succinate is a symmetric compound and because succinate dehydro-

genase does not differentiate between its two carboxyl groups, “randomization” of label occurs at this step such

that all four carbon atoms of oxaloacetate appear to be labeled after one turn of the cycle. During gluconeogene-

sis, some of the label in oxaloacetate is incorporated into glucose and glycogen (Figure 19-1). For a discussion of

the stereochemical aspects of the citric acid cycle, see Greville (1968). The sites of inhibition ( − ) by fluoroacetate,

malonate, and arsenite are indicated.

132

THE CITRIC ACID CYCLE: THE CATABOLISM OF ACETYL-CoA

133

When ketone bodies are being metabolized in extra-

the coenzyme for three dehydrogenases in the cycle—

hepatic tissues there is an alternative reaction catalyzed

isocitrate dehydrogenase, α-ketoglutarate dehydrogen-

by succinyl-CoA-acetoacetate-CoA transferase (thio-

ase, and malate dehydrogenase; (3) thiamin (vitamin

phorase)—involving transfer of CoA from succinyl-

B₁), as thiamin diphosphate, the coenzyme for decar-

CoA to acetoacetate, forming acetoacetyl-CoA (Chap-

boxylation in the α-ketoglutarate dehydrogenase reac-

ter 22).

tion; and (4) pantothenic acid, as part of coenzyme A,

The onward metabolism of succinate, leading to the

the cofactor attached to “active” carboxylic acid resi-

regeneration of oxaloacetate, is the same sequence of

dues such as acetyl-CoA and succinyl-CoA.

chemical reactions as occurs in the β-oxidation of fatty

acids: dehydrogenation to form a carbon-carbon double

bond, addition of water to form a hydroxyl group, and

THE CITRIC ACID CYCLE PLAYS A

a further dehydrogenation to yield the oxo- group of

PIVOTAL ROLE IN METABOLISM

oxaloacetate.

The citric acid cycle is not only a pathway for oxidation

The first dehydrogenation reaction, forming fu-

of two-carbon units—it is also a major pathway for in-

marate, is catalyzed by succinate dehydrogenase, which

terconversion of metabolites arising from transamina-

is bound to the inner surface of the inner mitochondrial

tion and deamination of amino acids. It also provides

membrane. The enzyme contains FAD and iron-sulfur

the substrates for amino acid synthesis by transamina-

(Fe:S) protein and directly reduces ubiquinone in the

tion, as well as for gluconeogenesis and fatty acid syn-

respiratory chain. Fumarase (fumarate hydratase) cat-

thesis. Because it functions in both oxidative and syn-

alyzes the addition of water across the double bond of

thetic processes, it is amphibolic (Figure 16-4).

fumarate, yielding malate. Malate is converted to ox-

aloacetate by malate dehydrogenase, a reaction requir-

ing NAD⁺. Although the equilibrium of this reaction

The Citric Acid Cycle Takes Part in

strongly favors malate, the net flux is toward the direc-

Gluconeogenesis, Transamination,

tion of oxaloacetate because of the continual removal of

& Deamination

oxaloacetate (either to form citrate, as a substrate for

gluconeogenesis, or to undergo transamination to as-

All the intermediates of the cycle are potentially gluco-

partate) and also because of the continual reoxidation

genic, since they can give rise to oxaloacetate and thus

of NADH.

net production of glucose (in the liver and kidney, the

organs that carry out gluconeogenesis; see Chapter 19).

The key enzyme that catalyzes net transfer out of the

cycle into gluconeogenesis is phosphoenolpyruvate

TWELVE ATP ARE FORMED PER TURN

carboxykinase, which decarboxylates oxaloacetate to

OF THE CITRIC ACID CYCLE

phosphoenolpyruvate, with GTP acting as the donor

As a result of oxidations catalyzed by the dehydrogen-

phosphate (Figure 16-4).

ases of the citric acid cycle, three molecules of NADH

Net transfer into the cycle occurs as a result of sev-

and one of FADH₂ are produced for each molecule of

eral different reactions. Among the most important of

acetyl-CoA catabolized in one turn of the cycle. These

such anaplerotic reactions is the formation of oxaloac-

reducing equivalents are transferred to the respiratory

etate by the carboxylation of pyruvate, catalyzed by

chain (Figure 16-2), where reoxidation of each NADH

pyruvate carboxylase. This reaction is important in

results in formation of

3 ATP and reoxidation of

maintaining an adequate concentration of oxaloacetate

FADH₂ in formation of 2 ATP. In addition, 1 ATP

for the condensation reaction with acetyl-CoA. If acetyl-

(or GTP) is formed by substrate-level phosphorylation

CoA accumulates, it acts both as an allosteric activator

catalyzed by succinate thiokinase.

of pyruvate carboxylase and as an inhibitor of pyruvate

dehydrogenase, thereby ensuring a supply of oxaloac-

etate. Lactate, an important substrate for gluconeogene-

sis, enters the cycle via oxidation to pyruvate and then

VITAMINS PLAY KEY ROLES

carboxylation to oxaloacetate.

IN THE CITRIC ACID CYCLE

Aminotransferase

(transaminase) reactions form

Four of the B vitamins are essential in the citric acid

pyruvate from alanine, oxaloacetate from aspartate, and

cycle and therefore in energy-yielding metabolism: (1)

α-ketoglutarate from glutamate. Because these reac-

riboflavin, in the form of flavin adenine dinucleotide

tions are reversible, the cycle also serves as a source of

(FAD), a cofactor in the α-ketoglutarate dehydrogenase

carbon skeletons for the synthesis of these amino acids.

complex and in succinate dehydrogenase; (2) niacin, in

Other amino acids contribute to gluconeogenesis be-

the form of nicotinamide adenine dinucleotide (NAD),

cause their carbon skeletons give rise to citric acid cycle

134

CHAPTER 16

Hydroxyproline

Lactate

Serine

Cysteine

Threonine

Glycine

TRANSAMINASE

Tryptophan

Alanine

Pyruvate

Acetyl-CoA

PYRUVATE

CARBOXYLASE

PHOSPHOENOLPYRUVATE

CARBOXYKINASE

Phosphoenol-

Glucose

Oxaloacetate

pyruvate

TRANSAMINASE

Tyrosine

Fumarate

Phenylalanine

Aspartate

Citrate

Isoleucine

Methionine

Succinyl-CoA

Valine

CO₂

α-Ketoglutarate

Propionate

CO₂

TRANSAMINASE

Histidine

Proline

Glutamate

Glutamine

Arginine

Figure 16-4. Involvement of the citric acid cycle in transamination and gluconeo-

genesis. The bold arrows indicate the main pathway of gluconeogenesis.

intermediates. Alanine, cysteine, glycine, hydroxypro-

Pyruvate dehydrogenase is a mitochondrial enzyme,

line, serine, threonine, and tryptophan yield pyruvate;

and fatty acid synthesis is a cytosolic pathway, but the

arginine, histidine, glutamine, and proline yield α-ke-

mitochondrial membrane is impermeable to acetyl-

toglutarate; isoleucine, methionine, and valine yield

CoA. Acetyl-CoA is made available in the cytosol from

succinyl-CoA; and tyrosine and phenylalanine yield fu-

citrate synthesized in the mitochondrion, transported

marate (Figure 16-4).

into the cytosol and cleaved in a reaction catalyzed by

In ruminants, whose main metabolic fuel is short-

ATP-citrate lyase.

chain fatty acids formed by bacterial fermentation, the

conversion of propionate, the major glucogenic product

of rumen fermentation, to succinyl-CoA via the

Regulation of the Citric Acid Cycle

methylmalonyl-CoA pathway (Figure 19-2) is espe-

Depends Primarily on a Supply

cially important.

of Oxidized Cofactors

In most tissues, where the primary role of the citric acid

The Citric Acid Cycle Takes Part

cycle is in energy-yielding metabolism, respiratory

in Fatty Acid Synthesis

control via the respiratory chain and oxidative phos-

(Figure 16-5)

phorylation regulates citric acid cycle activity (Chap-

Acetyl-CoA, formed from pyruvate by the action of

ter 14). Thus, activity is immediately dependent on the

pyruvate dehydrogenase, is the major building block for

supply of NAD⁺, which in turn, because of the tight

long-chain fatty acid synthesis in nonruminants. (In ru-

coupling between oxidation and phosphorylation, is de-

minants, acetyl-CoA is derived directly from acetate.)

pendent on the availability of ADP and hence, ulti-

THE CITRIC ACID CYCLE: THE CATABOLISM OF ACETYL-CoA

135

Fatty

regulated in the same way as is pyruvate dehydrogenase

Pyruvate

Glucose

acids

(Figure 17-6). Succinate dehydrogenase is inhibited by

oxaloacetate, and the availability of oxaloacetate, as

controlled by malate dehydrogenase, depends on the

[NADH]/[NAD⁺] ratio. Since the K_m for oxaloacetate

PYRUVATE

of citrate synthase is of the same order of magnitude as

DEHYDROGENASE

Acetyl-CoA

the intramitochondrial concentration, it is likely that

Acetyl-CoA

the concentration of oxaloacetate controls the rate of

citrate formation. Which of these mechanisms are im-

portant in vivo has still to be resolved.

Citric

ATP-CITRATE

acid

LYASE

cycle

SUMMARY

Oxaloacetate

Citrate

• The citric acid cycle is the final pathway for the oxi-

dation of carbohydrate, lipid, and protein whose

common end-metabolite, acetyl-CoA, reacts with ox-

aloacetate to form citrate. By a series of dehydrogena-

tions and decarboxylations, citrate is degraded,

releasing reduced coenzymes and 2CO₂ and regener-

CO₂

ating oxaloacetate.

MITOCHONDRIAL

• The reduced coenzymes are oxidized by the respira-

MEMBRANE

tory chain linked to formation of ATP. Thus, the

cycle is the major route for the generation of ATP

Figure 16-5. Participation of the citric acid cycle in

and is located in the matrix of mitochondria adjacent

fatty acid synthesis from glucose. See also Figure 21-5.

to the enzymes of the respiratory chain and oxidative

phosphorylation.

• The citric acid cycle is amphibolic, since in addition

mately, on the rate of utilization of ATP in chemical

to oxidation it is important in the provision of car-

and physical work. In addition, individual enzymes of

bon skeletons for gluconeogenesis, fatty acid synthe-

the cycle are regulated. The most likely sites for regula-

sis, and interconversion of amino acids.

tion are the nonequilibrium reactions catalyzed by

pyruvate dehydrogenase, citrate synthase, isocitrate de-

REFERENCES

hydrogenase, and α-ketoglutarate dehydrogenase. The

dehydrogenases are activated by Ca2+, which increases

Baldwin JE, Krebs HA: The evolution of metabolic cycles. Nature

1981;291:381.

in concentration during muscular contraction and se-

cretion, when there is increased energy demand. In a

Goodwin TW (editor): The Metabolic Roles of Citrate. Academic

Press, 1968.

tissue such as brain, which is largely dependent on car-

Greville GD: Vol 1, p 297, in: Carbohydrate Metabolism and Its

bohydrate to supply acetyl-CoA, control of the citric

Disorders. Dickens F, Randle PJ, Whelan WJ (editors). Acad-

acid cycle may occur at pyruvate dehydrogenase. Sev-

emic Press, 1968.

eral enzymes are responsive to the energy status, as

Kay J, Weitzman PDJ (editors): Krebs’ Citric Acid Cycle—Half a

shown by the [ATP]/[ADP] and [NADH]/[NAD⁺] ra-

Century and Still Turning. Biochemical Society, London,

tios. Thus, there is allosteric inhibition of citrate syn-

1987.

thase by ATP and long-chain fatty acyl-CoA. Allosteric

Srere PA: The enzymology of the formation and breakdown of cit-

activation of mitochondrial NAD-dependent isocitrate

rate. Adv Enzymol 1975;43:57.

dehydrogenase by ADP is counteracted by ATP and

Tyler DD: The Mitochondrion in Health and Disease. VCH Pub-

NADH. The α-ketoglutarate dehydrogenase complex is

lishers, 1992.

Glycolysis & the Oxidation

of Pyruvate

Peter A. Mayes, PhD, DSc, & David A. Bender, PhD

BIOMEDICAL IMPORTANCE

GLYCOLYSIS CAN FUNCTION UNDER

ANAEROBIC CONDITIONS

Most tissues have at least some requirement for glucose.

In brain, the requirement is substantial. Glycolysis, the

When a muscle contracts in an anaerobic medium, ie,

major pathway for glucose metabolism, occurs in the

one from which oxygen is excluded, glycogen disap-

cytosol of all cells. It is unique in that it can function ei-

pears and lactate appears as the principal end product.

ther aerobically or anaerobically. Erythrocytes, which

When oxygen is admitted, aerobic recovery takes place

lack mitochondria, are completely reliant on glucose as

and lactate disappears. However, if contraction occurs

their metabolic fuel and metabolize it by anaerobic gly-

under aerobic conditions, lactate does not accumulate

colysis. However, to oxidize glucose beyond pyruvate

and pyruvate is the major end product of glycolysis.

(the end product of glycolysis) requires both oxygen

Pyruvate is oxidized further to CO₂ and water (Figure

and mitochondrial enzyme systems such as the pyruvate

17-1). When oxygen is in short supply, mitochondrial

dehydrogenase complex, the citric acid cycle, and the

reoxidation of NADH formed from NAD⁺ during gly-

respiratory chain.

colysis is impaired, and NADH is reoxidized by reduc-

Glycolysis is both the principal route for glucose

ing pyruvate to lactate, so permitting glycolysis to pro-

metabolism and the main pathway for the metabolism

ceed (Figure 17-1). While glycolysis can occur under

of fructose, galactose, and other carbohydrates derived

anaerobic conditions, this has a price, for it limits the

from the diet. The ability of glycolysis to provide ATP

amount of ATP formed per mole of glucose oxidized,

in the absence of oxygen is especially important because

so that much more glucose must be metabolized under

it allows skeletal muscle to perform at very high levels

anaerobic than under aerobic conditions.

when oxygen supply is insufficient and because it allows

tissues to survive anoxic episodes. However, heart mus-

THE REACTIONS OF GLYCOLYSIS

cle, which is adapted for aerobic performance, has rela-

CONSTITUTE THE MAIN PATHWAY

tively low glycolytic activity and poor survival under

conditions of ischemia. Diseases in which enzymes of

OF GLUCOSE UTILIZATION

glycolysis (eg, pyruvate kinase) are deficient are mainly

The overall equation for glycolysis from glucose to lac-

seen as hemolytic anemias or, if the defect affects

tate is as follows:

skeletal muscle (eg, phosphofructokinase), as fatigue.

In fast-growing cancer cells, glycolysis proceeds at a

Glucose+2ADP+2P

→2

(+)−Lactate+2ATP+2H O

higher rate than is required by the citric acid cycle,

forming large amounts of pyruvate, which is reduced to

All of the enzymes of glycolysis (Figure 17-2) are

lactate and exported. This produces a relatively acidic

found in the cytosol. Glucose enters glycolysis by phos-

local environment in the tumor which may have impli-

phorylation to glucose 6-phosphate, catalyzed by hexo-

cations for cancer therapy. The lactate is used for gluco-

kinase, using ATP as the phosphate donor. Under

neogenesis in the liver, an energy-expensive process re-

physiologic conditions, the phosphorylation of glucose

sponsible for much of the hypermetabolism seen in

to glucose 6-phosphate can be regarded as irreversible.

cancer cachexia. Lactic acidosis results from several

Hexokinase is inhibited allosterically by its product,

causes, including impaired activity of pyruvate dehy-

glucose 6-phosphate. In tissues other than the liver and

drogenase.

pancreatic B islet cells, the availability of glucose for

136

GLYCOLYSIS & THE OXIDATION OF PYRUVATE

137

Glucose

Glycogen

This reaction is followed by another phosphorylation

C₆

(C₆ )_n

with ATP catalyzed by the enzyme phosphofructoki-

nase (phosphofructokinase-1), forming fructose 1,6-

bisphosphate. The phosphofructokinase reaction may

be considered to be functionally irreversible under

physiologic conditions; it is both inducible and subject

to allosteric regulation and has a major role in regulat-

Hexose phosphates

ing the rate of glycolysis. Fructose 1,6-bisphosphate is

C₆

cleaved by aldolase (fructose 1,6-bisphosphate aldolase)

into two triose phosphates, glyceraldehyde 3-phosphate

and dihydroxyacetone phosphate. Glyceraldehyde

3-phosphate and dihydroxyacetone phosphate are inter-

converted by the enzyme phosphotriose isomerase.

Glycolysis continues with the oxidation of glycer-

Triose phosphate

aldehyde 3-phosphate to 1,3-bisphosphoglycerate. The

C₃

enzyme catalyzing this oxidation, glyceraldehyde

NAD⁺

H₂O

3-phosphate dehydrogenase, is NAD-dependent.

Structurally, it consists of four identical polypeptides

(monomers) forming a tetramer.

SH groups are

O₂

NADH

¹/2O₂

present on each polypeptide, derived from cysteine

+ H⁺

residues within the polypeptide chain. One of the

Pyruvate

Lactate

SH groups at the active site of the enzyme (Figure

C₃

H₂O

17-3) combines with the substrate forming a thiohemi-

acetal that is oxidized to a thiol ester; the hydrogens re-

moved in this oxidation are transferred to NAD⁺. The

Figure 17-1. Summary of glycolysis. − , blocked by

thiol ester then undergoes phosphorolysis; inorganic

anaerobic conditions or by absence of mitochondria

phosphate (P_i) is added, forming 1,3-bisphosphoglycer-

containing key respiratory enzymes, eg, as in erythro-

ate, and the SH group is reconstituted.

cytes.

In the next reaction, catalyzed by phosphoglycerate

kinase, phosphate is transferred from 1,3-bisphospho-

glycerate onto ADP, forming ATP

(substrate-level

glycolysis (or glycogen synthesis in muscle and lipogen-

phosphorylation) and 3-phosphoglycerate. Since two

esis in adipose tissue) is controlled by transport into the

molecules of triose phosphate are formed per molecule

cell, which in turn is regulated by insulin. Hexokinase

of glucose, two molecules of ATP are generated at this

has a high affinity (low K_m) for its substrate, glucose,

stage per molecule of glucose undergoing glycolysis.

and in the liver and pancreatic B islet cells is saturated

The toxicity of arsenic is due to competition of arsenate

under all normal conditions and so acts at a constant

with inorganic phosphate (P_i) in the above reactions to

rate to provide glucose 6-phosphate to meet the cell’s

give

1-arseno-3-phosphoglycerate, which hydrolyzes

need. Liver and pancreatic B islet cells also contain an

spontaneously to give

3-phosphoglycerate plus heat,

isoenzyme of hexokinase, glucokinase, which has a K_m

without generating ATP. 3-Phosphoglycerate is isomer-

very much higher than the normal intracellular concen-

ized to 2-phosphoglycerate by phosphoglycerate mu-

tration of glucose. The function of glucokinase in the

tase. It is likely that 2,3-bisphosphoglycerate (diphos-

liver is to remove glucose from the blood following a

phoglycerate; DPG) is an intermediate in this reaction.

meal, providing glucose 6-phosphate in excess of re-

The subsequent step is catalyzed by enolase and in-

quirements for glycolysis, which will be used for glyco-

volves a dehydration, forming phosphoenolpyruvate.

gen synthesis and lipogenesis. In the pancreas, the

Enolase is inhibited by fluoride. To prevent glycolysis

glucose 6-phosphate formed by glucokinase signals in-

in the estimation of glucose, blood is collected in

creased glucose availability and leads to the secretion of

tubes containing fluoride. The enzyme is also depen-

insulin.

dent on the presence of either Mg2+ or Mn2+. The

Glucose 6-phosphate is an important compound at

phosphate of phosphoenolpyruvate is transferred to

the junction of several metabolic pathways (glycolysis,

ADP by pyruvate kinase to generate, at this stage,

gluconeogenesis, the pentose phosphate pathway, gly-

two molecules of ATP per molecule of glucose oxi-

cogenesis, and glycogenolysis). In glycolysis, it is con-

dized. The product of the enzyme-catalyzed reaction,

verted to fructose

6-phosphate by phosphohexose-

enolpyruvate, undergoes spontaneous

(nonenzymic)

isomerase, which involves an aldose-ketose isomerization.

isomerization to pyruvate and so is not available to

Glycogen

Glucose 1-phosphate

HEXOKINASE

CH₂OH

CH₂

O P

GLUCOKINASE

CH₂

O P

PHOSPHOHEXOSE

ISOMERASE

CH₂OH

Mg2+

H OH

ATP

ADP

α-D-Glucose

α-D-Glucose 6-phosphate

D-Fructose 6-phosphate

ATP

PHOSPHOFRUCTO-

ADP

KINASE

CH₂

O P

CH₂

O P

D-Fructose 1,6-bisphosphate

ALDOLASE

Iodoacetate

CH₂

O P

GLYCERALDEHYDE-3-PHOSPHATE

PHOSPHOGLYCERATE

C O

DEHYDROGENASE

KINASE

COO

C O

CH₂OH

Mg2+

Dihydroxyacetone phosphate

CH₂

O P

CH₂

O P

CH₂

O P

PHOSPHOTRIOSE

ATP

ADP

NADH NAD⁺

ISOMERASE

3-Phosphoglycerate

1,3-Bisphosphoglycerate

+ H⁺

Glyceraldehyde

3-phosphate

¹/2O

PHOSPHOGLYCERATE MUTASE

Mitochondrial

respiratory chain

COO^-

3ADP

3ATP

2-Phosphoglycerate

+ P

CH₂OH

Anaerobiosis

Fluoride

Mg2+

H₂O

ENOLASE

COO^-

Phosphoenolpyruvate

O P

Oxidation

in citric

CH₂

acid cycle

ADP

PYRUVATE

Mg²

KINASE

NADH + H⁺

NAD⁺

ATP

COO

COO^-

Spontaneous

C OH

C O

LACTATE

CH₂

CH₃

DEHYDROGENASE

(Enol)

(Keto)

L(+)-Lactate

Pyruvate

Figure 17-2. The pathway of glycolysis. ( P , PO32−; P_i, HOPO32−;

− , inhibition.) At asterisk: Carbon

atoms 1-3 of fructose bisphosphate form dihydroxyacetone phosphate, whereas carbons 4-6 form

glyceraldehyde 3-phosphate. The term “bis-,” as in bisphosphate, indicates that the phosphate groups

are separated, whereas diphosphate, as in adenosine diphosphate, indicates that they are joined.

138

GLYCOLYSIS & THE OXIDATION OF PYRUVATE

139

S Enz

H C O

H C OH

NAD⁺

CH₂

Glyceraldehyde 3-phosphate

Enzyme-substrate complex

HS Enz

NAD⁺

Bound

coenzyme

Substrate

oxidation

by bound

NAD⁺

C O

CH₂

1,3-Bisphosphoglycerate

S Enz

Enz

C O

NADH + H

NAD⁺

NADH + H⁺

NAD⁺

CH₂

Energy-rich intermediate

Figure 17-3. Mechanism of oxidation of glyceraldehyde 3-phosphate. (Enz, glycer-

aldehyde-3-phosphate dehydrogenase.) The enzyme is inhibited by the SH poison

iodoacetate, which is thus able to inhibit glycolysis. The NADH produced on the enzyme

is not as firmly bound to the enzyme as is NAD⁺. Consequently, NADH is easily displaced

by another molecule of NAD⁺.

undergo the reverse reaction. The pyruvate kinase re-

up into mitochondria for oxidation via one of the two

action is thus also irreversible under physiologic con-

shuttles described in Chapter 12.

ditions.

The redox state of the tissue now determines which

of two pathways is followed. Under anaerobic condi-

Tissues That Function Under Hypoxic

tions, the reoxidation of NADH through the respira-

Circumstances Tend to Produce Lactate

tory chain to oxygen is prevented. Pyruvate is reduced

(Figure 17-2)

by the NADH to lactate, the reaction being catalyzed

by lactate dehydrogenase. Several tissue-specific isoen-

This is true of skeletal muscle, particularly the white

zymes of this enzyme have been described and have

fibers, where the rate of work output—and therefore

clinical significance

(Chapter 7). The reoxidation of

the need for ATP formation—may exceed the rate at

NADH via lactate formation allows glycolysis to pro-

which oxygen can be taken up and utilized. Glycolysis

ceed in the absence of oxygen by regenerating sufficient

in erythrocytes, even under aerobic conditions, always

NAD⁺ for another cycle of the reaction catalyzed by

terminates in lactate, because the subsequent reactions

glyceraldehyde-3-phosphate dehydrogenase. Under aer-

of pyruvate are mitochondrial, and erythrocytes lack

obic conditions, pyruvate is taken up into mitochon-

mitochondria. Other tissues that normally derive much

dria and after conversion to acetyl-CoA is oxidized to

of their energy from glycolysis and produce lactate in-

CO₂ by the citric acid cycle. The reducing equivalents

clude brain, gastrointestinal tract, renal medulla, retina,

from the NADH + H⁺ formed in glycolysis are taken

and skin. The liver, kidneys, and heart usually take up

140

CHAPTER 17

lactate and oxidize it but will produce it under hypoxic

H C O

Glucose

conditions.

H C OH

CH₂

Glycolysis Is Regulated at Three Steps

Involving Nonequilibrium Reactions

Glyceraldehyde 3-phosphate

NAD⁺

Although most of the reactions of glycolysis are re-

versible, three are markedly exergonic and must there-

GLYCERALDEHYDE-3-PHOSPHATE

fore be considered physiologically irreversible. These re-

DEHYDROGENASE

actions, catalyzed by hexokinase

(and glucokinase),

NADH + H⁺

phosphofructokinase, and pyruvate kinase, are the

major sites of regulation of glycolysis. Cells that are ca-

pable of reversing the glycolytic pathway (gluconeoge-

C O

nesis) have different enzymes that catalyze reactions

BISPHOSPHOGLYCERATE

MUTASE

which effectively reverse these irreversible reactions.

The importance of these steps in the regulation of gly-

CH₂

colysis and gluconeogenesis is discussed in Chapter 19.

1,3-Bisphosphoglycerate

In Erythrocytes, the First Site in Glycolysis

ADP

COO-

for ATP Generation May Be Bypassed

PHOSPHOGLYCERATE

KINASE

In the erythrocytes of many mammals, the reaction cat-

CH₂

alyzed by phosphoglycerate kinase may be bypassed

ATP

by a process that effectively dissipates as heat the free

2,3-Bisphosphoglycerate

energy associated with the high-energy phosphate of

COO-

1,3-bisphosphoglycerate

(Figure

17-4). Bisphospho-

glycerate mutase catalyzes the conversion of 1,3-bis-

phosphoglycerate to 2,3-bisphosphoglycerate, which is

2,3-BISPHOSPHOGLYCERATE

CH₂

PHOSPHATASE

converted to 3-phosphoglycerate by 2,3-bisphospho-

glycerate phosphatase (and possibly also phosphoglyc-

3-Phosphoglycerate

erate mutase). This alternative pathway involves no net

Pyruvate

yield of ATP from glycolysis. However, it does serve to

Figure 17-4.

2,3-Bisphosphoglycerate pathway in

provide 2,3-bisphosphoglycerate, which binds to hemo-

erythrocytes.

globin, decreasing its affinity for oxygen and so making

oxygen more readily available to tissues (see Chapter 6).

in thiamin deficiency glucose metabolism is impaired

THE OXIDATION OF PYRUVATE TO

and there is significant (and potentially life-threatening)

ACETYL-CoA IS THE IRREVERSIBLE

lactic and pyruvic acidosis. Acetyl lipoamide reacts with

ROUTE FROM GLYCOLYSIS TO THE

coenzyme A to form acetyl-CoA and reduced lipoamide.

CITRIC ACID CYCLE

The cycle of reaction is completed when the reduced

lipoamide is reoxidized by a flavoprotein, dihydrolipoyl

Pyruvate, formed in the cytosol, is transported into the

dehydrogenase, containing FAD. Finally, the reduced

mitochondrion by a proton symporter (Figure 12-10).

flavoprotein is oxidized by NAD⁺, which in turn trans-

Inside the mitochondrion, pyruvate is oxidatively decar-

fers reducing equivalents to the respiratory chain.

boxylated to acetyl-CoA by a multienzyme complex that

is associated with the inner mitochondrial membrane.

Pyruvate + NAD

+ CoA → Acetyl−CoA + NADH + H

+ CO

This pyruvate dehydrogenase complex is analogous to

the α-ketoglutarate dehydrogenase complex of the citric

acid cycle (Figure 16-3). Pyruvate is decarboxylated by

The pyruvate dehydrogenase complex consists of a

the pyruvate dehydrogenase component of the enzyme

number of polypeptide chains of each of the three com-

complex to a hydroxyethyl derivative of the thiazole ring

ponent enzymes, all organized in a regular spatial con-

of enzyme-bound thiamin diphosphate, which in turn

figuration. Movement of the individual enzymes ap-

reacts with oxidized lipoamide, the prosthetic group of

pears to be restricted, and the metabolic intermediates

dihydrolipoyl transacetylase, to form acetyl lipoamide

do not dissociate freely but remain bound to the en-

(Figure 17-5). Thiamin is vitamin B₁ (Chapter 45), and

zymes. Such a complex of enzymes, in which the sub-

GLYCOLYSIS & THE OXIDATION OF PYRUVATE

141

CH₃

COO^-+ H⁺

TDP

Pyruvate

Acetyl

lipoamide

CoA-SH

PYRUVATE

DEHYDROGENASE

CO₂

TDP

H₃C C OH

Hydroxyethyl

H₂

H₂C

DIHYDROLIPOYL

Oxidized lipoamide

TRANSACETYLASE

Lipoic acid

Lysine

side chain

NAD⁺

FADH₂

DIHYDROLIPOYL

DEHYDROGENASE

CH₃

CO S CoA

Acetyl-CoA

Dihydrolipoamide

FAD

NADH + H⁺

Figure 17-5. Oxidative decarboxylation of pyruvate by the pyruvate dehydrogenase complex. Lipoic acid is

joined by an amide link to a lysine residue of the transacetylase component of the enzyme complex. It forms a long

flexible arm, allowing the lipoic acid prosthetic group to rotate sequentially between the active sites of each of the

enzymes of the complex. (NAD⁺, nicotinamide adenine dinucleotide; FAD, flavin adenine dinucleotide; TDP, thiamin

diphosphate.)

strates are handed on from one enzyme to the next, in-

lated by phosphorylation by a kinase of three serine

creases the reaction rate and eliminates side reactions,

residues on the pyruvate dehydrogenase component of

increasing overall efficiency.

the multienzyme complex, resulting in decreased activ-

ity, and by dephosphorylation by a phosphatase that

Pyruvate Dehydrogenase Is Regulated

causes an increase in activity. The kinase is activated by

increases in the

[ATP]/[ADP],

[acetyl-CoA]/[CoA],

by End-Product Inhibition

and [NADH]/[NAD⁺] ratios. Thus, pyruvate dehydro-

& Covalent Modification

genase—and therefore glycolysis—is inhibited not only

Pyruvate dehydrogenase is inhibited by its products,

by a high-energy potential but also when fatty acids are

acetyl-CoA and NADH (Figure 17-6). It is also regu-

being oxidized. Thus, in starvation, when free fatty acid

142

CHAPTER 17

[ Acetyl-CoA ]

[ NADH ]

[ ATP ]

[ CoA ]

[ NAD⁺ ]

[ ADP ]

Dichloroacetate

Acetyl-CoA

Ca2+

PDH KINASE

Pyruvate

NADH + H⁺

CO₂

Mg2+

ATP

ADP

–

PDH

PDH-a

PDH-b

(Active DEPHOSPHO-ENZYME)

(Inactive PHOSPHO-ENZYME)

NAD⁺

CoA

P_i

H₂O

Pyruvate

PDH PHOSPHATASE

Mg2+, Ca2+

Insulin

(in adipose tissue)

Figure 17-6. Regulation of pyruvate dehydrogenase (PDH). Arrows with wavy shafts indicate allosteric ef-

fects. A: Regulation by end-product inhibition. B: Regulation by interconversion of active and inactive forms.

concentrations increase, there is a decrease in the pro-

ATP synthase reaction has been calculated as approxi-

portion of the enzyme in the active form, leading to a

mately 51.6 kJ. It follows that the total energy captured

sparing of carbohydrate. In adipose tissue, where glu-

in ATP per mole of glucose oxidized is 1961 kJ, or ap-

cose provides acetyl CoA for lipogenesis, the enzyme is

proximately 68% of the energy of combustion. Most of

activated in response to insulin.

the ATP is formed by oxidative phosphorylation result-

ing from the reoxidation of reduced coenzymes by the

respiratory chain. The remainder is formed by substrate-

Oxidation of Glucose Yields Up to 38 Mol

level phosphorylation (Table 17-1).

of ATP Under Aerobic Conditions But Only

2 Mol When O₂ Is Absent

CLINICAL ASPECTS

When 1 mol of glucose is combusted in a calorimeter

Inhibition of Pyruvate Metabolism

to CO₂ and water, approximately 2870 kJ are liberated

Leads to Lactic Acidosis

as heat. When oxidation occurs in the tissues, approxi-

mately 38 mol of ATP are generated per molecule of

Arsenite and mercuric ions react with the SH groups

glucose oxidized to CO₂ and water. In vivo, ∆G for the

of lipoic acid and inhibit pyruvate dehydrogenase, as

GLYCOLYSIS & THE OXIDATION OF PYRUVATE

143

Table 17-1. Generation of high-energy phosphate in the catabolism of glucose.

Number of ~ P

Formed per

Pathway

Reaction Catalyzed by

Method of ~ P Production

Mole of Glucose

Glycolysis

Glyceraldehyde-3-phosphate dehydrogenase Respiratory chain oxidation of 2 NADH

Phosphoglycerate kinase

Phosphorylation at substrate level

Pyruvate kinase

Phosphorylation at substrate level

Allow for consumption of ATP by reactions catalyzed by hexokinase and phosphofructokinase

−2

Net 8

Pyruvate dehydrogenase

Respiratory chain oxidation of 2 NADH

Isocitrate dehydrogenase

Respiratory chain oxidation of 2 NADH

α-Ketoglutarate dehydrogenase

Respiratory chain oxidation of 2 NADH

Citric acid cycle Succinate thiokinase

Phosphorylation at substrate level

Succinate dehydrogenase

Respiratory chain oxidation of 2 FADH₂

Malate dehydrogenase

Respiratory chain oxidation of 2 NADH

Net 30

Total per mole of glucose under aerobic conditions

Total per mole of glucose under anaerobic conditions

*It is assumed that NADH formed in glycolysis is transported into mitochondria via the malate shuttle (see Figure 12-13). If the glyc-

erophosphate shuttle is used, only 2 ~ P would be formed per mole of NADH, the total net production being 26 instead of 38. The

calculation ignores the small loss of ATP due to a transport of H⁺ into the mitochondrion with pyruvate and a similar transport of H⁺

in the operation of the malate shuttle, totaling about 1 mol of ATP. Note that there is a substantial benefit under anaerobic condi-

tions if glycogen is the starting point, since the net production of high-energy phosphate in glycolysis is increased from 2 to 3, as ATP

is no longer required by the hexokinase reaction.

does a dietary deficiency of thiamin, allowing pyru-

• It can function anaerobically by regenerating oxidized

vate to accumulate. Nutritionally deprived alcoholics

NAD⁺ (required in the glyceraldehyde-3-phosphate de-

are thiamin-deficient and may develop potentially fatal

hydrogenase reaction) by reducing pyruvate to lactate.

pyruvic and lactic acidosis. Patients with inherited

• Lactate is the end product of glycolysis under anaero-

pyruvate dehydrogenase deficiency, which can be due

bic conditions (eg, in exercising muscle) or when the

to defects in one or more of the components of the en-

metabolic machinery is absent for the further oxida-

zyme complex, also present with lactic acidosis, particu-

tion of pyruvate (eg, in erythrocytes).

larly after a glucose load. Because of its dependence on

• Glycolysis is regulated by three enzymes catalyzing

glucose as a fuel, brain is a prominent tissue where these

nonequilibrium reactions: hexokinase, phosphofruc-

metabolic defects manifest themselves in neurologic

tokinase, and pyruvate kinase.

disturbances.

• In erythrocytes, the first site in glycolysis for genera-

Inherited aldolase A deficiency and pyruvate kinase

tion of ATP may be bypassed, leading to the forma-

deficiency in erythrocytes cause hemolytic anemia.

tion of 2,3-bisphosphoglycerate, which is important

The exercise capacity of patients with muscle phos-

in decreasing the affinity of hemoglobin for O₂.

phofructokinase deficiency is low, particularly on

high-carbohydrate diets. By providing an alternative

• Pyruvate is oxidized to acetyl-CoA by a multienzyme

complex, pyruvate dehydrogenase, that is dependent

lipid fuel, eg, during starvation, when blood free fatty

acids and ketone bodies are increased, work capacity is

on the vitamin cofactor thiamin diphosphate.

improved.

• Conditions that involve an inability to metabolize

pyruvate frequently lead to lactic acidosis.

SUMMARY

REFERENCES

• Glycolysis is the cytosolic pathway of all mammalian

cells for the metabolism of glucose (or glycogen) to

Behal RH et al: Regulation of the pyruvate dehydrogenase multien-

pyruvate and lactate.

zyme complex. Annu Rev Nutr 1993;13:497.

Metabolism of Glycogen

Peter A. Mayes, PhD, DSc, & David A. Bender, PhD

BIOMEDICAL IMPORTANCE

phatase catalyzes hydrolysis of pyrophosphate to 2 mol

of inorganic phosphate, shifting the equilibrium of the

Glycogen is the major storage carbohydrate in animals,

main reaction by removing one of its products.

corresponding to starch in plants; it is a branched poly-

Glycogen synthase catalyzes the formation of a gly-

mer of α-D-glucose. It occurs mainly in liver (up to 6%)

coside bond between C₁ of the activated glucose of

and muscle, where it rarely exceeds 1%. However, be-

UDPGlc and C₄ of a terminal glucose residue of glyco-

cause of its greater mass, muscle contains about three to

gen, liberating uridine diphosphate (UDP). A preexist-

four times as much glycogen as does liver (Table 18-1).

ing glycogen molecule, or “glycogen primer,” must be

Muscle glycogen is a readily available source of glu-

present to initiate this reaction. The glycogen primer

cose for glycolysis within the muscle itself. Liver glyco-

may in turn be formed on a primer known as glyco-

gen functions to store and export glucose to maintain

genin, which is a 37-kDa protein that is glycosylated

blood glucose between meals. After 12-18 hours of

on a specific tyrosine residue by UDPGlc. Further glu-

fasting, the liver glycogen is almost totally depleted.

cose residues are attached in the 1→4 position to make

Glycogen storage diseases are a group of inherited dis-

a short chain that is a substrate for glycogen synthase.

orders characterized by deficient mobilization of glyco-

In skeletal muscle, glycogenin remains attached in the

gen or deposition of abnormal forms of glycogen, lead-

center of the glycogen molecule

(Figure

13-15),

ing to muscular weakness or even death.

whereas in liver the number of glycogen molecules is

greater than the number of glycogenin molecules.

GLYCOGENESIS OCCURS MAINLY

IN MUSCLE & LIVER

Branching Involves Detachment

of Existing Glycogen Chains

The Pathway of Glycogen Biosynthesis

Involves a Special Nucleotide of Glucose

The addition of a glucose residue to a preexisting glyco-

(Figure 18-1)

gen chain, or

“primer,” occurs at the nonreducing,

outer end of the molecule so that the “branches” of the

As in glycolysis, glucose is phosphorylated to glucose

glycogen “tree” become elongated as successive 1→4

6-phosphate, catalyzed by hexokinase in muscle and

linkages are formed (Figure 18-3). When the chain has

glucokinase in liver. Glucose 6-phosphate is isomer-

been lengthened to at least 11 glucose residues, branch-

ized to glucose 1-phosphate by phosphoglucomutase.

ing enzyme transfers a part of the 1→4 chain (at least

The enzyme itself is phosphorylated, and the phospho-

six glucose residues) to a neighboring chain to form a

group takes part in a reversible reaction in which glu-

1→6 linkage, establishing a branch point. The

cose 1,6-bisphosphate is an intermediate. Next, glucose

branches grow by further additions of 1→4-glucosyl

1-phosphate reacts with uridine triphosphate (UTP) to

units and further branching.

form the active nucleotide uridine diphosphate glu-

cose (UDPGlc)* and pyrophosphate (Figure 18-2),

GLYCOGENOLYSIS IS NOT THE REVERSE

catalyzed by UDPGlc pyrophosphorylase. Pyrophos-

OF GLYCOGENESIS BUT IS A SEPARATE

* Other nucleoside diphosphate sugar compounds are known, eg,

PATHWAY (Figure 18-1)

UDPGal. In addition, the same sugar may be linked to different

Glycogen phosphorylase catalyzes the rate-limiting

nucleotides. For example, glucose may be linked to uridine (as

shown above) as well as to guanosine, thymidine, adenosine, or cy-

step in glycogenolysis by promoting the phosphorylytic

tidine nucleotides.

cleavage by inorganic phosphate (phosphorylysis; cf hy-

145

146

CHAPTER 18

Glycogen

(1→ 4 and 1→ 6 glucosyl units)_x

BRANCHING ENZYME

P_i

(1→ 4 Glucosyl units)_x

Insulin

UDP

GLYCOGEN

cAMP

SYNTHASE

PHOSPHORYLASE

Glycogen

primer

Glucagon

Epinephrine

GLUCAN

TRANSFERASE

Glycogenin

DEBRANCHING

ENZYME

Uridine

disphosphate

glucose (UDPGlc)

To uronic acid

Free glucose from

pathway

debranching

UDPGlc PYROPHOSPHORYLASE

enzyme

INORGANIC

PYROPHOSPHATASE

PPi

P_i

Uridine

UDP

triphosphate (UTP)

Glucose 1-phosphate

Mg2+

PHOSPHOGLUCOMUTASE

Glucose 6-phosphate

To glycolysis and pentose

phosphate pathway

ADP

NUCLEOSIDE

DIPHOSPHO-

ATP

ADP

GLUCOSE-6-

KINASE

Mg2+

GLUCOKINASE

PHOSPHATASE

P_i

ATP

Glucose

Figure 18-1. Pathway of glycogenesis and of glycogenolysis in the liver. Two high-energy phosphates are

used in the incorporation of 1 mol of glucose into glycogen. + , stimulation; − , inhibition. Insulin decreases the

level of cAMP only after it has been raised by glucagon or epinephrine—ie, it antagonizes their action. Glucagon

is active in heart muscle but not in skeletal muscle. At asterisk: Glucan transferase and debranching enzyme ap-

pear to be two separate activities of the same enzyme.

Table 18-1. Storage of carbohydrate in

drolysis) of the 1→4 linkages of glycogen to yield glu-

postabsorptive normal adult humans (70 kg).

cose 1-phosphate. The terminal glucosyl residues from

the outermost chains of the glycogen molecule are re-

Liver glycogen

4.0%

72 g¹

moved sequentially until approximately four glucose

Muscle glycogen

0.7%

245 g²

residues remain on either side of a 1→6 branch (Figure

Extracellular glucose

0.1%

10 g³

18-4). Another enzyme (

-[1v4]v -[1v4] glucan

327 g

transferase) transfers a trisaccharide unit from one

branch to the other, exposing the 1→6 branch point.

¹Liver weight 1800 g.

²Muscle mass 35 kg.

Hydrolysis of the

1→6 linkages requires the de-

³Total volume 10 L.

branching enzyme. Further phosphorylase action can

METABOLISM OF GLYCOGEN

147

dephosphorylation of enzyme protein in response to

6CH2

hormone action (Chapter 9).

Uracil

Cyclic AMP (cAMP) (Figure 18-5) is formed from

ATP by adenylyl cyclase at the inner surface of cell

OH H

membranes and acts as an intracellular second messen-

O P O P O CH2

ger in response to hormones such as epinephrine, nor-

H OH

O^-

epinephrine, and glucagon. cAMP is hydrolyzed by

phosphodiesterase, so terminating hormone action. In

liver, insulin increases the activity of phosphodiesterase.

H H

Ribose

HO OH

Phosphorylase Differs Between

Liver & Muscle

Glucose Diphosphate

Uridine

In liver, one of the serine hydroxyl groups of active

Figure 18-2. Uridine diphosphate glucose (UDPGlc).

phosphorylase a is phosphorylated. It is inactivated by

hydrolytic removal of the phosphate by protein phos-

phatase-1 to form phosphorylase b. Reactivation re-

then proceed. The combined action of phosphorylase

quires rephosphorylation catalyzed by phosphorylase

and these other enzymes leads to the complete break-

kinase.

down of glycogen. The reaction catalyzed by phospho-

Muscle phosphorylase is distinct from that of liver. It

glucomutase is reversible, so that glucose 6-phosphate

is a dimer, each monomer containing 1 mol of pyridoxal

can be formed from glucose 1-phosphate. In liver (and

phosphate (vitamin B₆). It is present in two forms: phos-

kidney), but not in muscle, there is a specific enzyme,

phorylase a, which is phosphorylated and active in either

glucose-6-phosphatase, that hydrolyzes glucose

the presence or absence of 5′-AMP (its allosteric modi-

6-phosphate, yielding glucose that is exported, leading

fier); and phosphorylase b, which is dephosphorylated

to an increase in the blood glucose concentration.

and active only in the presence of 5′-AMP. This occurs

during exercise when the level of 5′-AMP rises, providing,

CYCLIC AMP INTEGRATES THE

by this mechanism, fuel for the muscle. Phosphorylase a is

the normal physiologically active form of the enzyme.

REGULATION OF GLYCOGENOLYSIS

& GLYCOGENESIS

cAMP Activates Muscle Phosphorylase

The principal enzymes controlling glycogen metabo-

lism—glycogen phosphorylase and glycogen synthase—

Phosphorylase in muscle is activated in response to epi-

are regulated by allosteric mechanisms and covalent

nephrine (Figure 18-6) acting via cAMP. Increasing

modifications due to reversible phosphorylation and

the concentration of cAMP activates cAMP-dependent

1→ 4- Glucosidic bond

Unlabeled glucose residue

1→ 6- Glucosidic bond

¹⁴C-labeled glucose residue

¹⁴C-Glucose

New 1→ 6- bond

added

GLYCOGEN

BRANCHING

SYNTHASE

ENZYME

Figure 18-3. The biosynthesis of glycogen. The mechanism of branching as revealed

by adding ¹⁴C-labeled glucose to the diet in the living animal and examining the liver

glycogen at further intervals.

148

CHAPTER 18

types of subunits—α, β, γ, and δ—in a structure repre-

sented as (αβγδ)₄. The α and β subunits contain serine

residues that are phosphorylated by cAMP-dependent

protein kinase. The δ subunit binds four Ca2+ and is

identical to the Ca2+-binding protein calmodulin

(Chapter 43). The binding of Ca2+ activates the cat-

alytic site of the γ subunit while the molecule remains

in the dephosphorylated b configuration. However, the

phosphorylated a form is only fully activated in the

presence of Ca2+. A second molecule of calmodulin, or

TpC (the structurally similar Ca2+-binding protein in

muscle), can interact with phosphorylase kinase, caus-

ing further activation. Thus, activation of muscle con-

traction and glycogenolysis are carried out by the same

PHOSPHORYLASE

GLUCAN

DEBRANCHING

TRANSFERASE

ENZYME

Ca2+-binding protein, ensuring their synchronization.

Glucose residues joined by

Glycogenolysis in Liver Can

1 → 4- glucosidic bonds

Glucose residues joined by

Be cAMP-Independent

1 → 6- glucosidic bonds

In addition to the action of glucagon in causing forma-

tion of cAMP and activation of phosphorylase in liver,

Figure 18-4. Steps in glycogenolysis.

1-adrenergic receptors mediate stimulation of glyco-

genolysis by epinephrine and norepinephrine. This in-

volves a cAMP-independent mobilization of Ca2+

protein kinase, which catalyzes the phosphorylation by

from mitochondria into the cytosol, followed by the

ATP of inactive phosphorylase kinase b to active

stimulation of a Ca2+/calmodulin-sensitive phosphory-

phosphorylase kinase a, which in turn, by means of a

lase kinase. cAMP-independent glycogenolysis is also

further phosphorylation, activates phosphorylase b to

caused by vasopressin, oxytocin, and angiotensin II act-

phosphorylase a.

ing through calcium or the phosphatidylinositol bis-

phosphate pathway (Figure 43-7).

Ca²⁺ Synchronizes the Activation of

Phosphorylase With Muscle Contraction

Protein Phosphatase-1

Glycogenolysis increases in muscle several hundred-fold

Inactivates Phosphorylase

immediately after the onset of contraction. This in-

Both phosphorylase a and phosphorylase kinase a are

volves the rapid activation of phosphorylase by activa-

dephosphorylated and inactivated by protein phos-

tion of phosphorylase kinase by Ca2+, the same signal as

phatase-1. Protein phosphatase-1 is inhibited by a

that which initiates contraction in response to nerve

protein, inhibitor-1, which is active only after it has

stimulation. Muscle phosphorylase kinase has four

been phosphorylated by cAMP-dependent protein ki-

nase. Thus, cAMP controls both the activation and in-

activation of phosphorylase (Figure 18-6). Insulin re-

NH₂

inforces this effect by inhibiting the activation of

phosphorylase b. It does this indirectly by increasing

uptake of glucose, leading to increased formation of

glucose 6-phosphate, which is an inhibitor of phosphor-

5′

ylase kinase.

CH₂

Glycogen Synthase & Phosphorylase

^-O

P O

Activity Are Reciprocally Regulated

(Figure 18-7)

H H

Like phosphorylase, glycogen synthase exists in either a

3′

phosphorylated or nonphosphorylated state. However,

unlike phosphorylase, the active form is dephosphory-

Figure 18-5.

3′,5′-Adenylic acid (cyclic AMP; cAMP).

lated (glycogen synthase a) and may be inactivated to

150

CHAPTER 18

Epinephrine

β Receptor

Inactive

Active

adenylyl

cyclase

PHOSPHODIESTERASE

ATP

cAMP

5′-AMP

PHOSPHORYLASE

KINASE

Ca2+

Inactive

Active

cAMP-DEPENDENT

PROTEIN KINASE

ATP

Glycogen_(n+1)

Inhibitor-1

GSK

ADP

(inactive)

CALMODULIN-DEPENDENT

PROTEIN KINASE

ATP

GLYCOGEN SYNTHASE

(inactive)

Ca2+

(active)

Insulin

G6P

ADP

PROTEIN

PHOSPHATASE

Glycogen_(n)

H₂O

P_i

+ UDPG

Inhibitor-1-phosphate

PROTEIN

PHOSPHATASE-1

(active)

Figure 18-7. Control of glycogen synthase in muscle (n = number of glucose residues). The sequence of reac-

tions arranged in a cascade causes amplification at each step, allowing only nanomole quantities of hormone to

cause major changes in glycogen concentration. (GSK, glycogen synthase kinase-3, -4, and -5; G6P, glucose

6-phosphate.)

glycogen synthase b by phosphorylation on serine

REGULATION OF GLYCOGEN

residues by no fewer than six different protein kinases.

METABOLISM IS EFFECTED BY

Two of the protein kinases are Ca2+/calmodulin-

A BALANCE IN ACTIVITIES

dependent (one of these is phosphorylase kinase). An-

BETWEEN GLYCOGEN

other kinase is cAMP-dependent protein kinase, which

allows cAMP-mediated hormonal action to inhibit

SYNTHASE & PHOSPHORYLASE

glycogen synthesis synchronously with the activation of

(Figure 18-8)

glycogenolysis. Insulin also promotes glycogenesis in

muscle at the same time as inhibiting glycogenolysis by

Not only is phosphorylase activated by a rise in concen-

raising glucose

6-phosphate concentrations, which

tration of cAMP (via phosphorylase kinase), but glyco-

stimulates the dephosphorylation and activation of

gen synthase is at the same time converted to the

glycogen synthase. Dephosphorylation of glycogen syn-

inactive form; both effects are mediated via cAMP-

thase b is carried out by protein phosphatase-1, which

dependent protein kinase. Thus, inhibition of gly-

is under the control of cAMP-dependent protein ki-

cogenolysis enhances net glycogenesis, and inhibition of

nase.

glycogenesis enhances net glycogenolysis. Furthermore,

METABOLISM OF GLYCOGEN

151

Epinephrine

PHOSPHODIESTERASE

(liver, muscle)

Inhibitor-1

cAMP

5′-AMP

Inhibitor-1

Glucagon

phosphate

(liver)

GLYCOGEN

PHOSPHORYLASE

SYNTHASE b

KINASE b

cAMP-

PROTEIN

DEPENDENT

PHOSPHATASE-1

PROTEIN KINASE

GLYCOGEN

PHOSPHORYLASE

SYNTHASE a

KINASE a

Glycogen

PHOSPHORYLASE

UDPGIc

cycle

Glucose 1-phosphate

PROTEIN

PHOSPHATASE-1

Glucose (liver)

Glucose Lactate (muscle)

Figure 18-8. Coordinated control of glycogenolysis and glycogenesis by cAMP-dependent protein ki-

nase. The reactions that lead to glycogenolysis as a result of an increase in cAMP concentrations are shown

with bold arrows, and those that are inhibited by activation of protein phosphatase-1 are shown as broken

arrows. The reverse occurs when cAMP concentrations decrease as a result of phosphodiesterase activity,

leading to glycogenesis.

the dephosphorylation of phosphorylase a, phosphory-

more, they allow insulin, via glucose 6-phosphate eleva-

lase kinase a, and glycogen synthase b is catalyzed by

tion, to have effects that act reciprocally to those of

a single enzyme of wide specificity—protein phos-

cAMP (Figures 18-6 and 18-7).

phatase-1. In turn, protein phosphatase-1 is inhibited

by cAMP-dependent protein kinase via inhibitor-1.

Thus, glycogenolysis can be terminated and glycogenesis

CLINICAL ASPECTS

can be stimulated synchronously, or vice versa, because

Glycogen Storage Diseases Are Inherited

both processes are keyed to the activity of cAMP-depen-

dent protein kinase. Both phosphorylase kinase and

“Glycogen storage disease” is a generic term to describe

glycogen synthase may be reversibly phosphorylated in

a group of inherited disorders characterized by deposi-

more than one site by separate kinases and phosphatases.

tion of an abnormal type or quantity of glycogen in the

These secondary phosphorylations modify the sensitivity

tissues. The principal glycogenoses are summarized in

of the primary sites to phosphorylation and dephos-

Table

18-2. Deficiencies of adenylyl kinase and

phorylation

(multisite phosphorylation). What is

cAMP-dependent protein kinase have also been re-

152

CHAPTER 18

Table 18-2. Glycogen storage diseases.

Glycogenosis

Name

Cause of Disorder

Characteristics

Type I

Von Gierke’s disease

Deficiency of glucose-6-phosphatase

Liver cells and renal tubule cells loaded

with glycogen. Hypoglycemia, lactic-

acidemia, ketosis, hyperlipemia.

Type II

Pompe’s disease

Deficiency of lysosomal α-1→4- and

Fatal, accumulation of glycogen in lyso-

1→6-glucosidase (acid maltase)

somes, heart failure.

Type III

Limit dextrinosis, Forbes’ or

Absence of debranching enzyme

Accumulation of a characteristic

Cori’s disease

branched polysaccharide.

Type IV

Amylopectinosis, Andersen’s

Absence of branching enzyme

Accumulation of a polysaccharide hav-

disease

ing few branch points. Death due to

cardiac or liver failure in first year of life.

Type V

Myophosphorylase deficiency,

Absence of muscle phosphorylase

Diminished exercise tolerance; muscles

McArdle’s syndrome

have abnormally high glycogen con-

tent (2.5-4.1%). Little or no lactate in

blood after exercise.

Type VI

Hers’ disease

Deficiency of liver phosphorylase

High glycogen content in liver, ten-

dency toward hypoglycemia.

Type VII

Tarui’s disease

Deficiency of phosphofructokinase

As for type V but also possibility of he-

in muscle and erythrocytes

molytic anemia.

Type VIII

Deficiency of liver phosphorylase

As for type VI.

kinase

ported. Some of the conditions described have bene-

REFERENCES

fited from liver transplantation.

Bollen M, Keppens S, Stalmans W: Specific features of glycogen

metabolism in the liver. Biochem J 1998;336:19.

SUMMARY

Cohen P: The role of protein phosphorylation in the hormonal

control of enzyme activity. Eur J Biochem 1985;151:439.

• Glycogen represents the principal storage form of

Ercan N, Gannon MC, Nuttall FQ: Incorporation of glycogenin

carbohydrate in the mammalian body, mainly in the

into a hepatic proteoglycogen after oral glucose administra-

liver and muscle.

tion. J Biol Chem 1994;269:22328.

• In the liver, its major function is to provide glucose

Geddes R: Glycogen: a metabolic viewpoint. Bioscience Rep

for extrahepatic tissues. In muscle, it serves mainly as

1986;6:415.

a ready source of metabolic fuel for use in muscle.

McGarry JD et al: From dietary glucose to liver glycogen: the full

• Glycogen is synthesized from glucose by the pathway

circle round. Annu Rev Nutr 1987;7:51.

of glycogenesis. It is broken down by a separate path-

Meléndez-Hevia E, Waddell TG, Shelton ED: Optimization of

molecular design in the evolution of metabolism: the glyco-

way known as glycogenolysis. Glycogenolysis leads to

gen molecule. Biochem J 1993;295:477.

glucose formation in liver and lactate formation in

Raz I, Katz A, Spencer MK: Epinephrine inhibits insulin-mediated

muscle owing to the respective presence or absence of

glycogenesis but enhances glycolysis in human skeletal mus-

glucose-6-phosphatase.

cle. Am J Physiol 1991;260:E430.

• Cyclic AMP integrates the regulation of glycogenoly-

Scriver CR et al (editors): The Metabolic and Molecular Bases of In-

sis and glycogenesis by promoting the simultaneous

herited Disease, 8th ed. McGraw-Hill, 2001.

activation of phosphorylase and inhibition of glyco-

Shulman GI, Landau BR: Pathways of glycogen repletion. Physiol

gen synthase. Insulin acts reciprocally by inhibiting

Rev 1992;72:1019.

glycogenolysis and stimulating glycogenesis.

Villar-Palasi C: On the mechanism of inactivation of muscle glyco-

gen phosphorylase by insulin. Biochim Biophys Acta 1994;

• Inherited deficiencies in specific enzymes of glycogen

1224:384.

metabolism in both liver and muscle are the causes of

glycogen storage diseases.

Gluconeogenesis & Control

of the Blood Glucose

Peter A. Mayes, PhD, DSc, & David A. Bender, PhD

BIOMEDICAL IMPORTANCE

vate

(Figure

45-17). A second enzyme, phospho-

enolpyruvate carboxykinase, catalyzes the decarboxy-

Gluconeogenesis is the term used to include all path-

lation and phosphorylation of oxaloacetate to phospho-

ways responsible for converting noncarbohydrate pre-

enolpyruvate using GTP (or ITP) as the phosphate

cursors to glucose or glycogen. The major substrates are

donor. Thus, reversal of the reaction catalyzed by pyru-

the glucogenic amino acids and lactate, glycerol, and

vate kinase in glycolysis involves two endergonic reac-

propionate. Liver and kidney are the major gluco-

tions.

neogenic tissues. Gluconeogenesis meets the needs of

In pigeon, chicken, and rabbit liver, phospho-

the body for glucose when carbohydrate is not available

enolpyruvate carboxykinase is a mitochondrial enzyme,

in sufficient amounts from the diet or from glycogen

and phosphoenolpyruvate is transported into the cy-

reserves. A supply of glucose is necessary especially for

tosol for gluconeogenesis. In the rat and the mouse, the

the nervous system and erythrocytes. Failure of gluco-

enzyme is cytosolic. Oxaloacetate does not cross the mi-

neogenesis is usually fatal. Hypoglycemia causes brain

tochondrial inner membrane; it is converted to malate,

dysfunction, which can lead to coma and death. Glu-

which is transported into the cytosol, and converted

cose is also important in maintaining the level of inter-

back to oxaloacetate by cytosolic malate dehydrogenase.

mediates of the citric acid cycle even when fatty acids

In humans, the guinea pig, and the cow, the enzyme is

are the main source of acetyl-CoA in the tissues. In ad-

equally distributed between mitochondria and cytosol.

dition, gluconeogenesis clears lactate produced by mus-

The main source of GTP for phosphoenolpyruvate

cle and erythrocytes and glycerol produced by adipose

carboxykinase inside the mitochondrion is the reaction

tissue. Propionate, the principal glucogenic fatty acid

of succinyl-CoA synthetase (Chapter 16). This provides

produced in the digestion of carbohydrates by rumi-

a link and limit between citric acid cycle activity and

nants, is a major substrate for gluconeogenesis in these

the extent of withdrawal of oxaloacetate for gluconeo-

species.

genesis.

GLUCONEOGENESIS INVOLVES

B. FRUCTOSE 1,6-BISPHOSPHATE

GLYCOLYSIS, THE CITRIC ACID CYCLE,

& FRUCTOSE 6-PHOSPHATE

& SOME SPECIAL REACTIONS

The conversion of fructose 1,6-bisphosphate to fructose

(Figure 19-1)

6-phosphate, to achieve a reversal of glycolysis, is cat-

alyzed by fructose-1,6-bisphosphatase. Its presence

Thermodynamic Barriers Prevent

determines whether or not a tissue is capable of synthe-

a Simple Reversal of Glycolysis

sizing glycogen not only from pyruvate but also from

Three nonequilibrium reactions catalyzed by hexoki-

triosephosphates. It is present in liver, kidney, and

nase, phosphofructokinase, and pyruvate kinase prevent

skeletal muscle but is probably absent from heart and

simple reversal of glycolysis for glucose synthesis

smooth muscle.

(Chapter 17). They are circumvented as follows:

A. PYRUVATE & PHOSPHOENOLPYRUVATE

C. GLUCOSE 6-PHOSPHATE & GLUCOSE

Mitochondrial pyruvate carboxylase catalyzes the car-

The conversion of glucose 6-phosphate to glucose is

boxylation of pyruvate to oxaloacetate, an ATP-requir-

catalyzed by glucose-6-phosphatase. It is present in

ing reaction in which the vitamin biotin is the co-

liver and kidney but absent from muscle and adipose

enzyme. Biotin binds CO₂

from bicarbonate as

tissue, which, therefore, cannot export glucose into the

carboxybiotin prior to the addition of the CO₂ to pyru-

bloodstream.

153

Glucose

ATP

GLUCOKINASE

GLUCOSE-6-PHOSPHATASE

HEXOKINASE

Glucose 6-

H₂O

ADP

phosphate

Glycogen

AMP

Fructose 6-

ATP

phosphate

FRUCTOSE-1,6-

PHOSPHOFRUCTOKINASE

BISPHOSPHATASE

Fructose 1,6-

H₂O

ADP

bisphosphate

Fructose

cAMP

2,6-bisphosphate

(glucagon)

Fructose

2,6-bisphosphate

Glyceraldehyde 3-phosphate

Dihydroxyacetone phosphate

NAD+

NADH + H⁺

GLYCEROL 3-PHOSPHATE

NADH + H

DEHYDROGENASE

cAMP

(glucagon)

1,3-Bisphosphoglycerate

NAD⁺

ADP

Glycerol 3-phosphate

ADP

ATP

GLYCEROL KINASE

3-Phosphoglycerate

ATP

Glycerol

2-Phosphoglycerate

cAMP

(glucagon)

Phosphoenolpyruvate

ADP

PYRUVATE KINASE

Alanine

GDP + CO

Fatty

ATP

acids

PHOSPHOENOLPYRUVATE

Pyruvate

Lactate

CARBOXYKINASE

Citrate

GTP

NADH + H⁺ NAD⁺

PYRUVATE

Oxaloacetate

DEHYDROGENASE

Pyruvate

Acetyl-CoA

NADH + H⁺

CO₂ + ATP

PYRUVATE CARBOXYLASE

NAD⁺

ADP + P

NADH + H⁺

Oxaloacetate

NAD⁺

Malate

Citrate

Citric acid cycle

α-Ketoglutarate

Fumarate

Succinyl-CoA

Propionate

Figure 19-1. Major pathways and regulation of gluconeogenesis and glycolysis in the liver. Entry

points of glucogenic amino acids after transamination are indicated by arrows extended from circles.

(See also Figure 16-4.) The key gluconeogenic enzymes are enclosed in double-bordered boxes. The

ATP required for gluconeogenesis is supplied by the oxidation of long-chain fatty acids. Propionate is

of quantitative importance only in ruminants. Arrows with wavy shafts signify allosteric effects; dash-

shafted arrows, covalent modification by reversible phosphorylation. High concentrations of alanine

act as a “gluconeogenic signal” by inhibiting glycolysis at the pyruvate kinase step.

154

GLUCONEOGENESIS & CONTROL OF THE BLOOD GLUCOSE

155

D. GLUCOSE 1-PHOSPHATE & GLYCOGEN

phosphate by NAD⁺ catalyzed by glycerol-3-phos-

The breakdown of glycogen to glucose 1-phosphate is

phate dehydrogenase.

catalyzed by phosphorylase. Glycogen synthesis in-

volves a different pathway via uridine diphosphate glu-

SINCE GLYCOLYSIS & GLUCONEOGENESIS

cose and glycogen synthase (Figure 18-1).

The relationships between gluconeogenesis and the

SHARE THE SAME PATHWAY BUT IN

glycolytic pathway are shown in Figure 19-1. After

OPPOSITE DIRECTIONS, THEY MUST

transamination or deamination, glucogenic amino acids

BE REGULATED RECIPROCALLY

yield either pyruvate or intermediates of the citric acid

Changes in the availability of substrates are responsible

cycle. Therefore, the reactions described above can ac-

for most changes in metabolism either directly or indi-

count for the conversion of both glucogenic amino

rectly acting via changes in hormone secretion. Three

acids and lactate to glucose or glycogen. Propionate is a

mechanisms are responsible for regulating the activity

major source of glucose in ruminants and enters gluco-

of enzymes in carbohydrate metabolism: (1) changes in

neogenesis via the citric acid cycle. Propionate is esteri-

the rate of enzyme synthesis, (2) covalent modification

fied with CoA, then propionyl-CoA, is carboxylated to

by reversible phosphorylation, and (3) allosteric effects.

D-methylmalonyl-CoA, catalyzed by propionyl-CoA

carboxylase, a biotin-dependent enzyme (Figure 19-2).

Methylmalonyl-CoA racemase catalyzes the conver-

Induction & Repression of Key Enzyme

sion of D-methylmalonyl-CoA to L-methylmalonyl-

Synthesis Requires Several Hours

CoA, which then undergoes isomerization to succinyl-

CoA catalyzed by methylmalonyl-CoA isomerase.

The changes in enzyme activity in the liver that occur

This enzyme requires vitamin B₁₂ as a coenzyme, and

under various metabolic conditions are listed in Table

deficiency of this vitamin results in the excretion of

19-1. The enzymes involved catalyze nonequilibrium

methylmalonate (methylmalonic aciduria).

(physiologically irreversible) reactions. The effects are

C₁₅ and C₁₇ fatty acids are found particularly in the

generally reinforced because the activity of the enzymes

lipids of ruminants. Dietary odd-carbon fatty acids

catalyzing the changes in the opposite direction varies

upon oxidation yield propionate (Chapter 22), which is

reciprocally (Figure 19-1). The enzymes involved in

a substrate for gluconeogenesis in human liver.

the utilization of glucose (ie, those of glycolysis and li-

Glycerol is released from adipose tissue as a result of

pogenesis) all become more active when there is a su-

lipolysis, and only tissues such as liver and kidney that

perfluity of glucose, and under these conditions the en-

possess glycerol kinase, which catalyzes the conversion

zymes responsible for gluconeogenesis all have low

of glycerol to glycerol 3-phosphate, can utilize it. Glyc-

activity. The secretion of insulin, in response to in-

erol 3-phosphate may be oxidized to dihydroxyacetone

creased blood glucose, enhances the synthesis of the key

CoA SH

CO₂ + H₂O

ACYL-CoA

PROPIONYL-CoA

CH₃

SYNTHETASE

CH₃

CARBOXYLASE

CH₃

CH₂

COO^-

Mg2+

Biotin

COO^-

S CoA

ATP

AMP + PP

ATP

ADP + P

Propionate

Propionyl-CoA

D-Methyl-

malonyl-CoA

METHYLMALONYL-CoA

RACEMASE

COO^-

METHYLMALONYL-

CoA ISOMERASE

CH₂

Intermediates

^-OOC

C H

of citric acid cycle

CH₂

B₁₂

coenzyme

CO S CoA

S CoA

L-Methyl-

Succinyl-CoA

malonyl-CoA

Figure 19-2. Metabolism of propionate.

156

CHAPTER 19

Table 19-1. Regulatory and adaptive enzymes of the rat (mainly liver).

Activity In

Carbo-

Starva-

hydrate

tion and

Feeding

Diabetes

Inducer

Repressor

Activator

Inhibitor

Enzymes of glycogenesis, glycolysis, and pyruvate oxidation

Glycogen synthase

↑

↓

Insulin

Glucagon (cAMP) phos-

system

Glucose 6-

phorylase, glycogen

phosphate¹

Hexokinase

Glucose 6-phosphate¹

Glucokinase

↑

↓

Insulin

Glucagon

(cAMP)

Phosphofructokinase-1

↑

↓

Insulin

Glucagon

AMP, fructose 6-

Citrate (fatty acids, ketone

(cAMP)

phosphate, P_i, fruc-

bodies),¹ATP,¹ glucagon

tose 2,6-bisphos-

(cAMP)

phate¹

Pyruvate kinase

↑

↓

Insulin, fructose

Glucagon

Fructose 1,6-

ATP, alanine, glucagon

(cAMP)

bisphosphate¹, in-

(cAMP), epinephrine

sulin

Pyruvate dehydro-

↑

↓

CoA, NAD⁺, insu-

Acetyl-CoA, NADH, ATP

genase

lin,² ADP, pyruvate

(fatty acids, ketone bodies)

Enzymes of gluconeogenesis

Pyruvate carboxylase

↓

↑

Glucocorticoids,

Insulin

Acetyl-CoA¹

ADP¹

glucagon, epi-

nephrine (cAMP)

Phosphoenolpyruvate

↓

↑

Glucocorticoids,

Insulin

Glucagon?

carboxykinase

glucagon, epi-

nephrine (cAMP)

Fructose-1,6-

↓

↑

Glucocorticoids,

Insulin

Glucagon (cAMP)

Fructose 1,6- bisphosphate,

bisphosphatase

glucagon, epi-

AMP, fructose 2,6-bisphos-

nephrine (cAMP)

phate¹

Glucose-6-phosphatase

↓

↑

Glucocorticoids,

Insulin

glucagon, epi-

nephrine (cAMP)

Enzymes of the pentose phosphate pathway and lipogenesis

Glucose-6-phosphate

↑

↓

Insulin

dehydrogenase

6-Phosphogluconate

↑

↓

Insulin

dehydrogenase

“Malic enzyme”

↑

↓

Insulin

ATP-citrate lyase

↑

↓

Insulin

Acetyl-CoA carboxylase

↑

↓

Insulin?

Citrate,¹ insulin

Long-chain acyl-CoA, cAMP,

glucagon

Fatty acid synthase

↑

↓

Insulin?

¹Allosteric.

²In adipose tissue but not in liver.

GLUCONEOGENESIS & CONTROL OF THE BLOOD GLUCOSE

157

enzymes in glycolysis. Likewise, it antagonizes the effect

presence of adenylyl kinase in liver and many other

of the glucocorticoids and glucagon-stimulated cAMP,

tissues allows rapid equilibration of the reaction:

which induce synthesis of the key enzymes responsible

for gluconeogenesis.

ATP + AMP ↔ 2ADP

Both dehydrogenases of the pentose phosphate

pathway can be classified as adaptive enzymes, since

they increase in activity in the well-fed animal and

Thus, when ATP is used in energy-requiring processes

when insulin is given to a diabetic animal. Activity is

resulting in formation of ADP, [AMP] increases. As

low in diabetes or starvation.

“Malic enzyme” and

[ATP] may be 50 times [AMP] at equilibrium, a small

ATP-citrate lyase behave similarly, indicating that these

fractional decrease in [ATP] will cause a severalfold in-

two enzymes are involved in lipogenesis rather than

crease in [AMP]. Thus, a large change in [AMP] acts as

gluconeogenesis (Chapter 21).

a metabolic amplifier of a small change in [ATP]. This

mechanism allows the activity of phosphofructokinase-1

to be highly sensitive to even small changes in energy

Covalent Modification by Reversible

status of the cell and to control the quantity of carbohy-

Phosphorylation Is Rapid

drate undergoing glycolysis prior to its entry into the

citric acid cycle. The increase in [AMP] can also explain

Glucagon, and to a lesser extent epinephrine, hor-

why glycolysis is increased during hypoxia when [ATP]

mones that are responsive to decreases in blood glucose,

decreases. Simultaneously, AMP activates phosphory-

inhibit glycolysis and stimulate gluconeogenesis in the

lase, increasing glycogenolysis. The inhibition of phos-

liver by increasing the concentration of cAMP. This in

phofructokinase-1 by citrate and ATP is another expla-

turn activates cAMP-dependent protein kinase, leading

nation of the sparing action of fatty acid oxidation on

to the phosphorylation and inactivation of pyruvate

glucose oxidation and also of the Pasteur effect,

kinase. They also affect the concentration of fructose

whereby aerobic oxidation (via the citric acid cycle) in-

2,6-bisphosphate and therefore glycolysis and gluco-

hibits the anaerobic degradation of glucose. A conse-

neogenesis, as explained below.

quence of the inhibition of phosphofructokinase-1 is an

accumulation of glucose 6-phosphate that, in turn, in-

hibits further uptake of glucose in extrahepatic tissues

Allosteric Modification Is Instantaneous

by allosteric inhibition of hexokinase.

In gluconeogenesis, pyruvate carboxylase, which cata-

lyzes the synthesis of oxaloacetate from pyruvate, re-

quires acetyl-CoA as an allosteric activator. The pres-

Fructose 2,6-Bisphosphate Plays a Unique

ence of acetyl-CoA results in a change in the tertiary

Role in the Regulation of Glycolysis &

structure of the protein, lowering the K_m value for bi-

Gluconeogenesis in Liver

carbonate. This means that as acetyl-CoA is formed

from pyruvate, it automatically ensures the provision of

The most potent positive allosteric effector of phospho-

oxaloacetate and, therefore, its further oxidation in the

fructokinase-1 and inhibitor of fructose-1,6-bisphos-

citric acid cycle. The activation of pyruvate carboxylase

phatase in liver is fructose

2,6-bisphosphate. It re-

and the reciprocal inhibition of pyruvate dehydrogen-

lieves inhibition of phosphofructokinase-1 by ATP and

ase by acetyl-CoA derived from the oxidation of fatty

increases affinity for fructose 6-phosphate. It inhibits

acids explains the action of fatty acid oxidation in spar-

fructose-1,6-bisphosphatase by increasing the K_m for

ing the oxidation of pyruvate and in stimulating gluco-

fructose 1,6-bisphosphate. Its concentration is under

neogenesis. The reciprocal relationship between these

both substrate (allosteric) and hormonal control (cova-

two enzymes in both liver and kidney alters the meta-

lent modification) (Figure 19-3).

bolic fate of pyruvate as the tissue changes from carbo-

Fructose 2,6-bisphosphate is formed by phosphory-

hydrate oxidation, via glycolysis, to gluconeogenesis

lation of fructose

6-phosphate by phosphofructoki-

during transition from a fed to a starved state (Figure

nase-2. The same enzyme protein is also responsible for

19-1). A major role of fatty acid oxidation in promot-

its breakdown, since it has fructose-2,6-bisphos-

ing gluconeogenesis is to supply the requirement for

phatase activity. This bifunctional enzyme is under

ATP. Phosphofructokinase (phosphofructokinase-1)

the allosteric control of fructose

6-phosphate, which

occupies a key position in regulating glycolysis and is

stimulates the kinase and inhibits the phosphatase.

also subject to feedback control. It is inhibited by cit-

Hence, when glucose is abundant, the concentration of

rate and by ATP and is activated by 5′-AMP. 5′-AMP

fructose 2,6-bisphosphate increases, stimulating glycol-

acts as an indicator of the energy status of the cell. The

ysis by activating phosphofructokinase-1 and inhibiting

158

CHAPTER 19

Glycogen

Substrate (Futile) Cycles Allow Fine Tuning

Glucose

It will be apparent that the control points in glycolysis

and glycogen metabolism involve a cycle of phosphory-

Fructose 6-phosphate

lation and dephosphorylation catalyzed by: glucokinase

Glucagon

and glucose-6-phosphatase; phosphofructokinase-1 and

fructose-1,6-bisphosphatase; pyruvate kinase, pyruvate

cAMP

carboxylase, and phosphoenolypyruvate carboxykinase;

P_i

and glycogen synthase and phosphorylase. If these were

cAMP-DEPENDENT

allowed to cycle unchecked, they would amount to fu-

PROTEIN KINASE

tile cycles whose net result was hydrolysis of ATP. This

does not occur extensively due to the various control

ADP

ATP

mechanisms, which ensure that one reaction is inhib-

ited as the other is stimulated. However, there is a phys-

iologic advantage in allowing some cycling. The rate of

Active

Inactive

net glycolysis may increase several thousand-fold in re-

F-2,6-Pase

sponse to stimulation, and this is more readily achieved

Inactive

Active

PFK-2

by both increasing the activity of phosphofructokinase

and decreasing that of fructose bisphosphatase if both

are active, than by switching one enzyme “on” and the

H₂O

P_i

other “off” completely. This “fine tuning” of metabolic

control occurs at the expense of some loss of ATP.

PROTEIN

ADP

PHOSPHATASE-2

Citrate

THE CONCENTRATION OF BLOOD

Fructose 2,6-bisphosphate

P_i

ATP

GLUCOSE IS REGULATED WITHIN

NARROW LIMITS

F-1,6-Pase

PFK-1

In the postabsorptive state, the concentration of blood

H₂O

ADP

glucose in most mammals is maintained between 4.5

and 5.5 mmol/L. After the ingestion of a carbohydrate

meal, it may rise to 6.5-7.2 mmol/L, and in starvation,

Fructose 1,6-bisphosphate

it may fall to 3.3-3.9 mmol/L. A sudden decrease in

blood glucose will cause convulsions, as in insulin over-

Pyruvate

dose, owing to the immediate dependence of the brain

on a supply of glucose. However, much lower concen-

Figure 19-3. Control of glycolysis and gluconeoge-

trations can be tolerated, provided progressive adapta-

nesis in the liver by fructose 2,6-bisphosphate and the

tion is allowed. The blood glucose level in birds is con-

bifunctional enzyme PFK-2/F-2,6-Pase (6-phospho-

siderably higher

(14.0 mmol/L) and in ruminants

fructo-2-kinase/fructose-2,6-bisphosphatase). (PFK-1,

considerably lower

(approximately

2.2 mmol/L in

phosphofructokinase-1 [6-phosphofructo-1-kinase];

sheep and 3.3 mmol/L in cattle). These lower normal

F-1,6-Pase, fructose-1,6-bisphosphatase. Arrows with

levels appear to be associated with the fact that rumi-

wavy shafts indicate allosteric effects.)

nants ferment virtually all dietary carbohydrate to lower

(volatile) fatty acids, and these largely replace glucose as

the main metabolic fuel of the tissues in the fed condi-

fructose-1,6-bisphosphatase. When glucose is short,

tion.

glucagon stimulates the production of cAMP, activat-

ing cAMP-dependent protein kinase, which in turn in-

activates phosphofructokinase-2 and activates fructose

BLOOD GLUCOSE IS DERIVED FROM

2,6-bisphosphatase by phosphorylation. Therefore, glu-

THE DIET, GLUCONEOGENESIS,

coneogenesis is stimulated by a decrease in the concen-

& GLYCOGENOLYSIS

tration of fructose 2,6-bisphosphate, which deactivates

phosphofructokinase-1 and deinhibits fructose-1,6-bis-

The digestible dietary carbohydrates yield glucose,

phosphatase. This mechanism also ensures that glu-

galactose, and fructose that are transported via the

cagon stimulation of glycogenolysis in liver results in

hepatic portal vein to the liver where galactose and

glucose release rather than glycolysis.

fructose are readily converted to glucose (Chapter 20).

GLUCONEOGENESIS & CONTROL OF THE BLOOD GLUCOSE

159

Glucose is formed from two groups of compounds

Metabolic & Hormonal Mechanisms

that undergo gluconeogenesis (Figures 16-4 and 19-1):

Regulate the Concentration

(1) those which involve a direct net conversion to glu-

of the Blood Glucose

cose without significant recycling, such as some amino

The maintenance of stable levels of glucose in the blood

acids and propionate; and (2) those which are the

products of the metabolism of glucose in tissues. Thus,

is one of the most finely regulated of all homeostatic

mechanisms, involving the liver, extrahepatic tissues,

lactate, formed by glycolysis in skeletal muscle and

erythrocytes, is transported to the liver and kidney

and several hormones. Liver cells are freely permeable

to glucose (via the GLUT 2 transporter), whereas cells

where it re-forms glucose, which again becomes avail-

able via the circulation for oxidation in the tissues. This

of extrahepatic tissues (apart from pancreatic B islets)

are relatively impermeable, and their glucose trans-

process is known as the Cori cycle, or lactic acid cycle

(Figure 19-4). Triacylglycerol glycerol in adipose tissue

porters are regulated by insulin. As a result, uptake

from the bloodstream is the rate-limiting step in the

is derived from blood glucose. This triacylglycerol is

continuously undergoing hydrolysis to form free glyc-

utilization of glucose in extrahepatic tissues. The role of

various glucose transporter proteins found in cell mem-

erol, which cannot be utilized by adipose tissue and is

converted back to glucose by gluconeogenic mecha-

branes, each having

12 transmembrane domains, is

shown in Table 19-2.

nisms in the liver and kidney (Figure 19-1).

Of the amino acids transported from muscle to the

liver during starvation, alanine predominates. The glu-

Glucokinase Is Important in Regulating

cose-alanine cycle

(Figure

19-4) transports glucose

Blood Glucose After a Meal

from liver to muscle with formation of pyruvate, fol-

lowed by transamination to alanine, then transports

Hexokinase has a low K_m for glucose and in the liver is

alanine to the liver, followed by gluconeogenesis back

saturated and acting at a constant rate under all normal

to glucose. A net transfer of amino nitrogen from mus-

conditions. Glucokinase has a considerably higher K_m

cle to liver and of free energy from liver to muscle is ef-

(lower affinity) for glucose, so that its activity increases

fected. The energy required for the hepatic synthesis of

over the physiologic range of glucose concentrations

glucose from pyruvate is derived from the oxidation of

(Figure

19-5). It promotes hepatic uptake of large

fatty acids.

amounts of glucose at the high concentrations found in

Glucose is also formed from liver glycogen by

the hepatic portal vein after a carbohydrate meal. It is

glycogenolysis (Chapter 18).

absent from the liver of ruminants, which have little

BLOOD

Glucose

LIVER

MUSCLE

Glucose 6-phosphate

Glycogen

Glucose 6-phosphate

Urea

Pyruvate

Lactate

Pyruvate

-NH₂

Lactate

BLOOD

Pyruvate

Alanine

Figure 19-4. The lactic acid (Cori) cycle and glucose-alanine cycle.

160

CHAPTER 19

Table 19-2. Glucose transporters.

Tissue Location

Functions

Facilitative bidirectional transporters

GLUT 1

Brain, kidney, colon, placenta, erythrocyte

Uptake of glucose

GLUT 2

Liver, pancreatic B cell, small intestine, kidney

Rapid uptake and release of glucose

GLUT 3

Brain, kidney, placenta

Uptake of glucose

GLUT 4

Heart and skeletal muscle, adipose tissue

Insulin-stimulated uptake of glucose

GLUT 5

Small intestine

Absorption of glucose

Sodium-dependent unidirectional transporter

SGLT 1

Small intestine and kidney

Active uptake of glucose from lumen of intestine and

reabsorption of glucose in proximal tubule of kidney

against a concentration gradient

cose via the GLUT 2 transporter, and the glucose is

glucose entering the portal circulation from the intes-

phosphorylated by glucokinase. Therefore, increasing

tines.

blood glucose increases metabolic flux through glycoly-

At normal systemic-blood glucose concentrations

sis, the citric acid cycle, and the generation of ATP. In-

(4.5-5.5 mmol/L), the liver is a net producer of glu-

crease in

[ATP] inhibits ATP-sensitive K⁺ channels,

cose. However, as the glucose level rises, the output of

causing depolarization of the B cell membrane, which

glucose ceases, and there is a net uptake.

channels,

increases Ca2+ influx via voltage-sensitive Ca2+

Insulin Plays a Central Role in

stimulating exocytosis of insulin. Thus, the concentra-

tion of insulin in the blood parallels that of the blood

Regulating Blood Glucose

glucose. Other substances causing release of insulin from

In addition to the direct effects of hyperglycemia in en-

the pancreas include amino acids, free fatty acids, ketone

hancing the uptake of glucose into the liver, the hor-

bodies, glucagon, secretin, and the sulfonylurea drugs

mone insulin plays a central role in regulating blood glu-

tolbutamide and glyburide. These drugs are used to

cose. It is produced by the B cells of the islets of

stimulate insulin secretion in type 2 diabetes mellitus

Langerhans in the pancreas in response to hyper-

(NIDDM, non-insulin-dependent diabetes mellitus);

glycemia. The B islet cells are freely permeable to glu-

they act by inhibiting the ATP-sensitive K⁺ channels.

Epinephrine and norepinephrine block the release of in-

sulin. Insulin lowers blood glucose immediately by en-

max

100

hancing glucose transport into adipose tissue and muscle

Hexokinase

by recruitment of glucose transporters (GLUT 4) from

the interior of the cell to the plasma membrane. Al-

though it does not affect glucose uptake into the liver

directly, insulin does enhance long-term uptake as a re-

sult of its actions on the enzymes controlling glycolysis,

Glucokinase

glycogenesis, and gluconeogenesis (Chapter 18).

Glucagon Opposes the Actions of Insulin

Glucagon is the hormone produced by the A cells of

the pancreatic islets. Its secretion is stimulated by hypo-

Blood glucose (mmol/L)

glycemia. In the liver, it stimulates glycogenolysis by ac-

tivating phosphorylase. Unlike epinephrine, glucagon

Figure 19-5. Variation in glucose phosphorylating

does not have an effect on muscle phosphorylase.

activity of hexokinase and glucokinase with increase of

Glucagon also enhances gluconeogenesis from amino

blood glucose concentration. The K_m

for glucose of

acids and lactate. In all these actions, glucagon acts via

hexokinase is 0.05 mmol/L and of glucokinase is 10

generation of cAMP

(Table

19-1). Both hepatic

mmol/L.

glycogenolysis and gluconeogenesis contribute to the

GLUCONEOGENESIS & CONTROL OF THE BLOOD GLUCOSE

161

hyperglycemic effect of glucagon, whose actions op-

meals or at night. Furthermore, premature and low-

pose those of insulin. Most of the endogenous glucagon

birth-weight babies are more susceptible to hypo-

(and insulin) is cleared from the circulation by the liver.

glycemia, since they have little adipose tissue to gener-

ate alternative fuels such as free fatty acids or ketone

bodies during the transition from fetal dependency to

Other Hormones Affect Blood Glucose

the free-living state. The enzymes of gluconeogenesis

The anterior pituitary gland secretes hormones that

may not be completely functional at this time, and the

tend to elevate the blood glucose and therefore antago-

process is dependent on a supply of free fatty acids for

nize the action of insulin. These are growth hormone,

energy. Glycerol, which would normally be released

ACTH (corticotropin), and possibly other “diabeto-

from adipose tissue, is less available for gluconeogenesis.

genic” hormones. Growth hormone secretion is stimu-

lated by hypoglycemia; it decreases glucose uptake in

The Body’s Ability to Utilize Glucose

muscle. Some of this effect may not be direct, since it

May Be Ascertained by Measuring Its

stimulates mobilization of free fatty acids from adipose

Glucose Tolerance

tissue, which themselves inhibit glucose utilization. The

Glucose tolerance is the ability to regulate the blood

glucocorticoids (11-oxysteroids) are secreted by the

adrenal cortex and increase gluconeogenesis. This is a

glucose concentration after the administration of a test

dose of glucose (normally 1 g/kg body weight) (Figure

result of enhanced hepatic uptake of amino acids and

increased activity of aminotransferases and key enzymes

19-6). Diabetes mellitus (type 1, or insulin-dependent

diabetes mellitus; IDDM) is characterized by decreased

of gluconeogenesis. In addition, glucocorticoids inhibit

the utilization of glucose in extrahepatic tissues. In all

glucose tolerance due to decreased secretion of insulin

in response to the glucose challenge. Glucose tolerance

these actions, glucocorticoids act in a manner antago-

nistic to insulin.

is also impaired in type 2 diabetes mellitus (NIDDM),

which is often associated with obesity and raised levels

Epinephrine is secreted by the adrenal medulla as a

result of stressful stimuli (fear, excitement, hemorrhage,

of plasma free fatty acids and in conditions where the

liver is damaged; in some infections; and in response to

hypoxia, hypoglycemia, etc) and leads to glycogenolysis

in liver and muscle owing to stimulation of phosphory-

some drugs. Poor glucose tolerance can also be expected

lase via generation of cAMP. In muscle, glycogenolysis

results in increased glycolysis, whereas in liver glucose is

the main product leading to increase in blood glucose.

FURTHER CLINICAL ASPECTS

Glucosuria Occurs When the Renal

Threshold for Glucose Is Exceeded

When the blood glucose rises to relatively high levels,

the kidney also exerts a regulatory effect. Glucose is

continuously filtered by the glomeruli but is normally

completely reabsorbed in the renal tubules by active

transport. The capacity of the tubular system to reab-

sorb glucose is limited to a rate of about 350 mg/min,

and in hyperglycemia (as occurs in poorly controlled di-

abetes mellitus) the glomerular filtrate may contain

more glucose than can be reabsorbed, resulting in glu-

cosuria. Glucosuria occurs when the venous blood glu-

cose concentration exceeds 9.5-10.0 mmol/L; this is

termed the renal threshold for glucose.

Time (h)

Hypoglycemia May Occur During

Figure 19-6. Glucose tolerance test. Blood glucose

Pregnancy & in the Neonate

curves of a normal and a diabetic individual after oral

During pregnancy, fetal glucose consumption increases

administration of 50 g of glucose. Note the initial raised

and there is a risk of maternal and possibly fetal hypo-

concentration in the diabetic. A criterion of normality is

glycemia, particularly if there are long intervals between

the return of the curve to the initial value within 2 hours.

162

CHAPTER 19

due to hyperactivity of the pituitary or adrenal cortex

• Insulin is secreted as a direct response to hyper-

because of the antagonism of the hormones secreted by

glycemia; it stimulates the liver to store glucose as

these glands to the action of insulin.

glycogen and facilitates uptake of glucose into extra-

Administration of insulin (as in the treatment of di-

hepatic tissues.

abetes mellitus type 1) lowers the blood glucose and in-

• Glucagon is secreted as a response to hypoglycemia

creases its utilization and storage in the liver and muscle

and activates both glycogenolysis and gluconeogene-

as glycogen. An excess of insulin may cause hypo-

sis in the liver, causing release of glucose into the

glycemia, resulting in convulsions and even in death

blood.

unless glucose is administered promptly. Increased tol-

erance to glucose is observed in pituitary or adrenocor-

tical insufficiency—attributable to a decrease in the an-

tagonism to insulin by the hormones normally secreted

REFERENCES

by these glands.

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molecular regulation. Recent Prog Horm Res 1991;47:349.

SUMMARY

Krebs HA: Gluconeogenesis. Proc R Soc London (Biol) 1964;

159:545.

• Gluconeogenesis is the process of converting noncar-

Lenzen S: Hexose recognition mechanisms in pancreatic B-cells.

bohydrates to glucose or glycogen. It is of particular

Biochem Soc Trans 1990;18:105.

importance when carbohydrate is not available from

Newgard CB, McGarry JD: Metabolic coupling factors in pancre-

the diet. Significant substrates are amino acids, lac-

atic beta-cell signal transduction. Annu Rev Biochem 1995;

tate, glycerol, and propionate.

64:689.

• The pathway of gluconeogenesis in the liver and kid-

Newsholme EA, Start C: Regulation in Metabolism. Wiley, 1973.

ney utilizes those reactions in glycolysis which are re-

Nordlie RC, Foster JD, Lange AJ: Regulation of glucose produc-

versible plus four additional reactions that circum-

tion by the liver. Annu Rev Nutr 1999;19:379.

vent the irreversible nonequilibrium reactions.

Pilkis SJ, El-Maghrabi MR, Claus TH: Hormonal regulation of he-

patic gluconeogenesis and glycolysis. Annu Rev Biochem

• Since glycolysis and gluconeogenesis share the same

1988;57:755.

pathway but operate in opposite directions, their ac-

Pilkis SJ, Granner DK: Molecular physiology of the regulation of

tivities are regulated reciprocally.

hepatic gluconeogenesis and glycolysis. Annu Rev Physiol

• The liver regulates the blood glucose after a meal be-

1992;54:885.

cause it contains the high-K_m glucokinase that pro-

Yki-Jarvinen H: Action of insulin on glucose metabolism in vivo.

motes increased hepatic utilization of glucose.

Baillieres Clin Endocrinol Metab 1993;7:903.

The Pentose Phosphate

Pathway & Other Pathways

of Hexose Metabolism

Peter A. Mayes, PhD, DSc, & David A. Bender, PhD

BIOMEDICAL IMPORTANCE

REACTIONS OF THE PENTOSE

PHOSPHATE PATHWAY OCCUR

The pentose phosphate pathway is an alternative route

for the metabolism of glucose. It does not generate

IN THE CYTOSOL

ATP but has two major functions: (1) The formation of

The enzymes of the pentose phosphate pathway, as of

NADPH for synthesis of fatty acids and steroids and

glycolysis, are cytosolic. As in glycolysis, oxidation

(2) the synthesis of ribose for nucleotide and nucleic

is achieved by dehydrogenation; but NADP⁺ and not

acid formation. Glucose, fructose, and galactose are the

NAD⁺ is the hydrogen acceptor. The sequence of reac-

main hexoses absorbed from the gastrointestinal tract,

tions of the pathway may be divided into two phases: an

derived principally from dietary starch, sucrose, and

oxidative nonreversible phase and a nonoxidative re-

lactose, respectively. Fructose and galactose are con-

versible phase. In the first phase, glucose 6-phosphate

verted to glucose, mainly in the liver.

undergoes dehydrogenation and decarboxylation to yield

Genetic deficiency of glucose 6-phosphate dehydro-

a pentose, ribulose 5-phosphate. In the second phase,

genase, the first enzyme of the pentose phosphate path-

ribulose 5-phosphate is converted back to glucose 6-phos-

way, is a major cause of hemolysis of red blood cells, re-

phate by a series of reactions involving mainly two en-

sulting in hemolytic anemia and affecting approximately

zymes: transketolase and transaldolase (Figure 20-1).

100 million people worldwide. Glucuronic acid is synthe-

sized from glucose via the uronic acid pathway, of major

The Oxidative Phase Generates NADPH

significance for the excretion of metabolites and foreign

chemicals (xenobiotics) as glucuronides. A deficiency in

(Figures 20-1 and 20-2)

the pathway leads to essential pentosuria. The lack of

Dehydrogenation of glucose 6-phosphate to 6-phos-

one enzyme of the pathway (gulonolactone oxidase) in

phogluconate occurs via the formation of 6-phospho-

primates and some other animals explains why ascorbic

gluconolactone, catalyzed by glucose-6-phosphate

acid (vitamin C) is a dietary requirement for humans but

dehydrogenase, an NADP-dependent enzyme. The

not most other mammals. Deficiencies in the enzymes of

hydrolysis of 6-phosphogluconolactone is accomplished

fructose and galactose metabolism lead to essential fruc-

by the enzyme gluconolactone hydrolase. A second

tosuria and the galactosemias.

oxidative step is catalyzed by 6-phosphogluconate de-

hydrogenase, which also requires NADP⁺ as hydrogen

THE PENTOSE PHOSPHATE PATHWAY

acceptor and involves decarboxylation followed by for-

GENERATES NADPH & RIBOSE

mation of the ketopentose, ribulose 5-phosphate.

PHOSPHATE (Figure 20-1)

The Nonoxidative Phase Generates

The pentose phosphate pathway (hexose monophos-

Ribose Precursors

phate shunt) is a more complex pathway than glycoly-

sis. Three molecules of glucose 6-phosphate give rise to

Ribulose 5-phosphate is the substrate for two enzymes.

three molecules of CO₂ and three five-carbon sugars.

Ribulose 5-phosphate 3-epimerase alters the configu-

These are rearranged to regenerate two molecules of

ration about carbon 3, forming another ketopentose,

glucose 6-phosphate and one molecule of the glycolytic

xylulose 5-phosphate. Ribose 5-phosphate ketoisom-

intermediate, glyceraldehyde

3-phosphate. Since two

erase converts ribulose 5-phosphate to the correspond-

molecules of glyceraldehyde 3-phosphate can regenerate

ing aldopentose, ribose 5-phosphate, which is the pre-

glucose 6-phosphate, the pathway can account for the

cursor of the ribose required for nucleotide and nucleic

complete oxidation of glucose.

acid synthesis. Transketolase transfers the two-carbon

163

164

CHAPTER 20

Glucose 6-phosphate

C₆

NADP⁺ + H₂O

GLUCOSE-6-PHOSPHATE

DEHYDROGENASE

NADPH + H⁺

6-Phosphogluconate

C₆

NADP⁺

6-PHOSPHO-

GLUCONATE

DEHYDROGENASE

NADPH + H⁺

CO₂

Ribulose 5-phosphate

C₅

3-EPIMERASE

KETO-ISOMERASE

3-EPIMERASE

Xylulose 5-phosphate

Ribose 5-phosphate

Xylulose 5-phosphate

C₅

TRANSKETOLASE

Synthesis of

nucleotides,

RNA, DNA

Glyceraldehyde 3-phosphate

Sedoheptulose 7-phosphate

C₃

C₇

TRANSALDOLASE

Fructose 6-phosphate

Erythrose 4-phosphate

C₆

C₄

TRANSKETOLASE

Fructose 6-phosphate

Glyceraldehyde 3-phosphate

C₆

C₃

PHOSPHOTRIOSE

ALDOLASE

ISOMERASE

PHOSPHOHEXOSE

ISOMERASE

¹/2 Fructose 1,6-bisphosphate

C₆

FRUCTOSE-1,6-

BISPHOSPHATASE

¹/2 Fructose 6-phosphate

C₆

PHOSPHOHEXOSE

ISOMERASE

Glucose 6-phosphate

¹/2 Glucose 6-phosphate

C₆

Figure 20-1. Flow chart of pentose phosphate pathway and its connections with the pathway

of glycolysis. The full pathway, as indicated, consists of three interconnected cycles in which glu-

cose 6-phosphate is both substrate and end product. The reactions above the broken line are

nonreversible, whereas all reactions under that line are freely reversible apart from that catalyzed

by fructose-1,6-bisphosphatase.

THE PENTOSE PHOSPHATE PATHWAY & OTHER PATHWAYS OF HEXOSE METABOLISM

165

NADP⁺ NADPH + H⁺

H₂O

COO-

Mg2+, Mn2+,

or Ca2+

GLUCOSE-6-PHOSPHATE

GLUCONOLACTONE

DEHYDROGENASE

HYDROLASE

CH₂

O P

CH₂

O P

CH₂

O P

β-D-Glucose 6-phosphate

6-Phosphogluconolactone

6-Phosphogluconate

NADP⁺

6-PHOSPHOGLUCONATE

Mg2+, Mn2+,

DEHYDROGENASE

or Ca2+

NADP⁺+ H⁺

COO-

CHOH

CH₂OH

RIBOSE 5-PHOSPHATE

KETOISOMERASE

C O

CO₂

CH₂

O P

CH₂

O P

CH₂

O P

Enediol form

Ribulose 5-phosphate

3-Keto 6-phosphogluconate

RIBULOSE 5-PHOSPHATE

3-EPIMERASE

CH₂OH

C O

*CH2

O P

Xylulose 5-phosphate

CH₂

O P

CH₂

O P

Ribose 5-phosphate

Sedoheptulose 7-phosphate

ATP

TRANSKETOLASE

PRPP

Mg2+

Thiamin- P

SYNTHETASE

AMP

Mg2+

CH₂OH

O P

*CH2

O P

C O

Glyceraldehyde 3-phosphate

TRANSALDOLASE

CH₂

O P

H C

*CH2

PRPP

Fructose 6-phosphate

CH₂

O P

Erythrose 4-phosphate

CH₂OH

C O

TRANSKETOLASE

Thiamin- P₂

H C O

CH₂

O P

CH₂

O P

CH₂

O P

Xylulose 5-phosphate

Glyceraldehyde 3-phosphate

Fructose 6-phosphate

Figure 20-2. The pentose phosphate pathway. (

,  PO_32-; PRPP, 5-phosphoribosyl 1-pyrophosphate.)

166

CHAPTER 20

unit comprising carbons 1 and 2 of a ketose onto the

Ribose Can Be Synthesized in Virtually

aldehyde carbon of an aldose sugar. It therefore effects

All Tissues

the conversion of a ketose sugar into an aldose with two

Little or no ribose circulates in the bloodstream, so tis-

carbons less and simultaneously converts an aldose sugar

sues must synthesize the ribose required for nucleotide

into a ketose with two carbons more. The reaction re-

and nucleic acid synthesis (Chapter 34). The source of

quires Mg2+ and thiamin diphosphate (vitamin B₁) as

ribose 5-phosphate is the pentose phosphate pathway

coenzyme. Thus, transketolase catalyzes the transfer of

(Figure 20-2). Muscle has only low activity of glucose-

the two-carbon unit from xylulose 5-phosphate to ribose

6-phosphate dehydrogenase and

6-phosphogluconate

5-phosphate, producing the seven-carbon ketose sedo-

dehydrogenase. Nevertheless, like most other tissues, it

heptulose 7-phosphate and the aldose glyceraldehyde

is capable of synthesizing ribose 5-phosphate by reversal

3-phosphate. Transaldolase allows the transfer of a

of the nonoxidative phase of the pentose phosphate

three-carbon dihydroxyacetone moiety

(carbons

1-3)

pathway utilizing fructose 6-phosphate. It is not neces-

from the ketose sedoheptulose 7-phosphate onto the al-

sary to have a completely functioning pentose phosphate

dose glyceraldehyde

3-phosphate to form the ketose

pathway for a tissue to synthesize ribose phosphates.

fructose 6-phosphate and the four-carbon aldose erythrose

4-phosphate. In a further reaction catalyzed by transke-

tolase, xylulose 5-phosphate donates a two-carbon unit

THE PENTOSE PHOSPHATE PATHWAY

to erythrose 4-phosphate to form fructose 6-phosphate

& GLUTATHIONE PEROXIDASE PROTECT

and glyceraldehyde 3-phosphate.

ERYTHROCYTES AGAINST HEMOLYSIS

In order to oxidize glucose completely to CO₂ via

the pentose phosphate pathway, there must be enzymes

In erythrocytes, the pentose phosphate pathway pro-

present in the tissue to convert glyceraldehyde 3-phos-

vides NADPH for the reduction of oxidized glu-

phate to glucose 6-phosphate. This involves reversal of

tathione catalyzed by glutathione reductase, a flavo-

glycolysis and the gluconeogenic enzyme fructose 1,6-

protein containing FAD. Reduced glutathione removes

bisphosphatase. In tissues that lack this enzyme, glyc-

H₂O₂ in a reaction catalyzed by glutathione peroxi-

eraldehyde 3-phosphate follows the normal pathway of

dase, an enzyme that contains the selenium analogue

glycolysis to pyruvate.

of cysteine (selenocysteine) at the active site

(Figure

20-3). This reaction is important, since accumulation

of H₂O₂ may decrease the life span of the erythrocyte

The Two Major Pathways for the

by causing oxidative damage to the cell membrane,

Catabolism of Glucose Have

leading to hemolysis.

Little in Common

Although glucose

6-phosphate is common to both

GLUCURONATE, A PRECURSOR OF

pathways, the pentose phosphate pathway is markedly

PROTEOGLYCANS & CONJUGATED

different from glycolysis. Oxidation utilizes NADP

GLUCURONIDES, IS A PRODUCT OF

rather than NAD, and CO₂, which is not produced in

THE URONIC ACID PATHWAY

glycolysis, is a characteristic product. No ATP is gener-

ated in the pentose phosphate pathway, whereas ATP is

In liver, the uronic acid pathway catalyzes the conver-

a major product of glycolysis.

sion of glucose to glucuronic acid, ascorbic acid, and

pentoses (Figure 20-4). It is also an alternative oxidative

pathway for glucose, but—like the pentose phosphate

Reducing Equivalents Are Generated

pathway—it does not lead to the generation of ATP.

in Those Tissues Specializing

Glucose 6-phosphate is isomerized to glucose 1-phos-

in Reductive Syntheses

phate, which then reacts with uridine triphosphate

The pentose phosphate pathway is active in liver, adipose

(UTP) to form uridine diphosphate glucose (UDPGlc)

tissue, adrenal cortex, thyroid, erythrocytes, testis, and

in a reaction catalyzed by UDPGlc pyrophosphorylase,

lactating mammary gland. Its activity is low in nonlactat-

as occurs in glycogen synthesis (Chapter 18). UDPGlc is

ing mammary gland and skeletal muscle. Those tissues in

oxidized at carbon 6 by NAD-dependent UDPGlc de-

which the pathway is active use NADPH in reductive

hydrogenase in a two-step reaction to yield UDP-glu-

syntheses, eg, of fatty acids, steroids, amino acids via glu-

curonate. UDP-glucuronate is the “active” form of glu-

tamate dehydrogenase, and reduced glutathione. The

curonate for reactions involving incorporation of

synthesis of glucose-6-phosphate dehydrogenase and

glucuronic acid into proteoglycans or for reactions in

6-phosphogluconate dehydrogenase may also be induced

which substrates such as steroid hormones, bilirubin, and

by insulin during conditions associated with the “fed

a number of drugs are conjugated with glucuronate for

state” (Table 19-1), when lipogenesis increases.

excretion in urine or bile (Figure 32-14).

THE PENTOSE PHOSPHATE PATHWAY & OTHER PATHWAYS OF HEXOSE METABOLISM

167

NADPH + H⁺

S S

2H₂O

PENTOSE

GLUTATHIONE

PHOSPHATE

FAD

REDUCTASE

PEROXIDASE

PATHWAY

NADP⁺

2G SH

H₂O₂

Figure 20-3. Role of the pentose phosphate pathway in the glutathione peroxidase re-

action of erythrocytes. (G-S-S-G, oxidized glutathione; G-SH, reduced glutathione; Se, sele-

nium cofactor.)

Glucuronate is reduced to L-gulonate in an NADPH-

tose. However, glucose inhibits the phosphorylation of

dependent reaction; L-gulonate is the direct precursor of

fructose since it is a better substrate for hexokinase.

ascorbate in those animals capable of synthesizing this

Nevertheless, some fructose can be metabolized in adi-

vitamin. In humans and other primates as well as guinea

pose tissue and muscle. Fructose, a potential fuel, is

pigs, ascorbic acid cannot be synthesized because of the

found in seminal plasma and in the fetal circulation of

absence of L-gulonolactone oxidase. L-Gulonate is me-

ungulates and whales. Aldose reductase is found in the

tabolized ultimately to D-xylulose 5-phosphate, a con-

placenta of the ewe and is responsible for the secretion

stituent of the pentose phosphate pathway.

of sorbitol into the fetal blood. The presence of sor-

bitol dehydrogenase in the liver, including the fetal

liver, is responsible for the conversion of sorbitol into

INGESTION OF LARGE QUANTITIES

fructose. This pathway is also responsible for the occur-

OF FRUCTOSE HAS PROFOUND

rence of fructose in seminal fluid.

METABOLIC CONSEQUENCES

Diets high in sucrose or in high-fructose syrups used in

manufactured foods and beverages lead to large amounts

GALACTOSE IS NEEDED FOR THE

of fructose (and glucose) entering the hepatic portal vein.

SYNTHESIS OF LACTOSE, GLYCOLIPIDS,

Fructose undergoes more rapid glycolysis in the liver than

PROTEOGLYCANS, & GLYCOPROTEINS

does glucose because it bypasses the regulatory step cat-

alyzed by phosphofructokinase (Figure 20-5). This allows

Galactose is derived from intestinal hydrolysis of the

fructose to flood the pathways in the liver, leading to en-

disaccharide lactose, the sugar of milk. It is readily con-

hanced fatty acid synthesis, increased esterification of fatty

verted in the liver to glucose. Galactokinase catalyzes

acids, and increased VLDL secretion, which may raise

the phosphorylation of galactose, using ATP as phos-

serum triacylglycerols and ultimately raise LDL choles-

phate donor (Figure 20-6A). Galactose 1-phosphate re-

terol concentrations

(Figure

25-6). A specific kinase,

acts with uridine diphosphate glucose (UDPGlc) to

fructokinase, in liver (and kidney and intestine) catalyzes

form uridine diphosphate galactose (UDPGal) and glu-

the phosphorylation of fructose to fructose 1-phosphate.

cose 1-phosphate, in a reaction catalyzed by galactose

This enzyme does not act on glucose, and, unlike glucoki-

1-phosphate uridyl transferase. The conversion of

nase, its activity is not affected by fasting or by insulin,

UDPGal to UDPGlc is catalyzed by UDPGal 4-epim-

which may explain why fructose is cleared from the blood

erase. Epimerization involves an oxidation and reduc-

of diabetic patients at a normal rate. Fructose 1-phos-

tion at carbon 4 with NAD⁺ as coenzyme. Finally, glu-

phate is cleaved to D-glyceraldehyde and dihydroxyace-

cose is liberated from UDPGlc after conversion to

tone phosphate by aldolase B, an enzyme found in the

glucose 1-phosphate, probably via incorporation into

liver, which also functions in glycolysis by cleaving fruc-

glycogen followed by phosphorolysis (Chapter 18).

tose 1,6-bisphosphate. D-Glyceraldehyde enters glycolysis

Since the epimerase reaction is freely reversible, glu-

via phosphorylation to glyceraldehyde 3-phosphate, cat-

cose can be converted to galactose, so that galactose is

alyzed by triokinase. The two triose phosphates, dihy-

not a dietary essential. Galactose is required in the body

droxyacetone phosphate and glyceraldehyde 3-phosphate,

not only in the formation of lactose but also as a con-

may be degraded by glycolysis or may be substrates for al-

stituent of glycolipids

(cerebrosides), proteoglycans,

dolase and hence gluconeogenesis, which is the fate of

and glycoproteins. In the synthesis of lactose in the

much of the fructose metabolized in the liver.

mammary gland, UDPGal condenses with glucose to

In extrahepatic tissues, hexokinase catalyzes the

yield lactose, catalyzed by lactose synthase

(Figure

phosphorylation of most hexose sugars, including fruc-

20-6B).

168

CHAPTER 20

O P

O UDP

PHOSPHO-

UDPGlc PYRO-

UDPGlc

GLUCOMUTASE

PHOSPHORYLASE

DEHYDROGENASE

UTP

PP_i

2NAD

2NADH

+ H₂O

+ 2H⁺

CH₂

O P

CH₂OH

O^-

α-D-Glucose

Glucose

Uridine diphosphate

6-phosphate

1-phosphate

glucose (UDPGlc)

glucuronate

Glucuronides

H₂O

Proteoglycans

UDP

O^-

NADH

NADPH

CH₂OH

C H

HO C H

+ H⁺

NAD⁺

NADP⁺

+ H⁺

C O

HO C H

CH₂OH

*CH₂OH

O^-

L-Xylulose

3-Keto-L-gulonate

L-Gulonate

D-Glucuronate

H₂O

NADPH + H⁺

Oxalate

Glycolate

L-Gulonolactone

CO₂

O₂

NADP⁺

BLOCK IN PRIMATES

AND GUINEA PIGS

BLOCK IN HUMANS

Glycolaldehyde

2-Keto-L-gulonolactone

BLOCK IN

PENTOSURIA

D-Xylulose 1-phosphate

CH₂OH

NADH

C O

NAD

+ H⁺

[2H]

H D-Xylulose

HO C

CH₂OH

D-XYLULOSE

CH₂OH

REDUCTASE

Oxalate

Xylitol

ATP

HO C H

Diet

Mg2+

CH₂OH

ADP

L-Ascorbate

L-Dehydroascorbate

D-Xylulose 5-phosphate

Pentose phosphate pathway

Figure 20-4. Uronic acid pathway. (Asterisk indicates the fate of carbon 1 of glucose;

,  PO_32-.)

THE PENTOSE PHOSPHATE PATHWAY & OTHER PATHWAYS OF HEXOSE METABOLISM

169

ATP

HEXOKINASE

Glycogen

GLUCOKINASE

ALDOSE

REDUCTASE

Glucose 6-phosphate

D-Glucose

D-Sorbitol

NAD

NADPH

NADP

GLUCOSE-6-PHOSPHATASE

PHOSPHOHEXOSE

+ H⁺

ISOMERASE

SORBITOL

DEHYDROGENASE

NADH

+ H⁺

HEXOKINASE

Fructose 6-phosphate

D-Fructose

Diet

ATP

FRUCTOSE-1,6-

FRUCTOKINASE

ATP

PHOSPHOFRUCTOKINASE

ATP

BISPHOSPHATASE

BLOCK IN ESSENTIAL

FRUCTOSURIA

Fructose 1,6-bisphosphate

Fructose 1-phosphate

BLOCK IN HEREDITARY

FRUCTOSE INTOLERANCE

ALDOLASE B

Dihydroxyacetone-phosphate

ALDOLASE A

PHOSPHO-

Fatty acid

ALDOLASE B

TRIOSE

esterification

ISOMERASE

ATP

Glyceraldehyde 3-phosphate

D-Glyceraldehyde

TRIOKINASE

2-Phosphoglycerate

Pyruvate

Fatty acid synthesis

Figure 20-5. Metabolism of fructose. Aldolase A is found in all tissues, whereas aldolase B is

the predominant form in liver. (*, not found in liver.)

Glucose Is the Precursor of All

CLINICAL ASPECTS

Amino Sugars (Hexosamines)

Impairment of the Pentose Phosphate

Amino sugars are important components of glycopro-

Pathway Leads to Erythrocyte Hemolysis

teins (Chapter 47), of certain glycosphingolipids (eg,

gangliosides) (Chapter 14), and of glycosaminoglycans

Genetic deficiency of glucose-6-phosphate dehydrogen-

(Chapter 48). The major amino sugars are glucosa-

ase, with consequent impairment of the generation of

mine, galactosamine, and mannosamine and the

NADPH, is common in populations of Mediterranean

nine-carbon compound sialic acid. The principal sialic

and Afro-Caribbean origin. The defect is manifested as

acid found in human tissues is N-acetylneuraminic acid

red cell hemolysis (hemolytic anemia) when suscepti-

(NeuAc). A summary of the metabolic interrelationships

ble individuals are subjected to oxidants, such as the an-

among the amino sugars is shown in Figure 20-7.

timalarial primaquine, aspirin, or sulfonamides or when

170

CHAPTER 20

Galactose

Glycogen

GLYCOGEN SYNTHASE

ATP

PHOSPHORYLASE

Mg2+

GALACTOKINASE

Glucose 1-phosphate

ADP

BLOCK IN

Galactose

GALACTOSEMIA

PHOSPHOGLUCOMUTASE

1-phosphate

UDPGlc

GALACTOSE

URIDINE

1-PHOSPHATE

NAD⁺

DIPHOSPHOGALACTOSE

GLUCOSE-

URIDYL TRANSFERASE

4-EPIMERASE

6-PHOSPHATASE

Glucose

1-phosphate

UDPGal

Glucose 6-phosphate

Glucose

NAD⁺

Glucose

UDPGlc

UDPGal

URIDINE

ATP

DIPHOSPHOGALACTOSE

4-EPIMERASE

UDPGlc

Mg2+

HEXOKINASE

Lactose

PYROPHOSPHORYLASE

LACTOSE

SYNTHASE

ADP

PHOSPHOGLUCOMUTASE

Glucose 6-phosphate

Glucose 1-phosphate

Glucose

Figure 20-6. Pathway of conversion of (A) galactose to glucose in the liver and (B) glucose to lactose in

the lactating mammary gland.

they have eaten fava beans (Vicia fava—hence the term

uronic acid pathway. For example, administration of

favism). Glutathione peroxidase is dependent upon a

barbital or of chlorobutanol to rats results in a signifi-

supply of NADPH, which in erythrocytes can be

cant increase in the conversion of glucose to glu-

formed only via the pentose phosphate pathway. It re-

curonate, L-gulonate, and ascorbate.

duces organic peroxides and H₂O₂ as part of the body’s

defense against lipid peroxidation

(Figure

14-21).

Measurement of erythrocyte transketolase and its acti-

Loading of the Liver With Fructose

vation by thiamin diphosphate is used to assess thiamin

May Potentiate Hyperlipidemia

nutritional status (Chapter 45).

& Hyperuricemia

In the liver, fructose increases triacylglycerol synthesis

Disruption of the Uronic Acid Pathway Is

and VLDL secretion, leading to hypertriacylglyc-

Caused by Enzyme Defects & Some Drugs

erolemia—and increased LDL cholesterol—which can

In the rare hereditary disease essential pentosuria, con-

be regarded as potentially atherogenic (Chapter 26). In

siderable quantities of L-xylulose appear in the urine

addition, acute loading of the liver with fructose, as can

because of absence of the enzyme necessary to reduce

occur with intravenous infusion or following very high

L-xylulose to xylitol. Parenteral administration of xylitol

fructose intakes, causes sequestration of inorganic phos-

may lead to oxalosis, involving calcium oxalate deposi-

phate in fructose

1-phosphate and diminished ATP

tion in brain and kidneys (Figure 20-4). Various drugs

synthesis. As a result there is less inhibition of de novo

markedly increase the rate at which glucose enters the

purine synthesis by ATP and uric acid formation is in-

THE PENTOSE PHOSPHATE PATHWAY & OTHER PATHWAYS OF HEXOSE METABOLISM

171

Glycogen

Glucose 1-phosphate

ATP ADP

Glucose

Glucose 6-phosphate

Fructose 6-phosphate

Glutamine

AMIDOTRANSFERASE

ATP ADP

UTP

Glutamate

Glucosamine

UDP-

6-phosphate

PHOSPHOGLUCO-

1-phosphate

glucosamine*

MUTASE

Acetyl-CoA

ATP ADP

N -Acetyl-

glucosamine

Glycosaminoglycans

6-phosphate

1-phosphate

(eg, heparin)

UTP

EPIMERASE

N -Acetyl-

UDP-

Glycosaminoglycans

mannosamine

6-phosphate

N -acetylglucosamine*

(hyaluronic acid),

glycoproteins

Phosphoenolpyruvate

NAD⁺

EPIMERASE

N -Acetyl-

UDP-

neuraminic acid

N -acetylgalactosamine*

9-phosphate

Inhibiting

allosteric

effect

Sialic acid,

Glycosaminoglycans

gangliosides,

(chondroitins),

glycoproteins

Figure 20-7. Summary of the interrelationships in metabolism of amino sugars. (At asterisk: Analo-

gous to UDPGlc.) Other purine or pyrimidine nucleotides may be similarly linked to sugars or amino sug-

ars. Examples are thymidine diphosphate (TDP)-glucosamine and TDP-N-acetylglucosamine.

creased, causing hyperuricemia, which is a cause of gout

fructose 1-phosphate, leads to hereditary fructose in-

(Chapter 34).

tolerance. Diets low in fructose, sorbitol, and sucrose

are beneficial for both conditions. One consequence of

hereditary fructose intolerance and of another condi-

Defects in Fructose Metabolism Cause

tion due to fructose-1,6-bisphosphatase deficiency is

Disease (Figure 20-5)

fructose-induced hypoglycemia despite the presence of

Lack of hepatic fructokinase causes essential fructo-

high glycogen reserves. The accumulation of fructose

suria, and absence of hepatic aldolase B, which cleaves

1-phosphate and fructose 1,6-bisphosphate allosterically

172

CHAPTER 20

inhibits the activity of liver phosphorylase. The seques-

SUMMARY

tration of inorganic phosphate also leads to depletion of

•

The pentose phosphate pathway, present in the cy-

ATP and hyperuricemia.

tosol, can account for the complete oxidation of glu-

cose, producing NADPH and CO₂ but not ATP.

Fructose & Sorbitol in the Lens Are

•

The pathway has an oxidative phase, which is irre-

Associated With Diabetic Cataract

versible and generates NADPH; and a nonoxidative

Both fructose and sorbitol are found in the lens of the

phase, which is reversible and provides ribose precur-

eye in increased concentrations in diabetes mellitus and

sors for nucleotide synthesis. The complete pathway

may be involved in the pathogenesis of diabetic

is present only in those tissues having a requirement

cataract. The sorbitol (polyol) pathway (not found in

for NADPH for reductive syntheses, eg, lipogenesis

liver) is responsible for fructose formation from glucose

or steroidogenesis, whereas the nonoxidative phase is

(Figure 20-5) and increases in activity as the glucose

present in all cells requiring ribose.

concentration rises in diabetes in those tissues that are

•

In erythrocytes, the pathway has a major function in

not insulin-sensitive, ie, the lens, peripheral nerves, and

preventing hemolysis by providing NADPH to

renal glomeruli. Glucose is reduced to sorbitol by al-

maintain glutathione in the reduced state as the sub-

dose reductase, followed by oxidation of sorbitol to

strate for glutathione peroxidase.

fructose in the presence of NAD⁺ and sorbitol dehydro-

•

The uronic acid pathway is the source of glucuronic

genase (polyol dehydrogenase). Sorbitol does not dif-

acid for conjugation of many endogenous and exoge-

fuse through cell membranes easily and accumulates,

nous substances before excretion as glucuronides in

causing osmotic damage. Simultaneously, myoinositol

urine and bile.

levels fall. Sorbitol accumulation, myoinositol deple-

•

Fructose bypasses the main regulatory step in glycol-

tion, and diabetic cataract can be prevented by aldose

ysis, catalyzed by phosphofructokinase, and stimu-

reductase inhibitors in diabetic rats, and promising re-

lates fatty acid synthesis and hepatic triacylglycerol

sults have been obtained in clinical trials.

secretion.

When sorbitol is administered intravenously, it is

•

Galactose is synthesized from glucose in the lactating

converted to fructose rather than to glucose. It is poorly

absorbed in the small intestine, and much is fermented

mammary gland and in other tissues where it is re-

quired for the synthesis of glycolipids, proteoglycans,

by colonic bacteria to short-chain fatty acids, CO₂, and

H₂, leading to abdominal pain and diarrhea (sorbitol

and glycoproteins.

intolerance).

Enzyme Deficiencies in the Galactose

REFERENCES

Pathway Cause Galactosemia

Couet C, Jan P, Debry G: Lactose and cataract in humans: a re-

view. J Am Coll Nutr 1991;10:79.

Inability to metabolize galactose occurs in the galac-

Cox TM: Aldolase B and fructose intolerance. FASEB J 1994;8:62.

tosemias, which may be caused by inherited defects in

Cross NCP, Cox TM: Hereditary fructose intolerance. Int J

galactokinase, uridyl transferase, or 4-epimerase (Figure

Biochem 1990;22:685.

20-6A), though a deficiency in uridyl transferase is

Kador PF: The role of aldose reductase in the development of dia-

the best known cause. The galactose concentration in

betic complications. Med Res Rev 1988;8:325.

the blood and in the eye is reduced by aldose reductase

Kaufman FR, Devgan S: Classical galactosemia: a review. Endocri-

to galactitol, which accumulates, causing cataract. In

nologist 1995;5:189.

uridyl transferase deficiency, galactose 1-phosphate ac-

Macdonald I, Vrana A (editors): Metabolic Effects of Dietary Carbo-

cumulates and depletes the liver of inorganic phos-

hydrates. Karger, 1986.

phate. Ultimately, liver failure and mental deterioration

Mayes PA: Intermediary metabolism of fructose. Am J Clin Nutr

result. As the epimerase is present in adequate amounts,

1993(5 Suppl);58:754S.

the galactosemic individual can still form UDPGal

Van den Berghe G: Inborn errors of fructose metabolism. Annu

from glucose, and normal growth and development can

Rev Nutr 1994;14:41.

occur regardless of the galactose-free diets used to con-

Wood T: Physiological functions of the pentose phosphate path-

trol the symptoms of the disease.

way. Cell Biol Funct 1986;4:241.

Biosynthesis of Fatty Acids

Peter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DSc

BIOMEDICAL IMPORTANCE

The Fatty Acid Synthase Complex

Is a Polypeptide Containing

Fatty acids are synthesized by an extramitochondrial

Seven Enzyme Activities

system, which is responsible for the complete synthesis

of palmitate from acetyl-CoA in the cytosol. In the rat,

In bacteria and plants, the individual enzymes of the

the pathway is well represented in adipose tissue and

fatty acid synthase system are separate, and the acyl

liver, whereas in humans adipose tissue may not be an

radicals are found in combination with a protein called

important site, and liver has only low activity. In birds,

the acyl carrier protein (ACP). However, in yeast,

lipogenesis is confined to the liver, where it is particu-

mammals, and birds, the synthase system is a multien-

larly important in providing lipids for egg formation. In

zyme polypeptide complex that incorporates ACP,

most mammals, glucose is the primary substrate for

which takes over the role of CoA. It contains the vita-

lipogenesis, but in ruminants it is acetate, the main fuel

min pantothenic acid in the form of 4′-phosphopan-

molecule produced by the diet. Critical diseases of the

tetheine (Figure 45-18). The use of one multienzyme

pathway have not been reported in humans. However,

functional unit has the advantages of achieving the ef-

inhibition of lipogenesis occurs in type 1 (insulin-de-

fect of compartmentalization of the process within the

pendent) diabetes mellitus, and variations in its activ-

cell without the erection of permeability barriers, and

ity may affect the nature and extent of obesity.

synthesis of all enzymes in the complex is coordinated

since it is encoded by a single gene.

THE MAIN PATHWAY FOR DE NOVO

In mammals, the fatty acid synthase complex is a

dimer comprising two identical monomers, each con-

SYNTHESIS OF FATTY ACIDS

taining all seven enzyme activities of fatty acid synthase

(LIPOGENESIS) OCCURS

on one polypeptide chain (Figure 21-2). Initially, a

IN THE CYTOSOL

priming molecule of acetyl-CoA combines with a cys-

This system is present in many tissues, including liver,

teine

SH group catalyzed by acetyl transacylase

kidney, brain, lung, mammary gland, and adipose tis-

(Figure

21-3, reaction

1a). Malonyl-CoA combines

sue. Its cofactor requirements include NADPH, ATP,

with the adjacent SH on the 4′-phosphopantetheine

Mn2+, biotin, and HCO3− (as a source of CO₂). Acetyl-

of ACP of the other monomer, catalyzed by malonyl

CoA is the immediate substrate, and free palmitate is

transacylase (reaction 1b), to form acetyl (acyl)-mal-

the end product.

onyl enzyme. The acetyl group attacks the methylene

group of the malonyl residue, catalyzed by 3-ketoacyl

synthase, and liberates CO₂, forming 3-ketoacyl en-

Production of Malonyl-CoA Is

zyme (acetoacetyl enzyme) (reaction 2), freeing the cys-

the Initial & Controlling Step

teine SH group. Decarboxylation allows the reaction

in Fatty Acid Synthesis

to go to completion, pulling the whole sequence of re-

Bicarbonate as a source of CO₂ is required in the initial

actions in the forward direction. The 3-ketoacyl group

reaction for the carboxylation of acetyl-CoA to mal-

is reduced, dehydrated, and reduced again (reactions 3,

onyl-CoA in the presence of ATP and acetyl-CoA car-

5) to form the corresponding saturated acyl-S-

boxylase. Acetyl-CoA carboxylase has a requirement

enzyme. A new malonyl-CoA molecule combines with

for the vitamin biotin (Figure 21-1). The enzyme is a

the SH of 4′-phosphopantetheine, displacing the sat-

multienzyme protein containing a variable number of

urated acyl residue onto the free cysteine SH group.

identical subunits, each containing biotin, biotin car-

The sequence of reactions is repeated six more times

boxylase, biotin carboxyl carrier protein, and transcar-

until a saturated 16-carbon acyl radical (palmityl) has

boxylase, as well as a regulatory allosteric site. The reac-

been assembled. It is liberated from the enzyme com-

tion takes place in two steps: (1) carboxylation of biotin

plex by the activity of a seventh enzyme in the complex,

involving ATP and (2) transfer of the carboxyl to

thioesterase (deacylase). The free palmitate must be ac-

acetyl-CoA to form malonyl-CoA.

tivated to acyl-CoA before it can proceed via any other

173

174

CHAPTER 21

CH₃

CO S CoA

^-OOC

CH₂

S CoA

Acetyl-CoA

Malonyl-CoA

Enz biotin COO^-

Enz biotin

ADP + P _i

ATP + HCO3-

Figure 21-1. Biosynthesis of malonyl-CoA. (Enz, acetyl-CoA carboxylase.)

metabolic pathway. Its usual fate is esterification into

CH CO⋅S⋅CoA+7HOOC⋅CH CO⋅S⋅CoA+14NADPH+14H

acylglycerols, chain elongation or desaturation, or ester-

ification to cholesteryl ester. In mammary gland, there

CH (CH ) COOH+7CO +6H O+8CoA⋅SH+14NADP

2 14

→

is a separate thioesterase specific for acyl residues of C₈,

C₁₀, or C₁₂, which are subsequently found in milk

The acetyl-CoA used as a primer forms carbon

lipids.

atoms 15 and 16 of palmitate. The addition of all the

The equation for the overall synthesis of palmitate

subsequent C₂ units is via malonyl-CoA. Propionyl-

from acetyl-CoA and malonyl-CoA is:

CoA acts as primer for the synthesis of long-chain fatty

Hydratase

Malonyl

Enoyl

transacylase

reductase

Acetyl

Ketoacyl

transacylase

reductase

Ketoacyl

ACP

Thioesterase

synthase

4′-Phospho-

Cys

pantetheine

Subunit

division

4′-Phospho-

Cys

pantetheine

Ketoacyl

Thioesterase

ACP

synthase

Ketoacyl

Acetyl

reductase

transacylase

Enoyl

Malonyl

reductase

transacylase

Hydratase

Figure 21-2. Fatty acid synthase multienzyme complex. The complex is a dimer of two identical polypeptide

monomers, 1 and 2, each consisting of seven enzyme activities and the acyl carrier protein (ACP). (CysSH, cys-

teine thiol.) The SH of the 4′-phosphopantetheine of one monomer is in close proximity to the SH of the cys-

teine residue of the ketoacyl synthase of the other monomer, suggesting a “head-to-tail” arrangement of the two

monomers. Though each monomer contains all the partial activities of the reaction sequence, the actual func-

tional unit consists of one-half of one monomer interacting with the complementary half of the other. Thus, two

acyl chains are produced simultaneously. The sequence of the enzymes in each monomer is based on Wakil.

*CO₂

Acetyl-CoA

*Malonyl-CoA

C₂

ACETYL-CoA

C₃

CARBOXYLASE

HS Pan

Cys SH

CoA

ACETYL

C_n transfer from

TRANSACYLASE

MALONYL

TRANSACYLASE

CoA

Cys

Pan SH

C₂

Fatty acid synthase

multienzyme complex

Cys S

C CH

Pan S

C CH₂

*COO^-

^(C3 )

Acyl(acetyl)-malonyl enzyme

3-KETOACYL

SYNTHASE

*CO₂

Cys SH

Pan S

C CH₂

CH₃

3-Ketoacyl enzyme

(acetoacetyl enzyme)

NADPH + H⁺

3-KETOACYL

REDUCTASE

NADP⁺

Cys SH

Pan S

C CH₂

CH CH₃

NADPH

GENERATORS

(-)-3-Hydroxyacyl enzyme

Pentose phosphate

pathway

HYDRATASE

Isocitrate

H₂O

dehydrogenase

Malic

enzyme

Cys SH

Pan S

C CH CH CH₃

2,3-Unsaturated acyl enzyme

NADPH + H

ENOYL REDUCTASE

NADP⁺

H₂O

Cys SH

THIOESTERASE

After cycling through

Pan S

C CH₂

CH₂

CH₃

)

steps

seven times

^(Cn

Acyl enzyme

Palmitate

KEY:

, individual monomers of fatty acid synthase

Figure 21-3. Biosynthesis of long-chain fatty acids. Details of how addition of a malonyl residue

causes the acyl chain to grow by two carbon atoms. (Cys, cysteine residue; Pan, 4′-phosphopante-

theine.) The blocks shown in dark blue contain initially a C₂ unit derived from acetyl-CoA (as illustrated)

and subsequently the C_n unit formed in reaction 5.

175

176

CHAPTER 21

acids having an odd number of carbon atoms, found

phate pathway. Moreover, both metabolic pathways are

particularly in ruminant fat and milk.

found in the cytosol of the cell, so there are no mem-

branes or permeability barriers against the transfer of

The Main Source of NADPH

NADPH. Other sources of NADPH include the reac-

for Lipogenesis Is the Pentose

tion that converts malate to pyruvate catalyzed by the

“malic enzyme” (NADP malate dehydrogenase) (Fig-

Phosphate Pathway

ure 21-4) and the extramitochondrial isocitrate dehy-

NADPH is involved as donor of reducing equivalents

drogenase reaction (probably not a substantial source,

in both the reduction of the 3-ketoacyl and of the 2,3-

except in ruminants).

unsaturated acyl derivatives (Figure 21-3, reactions 3

and 5). The oxidative reactions of the pentose phos-

Acetyl-CoA Is the Principal Building

phate pathway (see Chapter 20) are the chief source of

Block of Fatty Acids

the hydrogen required for the reductive synthesis of

fatty acids. Significantly, tissues specializing in active

Acetyl-CoA is formed from glucose via the oxidation of

lipogenesis—ie, liver, adipose tissue, and the lactating

pyruvate within the mitochondria. However, it does

mammary gland—also possess an active pentose phos-

not diffuse readily into the extramitochondrial cytosol,

Glucose

Palmitate

Glucose 6-phosphate

NADP⁺

PPP

NADP⁺

Fructose 6-phosphate

NADPH

NADPH + H⁺

Malic

+ H⁺

enzyme

MALATE

Glyceraldehyde

DEHYDROGENASE

Malonyl-CoA

3-phosphate

NAD⁺

Malate

GLYCERALDEHYDE-

ACETYL-

ATP

3-PHOSPHATE

CoA

DEHYDROGENASE

CARBOXY-

NADH + H⁺

Oxaloacetate

LASE

Pyruvate

Acetyl-CoA

CoA

CYTOSOL

ATP-

CITRATE

CoA

ATP

Acetate

LYASE

ATP

Citrate

H⁺

Citrate

Isocitrate

ISOCITRATE

Outside

DEHYDROGENASE

INNER MITOCHONDRIAL MEMBRANE

Inside

PYRUVATE

DEHYDROGENASE

Malate

Pyruvate

Acetyl-CoA

MITOCHONDRION

α-Ketoglutarate

NADH + H⁺

Oxaloacetate

Citrate

Citric acid cycle

NAD⁺

Malate

α-Ketoglutarate

Figure 21-4. The provision of acetyl-CoA and NADPH for lipogenesis. (PPP, pentose phosphate path-

way; T, tricarboxylate transporter; K, α-ketoglutarate transporter; P, pyruvate transporter.)

BIOSYNTHESIS OF FATTY ACIDS

177

the principal site of fatty acid synthesis. Citrate, formed

after condensation of acetyl-CoA with oxaloacetate in

CH₂

C S CoA

+ CH₂

C S CoA

the citric acid cycle within mitochondria, is translo-

cated into the extramitochondrial compartment via the

COOH

tricarboxylate transporter, where in the presence of

Acyl-CoA

Malonyl-CoA

CoA and ATP it undergoes cleavage to acetyl-CoA and

oxaloacetate catalyzed by ATP-citrate lyase, which in-

creases in activity in the well-fed state. The acetyl-CoA

3-KETOACYL-CoA

is then available for malonyl-CoA formation and syn-

SYNTHASE

CoA SH + CO₂

thesis to palmitate

(Figure 21-4). The resulting ox-

aloacetate can form malate via NADH-linked malate

dehydrogenase, followed by the generation of NADPH

via the malic enzyme. The NADPH becomes available

CH₂

C S CoA

for lipogenesis, and the pyruvate can be used to regen-

erate acetyl-CoA after transport into the mitochon-

3-Ketoacyl-CoA

drion. This pathway is a means of transferring reducing

NADPH + H⁺

equivalents from extramitochondrial NADH to NADP.

Alternatively, malate itself can be transported into the

3-KETOACYL-CoA

REDUCTASE

mitochondrion, where it is able to re-form oxaloacetate.

Note that the citrate (tricarboxylate) transporter in the

NADP⁺

mitochondrial membrane requires malate to exchange

with citrate (see Figure 12-10). There is little ATP-

citrate lyase or malic enzyme in ruminants, probably

CH₂

C S CoA

because in these species acetate

(derived from the

3-Hydroxyacyl-CoA

rumen and activated to acetyl CoA extramitochondri-

ally) is the main source of acetyl-CoA.

3-HYDROXYACYL-CoA

DEHYDRASE

Elongation of Fatty Acid Chains Occurs

in the Endoplasmic Reticulum

This pathway (the “microsomal system”) elongates sat-

urated and unsaturated fatty acyl-CoAs (from C₁₀ up-

C S CoA

ward) by two carbons, using malonyl-CoA as acetyl

2-trans-Enoyl-CoA

donor and NADPH as reductant, and is catalyzed by

the microsomal fatty acid elongase system of enzymes

NADPH + H⁺

(Figure 21-5). Elongation of stearyl-CoA in brain in-

2-trans-ENOYL-CoA

creases rapidly during myelination in order to provide

REDUCTASE

C₂₂ and C₂₄ fatty acids for sphingolipids.

NADP⁺

THE NUTRITIONAL STATE

REGULATES LIPOGENESIS

CH₂

C S CoA

Acyl-CoA

Excess carbohydrate is stored as fat in many animals in

anticipation of periods of caloric deficiency such as star-

Figure 21-5. Microsomal elongase system for fatty

vation, hibernation, etc, and to provide energy for use

acid chain elongation. NADH is also used by the reduc-

between meals in animals, including humans, that take

tases, but NADPH is preferred.

their food at spaced intervals. Lipogenesis converts sur-

plus glucose and intermediates such as pyruvate, lactate,

and acetyl-CoA to fat, assisting the anabolic phase of

a fat diet, or when there is a deficiency of insulin, as in

this feeding cycle. The nutritional state of the organism

diabetes mellitus. These latter conditions are associated

is the main factor regulating the rate of lipogenesis.

with increased concentrations of plasma free fatty acids,

Thus, the rate is high in the well-fed animal whose diet

and an inverse relationship has been demonstrated be-

contains a high proportion of carbohydrate. It is de-

tween hepatic lipogenesis and the concentration of

pressed under conditions of restricted caloric intake, on

serum-free fatty acids. Lipogenesis is increased when su-

178

CHAPTER 21

crose is fed instead of glucose because fructose bypasses

PROTEIN

the phosphofructokinase control point in glycolysis and

P_i

PHOSPHATASE

floods the lipogenic pathway (Figure 20-5).

H₂O

SHORT- & LONG-TERM MECHANISMS

ACETYL-CoA

REGULATE LIPOGENESIS

CARBOXYLASE

(active)

(inactive)

Long-chain fatty acid synthesis is controlled in the

short term by allosteric and covalent modification of

Acetyl-

enzymes and in the long term by changes in gene ex-

CoA

pression governing rates of synthesis of enzymes.

ATP

AMPK

ADP

Acetyl-CoA Carboxylase Is the Most

H₂O

(active)

Important Enzyme in the Regulation

Malonyl-

CoA

of Lipogenesis

AMPKK

Acetyl-CoA carboxylase is an allosteric enzyme and is

activated by citrate, which increases in concentration in

AMPK

the well-fed state and is an indicator of a plentiful sup-

(inactive)

P_i

ATP

ply of acetyl-CoA. Citrate converts the enzyme from an

Acyl-CoA

inactive dimer to an active polymeric form, having a

molecular mass of several million. Inactivation is pro-

cAMP-DEPENDENT

Glucagon

cAMP

moted by phosphorylation of the enzyme and by long-

PROTEIN KINASE

chain acyl-CoA molecules, an example of negative feed-

back inhibition by a product of a reaction. Thus, if

Figure 21-6. Regulation of acetyl-CoA carboxylase

acyl-CoA accumulates because it is not esterified

by phosphorylation/dephosphorylation. The enzyme is

quickly enough or because of increased lipolysis or an

inactivated by phosphorylation by AMP-activated pro-

influx of free fatty acids into the tissue, it will automati-

tein kinase (AMPK), which in turn is phosphorylated

cally reduce the synthesis of new fatty acid. Acyl-CoA

and activated by AMP-activated protein kinase kinase

may also inhibit the mitochondrial tricarboxylate

(AMPKK). Glucagon (and epinephrine), after increasing

transporter, thus preventing activation of the enzyme

by egress of citrate from the mitochondria into the cy-

cAMP, activate this latter enzyme via cAMP-dependent

tosol.

protein kinase. The kinase kinase enzyme is also be-

Acetyl-CoA carboxylase is also regulated by hor-

lieved to be activated by acyl-CoA. Insulin activates

mones such as glucagon, epinephrine, and insulin via

acetyl-CoA carboxylase, probably through an “activa-

changes in its phosphorylation state (details in Figure

tor” protein and an insulin-stimulated protein kinase.

21-6).

Pyruvate Dehydrogenase Is Also

Insulin Also Regulates Lipogenesis

Regulated by Acyl-CoA

by Other Mechanisms

Acyl-CoA causes an inhibition of pyruvate dehydrogen-

Insulin stimulates lipogenesis by several other mecha-

ase by inhibiting the ATP-ADP exchange transporter of

nisms as well as by increasing acetyl-CoA carboxylase

the inner mitochondrial membrane, which leads to in-

activity. It increases the transport of glucose into the

creased intramitochondrial

[ATP]/[ADP] ratios and

cell (eg, in adipose tissue), increasing the availability of

therefore to conversion of active to inactive pyruvate

both pyruvate for fatty acid synthesis and glycerol

dehydrogenase (see Figure 17-6), thus regulating the

3-phosphate for esterification of the newly formed fatty

availability of acetyl-CoA for lipogenesis. Furthermore,

acids, and also converts the inactive form of pyruvate

oxidation of acyl-CoA due to increased levels of free

dehydrogenase to the active form in adipose tissue but

fatty acids may increase the ratios of

[acetyl-CoA]/

not in liver. Insulin also—by its ability to depress the

[CoA] and [NADH]/[NAD⁺] in mitochondria, inhibit-

level of intracellular cAMP—inhibits lipolysis in adi-

ing pyruvate dehydrogenase.

pose tissue and thereby reduces the concentration of

BIOSYNTHESIS OF FATTY ACIDS

179

plasma free fatty acids and therefore long-chain acyl-

• Acetyl-CoA carboxylase is required to convert acetyl-

CoA, an inhibitor of lipogenesis.

CoA to malonyl-CoA. In turn, fatty acid synthase, a

multienzyme complex of one polypeptide chain with

The Fatty Acid Synthase Complex

seven separate enzymatic activities, catalyzes the as-

sembly of palmitate from one acetyl-CoA and seven

& Acetyl-CoA Carboxylase Are

malonyl-CoA molecules.

Adaptive Enzymes

• Lipogenesis is regulated at the acetyl-CoA carboxy-

These enzymes adapt to the body’s physiologic needs

lase step by allosteric modifiers, phosphorylation/de-

by increasing in total amount in the fed state and by

phosphorylation, and induction and repression of en-

decreasing in starvation, feeding of fat, and in diabetes.

zyme synthesis. Citrate activates the enzyme, and

Insulin is an important hormone causing gene expres-

long-chain acyl-CoA inhibits its activity. Insulin acti-

sion and induction of enzyme biosynthesis, and

vates acetyl-CoA carboxylase whereas glucagon and

glucagon (via cAMP) antagonizes this effect. Feeding

epinephrine have opposite actions.

fats containing polyunsaturated fatty acids coordinately

regulates the inhibition of expression of key enzymes of

glycolysis and lipogenesis. These mechanisms for

REFERENCES

longer-term regulation of lipogenesis take several days

to become fully manifested and augment the direct and

Hudgins LC et al: Human fatty acid synthesis is stimulated by a

eucaloric low fat, high carbohydrate diet. J Clin Invest

immediate effect of free fatty acids and hormones such

1996;97:2081.

as insulin and glucagon.

Jump DB et al: Coordinate regulation of glycolytic and lipogenic

gene expression by polyunsaturated fatty acids. J Lipid Res

SUMMARY

1994;35:1076.

Kim KH: Regulation of mammalian acetyl-coenzyme A carboxy-

• The synthesis of long-chain fatty acids (lipogenesis) is

lase. Annu Rev Nutr 1997;17:77.

carried out by two enzyme systems: acetyl-CoA car-

Salati LM, Goodridge AG: Fatty acid synthesis in eukaryotes. In:

boxylase and fatty acid synthase.

Biochemistry of Lipids, Lipoproteins and Membranes. Vance

• The pathway converts acetyl-CoA to palmitate and

DE, Vance JE (editors). Elsevier, 1996.

requires NADPH, ATP, Mn2+, biotin, pantothenic

Wakil SJ: Fatty acid synthase, a proficient multifunctional enzyme.

acid, and HCO3− as cofactors.

Biochemistry 1989;28:4523.

Oxidation of Fatty Acids:

Ketogenesis

Peter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DSc

BIOMEDICAL IMPORTANCE

more water-soluble and exist as the un-ionized acid or

as a fatty acid anion.

Although fatty acids are both oxidized to acetyl-CoA

and synthesized from acetyl-CoA, fatty acid oxidation is

Fatty Acids Are Activated Before

not the simple reverse of fatty acid biosynthesis but an

Being Catabolized

entirely different process taking place in a separate com-

partment of the cell. The separation of fatty acid oxida-

Fatty acids must first be converted to an active interme-

tion in mitochondria from biosynthesis in the cytosol

diate before they can be catabolized. This is the only

allows each process to be individually controlled and

step in the complete degradation of a fatty acid that re-

integrated with tissue requirements. Each step in fatty

quires energy from ATP. In the presence of ATP and

acid oxidation involves acyl-CoA derivatives catalyzed

coenzyme A, the enzyme acyl-CoA synthetase (thioki-

by separate enzymes, utilizes NAD⁺ and FAD as coen-

nase) catalyzes the conversion of a fatty acid (or free

zymes, and generates ATP. It is an aerobic process, re-

fatty acid) to an “active fatty acid” or acyl-CoA, which

quiring the presence of oxygen.

uses one high-energy phosphate with the formation of

Increased fatty acid oxidation is a characteristic of

AMP and PP_i (Figure 22-1). The PP_i is hydrolyzed by

starvation and of diabetes mellitus, leading to ketone

inorganic pyrophosphatase with the loss of a further

body production by the liver (ketosis). Ketone bodies

high-energy phosphate, ensuring that the overall reac-

are acidic and when produced in excess over long peri-

tion goes to completion. Acyl-CoA synthetases are

ods, as in diabetes, cause ketoacidosis, which is ulti-

found in the endoplasmic reticulum, peroxisomes, and

mately fatal. Because gluconeogenesis is dependent

inside and on the outer membrane of mitochondria.

upon fatty acid oxidation, any impairment in fatty acid

oxidation leads to hypoglycemia. This occurs in vari-

ous states of carnitine deficiency or deficiency of es-

Long-Chain Fatty Acids Penetrate the

sential enzymes in fatty acid oxidation, eg, carnitine

Inner Mitochondrial Membrane as

palmitoyltransferase, or inhibition of fatty acid oxida-

Carnitine Derivatives

tion by poisons, eg, hypoglycin.

Carnitine

(β-hydroxy-γ-trimethylammonium buty-

rate), (CH₃)₃N⁺CH₂CH(OH)CH₂COO⁻, is

widely distributed and is particularly abundant in mus-

OXIDATION OF FATTY ACIDS OCCURS

cle. Long-chain acyl-CoA (or FFA) will not penetrate

IN MITOCHONDRIA

the inner membrane of mitochondria. However, car-

nitine palmitoyltransferase-I, present in the outer

Fatty Acids Are Transported in the

mitochondrial membrane, converts long-chain acyl-

Blood as Free Fatty Acids (FFA)

CoA to acylcarnitine, which is able to penetrate the

Free fatty acids—also called unesterified (UFA) or non-

inner membrane and gain access to the β-oxidation

esterified (NEFA) fatty acids—are fatty acids that are in

system of enzymes (Figure 22-1). Carnitine-acylcar-

the unesterified state. In plasma, longer-chain FFA are

nitine translocase acts as an inner membrane ex-

combined with albumin, and in the cell they are at-

change transporter. Acylcarnitine is transported in,

tached to a fatty acid-binding protein, so that in fact

coupled with the transport out of one molecule of car-

they are never really “free.” Shorter-chain fatty acids are

nitine. The acylcarnitine then reacts with CoA, cat-

180

OXIDATION OF FATTY ACIDS: KETOGENESIS

181

CoA SH

ATP

AMP + PP

H₃C

CoA

FFA

Acyl-CoA

CO S CoA

Palmitoyl-CoA

CARNITINE

Acyl-CoA

PALMITOYL-

OUTER

H₃C

SYNTHETASE

TRANSFERASE

MITOCHONDRIAL

MEMBRANE

S CoA

CH₃

CO S CoA

Acyl-CoA

CoA

Acetyl-CoA

Successive removal of acetyl-CoA (C₂) units

Carnitine

Acylcarnitine

8 CH₃

CO S CoA

Acetyl-CoA

CARNITINE

INNER

CARNITINE

ACYLCAR-

MITOCHONDRIAL

PALMITOYL-

NITINE

Figure 22-2. Overview of β-oxidation of fatty acids.

MEMBRANE

TRANSFERASE

TRANSLOCASE

The Cyclic Reaction Sequence Generates

CoA

FADH₂ & NADH

Acylcarnitine

Carnitine

Several enzymes, known collectively as “fatty acid oxi-

dase,” are found in the mitochondrial matrix or inner

Acylcarnitine

Acyl-CoA

β-Oxidation

membrane adjacent to the respiratory chain. These cat-

alyze the oxidation of acyl-CoA to acetyl-CoA, the sys-

tem being coupled with the phosphorylation of ADP to

Figure 22-1. Role of carnitine in the transport of

ATP (Figure 22-3).

long-chain fatty acids through the inner mitochondrial

The first step is the removal of two hydrogen atoms

membrane. Long-chain acyl-CoA cannot pass through

from the 2(α)- and 3(β)-carbon atoms, catalyzed by

the inner mitochondrial membrane, but its metabolic

acyl-CoA dehydrogenase and requiring FAD. This re-

product, acylcarnitine, can.

sults in the formation of

∆²-trans-enoyl-CoA and

FADH₂. The reoxidation of FADH₂ by the respiratory

chain requires the mediation of another flavoprotein,

alyzed by carnitine palmitoyltransferase-II, located

termed electron-transferring flavoprotein

(Chapter

on the inside of the inner membrane. Acyl-CoA is re-

11). Water is added to saturate the double bond and

formed in the mitochondrial matrix, and carnitine is

form 3-hydroxyacyl-CoA, catalyzed by

²-enoyl-CoA

liberated.

hydratase. The 3-hydroxy derivative undergoes further

dehydrogenation on the 3-carbon catalyzed by L(+)-3-

hydroxyacyl-CoA dehydrogenase to form the corre-

-OXIDATION OF FATTY ACIDS

sponding

3-ketoacyl-CoA compound. In this case,

NAD⁺ is the coenzyme involved. Finally, 3-ketoacyl-

INVOLVES SUCCESSIVE CLEAVAGE

CoA is split at the 2,3- position by thiolase (3-keto-

WITH RELEASE OF ACETYL-CoA

acyl-CoA-thiolase), forming acetyl-CoA and a new acyl-

In β-oxidation (Figure 22-2), two carbons at a time are

CoA two carbons shorter than the original acyl-CoA

cleaved from acyl-CoA molecules, starting at the car-

molecule. The acyl-CoA formed in the cleavage reac-

boxyl end. The chain is broken between the α(2)- and

tion reenters the oxidative pathway at reaction 2 (Fig-

β(3)-carbon atoms—hence the name β-oxidation. The

ure 22-3). In this way, a long-chain fatty acid may be

two-carbon units formed are acetyl-CoA; thus, palmi-

degraded completely to acetyl-CoA (C₂ units). Since

toyl-CoA forms eight acetyl-CoA molecules.

acetyl-CoA can be oxidized to CO₂ and water via the

182

CHAPTER 22

Figure 22-3. β-Oxidation of fatty acids. Long-chain

³CH

²CH₂

C O^-

acyl-CoA is cycled through reactions 2-5, acetyl-CoA

Fatty acid

being split off, each cycle, by thiolase (reaction 5).

CoA SH

ATP

When the acyl radical is only four carbon atoms in

ACYL-CoA

Mg2+

length, two acetyl-CoA molecules are formed in reac-

SYNTHETASE

tion 5.

AMP + PP

³CH₂

²CH

S CoA

citric acid cycle (which is also found within the mito-

Acyl-CoA

chondria), the complete oxidation of fatty acids is

achieved.

(outside)

C side

Oxidation of a Fatty Acid With an Odd

INNER MITOCHONDRIAL MEMBRANE

C CARNITINE TRANSPORTER

Number of Carbon Atoms Yields Acetyl-

M side

(inside)

CoA Plus a Molecule of Propionyl-CoA

Fatty acids with an odd number of carbon atoms are oxi-

dized by the pathway of β-oxidation, producing acetyl-

³CH₂

²CH₂

S CoA

CoA, until a three-carbon (propionyl-CoA) residue re-

mains. This compound is converted to succinyl-CoA, a

Acyl-CoA

constituent of the citric acid cycle (Figure 19-2). Hence,

FAD

the propionyl residue from an odd-chain fatty acid is

ACYL-CoA

the only part of a fatty acid that is glucogenic.

DEHYDROGENASE

FADH₂

H₂O

Respiratory

chain

Oxidation of Fatty Acids Produces

²CH C

S CoA

a Large Quantity of ATP

∆²-trans-Enoyl-CoA

H₂O

Transport in the respiratory chain of electrons from

∆²-ENOYL-CoA

FADH₂

and NADH will lead to the synthesis of five

HYDRATASE

high-energy phosphates (Chapter 12) for each of the

first seven acetyl-CoA molecules formed by β-oxidation

of palmitate (7 × 5 = 35). A total of 8 mol of acetyl-

R CH

²CH

S CoA

CoA is formed, and each will give rise to 12 mol of

L(+)-3-Hydroxy-

acyl-CoA

ATP on oxidation in the citric acid cycle, making 8 ×

12 = 96 mol. Two must be subtracted for the initial ac-

NAD⁺

tivation of the fatty acid, yielding a net gain of 129 mol

L(+)-3-HYDROXYACYL-

CoA DEHYDROGENASE

of ATP per mole of palmitate, or 129 × 51.6* = 6656

kJ. This represents 68% of the free energy of combus-

NADH + H⁺

H₂O

Respiratory

tion of palmitic acid.

chain

³C

²CH₂

S CoA

3-Ketoacyl-CoA

Peroxisomes Oxidize Very Long

CoA SH

Chain Fatty Acids

THIOLASE

A modified form of β-oxidation is found in peroxi-

somes and leads to the formation of acetyl-CoA and

H₂O₂ (from the flavoprotein-linked dehydrogenase

R C

CoA +CH

S CoA

step), which is broken down by catalase. Thus, this de-

Acyl-CoA

Acetyl-CoA

hydrogenation in peroxisomes is not linked directly to

phosphorylation and the generation of ATP. The sys-

tem facilitates the oxidation of very long chain fatty

Citric

acid

acids

(eg, C₂₀, C₂₂). These enzymes are induced by

cycle

2CO₂

* ∆G for the ATP reaction, as explained in Chapter 17.

OXIDATION OF FATTY ACIDS: KETOGENESIS

183

Figure 22-4. Sequence of reactions in the oxidation

cis

of unsaturated fatty acids, eg, linoleic acid. ∆⁴-cis-fatty

S CoA

acids or fatty acids forming ∆⁴-cis-enoyl-CoA enter the

pathway at the position shown. NADPH for the dienoyl-

Linoleyl-CoA

CoA reductase step is supplied by intramitochondrial

3 Cycles of

sources such as glutamate dehydrogenase, isocitrate

β-oxidation

3 Acetyl-CoA

dehydrogenase, and NAD(P)H transhydrogenase.

cis

S CoA

high-fat diets and in some species by hypolipidemic

drugs such as clofibrate.

The enzymes in peroxisomes do not attack shorter-

∆³-cis-∆⁶-cis-Dienoyl-CoA

chain fatty acids; the β-oxidation sequence ends at oc-

∆³-cis (or trans) → ∆²-trans-ENOYL-CoA

tanoyl-CoA. Octanoyl and acetyl groups are both further

ISOMERASE

oxidized in mitochondria. Another role of peroxisomal

β-oxidation is to shorten the side chain of cholesterol in

cis

bile acid formation (Chapter 26). Peroxisomes also take

part in the synthesis of ether glycerolipids (Chapter 24),

C S CoA

cholesterol, and dolichol (Figure 26-2).

∆²-trans-∆⁶-cis-Dienoyl-CoA

OXIDATION OF UNSATURATED FATTY

(∆²-trans-Enoyl-CoA stage of β-oxidation)

ACIDS OCCURS BY A MODIFIED

1 Cycle of

-OXIDATION PATHWAY

β-oxidation

Acetyl-CoA

The CoA esters of these acids are degraded by the en-

cis

zymes normally responsible for β-oxidation until either

a ∆³-cis-acyl-CoA compound or a ∆⁴-cis-acyl-CoA com-

C S CoA

pound is formed, depending upon the position of the

ACYL-CoA

DEHYDROGENASE

double bonds (Figure 22-4). The former compound is

isomerized (

³cis v

²-trans-enoyl-CoA isomerase)

∆2-trans-∆4-cis- Dienoyl-CoA

∆⁴-cis-Enoyl-CoA

to the corresponding ∆²-trans-CoA stage of β-oxidation

H⁺ + NADPH

for subsequent hydration and oxidation. Any ∆⁴-cis-acyl-

∆²-trans-∆⁴-cis-DIENOYL-CoA

REDUCTASE

CoA either remaining, as in the case of linoleic acid, or

NADP⁺

entering the pathway at this point after conversion by

acyl-CoA dehydrogenase to

∆²-trans-∆⁴-cis-dienoyl-

CoA, is then metabolized as indicated in Figure 22-4.

C S CoA

KETOGENESIS OCCURS WHEN THERE

IS A HIGH RATE OF FATTY ACID

∆³-trans-Enoyl-CoA

OXIDATION IN THE LIVER

∆³-cis (or trans) → ∆²-trans-ENOYL-CoA

Under metabolic conditions associated with a high rate

ISOMERASE

of fatty acid oxidation, the liver produces considerable

quantities of acetoacetate and D(

)-3-hydroxybutyrate

(β-hydroxybutyrate). Acetoacetate continually under-

goes spontaneous decarboxylation to yield acetone.

C S CoA

These three substances are collectively known as the ke-

tone bodies (also called acetone bodies or [incorrectly*]

“ketones”) (Figure 22-5). Acetoacetate and 3-hydroxybu-

∆²-trans-Enoyl-CoA

4 Cycles of

β-oxidation

* The term “ketones” should not be used because 3-hydroxybu-

tyrate is not a ketone and there are ketones in blood that are not

ketone bodies, eg, pyruvate, fructose.

5 Acetyl-CoA

184

CHAPTER 22

tyrate are interconverted by the mitochondrial enzyme

)-3-hydroxybutyrate dehydrogenase; the equi-

CH₃

librium is controlled by the mitochondrial [NAD⁺]/

Acetone

CO₂

[NADH] ratio, ie, the redox state. The concentration

of total ketone bodies in the blood of well-fed mam-

mals does not normally exceed 0.2 mmol/L except in

ruminants, where 3-hydroxybutyrate is formed contin-

uously from butyric acid (a product of ruminal fermen-

tation) in the rumen wall. In vivo, the liver appears to

CH₃

CH₂

COO^-

be the only organ in nonruminants to add significant

Acetoacetate

quantities of ketone bodies to the blood. Extrahepatic

D(-)-3-HYDROXYBUTYRATE

tissues utilize them as respiratory substrates. The net

DEHYDROGENASE

NADH + H⁺

flow of ketone bodies from the liver to the extrahepatic

tissues results from active hepatic synthesis coupled

with very low utilization. The reverse situation occurs

NAD⁺

in extrahepatic tissues (Figure 22-6).

CH₃

CH₂

COO^-

D(-)-3-Hydroxybutyrate

3-Hydroxy-3-Methylglutaryl-CoA

(HMG-CoA) Is an Intermediate

Figure 22-5. Interrelationships of the ketone bod-

in the Pathway of Ketogenesis

ies. D(−)-3-hydroxybutyrate dehydrogenase is a mito-

chondrial enzyme.

Enzymes responsible for ketone body formation are as-

sociated mainly with the mitochondria. Two acetyl-

CoA molecules formed in β-oxidation condense with

one another to form acetoacetyl-CoA by a reversal of

the thiolase reaction. Acetoacetyl-CoA, which is the

EXTRAHEPATIC

LIVER

BLOOD

TISSUES

Acyl-CoA

FFA

Glucose

Acyl-CoA

URINE

Acetyl-CoA

Ketone

Ketone bodies

bodies

Acetone

Citric

acid

cycle

LUNGS

2CO₂

Figure 22-6. Formation, utilization, and excretion of ketone bodies. (The main

pathway is indicated by the solid arrows.)

OXIDATION OF FATTY ACIDS: KETOGENESIS

185

starting material for ketogenesis, also arises directly

predominant ketone body present in the blood and

from the terminal four carbons of a fatty acid during

urine in ketosis.

β-oxidation

(Figure

22-7). Condensation of ace-

toacetyl-CoA with another molecule of acetyl-CoA

Ketone Bodies Serve as a Fuel

by 3-hydroxy-3-methylglutaryl-CoA synthase forms

for Extrahepatic Tissues

HMG-CoA. 3-Hydroxy-3-methylglutaryl-CoA lyase

then causes acetyl-CoA to split off from the HMG-

While an active enzymatic mechanism produces ace-

CoA, leaving free acetoacetate. The carbon atoms split

toacetate from acetoacetyl-CoA in the liver, acetoac-

off in the acetyl-CoA molecule are derived from the

etate once formed cannot be reactivated directly except

original acetoacetyl-CoA molecule. Both enzymes

in the cytosol, where it is used in a much less active

must be present in mitochondria for ketogenesis to

pathway as a precursor in cholesterol synthesis. This ac-

take place. This occurs solely in liver and rumen ep-

counts for the net production of ketone bodies by the

ithelium. D(−)-3-Hydroxybutyrate is quantitatively the

liver.

FFA

ATP

CoA

ACYL-CoA

SYNTHETASE

Esterification

Triacylglycerol

Acyl-CoA

Phospholipid

β-Oxidation

(Acetyl-CoA)

CH₃

CH C S CoA

Acetoacetyl-CoA

HMG-CoA

SYNTHASE

CoA SH

THIOLASE

H₂O

CH₃

S CoA

*CH₂

*COO^-

*CH₃

S CoA

CoA SH

3-Hydroxy-3-methyl-

Acetyl-CoA

glutaryl-CoA (HMG-CoA)

HMG-CoA

CO S CoA

LYASE

Acetyl-CoA

Citric

acid

cycle

CH₃

*COO^-

*CH₂

Acetoacetate

2CO₂

NADH + H⁺

D(-)-3-HYDROXYBUTYRATE

DEHYDROGENASE

NAD⁺

CH₃

*CH₂

*COO^-

D(-)-3-Hydroxybutyrate

Figure 22-7.

Pathways of ketogenesis in the liver. (FFA, free fatty acids; HMG, 3-hy-

droxy-3-methylglutaryl.)

186

CHAPTER 22

In extrahepatic tissues, acetoacetate is activated to

ketonemia, not the ketonuria, is the preferred method

acetoacetyl-CoA by succinyl-CoA-acetoacetate CoA

of assessing the severity of ketosis.

transferase. CoA is transferred from succinyl-CoA to

form acetoacetyl-CoA (Figure 22-8). The acetoacetyl-

KETOGENESIS IS REGULATED

CoA is split to acetyl-CoA by thiolase and oxidized in

AT THREE CRUCIAL STEPS

the citric acid cycle. If the blood level is raised, oxida-

tion of ketone bodies increases until, at a concentration

(1) Ketosis does not occur in vivo unless there is an

of approximately 12 mmol/L, they saturate the oxida-

increase in the level of circulating free fatty acids that

tive machinery. When this occurs, a large proportion of

arise from lipolysis of triacylglycerol in adipose tissue.

the oxygen consumption may be accounted for by the

Free fatty acids are the precursors of ketone bodies

oxidation of ketone bodies.

in the liver. The liver, both in fed and in fasting condi-

In most cases, ketonemia is due to increased pro-

tions, extracts about 30% of the free fatty acids passing

duction of ketone bodies by the liver rather than to a

through it, so that at high concentrations the flux pass-

deficiency in their utilization by extrahepatic tissues.

ing into the liver is substantial. Therefore, the factors

While acetoacetate and D(−)-3-hydroxybutyrate are

regulating mobilization of free fatty acids from adi-

readily oxidized by extrahepatic tissues, acetone is diffi-

pose tissue are important in controlling ketogenesis

cult to oxidize in vivo and to a large extent is volatilized

(Figures 22-9 and 25-8).

in the lungs.

(2) After uptake by the liver, free fatty acids are ei-

In moderate ketonemia, the loss of ketone bodies via

ther

-oxidized to CO₂ or ketone bodies or esterified

the urine is only a few percent of the total ketone body

to triacylglycerol and phospholipid. There is regulation

production and utilization. Since there are renal thresh-

of entry of fatty acids into the oxidative pathway by car-

old-like effects (there is not a true threshold) that vary

nitine palmitoyltransferase-I (CPT-I), and the remain-

between species and individuals, measurement of the

der of the fatty acid uptake is esterified. CPT-I activity is

EXTRAHEPATIC TISSUES

eg, MUSCLE

FFA

Acyl-CoA

Acetyl-CoA

LIVER

β-Oxidation

THIOLASE

Acetyl-CoA

Acetoacetyl-CoA

Succinate

OAA

CoA

Citric acid cycle

HMG-CoA

TRANSFERASE

Succinyl-

Citrate

CoA

2CO₂

Acetoacetate

NADH + H⁺

NADH

+ H+

NAD⁺

3-Hydroxybutyrate

Figure 22-8. Transport of ketone bodies from the liver and pathways of utilization and oxidation in extrahe-

patic tissues.

OXIDATION OF FATTY ACIDS: KETOGENESIS

187

Triacylglycerol

(3) In turn, the acetyl-CoA formed in β-oxidation is

oxidized in the citric acid cycle, or it enters the pathway

of ketogenesis to form ketone bodies. As the level of

ADIPOSE TISSUE

serum free fatty acids is raised, proportionately more

Lipolysis

free fatty acid is converted to ketone bodies and less is

oxidized via the citric acid cycle to CO₂. The partition

FFA

of acetyl-CoA between the ketogenic pathway and the

is so regulated that the

pathway of oxidation to CO₂

BLOOD

total free energy captured in ATP which results from

the oxidation of free fatty acids remains constant. This

FFA

may be appreciated when it is realized that complete

oxidation of 1 mol of palmitate involves a net produc-

LIVER

tion of 129 mol of ATP via β-oxidation and CO₂ pro-

duction in the citric acid cycle (see above), whereas only

33 mol of ATP are produced when acetoacetate is the

Acyl-CoA

CPT-I

end product and only 21 mol when 3-hydroxybutyrate

gateway

Esterification

is the end product. Thus, ketogenesis may be regarded

as a mechanism that allows the liver to oxidize increas-

β-Oxidation

ing quantities of fatty acids within the constraints of a

tightly coupled system of oxidative phosphorylation—

Acylglycerols

without increasing its total energy expenditure.

Acetyl-CoA

Theoretically, a fall in concentration of oxaloacetate,

Citric acid

cycle

particularly within the mitochondria, could impair the

Ketogenesis

ability of the citric acid cycle to metabolize acetyl-CoA

and divert fatty acid oxidation toward ketogenesis.

CO₂

Such a fall may occur because of an increase in the

[NADH]/[NAD⁺] ratio caused by increased β-oxida-

Ketone bodies

tion affecting the equilibrium between oxaloacetate and

malate and decreasing the concentration of oxaloac-

Figure 22-9.

Regulation of ketogenesis. 1 - 3

show

etate. However, pyruvate carboxylase, which catalyzes

three crucial steps in the pathway of metabolism of free

the conversion of pyruvate to oxaloacetate, is activated

fatty acids (FFA) that determine the magnitude of keto-

by acetyl-CoA. Consequently, when there are signifi-

genesis. (CPT-I, carnitine palmitoyltransferase-I.)

cant amounts of acetyl-CoA, there should be sufficient

oxaloacetate to initiate the condensing reaction of the

citric acid cycle.

low in the fed state, leading to depression of fatty acid

oxidation, and high in starvation, allowing fatty acid ox-

CLINICAL ASPECTS

idation to increase. Malonyl-CoA, the initial intermedi-

ate in fatty acid biosynthesis (Figure 21-1), formed by

Impaired Oxidation of Fatty Acids

acetyl-CoA carboxylase in the fed state, is a potent in-

Gives Rise to Diseases Often

hibitor of CPT-I (Figure 22-10). Under these condi-

Associated With Hypoglycemia

tions, free fatty acids enter the liver cell in low concen-

trations and are nearly all esterified to acylglycerols and

Carnitine deficiency can occur particularly in the new-

transported out of the liver in very low density lipopro-

born—and especially in preterm infants—owing to in-

teins (VLDL). However, as the concentration of free

adequate biosynthesis or renal leakage. Losses can also

fatty acids increases with the onset of starvation, acetyl-

occur in hemodialysis. This suggests a vitamin-like di-

CoA carboxylase is inhibited directly by acyl-CoA, and

etary requirement for carnitine in some individuals.

[malonyl-CoA] decreases, releasing the inhibition of

Symptoms of deficiency include hypoglycemia, which

CPT-I and allowing more acyl-CoA to be β-oxidized.

is a consequence of impaired fatty acid oxidation and

These events are reinforced in starvation by decrease in

lipid accumulation with muscular weakness. Treatment

the [insulin]/[glucagon] ratio. Thus, β-oxidation from

is by oral supplementation with carnitine.

free fatty acids is controlled by the CPT-I gateway into

Inherited CPT-I deficiency affects only the liver,

the mitochondria, and the balance of the free fatty acid

resulting in reduced fatty acid oxidation and ketogene-

uptake not oxidized is esterified.

sis, with hypoglycemia. CPT-II deficiency affects pri-

188

CHAPTER 22

Glucose

FFA

VLDL

BLOOD

LIVER

Acetyl-CoA

Acylglycerols

Insulin

−

Acyl-CoA

Lipogenesis

ACETYL-CoA

CARBOXYLASE

Cytosol

−

Glucagon

Malonyl-CoA

CARNITINE

−

PALMITOYL-

TRANSFERASE I

Palmitate

Figure 22-10. Regulation of

Acyl-CoA

long-chain fatty acid oxidation in the

Mitochondrion

β-Oxidation

liver. (FFA, free fatty acids; VLDL,

very low density lipoprotein.) Posi-

Acetyl-CoA

tive ( + ) and negative ( − ) regulatory

effects are represented by broken

CO2

arrows and substrate flow by solid

Ketone bodies

arrows.

marily skeletal muscle and, when severe, the liver. The

syndrome occurs in individuals with a rare inherited

sulfonylurea drugs

(glyburide

[glibenclamide] and

absence of peroxisomes in all tissues. They accumulate

tolbutamide), used in the treatment of type 2 diabetes

C₂₆-C₃₈ polyenoic acids in brain tissue and also exhibit

mellitus, reduce fatty acid oxidation and, therefore, hy-

a generalized loss of peroxisomal functions, eg, im-

perglycemia by inhibiting CPT-I.

paired bile acid and ether lipid synthesis.

Inherited defects in the enzymes of β-oxidation and

ketogenesis also lead to nonketotic hypoglycemia, coma,

Ketoacidosis Results From

and fatty liver. Defects are known in long- and short-

Prolonged Ketosis

chain 3-hydroxyacyl-CoA dehydrogenase (deficiency of

the long-chain enzyme may be a cause of acute fatty

Higher than normal quantities of ketone bodies present

liver of pregnancy).

3-Ketoacyl-CoA thiolase and

in the blood or urine constitute ketonemia (hyperke-

HMG-CoA lyase deficiency also affect the degradation

tonemia) or ketonuria, respectively. The overall condi-

of leucine, a ketogenic amino acid (Chapter 30).

tion is called ketosis. Acetoacetic and 3-hydroxybutyric

Jamaican vomiting sickness is caused by eating the

acids are both moderately strong acids and are buffered

unripe fruit of the akee tree, which contains a toxin,

when present in blood or other tissues. However, their

hypoglycin, that inactivates medium- and short-chain

continual excretion in quantity progressively depletes

acyl-CoA dehydrogenase, inhibiting β-oxidation and

the alkali reserve, causing ketoacidosis. This may be

causing hypoglycemia. Dicarboxylic aciduria is char-

fatal in uncontrolled diabetes mellitus.

acterized by the excretion of C₆-C₁₀ ω-dicarboxylic

The basic form of ketosis occurs in starvation and

acids and by nonketotic hypoglycemia. It is caused by a

involves depletion of available carbohydrate coupled

lack of mitochondrial medium-chain acyl-CoA dehy-

with mobilization of free fatty acids. This general pat-

drogenase. Refsum’s disease is a rare neurologic disor-

tern of metabolism is exaggerated to produce the patho-

der due to a defect that causes the accumulation of phy-

logic states found in diabetes mellitus, twin lamb dis-

tanic acid, which is found in plant foodstuffs and

ease, and ketosis in lactating cattle. Nonpathologic

blocks β-oxidation. Zellweger’s (cerebrohepatorenal)

forms of ketosis are found under conditions of high-fat

OXIDATION OF FATTY ACIDS: KETOGENESIS

189

feeding and after severe exercise in the postabsorptive

• Diseases associated with impairment of fatty acid oxi-

state.

dation lead to hypoglycemia, fatty infiltration of or-

gans, and hypoketonemia.

SUMMARY

• Ketosis is mild in starvation but severe in diabetes

mellitus and ruminant ketosis.

• Fatty acid oxidation in mitochondria leads to the gen-

eration of large quantities of ATP by a process called

β-oxidation that cleaves acetyl-CoA units sequentially

from fatty acyl chains. The acetyl-CoA is oxidized in

REFERENCES

the citric acid cycle, generating further ATP.

Eaton S, Bartlett K, Pourfarzam M: Mammalian mitochondrial β-

• The ketone bodies (acetoacetate, 3-hydroxybutyrate,

oxidation. Biochem J 1996;320:345.

and acetone) are formed in hepatic mitochondria

Mayes PA, Laker ME: Regulation of ketogenesis in the liver.

when there is a high rate of fatty acid oxidation. The

Biochem Soc Trans 1981;9:339.

pathway of ketogenesis involves synthesis and break-

McGarry JD, Foster DW: Regulation of hepatic fatty acid oxida-

down of 3-hydroxy-3-methylglutaryl-CoA (HMG-

tion and ketone body production. Annu Rev Biochem

CoA) by two key enzymes, HMG-CoA synthase and

1980;49:395.

HMG-CoA lyase.

Osmundsen H, Hovik R: β-Oxidation of polyunsaturated fatty

acids. Biochem Soc Trans 1988;16:420.

• Ketone bodies are important fuels in extrahepatic tis-

Reddy JK, Mannaerts GP: Peroxisomal lipid metabolism. Annu

sues.

Rev Nutr 1994;14:343.

• Ketogenesis is regulated at three crucial steps:

(1)

Scriver CR et al (editors): The Metabolic and Molecular Bases of In-

control of free fatty acid mobilization from adipose

herited Disease, 8th ed. McGraw-Hill, 2001.

tissue;

(2) the activity of carnitine palmitoyltrans-

Treem WR et al: Acute fatty liver of pregnancy and long-chain 3-

ferase-I in liver, which determines the proportion of

hydroxyacyl-coenzyme A dehydrogenase deficiency. Hepatol-

the fatty acid flux that is oxidized rather than esteri-

ogy 1994;19:339.

fied; and (3) partition of acetyl-CoA between the

Wood PA: Defects in mitochondrial beta-oxidation of fatty acids.

pathway of ketogenesis and the citric acid cycle.

Curr Opin Lipidol 1999;10:107.

Metabolism of Unsaturated Fatty

Acids & Eicosanoids

Peter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DSc

BIOMEDICAL IMPORTANCE

duced at the ∆⁴, ∆⁵, ∆⁶, and ∆⁹ positions (see Chapter

14) in most animals, but never beyond the ∆⁹ position.

Unsaturated fatty acids in phospholipids of the cell

In contrast, plants are able to synthesize the nutrition-

membrane are important in maintaining membrane

ally essential fatty acids by introducing double bonds at

fluidity. A high ratio of polyunsaturated fatty acids to

the ∆¹²

and ∆¹⁵ positions.

saturated fatty acids (P:S ratio) in the diet is a major

factor in lowering plasma cholesterol concentrations

and is considered to be beneficial in preventing coro-

nary heart disease. Animal tissues have limited capacity

COOH

for desaturating fatty acids, and that process requires

Palmitoleic acid (ω7, 16:1, ∆⁹)

certain dietary polyunsaturated fatty acids derived

from plants. These essential fatty acids are used to

form eicosanoic (C₂₀) fatty acids, which in turn give

COOH

rise to the prostaglandins and thromboxanes and to

Oleic acid (ω 9, 18:1, ∆⁹)

leukotrienes and lipoxins—known collectively as

eicosanoids. The prostaglandins and thromboxanes are

COOH

local hormones that are synthesized rapidly when re-

quired. Prostaglandins mediate inflammation, produce

*Linoleic acid (ω 6, 18:2, ∆^9,12)

pain, and induce sleep as well as being involved in the

regulation of blood coagulation and reproduction.

Nonsteroidal anti-inflammatory drugs such as aspirin

COOH

act by inhibiting prostaglandin synthesis. Leukotrienes

have muscle contractant and chemotactic properties

*α-Linolenic acid (ω 3, 18:3, ∆^9,12,15)

and are important in allergic reactions and inflamma-

COOH

tion.

*Arachidonic acid (ω 6, 20:4, ∆^5,8,11,14)

SOME POLYUNSATURATED FATTY

ACIDS CANNOT BE SYNTHESIZED

BY MAMMALS & ARE

COOH

NUTRITIONALLY ESSENTIAL

Eicosapentaenoic acid (ω 3, 20:5, ∆^5,8,11,14,17)

Certain long-chain unsaturated fatty acids of metabolic

Figure 23-1. Structure of some unsaturated fatty

significance in mammals are shown in Figure 23-1.

acids. Although the carbon atoms in the molecules are

Other C₂₀, C₂₂, and C₂₄ polyenoic fatty acids may be

conventionally numbered—ie, numbered from the car-

derived from oleic, linoleic, and α-linolenic acids by

boxyl terminal—the ω numbers (eg, ω7 in palmitoleic

chain elongation. Palmitoleic and oleic acids are not es-

acid) are calculated from the reverse end (the methyl

sential in the diet because the tissues can introduce a

double bond at the ∆⁹ position of a saturated fatty acid.

terminal) of the molecules. The information in paren-

Linoleic and

-linolenic acids are the only fatty acids

theses shows, for instance, that α-linolenic acid con-

known to be essential for the complete nutrition of

tains double bonds starting at the third carbon from

many species of animals, including humans, and are

the methyl terminal, has 18 carbons and 3 double

known as the nutritionally essential fatty acids. In

bonds, and has these double bonds at the 9th, 12th,

most mammals, arachidonic acid can be formed from

and 15th carbons from the carboxyl terminal. (Asterisks:

linoleic acid (Figure 23-4). Double bonds can be intro-

Classified as “essential fatty acids.”)

190

METABOLISM OF UNSATURATED FATTY ACIDS & EICOSANOIDS

191

Stearoyl CoA

are able to synthesize the ω9 (oleic acid) family of unsat-

urated fatty acids completely by a combination of chain

O₂ + NADH + H⁺

elongation and desaturation (Figure 23-3). However, as

indicated above, linoleic (ω6) or α-linolenic (ω3) acids

∆⁹

DESATURASE

Cyt b₅

required for the synthesis of the other members of the

ω6 or ω3 families must be supplied in the diet.

NAD⁺+ 2H₂O

Linoleate may be converted to arachidonate via

Oleoyl CoA

linolenate by the pathway shown in Figure 23-4. The

nutritional requirement for arachidonate may thus be

Figure 23-2. Microsomal ∆⁹ desaturase.

dispensed with if there is adequate linoleate in the diet.

The desaturation and chain elongation system is greatly

diminished in the starving state, in response to glucagon

MONOUNSATURATED FATTY

and epinephrine administration, and in the absence of

ACIDS ARE SYNTHESIZED BY

insulin as in type 1 diabetes mellitus.

⁹ DESATURASE SYSTEM

Several tissues including the liver are considered to be re-

DEFICIENCY SYMPTOMS ARE PRODUCED

sponsible for the formation of nonessential monounsatu-

WHEN THE ESSENTIAL FATTY ACIDS

rated fatty acids from saturated fatty acids. The first dou-

(EFA) ARE ABSENT FROM THE DIET

ble bond introduced into a saturated fatty acid is nearly

always in the ∆⁹ position. An enzyme system—

⁹ desat-

Rats fed a purified nonlipid diet containing vitamins A

urase (Figure 23-2)—in the endoplasmic reticulum will

and D exhibit a reduced growth rate and reproductive

catalyze the conversion of palmitoyl-CoA or stearoyl-CoA

deficiency which may be cured by the addition of

to palmitoleoyl-CoA or oleoyl-CoA, respectively. Oxygen

linoleic,

-linolenic, and arachidonic acids to the diet.

and either NADH or NADPH are necessary for the reac-

These fatty acids are found in high concentrations in

tion. The enzymes appear to be similar to a monooxyge-

vegetable oils (Table 14-2) and in small amounts in ani-

nase system involving cytochrome b₅

(Chapter 11).

mal carcasses. These essential fatty acids are required for

prostaglandin, thromboxane, leukotriene, and lipoxin

SYNTHESIS OF POLYUNSATURATED

formation (see below), and they also have various other

functions which are less well defined. Essential fatty acids

FATTY ACIDS INVOLVES DESATURASE

are found in the structural lipids of the cell, often in the

& ELONGASE ENZYME SYSTEMS

2 position of phospholipids, and are concerned with the

Additional double bonds introduced into existing mo-

structural integrity of the mitochondrial membrane.

nounsaturated fatty acids are always separated from each

Arachidonic acid is present in membranes and ac-

other by a methylene group (methylene interrupted) ex-

counts for 5-15% of the fatty acids in phospholipids.

cept in bacteria. Since animals have a ∆⁹ desaturase, they

Docosahexaenoic acid (DHA; ω3, 22:6), which is syn-

ω9

Oleic acid

18:2

20:2

20:3

22:3

22:4

Family

18:1

Accumulates in essential

fatty acid deficiency

—

24:1

22:1

20:1

ω6

Linoleic acid

18:3

20:3

20:4

22:4

22:5

Family

18:2

—

20:2

ω3

α-Linolenic

18:3

20:3

20:4

22:4

22:5

Family

acid

18:3

Figure 23-3. Biosynthesis of the ω9, ω6, and ω3 families of polyunsaturated fatty

acids. Each step is catalyzed by the microsomal chain elongation or desaturase sys-

tem: 1, elongase; 2, ∆⁶ desaturase; 3, ∆⁵ desaturase; 4, ∆⁴ desaturase. (

, Inhibition.)

—

192

CHAPTER 23

fatty acids in phospholipids, other complex lipids, and

membranes, particularly with ∆^5,8,11-eicosatrienoic acid

S CoA

(ω9 20:3) (Figure 23-3). The triene:tetraene ratio in

plasma lipids can be used to diagnose the extent of es-

Linoleoyl-CoA (∆^9,12-octadecadienoyl-CoA)

sential fatty acid deficiency.

O₂ + NADH + H

Trans Fatty Acids Are Implicated

in Various Disorders

∆⁶

DESATURASE

Small amounts of trans-unsaturated fatty acids are found

2H₂O + NAD

in ruminant fat (eg, butter fat has 2-7%), where they

arise from the action of microorganisms in the rumen,

but the main source in the human diet is from partially

hydrogenated vegetable oils (eg, margarine). Trans fatty

S CoA

acids compete with essential fatty acids and may exacer-

bate essential fatty acid deficiency. Moreover, they are

γ-Linolenoyl-CoA (∆^6,9,12-octadecatrienoyl-CoA)

structurally similar to saturated fatty acids (Chapter 14)

and have comparable effects in the promotion of hyper-

C₂

cholesterolemia and atherosclerosis (Chapter 26).

(Malonyl-CoA,

MICROSOMAL CHAIN

NADPH)

ELONGATION SYSTEM

(ELONGASE)

EICOSANOIDS ARE FORMED FROM C₂₀

POLYUNSATURATED FATTY ACIDS

Arachidonate and some other C₂₀ polyunsaturated fatty

acids give rise to eicosanoids, physiologically and phar-

C S CoA

macologically active compounds known as prosta-

glandins

(PG), thromboxanes

(TX), leukotrienes

(LT), and lipoxins (LX) (Chapter 14). Physiologically,

they are considered to act as local hormones function-

Dihomo-γ-linolenoyl-CoA (∆^8,11,14-eicosatrienoyl-CoA)

ing through G-protein-linked receptors to elicit their

O₂

+ NADH + H

biochemical effects.

There are three groups of eicosanoids that are syn-

∆⁵

DESATURASE

thesized from C₂₀ eicosanoic acids derived from the es-

sential fatty acids linoleate and

-linolenate, or di-

2H₂O + NAD+

rectly from dietary arachidonate and eicosapentaenoate

(Figure 23-5). Arachidonate, usually derived from the

2 position of phospholipids in the plasma membrane by

S CoA

the action of phospholipase A₂ (Figure 24-6)—but also

from the diet—is the substrate for the synthesis of the

PG₂, TX₂ series (prostanoids) by the cyclooxygenase

Arachidonoyl-CoA (∆^5,8,11,14-eicosatetraenoyl-CoA)

pathway, or the LT₄ and LX₄ series by the lipoxyge-

Figure 23-4. Conversion of linoleate to arachido-

nase pathway, with the two pathways competing for

nate. Cats cannot carry out this conversion owing to ab-

the arachidonate substrate (Figure 23-5).

sence of ∆⁶ desaturase and must obtain arachidonate in

their diet.

THE CYCLOOXYGENASE

PATHWAY IS RESPONSIBLE FOR

PROSTANOID SYNTHESIS

thesized from α-linolenic acid or obtained directly from

fish oils, is present in high concentrations in retina,

Prostanoid synthesis (Figure 23-6) involves the con-

cerebral cortex, testis, and sperm. DHA is particularly

sumption of two molecules of O₂

catalyzed by

needed for development of the brain and retina and is

prostaglandin H synthase (PGHS), which consists of

supplied via the placenta and milk. Patients with retini-

two enzymes, cyclooxygenase and peroxidase. PGHS

tis pigmentosa are reported to have low blood levels of

is present as two isoenzymes, PGHS-1 and PGHS-2.

DHA. In essential fatty acid deficiency, nonessential

The product, an endoperoxide (PGH), is converted to

polyenoic acids of the ω9 family replace the essential

prostaglandins D, E, and F as well as to a thromboxane

METABOLISM OF UNSATURATED FATTY ACIDS & EICOSANOIDS

193

Diet

Membrane phospholipid

PHOSPHOLIPASE

Angiotensin II

Linoleate

A₂

Bradykinin

Epinephrine

Thrombin

-2H

γ-Linolenate

GROUP 1

Diet

GROUP 2

Prostanoids

+2C

PGE₁

PGD₂

PGF₁

PGE₂

TXA₁

PGF₂

COOH

-2H

PGI₂

TXA₂

Leukotrienes

Lipoxins

LTA₃

8,11,14-Eicosatrienoate

5,8,11,14-

LTA₄

LXA₄

LTC₃

(dihomo γ-linolenate)

Eicosatetraenoate

LTB₄

LXB₄

LTD₃

LTC₄

LXC₄

Arachidonate

LTD₄

LXD₄

LTE₄

LXE₄

GROUP 3

Prostanoids

PGD₃

PGE₃

PGF₃

COOH

-2H

PGI

Eicosatetraenoate

TXA₃

Leukotrienes

+2C

5,8,11,14,17-

LTA₅

Eicosapentaenoate

LTB₅

LTC₅

Octadecatetraenoate

-2H

Diet

α-Linolenate

Diet

Figure 23-5. The three groups of eicosanoids and their biosynthetic origins. (PG, prostaglandin; PGI, prosta-

cyclin; TX, thromboxane; LT, leukotriene; LX, lipoxin; 1 , cyclooxygenase pathway; 2 , lipoxygenase pathway.)

The subscript denotes the total number of double bonds in the molecule and the series to which the compound

belongs.

(TXA₂) and prostacyclin (PGI₂). Each cell type pro-

Essential Fatty Acids Do Not Exert

duces only one type of prostanoid. Aspirin, a nons-

All Their Physiologic Effects Via

teroidal anti-inflammatory drug (NSAID), inhibits cy-

Prostaglandin Synthesis

clooxygenase of both PGHS-1 and PGHS-2 by

acetylation. Most other NSAIDs, such as indomethacin

The role of essential fatty acids in membrane formation

and ibuprofen, inhibit cyclooxygenases by competing

is unrelated to prostaglandin formation. Prostaglandins

with arachidonate. Transcription of PGHS-2—but not

do not relieve symptoms of essential fatty acid defi-

of PGHS-1—is completely inhibited by anti-inflam-

ciency, and an essential fatty acid deficiency is not

matory corticosteroids.

caused by inhibition of prostaglandin synthesis.

194

CHAPTER 23

COOH

Arachidonate

2O₂

CYCLOOXYGENASE

Aspirin

- Indomethacin

Ibuprofen

COOH

PGI

OOH

PROSTACYCLIN

PGG₂

PEROXIDASE

SYNTHASE

COOH

PGH

Malondialdehyde + HHT

ISOMERASE

THROMBOXANE

Imidazole

COOH

O O

SYNTHASE

COOH

TXA₂

6-Keto PGF1α

PGE₂

ISOMERASE

REDUCTASE

COOH

PGF2 α

PGD₂

TXB₂

Figure 23-6. Conversion of arachidonic acid to prostaglandins and thromboxanes of series 2. (PG,

prostaglandin; TX, thromboxane; PGI, prostacyclin; HHT, hydroxyheptadecatrienoate.) (Asterisk: Both of these

starred activities are attributed to one enzyme: prostaglandin H synthase. Similar conversions occur in

prostaglandins and thromboxanes of series 1 and 3.)

Cyclooxygenase Is a “Suicide Enzyme”

nase pathway in response to both immunologic and

nonimmunologic stimuli. Three different lipoxygenases

“Switching off” of prostaglandin activity is partly achieved

(dioxygenases) insert oxygen into the 5, 12, and 15 po-

by a remarkable property of cyclooxygenase—that of

sitions of arachidonic acid, giving rise to hydroperox-

self-catalyzed destruction; ie, it is a “suicide enzyme.”

ides

(HPETE). Only 5-lipoxygenase forms leuko-

Furthermore, the inactivation of prostaglandins by 15-

trienes (details in Figure 23-7). Lipoxins are a family of

hydroxyprostaglandin dehydrogenase is rapid. Block-

conjugated tetraenes also arising in leukocytes. They are

ing the action of this enzyme with sulfasalazine or in-

formed by the combined action of more than one

domethacin can prolong the half-life of prostaglandins

lipoxygenase (Figure 23-7).

in the body.

CLINICAL ASPECTS

LEUKOTRIENES & LIPOXINS

ARE FORMED BY THE

Symptoms of Essential Fatty Acid

LIPOXYGENASE PATHWAY

Deficiency in Humans Include Skin

Lesions & Impairment of Lipid Transport

The leukotrienes are a family of conjugated trienes

formed from eicosanoic acids in leukocytes, mastocy-

In adults subsisting on ordinary diets, no signs of es-

toma cells, platelets, and macrophages by the lipoxyge-

sential fatty acid deficiencies have been reported. How-

METABOLISM OF UNSATURATED FATTY ACIDS & EICOSANOIDS

195

COOH

15-LIPOXYGENASE

12-LIPOXYGENASE

Arachidonate

COOH

O₂

HOO

OOH

12-HPETE

15-HPETE

5-LIPOXYGENASE

COOH

15-HETE

OOH

12-HETE

COOH

5-LIPOXYGENASE

5-HPETE

5-HETE

H₂O

OH OH

COOH

H₂O

COOH

15-LIPOXYGENASE

Leukotriene B₄

Leukotriene A₄

Lipoxins, eg, LXA₄

Glutathione

Glutamic acid

NH₂

Glycine

NH₂

Cysteine

Glutamic acid

Cysteine

Glycine

Cysteine

COOH

Leukotriene C₄

Leukotriene D₄

Leukotriene E₄

Figure 23-7. Conversion of arachidonic acid to leukotrienes and lipoxins of series 4 via the lipoxygenase path-

way. Some similar conversions occur in series 3 and 5 leukotrienes. (HPETE, hydroperoxyeicosatetraenoate; HETE,

hydroxyeicosatetraenoate; 1 , peroxidase; 2 , leukotriene A₄ epoxide hydrolase; 3 , glutathione S-transferase;

4 , γ-glutamyltranspeptidase; 5 , cysteinyl-glycine dipeptidase.)

ever, infants receiving formula diets low in fat and pa-

Abnormal Metabolism of Essential Fatty

tients maintained for long periods exclusively by intra-

Acids Occurs in Several Diseases

venous nutrition low in essential fatty acids show defi-

ciency symptoms that can be prevented by an essential

Abnormal metabolism of essential fatty acids, which

fatty acid intake of 1-2% of the total caloric require-

may be connected with dietary insufficiency, has been

ment.

noted in cystic fibrosis, acrodermatitis enteropathica,

196

CHAPTER 23

hepatorenal syndrome, Sjögren-Larsson syndrome,

immediate hypersensitivity reactions, such as asthma.

multisystem neuronal degeneration, Crohn’s disease,

Leukotrienes are vasoactive, and

5-lipoxygenase has

cirrhosis and alcoholism, and Reye’s syndrome. Ele-

been found in arterial walls. Evidence supports a role

vated levels of very long chain polyenoic acids have

for lipoxins in vasoactive and immunoregulatory func-

been found in the brains of patients with Zellweger’s

tion, eg, as counterregulatory compounds (chalones) of

syndrome (Chapter 22). Diets with a high P:S (polyun-

the immune response.

saturated:saturated fatty acid) ratio reduce serum cho-

lesterol levels and are considered to be beneficial in

terms of the risk of development of coronary heart dis-

SUMMARY

ease.

• Biosynthesis of unsaturated long-chain fatty acids is

achieved by desaturase and elongase enzymes, which

Prostanoids Are Potent Biologically

introduce double bonds and lengthen existing acyl

Active Substances

chains, respectively.

Thromboxanes are synthesized in platelets and upon

• Higher animals have ∆⁴, ∆⁵, ∆⁶, and ∆⁹ desaturases

release cause vasoconstriction and platelet aggregation.

but cannot insert new double bonds beyond the 9

Their synthesis is specifically inhibited by low-dose as-

position of fatty acids. Thus, the essential fatty acids

pirin. Prostacyclins (PGI₂) are produced by blood ves-

linoleic (ω6) and α-linolenic (ω3) must be obtained

sel walls and are potent inhibitors of platelet aggrega-

from the diet.

tion. Thus, thromboxanes and prostacyclins are

• Eicosanoids are derived from C₂₀ (eicosanoic) fatty

antagonistic. PG₃ and TX₃, formed from eicosapen-

acids synthesized from the essential fatty acids and

taenoic acid (EPA) in fish oils, inhibit the release of

comprise important groups of physiologically and

arachidonate from phospholipids and the formation

pharmacologically active compounds, including the

of PG₂ and TX₂. PGI₃ is as potent an antiaggregator of

prostaglandins, thromboxanes, leukotrienes, and

platelets as PGI₂, but TXA₃ is a weaker aggregator than

lipoxins.

TXA₂, changing the balance of activity and favoring

longer clotting times. As little as 1 ng/mL of plasma

prostaglandins causes contraction of smooth muscle in

REFERENCES

animals. Potential therapeutic uses include prevention

Connor WE: The beneficial effects of omega-3 fatty acids: cardio-

of conception, induction of labor at term, termination

vascular disease and neurodevelopment. Curr Opin Lipidol

of pregnancy, prevention or alleviation of gastric ulcers,

1997;8:1.

control of inflammation and of blood pressure, and re-

Fischer S: Dietary polyunsaturated fatty acids and eicosanoid for-

lief of asthma and nasal congestion. In addition, PGD₂

mation in humans. Adv Lipid Res 1989;23:169.

is a potent sleep-promoting substance. Prostaglandins

Lagarde M, Gualde N, Rigaud M: Metabolic interactions between

increase cAMP in platelets, thyroid, corpus luteum,

eicosanoids in blood and vascular cells. Biochem J 1989;

fetal bone, adenohypophysis, and lung but reduce

257:313.

cAMP in renal tubule cells and adipose tissue (Chap-

Neuringer M, Anderson GJ, Connor WE: The essentiality of n-3

ter 25).

fatty acids for the development and function of the retina and

brain. Annu Rev Nutr 1988;8:517.

Serhan CN: Lipoxin biosynthesis and its impact in inflammatory

Leukotrienes & Lipoxins Are Potent

and vascular events. Biochim Biophys Acta 1994;1212:1.

Regulators of Many Disease Processes

Smith WL, Fitzpatrick FA: The eicosanoids: Cyclooxygenase,

lipoxygenase, and epoxygenase pathways. In: Biochemistry of

Slow-reacting substance of anaphylaxis (SRS-A) is a

Lipids, Lipoproteins and Membranes. Vance DE, Vance JE

mixture of leukotrienes C₄, D₄, and E₄. This mixture of

(editors). Elsevier, 1996.

leukotrienes is a potent constrictor of the bronchial air-

Tocher DR, Leaver MJ, Hodgson PA: Recent advances in the bio-

way musculature. These leukotrienes together with

chemistry and molecular biology of fatty acyl desaturases.

leukotriene B₄ also cause vascular permeability and at-

Prog Lipid Res 1998;37:73.

traction and activation of leukocytes and are important

Valenzuela A, Morgado N: Trans fatty acid isomers in human

regulators in many diseases involving inflammatory or

health and the food industry. Biol Res 1999;32:273.

Metabolism of Acylglycerols

& Sphingolipids

Peter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DSc

BIOMEDICAL IMPORTANCE

possess glycerol kinase, found in significant amounts

in liver, kidney, intestine, brown adipose tissue, and

Acylglycerols constitute the majority of lipids in the

lactating mammary gland.

body. Triacylglycerols are the major lipids in fat de-

posits and in food, and their roles in lipid transport and

storage and in various diseases such as obesity, diabetes,

TRIACYLGLYCEROLS &

and hyperlipoproteinemia will be described in subse-

PHOSPHOGLYCEROLS ARE FORMED BY

quent chapters. The amphipathic nature of phospho-

ACYLATION OF TRIOSE PHOSPHATES

lipids and sphingolipids makes them ideally suitable as

the main lipid component of cell membranes. Phos-

The major pathways of triacylglycerol and phosphoglyc-

pholipids also take part in the metabolism of many

erol biosynthesis are outlined in Figure 24-1. Impor-

other lipids. Some phospholipids have specialized func-

tant substances such as triacylglycerols, phosphatidyl-

tions; eg, dipalmitoyl lecithin is a major component of

choline, phosphatidylethanolamine, phosphatidylinositol,

lung surfactant, which is lacking in respiratory distress

and cardiolipin, a constituent of mitochondrial mem-

syndrome of the newborn. Inositol phospholipids in the

branes, are formed from glycerol-3-phosphate. Significant

cell membrane act as precursors of hormone second

branch points in the pathway occur at the phosphati-

messengers, and platelet-activating factor is an alkyl-

date and diacylglycerol steps. From dihydroxyacetone

phospholipid. Glycosphingolipids, containing sphingo-

phosphate are derived phosphoglycerols containing an

sine and sugar residues as well as fatty acid and found in

ether link (COC), the best-known of which

the outer leaflet of the plasma membrane with their

are plasmalogens and platelet-activating factor (PAF).

oligosaccharide chains facing outward, form part of the

Glycerol 3-phosphate and dihydroxyacetone phosphate

glycocalyx of the cell surface and are important (1) in

are intermediates in glycolysis, making a very important

cell adhesion and cell recognition; (2) as receptors for

connection between carbohydrate and lipid metabo-

bacterial toxins (eg, the toxin that causes cholera); and

lism.

(3) as ABO blood group substances. A dozen or so gly-

colipid storage diseases have been described

(eg,

Gaucher’s disease, Tay-Sachs disease), each due to a ge-

netic defect in the pathway for glycolipid degradation

in the lysosomes.

Glycerol 3-phosphate

Dihydroxyacetone phosphate

HYDROLYSIS INITIATES CATABOLISM

OF TRIACYLGLYCEROLS

Phosphatidate

Plasmalogens

PAF

Triacylglycerols must be hydrolyzed by a lipase to their

constituent fatty acids and glycerol before further catab-

olism can proceed. Much of this hydrolysis (lipolysis)

Diacylglycerol

Cardiolipin

Phosphatidylinositol

occurs in adipose tissue with release of free fatty acids

into the plasma, where they are found combined with

serum albumin. This is followed by free fatty acid up-

Phosphatidylcholine

Triacylglycerol

Phosphatidylinositol

take into tissues (including liver, heart, kidney, muscle,

Phosphatidylethanolamine

4,5-bisphosphate

lung, testis, and adipose tissue, but not readily by

brain), where they are oxidized or reesterified. The uti-

Figure 24-1. Overview of acylglycerol biosynthesis.

lization of glycerol depends upon whether such tissues

(PAF, platelet-activating factor.)

197

ATP

ADP

NAD⁺

NADH + H⁺

H₂C OH

HO C H

C O

Glycolysis

H₂C

GLYCEROL KINASE

H₂C

GLYCEROL-

3-PHOSPHATE

Glycerol

sn-Glycerol

DEHYDROGENASE

Dihydroxyacetone

3-phosphate

phosphate

Acyl-CoA (mainly saturated)

GLYCEROL-

3-PHOSPHATE

ACYLTRANSFERASE

CoA

H₂C O

C R₁

HO CH

H₂C

R₂

1-Acylglycerol-

3-phosphate

H₂C OH

(lysophosphatidate)

2-Monoacylglycerol

Acyl-CoA (usually unsaturated)

1-ACYLGLYCEROL-

3-PHOSPHATE

ACYLTRANSFERASE

Acyl-CoA

CoA

MONOACYLGLYCEROL

ACYLTRANSFERASE

(INTESTINE)

H₂C O

C R₁

CoA

O C

H₂C

1,2-Diacylglycerol

phosphate

(phosphatidate)

Choline

H₂O

CTP

ATP

PHOSPHATIDATE

CDP-DG

CHOLINE

PHOSPHOHYDROLASE

SYNTHASE

KINASE

P₁

PP₁

ADP

Phosphocholine

H₂C O

C R₁

H₂C

C R₁

CTP

CTP:

PHOSPHOCHOLINE

H₂COH

H₂C

O P P

CYTIDYL

1,2-Diacylglycerol

TRANSFERASE

Cytidine

CDP-diacylglycerol

Cardiolipin

CDP-choline

Acyl-CoA

Inositol

CDP-CHOLINE:

DIACYLGLYCEROL

PHOSPHATIDYL-

PHOSPHOCHOLINE

ACYLTRANSFERASE

INOSITOL SYNTHASE

TRANSFERASE

CoA

CMP

ATP

ADP

KINASE

H₂C O

C R₁

H₂C O C R₁

H₂C O

R₁

O H C O

O H C O C R

H C O

O H C O

Inositol

Triacyglycerol

Choline

Inositol

Phosphatidylinositol 4-phosphate

Phosphatidylcholine

Phosphatidylinositol

ATP

PHOSPHATIDYLETHANOLAMINE

N-METHYLTRANSFERASE

(-CH₃)₃

KINASE

Phosphatidylethanolamine

Serine

ADP

H₂C O C R₁

Phosphatidylserine

Ethanolamine

Figure 24-2 . Biosynthesis of triacylglycerol and phospholipids.

O H C O

P Inositol

( 1 , Monoacylglycerol pathway; 2, glycerol phosphate pathway.)

Phosphatidylinositol 4,5-bisphosphate

Phosphatidylethanolamine may be formed from ethanolamine by a

pathway similar to that shown for the formation of phosphatidyl-

choline from choline.

METABOLISM OF ACYLGLYCEROLS & SPHINGOLIPIDS

199

Phosphatidate Is the Common Precursor

from phosphatidylglycerol, which in turn is synthesized

in the Biosynthesis of Triacylglycerols,

from CDP-diacylglycerol (Figure 24-2) and glycerol

Many Phosphoglycerols, & Cardiolipin

3-phosphate according to the scheme shown in Figure

24-3. Cardiolipin, found in the inner membrane of

Both glycerol and fatty acids must be activated by ATP

mitochondria, is specifically required for the function-

before they can be incorporated into acylglycerols.

ing of the phosphate transporter and for cytochrome

Glycerol kinase catalyzes the activation of glycerol to

oxidase activity.

sn-glycerol 3-phosphate. If the activity of this enzyme is

absent or low, as in muscle or adipose tissue, most of

B. BIOSYNTHESIS OF GLYCEROL ETHER PHOSPHOLIPIDS

the glycerol 3-phosphate is formed from dihydroxyace-

tone phosphate by glycerol-3-phosphate dehydrogen-

This pathway is located in peroxisomes. Dihydroxyace-

ase (Figure 24-2).

tone phosphate is the precursor of the glycerol moiety

of glycerol ether phospholipids

(Figure

24-4). This

A. BIOSYNTHESIS OF TRIACYLGLYCEROLS

compound combines with acyl-CoA to give 1-acyldihy-

Two molecules of acyl-CoA, formed by the activation

droxyacetone phosphate. The ether link is formed in

of fatty acids by acyl-CoA synthetase (Chapter 22),

the next reaction, producing 1-alkyldihydroxyacetone

combine with glycerol 3-phosphate to form phosphati-

phosphate, which is then converted to 1-alkylglycerol

date (1,2-diacylglycerol phosphate). This takes place in

3-phosphate. After further acylation in the 2 position,

two stages, catalyzed by glycerol-3-phosphate acyl-

the resulting 1-alkyl-2-acylglycerol 3-phosphate (analo-

transferase and 1-acylglycerol-3-phosphate acyltrans-

gous to phosphatidate in Figure 24-2) is hydrolyzed to

ferase. Phosphatidate is converted by phosphatidate

give the free glycerol derivative. Plasmalogens, which

phosphohydrolase and diacylglycerol acyltransferase

comprise much of the phospholipid in mitochondria,

to 1,2-diacylglycerol and then triacylglycerol. In intesti-

are formed by desaturation of the analogous 3-phos-

nal mucosa, monoacylglycerol acyltransferase con-

phoethanolamine derivative

(Figure

24-4). Platelet-

verts monoacylglycerol to

1,2-diacylglycerol in the

activating factor (PAF) (1-alkyl-2-acetyl-sn-glycerol-3-

monoacylglycerol pathway. Most of the activity of

phosphocholine) is synthesized from the corresponding

these enzymes resides in the endoplasmic reticulum of

3-phosphocholine derivative. It is formed by many

the cell, but some is found in mitochondria. Phosphati-

blood cells and other tissues and aggregates platelets at

date phosphohydrolase is found mainly in the cytosol,

concentrations as low as 10−11 mol/L. It also has hy-

but the active form of the enzyme is membrane-bound.

potensive and ulcerogenic properties and is involved in

In the biosynthesis of phosphatidylcholine and

a variety of biologic responses, including inflammation,

phosphatidylethanolamine

(Figure

24-2), choline or

chemotaxis, and protein phosphorylation.

ethanolamine must first be activated by phosphoryla-

tion by ATP followed by linkage to CTP. The resulting

CDP-choline or CDP-ethanolamine reacts with 1,2-di-

acylglycerol to form either phosphatidylcholine or

CDP-Diacyl-

sn-Glycerol

phosphatidylethanolamine, respectively. Phosphatidyl-

glycerol

3-phosphate

serine is formed from phosphatidylethanolamine di-

rectly by reaction with serine

(Figure

24-2). Phos-

CMP

phatidylserine may re-form phosphatidylethanolamine

Phosphatidylglycerol phosphate

by decarboxylation. An alternative pathway in liver en-

ables phosphatidylethanolamine to give rise directly to

H₂O

phosphatidylcholine by progressive methylation of the

ethanolamine residue. In spite of these sources of

choline, it is considered to be an essential nutrient in

P_i

many mammalian species, but this has not been estab-

Phosphatidylglycerol

lished in humans.

The regulation of triacylglycerol, phosphatidyl-

choline, and phosphatidylethanolamine biosynthesis is

driven by the availability of free fatty acids. Those that

escape oxidation are preferentially converted to phos-

CMP

pholipids, and when this requirement is satisfied they

Cardiolipin

are used for triacylglycerol synthesis.

(diphosphatidylglycerol)

A phospholipid present in mitochondria is cardio-

lipin (diphosphatidylglycerol; Figure 14-8). It is formed

Figure 24-3. Biosynthesis of cardiolipin.

200

CHAPTER 24

NADPH

R₂

(CH₂)₂

+ H⁺ NADP⁺

Acyl-CoA

H₂COH

H₂C

O C R₁

H₂C

(CH₂)₂

R₂

H₂C

(CH₂)₂

R₂

H₂C O P

ACYL-

H₂C O P

SYNTHASE

H₂C O P

REDUCTASE

H₂C O

TRANSFERASE

HOOC R₁

Dihydroxyacetone

1-Acyldihydroxyacetone

1-Alkyldihydroxyacetone

1-Alkylglycerol 3-phosphate

phosphate

Acyl-CoA

ACYL-

TRANSFERASE

CDP-

CMP Ethanolamine

P_i

H₂O

H₂C

(CH₂)₂

R₂

H₂C

(CH₂)₂

R₂

H₂C

(CH₂)₂

R₂

R₃

H₂C O P CH₂

CH₂

NH₂

CDP-ETHANOLAMINE:

H₂C OH

PHOSPHOHYDROLASE

H₂C O P

ALKYLACYLGLYCEROL

PHOSPHOETHANOLAMINE

1-Alkyl-2-acylglycerol

TRANSFERASE

3-phosphoethanolamine

1-Alkyl-2-acylglycerol 3-phosphate

1-Alkyl-2-acylglycerol

CDP-choline

NADPH, O₂,

CDP-CHOLINE:

DESATURASE

ALKYLACYLGLYCEROL

Cyt b₅

Alkyl, diacyl glycerols

PHOSPHOCHOLINE

TRANSFERASE

CMP

H₂C

CH CH

R₂

H₂C

(CH₂)₂

R₂

R₃

H2O R3

COOH

R₃

H₂C

(CH₂)₂

R₂

H₂C O P

(CH₂)₂

NH₂

H₂C O

1-Alkenyl-2-acylglycerol

PHOSPHOLIPASE A₂

H₂C O P

Choline

3-phosphoethanolamine

1-Alkyl-2-acylglycerol

Choline

plasmalogen

3-phosphocholine

1-Alkyl-2-lysoglycerol

Acetyl-CoA

3-phosphocholine

ACETYLTRANSFERASE

H₂C

(CH₂)₂

R₂

H₃C

H₂C O

Choline

1-Alkyl-2-acetylglycerol 3-phosphocholine

PAF

Figure 24-4. Biosynthesis of ether lipids, including plasmalogens, and platelet-activating factor (PAF). In the

de novo pathway for PAF synthesis, acetyl-CoA is incorporated at stage *, avoiding the last two steps in the path-

way shown here.

Phospholipases Allow Degradation

solecithin) is attacked by lysophospholipase, forming

& Remodeling of Phosphoglycerols

the corresponding glyceryl phosphoryl base, which in

turn may be split by a hydrolase liberating glycerol

Although phospholipids are actively degraded, each

3-phosphate plus base. Phospholipases A₁, A₂, B, C,

portion of the molecule turns over at a different rate—

and D attack the bonds indicated in Figure 24-6.

eg, the turnover time of the phosphate group is differ-

Phospholipase A₂ is found in pancreatic fluid and

ent from that of the 1-acyl group. This is due to the

snake venom as well as in many types of cells; phos-

presence of enzymes that allow partial degradation fol-

pholipase C is one of the major toxins secreted by bac-

lowed by resynthesis (Figure 24-5). Phospholipase A₂

teria; and phospholipase D is known to be involved in

catalyzes the hydrolysis of glycerophospholipids to form

mammalian signal transduction.

a free fatty acid and lysophospholipid, which in turn

Lysolecithin

(lysophosphatidylcholine) may be

may be reacylated by acyl-CoA in the presence of an

formed by an alternative route that involves lecithin:

acyltransferase. Alternatively, lysophospholipid (eg, ly-

cholesterol acyltransferase

(LCAT). This enzyme,

METABOLISM OF ACYLGLYCEROLS & SPHINGOLIPIDS

201

found in plasma, catalyzes the transfer of a fatty acid

residue from the 2 position of lecithin to cholesterol to

H₂C O C R₁

form cholesteryl ester and lysolecithin and is considered

R₂

to be responsible for much of the cholesteryl ester in

H₂C O P Choline

plasma lipoproteins. Long-chain saturated fatty acids

are found predominantly in the 1 position of phospho-

Phosphatidylcholine

lipids, whereas the polyunsaturated acids (eg, the pre-

H₂O

cursors of prostaglandins) are incorporated more into

the 2 position. The incorporation of fatty acids into

ACYLTRANSFERASE

PHOSPHOLIPASE A₂

lecithin occurs by complete synthesis of the phospho-

R COOH

lipid, by transacylation between cholesteryl ester and

lysolecithin, and by direct acylation of lysolecithin by

acyl-CoA. Thus, a continuous exchange of the fatty

H₂C O C R

acids is possible, particularly with regard to introducing

essential fatty acids into phospholipid molecules.

H₂C O P Choline

Acyl-CoA

Lysophosphatidylcholine (lysolecithin)

ALL SPHINGOLIPIDS ARE FORMED

H₂O

FROM CERAMIDE

LYSOPHOSPHOLIPASE

Ceramide is synthesized in the endoplasmic reticulum

from the amino acid serine according to Figure 24-7.

R COOH

Ceramide is an important signaling molecule (second

messenger) regulating pathways including apoptosis

H₂C OH

(processes leading to cell death), cell senescence, and

differentiation, and opposes some of the actions of di-

H₂C O P Choline

acylglycerol.

Sphingomyelins (Figure 14-11) are phospholipids

Glycerylphosphocholine

and are formed when ceramide reacts with phos-

H₂O

phatidylcholine to form sphingomyelin plus diacylglyc-

GLYCERYLPHOSPHO-

erol (Figure 24-8A). This occurs mainly in the Golgi

CHOLINE HYDROLASE

apparatus and to a lesser extent in the plasma mem-

brane.

H₂C OH

Glycosphingolipids Are a Combination

+ Choline

of Ceramide With One or More

C O P

H₂

Sugar Residues

sn-Glycerol 3-phosphate

The simplest glycosphingolipids

(cerebrosides) are

Figure 24-5.

Metabolism of phosphatidylcholine

galactosylceramide

(GalCer) and glucosylceramide

(lecithin).

(GlcCer). GalCer is a major lipid of myelin, whereas

GlcCer is the major glycosphingolipid of extraneural

tissues and a precursor of most of the more complex

PHOSPHOLIPASE B

PHOSPHOLIPASE A₁

glycosphingolipids. Galactosylceramide (Figure 24-8B)

is formed in a reaction between ceramide and UDPGal

(formed by epimerization from UDPGlc—Figure

H₂C O

C R

20-6). Sulfogalactosylceramide and other sulfolipids

PHOSPHOLIPASE D

such as the sulfo(galacto)-glycerolipids and the

steroid sulfates are formed after further reactions in-

volving 3′-phosphoadenosine-5′-phosphosulfate (PAPS;

H₂C O

N-BASE

“active sulfate”). Gangliosides are synthesized from

PHOSPHOLIPASE A₂

O^-

ceramide by the stepwise addition of activated sugars (eg,

PHOSPHOLIPASE C

UDPGlc and UDPGal) and a sialic acid, usually N-

acetylneuraminic acid (Figure 24-9). A large number

Figure 24-6. Sites of the hydrolytic activity of phos-

of gangliosides of increasing molecular weight may be

pholipases on a phospholipid substrate.

formed. Most of the enzymes transferring sugars from

202

CHAPTER 24

nucleotide sugars (glycosyl transferases) are found in

⁺NH

the Golgi apparatus.

CH₃

(CH₂)₁₄

S CoA

⁻OOC

CH₂

Glycosphingolipids are constituents of the outer

Palmitoyl-CoA

Serine

leaflet of plasma membranes and are important in cell

adhesion and cell recognition. Some are antigens, eg,

ABO blood group substances. Certain gangliosides

Pyridoxal phosphate, Mn²⁺

function as receptors for bacterial toxins (eg, for cholera

toxin, which subsequently activates adenylyl cyclase).

SERINE

PALMITOYLTRANSFERASE

CLINICAL ASPECTS

CoA SH

Deficiency of Lung Surfactant Causes

Respiratory Distress Syndrome

CH₃

(CH₂)₁₂

C CH

CH₂

Lung surfactant is composed mainly of lipid with

NH₃

3-Ketosphinganine

some proteins and carbohydrate and prevents the alve-

oli from collapsing. Surfactant activity is largely attrib-

NADPH + H⁺

uted to dipalmitoylphosphatidylcholine, which is

3-KETOSPHINGANINE

REDUCTASE

synthesized shortly before parturition in full-term in-

fants. Deficiency of lung surfactant in the lungs of

NADP⁺

many preterm newborns gives rise to respiratory dis-

CH₃(CH₂)₁₂

CH₂

CH CH

CH₂

tress syndrome. Administration of either natural or ar-

tificial surfactant has been of therapeutic benefit.

OH NH

Dihydrosphingosine (sphinganine)

Phospholipids & Sphingolipids

R CO

S CoA

Are Involved in Multiple Sclerosis

Acyl-CoA

DIHYDROSPHINGOSINE

N -ACYLTRANSFERASE

and Lipidoses

CoA SH

Certain diseases are characterized by abnormal quanti-

CH₃

(CH₂)₁₂

CH₂

ties of these lipids in the tissues, often in the nervous

system. They may be classified into two groups: (1) true

demyelinating diseases and (2) sphingolipidoses.

Dihydroceramide

In multiple sclerosis, which is a demyelinating dis-

DIHYDROCERAMIDE

ease, there is loss of both phospholipids (particularly

DESATURASE

ethanolamine plasmalogen) and of sphingolipids from

white matter. Thus, the lipid composition of white

matter resembles that of gray matter. The cerebrospinal

CH₃

(CH₂)₁₂

CH CH CH

CH₂

fluid shows raised phospholipid levels.

The sphingolipidoses (lipid storage diseases) are a

Ceramide

group of inherited diseases that are often manifested in

childhood. These diseases are part of a larger group of

Figure 24-7. Biosynthesis of ceramide.

lysosomal disorders and exhibit several constant fea-

tures: (1) Complex lipids containing ceramide accumu-

late in cells, particularly neurons, causing neurodegen-

A Ceramide

Sphingomyelin

Phosphatidylcholine

Diacylglycerol

Figure 24-8. Biosynthesis of sphingomyelin (A),

UDPGal UDP

PAPS

galactosylceramide and its sulfo derivative (B). (PAPS,

Sulfogalactosyl-

Galactosylceramide

ceramide

“active sulfate,” adenosine 3′-phosphate-5′-phospho-

Ceramide

(cerebroside)

(sulfatide)

sulfate.)

METABOLISM OF ACYLGLYCEROLS & SPHINGOLIPIDS

203

UDPGlc

UDP

UDPGal

UDP

CMP-NeuAc

CMP

Glucosyl

Ceramide

ceramide

Cer-Glc-Gal

(Cer-Glc)

NeuAc

(G_M3)

UDP-N-acetyl

galactosamine

UDP

UDPGal

UDP

Higher gangliosides

Cer-Glc-Gal-GalNAc-Gal

Cer-Glc-Gal-GalNAc

(disialo- and trisialo-

gangliosides)

NeuAc

(G_M1)

(G_M2)

Figure 24-9. Biosynthesis of gangliosides. (NeuAc, N-acetylneuraminic acid.)

eration and shortening the life span. (2) The rate of

dase) in the treatment of Gaucher’s disease. A recent

synthesis of the stored lipid is normal. (3) The enzy-

promising approach is substrate reduction therapy to

matic defect is in the lysosomal degradation pathway

inhibit the synthesis of sphingolipids, and gene therapy

of sphingolipids. (4) The extent to which the activity of

for lysosomal disorders is currently under investigation.

the affected enzyme is decreased is similar in all tissues.

Some examples of the more important lipid storage dis-

There is no effective treatment for many of the diseases,

eases are shown in Table 24-1.

though some success has been achieved with enzymes

Multiple sulfatase deficiency results in accumula-

that have been chemically modified to ensure binding

tion of sulfogalactosylceramide, steroid sulfates, and

to receptors of target cells, eg, to macrophages in the

proteoglycans owing to a combined deficiency of aryl-

liver in order to deliver β-glucosidase (glucocerebrosi-

sulfatases A, B, and C and steroid sulfatase.

Table 24-1. Examples of sphingolipidoses.

Disease

Enzyme Deficiency

Lipid Accumulating¹

Clinical Symptoms

Tay-Sachs disease

Hexosaminidase A

Cer—Glc—Gal(NeuAc)—

Mental retardation, blindness, muscular weakness.

:GalNAc

GM2 Ganglioside

Fabry’s disease

α-Galactosidase

Cer—Glc—Gal—

Skin rash, kidney failure (full symptoms only in

:Gal

Globotriaosylceramide

males; X-linked recessive).

Metachromatic

Arylsulfatase A

Cer—Gal—

Mental retardation and psychologic disturbances in

:OSO3

leukodystrophy

3-Sulfogalactosylceramide

adults; demyelination.

Krabbe’s disease

β-Galactosidase

Cer—

Mental retardation; myelin almost absent.

:Gal

Galactosylceramide

Gaucher’s disease

β-Glucosidase

Cer—

Enlarged liver and spleen, erosion of long bones,

:Glc

Glucosylceramide

mental retardation in infants.

Niemann-Pick

Sphingomyelinase

Cer—

Enlarged liver and spleen, mental retardation; fatal in

:P—choline

disease

Sphingomyelin

early life.

Farber’s disease

Ceramidase

Acyl—

Hoarseness, dermatitis, skeletal deformation, mental

:Sphingosine

Ceramide

retardation; fatal in early life.

¹NeuAc, N-acetylneuraminic acid; Cer, ceramide; Glc, glucose; Gal, galactose. —, site of deficient enzyme reaction.

204

CHAPTER 24

SUMMARY

(demyelination), and sphingolipidoses (inability to

break down sphingolipids in lysosomes due to inher-

•

Triacylglycerols are the major energy-storing lipids,

ited defects in hydrolase enzymes).

whereas phosphoglycerols, sphingomyelin, and gly-

cosphingolipids are amphipathic and have structural

functions in cell membranes as well as other special-

ized roles.

REFERENCES

•

Triacylglycerols and some phosphoglycerols are syn-

Griese M: Pulmonary surfactant in health and human lung dis-

thesized by progressive acylation of glycerol 3-phos-

eases: state of the art. Eur Respir J 1999;13:1455.

phate. The pathway bifurcates at phosphatidate,

Merrill AH, Sweeley CC: Sphingolipids: metabolism and cell sig-

forming inositol phospholipids and cardiolipin on

naling. In: Biochemistry of Lipids, Lipoproteins and Mem-

the one hand and triacylglycerol and choline and

branes. Vance DE, Vance JE (editors). Elsevier, 1996.

ethanolamine phospholipids on the other.

Prescott SM et al: Platelet-activating factor and related lipid media-

tors. Annu Rev Biochem 2000;69:419.

•

Plasmalogens and platelet-activating factor (PAF) are

Ruvolo PP: Ceramide regulates cellular homeostasis via diverse

ether phospholipids formed from dihydroxyacetone

stress signaling pathways. Leukemia 2001;15:1153.

phosphate.

Schuette CG et al: The glycosphingolipidoses—from disease to

•

Sphingolipids are formed from ceramide (N-acyl-

basic principles of metabolism. Biol Chem 1999;380:759.

sphingosine). Sphingomyelin is present in mem-

Scriver CR et al (editors): The Metabolic and Molecular Bases of In-

branes of organelles involved in secretory processes

herited Disease, 8th ed. McGraw-Hill, 2001.

(eg, Golgi apparatus). The simplest glycosphin-

Tijburg LBM, Geelen MJH, van Golde LMG: Regulation of the

golipids are a combination of ceramide plus a sugar

biosynthesis of triacylglycerol, phosphatidylcholine and phos-

residue

(eg, GalCer in myelin). Gangliosides are

phatidylethanolamine in the liver. Biochim Biophys Acta

1989;1004:1.

more complex glycosphingolipids containing more

sugar residues plus sialic acid. They are present in the

Vance DE: Glycerolipid biosynthesis in eukaryotes. In: Biochem-

istry of Lipids, Lipoproteins and Membranes. Vance DE, Vance

outer layer of the plasma membrane, where they con-

JE (editors). Elsevier, 1996.

tribute to the glycocalyx and are important as anti-

van Echten G, Sandhoff K: Ganglioside metabolism. Enzymology,

gens and cell receptors.

topology, and regulation. J Biol Chem 1993;268:5341.

•

Phospholipids and sphingolipids are involved in sev-

Waite M: Phospholipases. In: Biochemistry of Lipids, Lipoproteins

eral disease processes, including respiratory distress

and Membranes. Vance DE, Vance JE (editors). Elsevier,

syndrome (lack of lung surfactant), multiple sclerosis

1996.

Lipid Transport & Storage

Peter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DSc

BIOMEDICAL IMPORTANCE

Four Major Groups of Plasma Lipoproteins

Have Been Identified

Fat absorbed from the diet and lipids synthesized by the

liver and adipose tissue must be transported between

Because fat is less dense than water, the density of a

the various tissues and organs for utilization and stor-

lipoprotein decreases as the proportion of lipid to pro-

age. Since lipids are insoluble in water, the problem of

tein increases (Table 25-1). In addition to FFA, four

how to transport them in the aqueous blood plasma is

major groups of lipoproteins have been identified that

solved by associating nonpolar lipids

(triacylglycerol

are important physiologically and in clinical diagnosis.

and cholesteryl esters) with amphipathic lipids (phos-

These are (1) chylomicrons, derived from intestinal

pholipids and cholesterol) and proteins to make water-

absorption of triacylglycerol and other lipids; (2) very

miscible lipoproteins.

low density lipoproteins (VLDL, or pre-β-lipopro-

In a meal-eating omnivore such as the human, ex-

teins), derived from the liver for the export of triacyl-

cess calories are ingested in the anabolic phase of the

glycerol;

(3) low-density lipoproteins

(LDL, or β-

feeding cycle, followed by a period of negative caloric

lipoproteins), representing a final stage in the catabolism

balance when the organism draws upon its carbohy-

of VLDL; and (4) high-density lipoproteins (HDL, or

drate and fat stores. Lipoproteins mediate this cycle by

α-lipoproteins), involved in VLDL and chylomicron

transporting lipids from the intestines as chylomi-

metabolism and also in cholesterol transport. Triacyl-

crons—and from the liver as very low density lipopro-

glycerol is the predominant lipid in chylomicrons and

teins

(VLDL)—to most tissues for oxidation and to

VLDL, whereas cholesterol and phospholipid are the

adipose tissue for storage. Lipid is mobilized from adi-

predominant lipids in LDL and HDL, respectively

pose tissue as free fatty acids (FFA) attached to serum

(Table 25-1). Lipoproteins may be separated according

albumin. Abnormalities of lipoprotein metabolism

to their electrophoretic properties into

-, and pre-

cause various hypo- or hyperlipoproteinemias. The

-lipoproteins.

most common of these is diabetes mellitus, where in-

sulin deficiency causes excessive mobilization of FFA

and underutilization of chylomicrons and VLDL, lead-

Lipoproteins Consist of a Nonpolar

ing to hypertriacylglycerolemia. Most other patho-

Core & a Single Surface Layer of

logic conditions affecting lipid transport are due pri-

Amphipathic Lipids

marily to inherited defects, some of which cause

The nonpolar lipid core consists of mainly triacylglyc-

hypercholesterolemia, and premature atherosclerosis.

erol and cholesteryl ester and is surrounded by a sin-

Obesity—particularly abdominal obesity—is a risk fac-

gle surface layer of amphipathic phospholipid and

tor for increased mortality, hypertension, type 2 dia-

cholesterol molecules (Figure 25-1). These are oriented

betes mellitus, hyperlipidemia, hyperglycemia, and vari-

so that their polar groups face outward to the aqueous

ous endocrine dysfunctions.

medium, as in the cell membrane (Chapter 14). The

protein moiety of a lipoprotein is known as an apo-

LIPIDS ARE TRANSPORTED IN THE

lipoprotein or apoprotein, constituting nearly 70% of

some HDL and as little as 1% of chylomicrons. Some

PLASMA AS LIPOPROTEINS

apolipoproteins are integral and cannot be removed,

Four Major Lipid Classes Are Present

whereas others are free to transfer to other lipoproteins.

in Lipoproteins

Plasma lipids consist of triacylglycerols (16%), phos-

The Distribution of Apolipoproteins

pholipids (30%), cholesterol (14%), and cholesteryl

Characterizes the Lipoprotein

esters (36%) and a much smaller fraction of unesteri-

fied long-chain fatty acids (free fatty acids) (4%). This

One or more apolipoproteins (proteins or polypeptides)

latter fraction, the free fatty acids (FFA), is metaboli-

are present in each lipoprotein. The major apolipopro-

cally the most active of the plasma lipids.

teins of HDL (α-lipoprotein) are designated A (Table

205

206

CHAPTER 25

Table 25-1.

Composition of the lipoproteins in plasma of humans.

Composition

Diameter

Density

Protein

Lipid

Main Lipid

Lipoprotein

Source

(nm)

(g/mL)

(%)

Components

Apolipoproteins

Chylomicrons

Intestine

90-1000

< 0.95

1-2

98-99

Triacylglycerol

A-I, A-II, A-IV,¹ B-48, C-I, C-II, C-III,

Chylomicron

Chylomicrons

45-150

< 1.006

6-8

92-94

Triacylglycerol,

B-48, E

remnants

phospholipids,

cholesterol

VLDL

Liver (intestine)

30-90

0.95-1.006

7-10

90-93

Triacylglycerol

B-100, C-I, C-II, C-III

IDL

VLDL

25-35

1.006-1.019

Triacylglycerol,

B-100, E

cholesterol

LDL

VLDL

20-25

1.019-1.063

Cholesterol

B-100

HDL

Liver, intestine,

Phospholipids,

A-I, A-II, A-IV, C-I, C-II, C-III, D,² E

HDL₁

VLDL, chylo-

20-25

1.019-1.063

cholesterol

microns

HDL₂

10-20

1.063-1.125

HDL₃

5-10

1.125-1.210

Preβ-HDL³

< 5

> 1.210

A-I

Albumin/free

Adipose

> 1.281

Free fatty acids

fatty acids

tissue

Abbreviations: HDL, high-density lipoproteins; IDL, intermediate-density lipoproteins; LDL, low-density lipoproteins; VLDL, very low

density lipoproteins.

¹Secreted with chylomicrons but transfers to HDL.

²Associated with HDL₂ and HDL₃ subfractions.

³Part of a minor fraction known as very high density lipoproteins (VHDL).

25-1). The main apolipoprotein of LDL (β-lipopro-

tein receptors in tissues, eg, apo B-100 and apo E for

tein) is apolipoprotein B (B-100) and is found also in

the LDL receptor, apo E for the LDL receptor-related

VLDL. Chylomicrons contain a truncated form of apo

protein (LRP), which has been identified as the rem-

B (B-48) that is synthesized in the intestine, while

nant receptor, and apo A-I for the HDL receptor. The

B-100 is synthesized in the liver. Apo B-100 is one of

functions of apo A-IV and apo D, however, are not yet

the longest single polypeptide chains known, having

clearly defined.

4536 amino acids and a molecular mass of 550,000 Da.

Apo B-48 (48% of B-100) is formed from the same

mRNA as apo B-100 after the introduction of a stop sig-

FREE FATTY ACIDS ARE

nal by an RNA editing enzyme. Apo C-I, C-II, and

RAPIDLY METABOLIZED

C-III are smaller polypeptides (molecular mass 7000-

9000 Da) freely transferable between several different

The free fatty acids (FFA, nonesterified fatty acids, un-

lipoproteins. Apo E is found in VLDL, HDL, chylomi-

esterified fatty acids) arise in the plasma from lipolysis

crons, and chylomicron remnants; it accounts for 5-

of triacylglycerol in adipose tissue or as a result of the

10% of total VLDL apolipoproteins in normal subjects.

action of lipoprotein lipase during uptake of plasma tri-

Apolipoproteins carry out several roles: (1) they can

acylglycerols into tissues. They are found in combina-

form part of the structure of the lipoprotein, eg, apo B;

tion with albumin, a very effective solubilizer, in con-

(2) they are enzyme cofactors, eg, C-II for lipoprotein

centrations varying between 0.1 and 2.0 µeq/mL of

lipase, A-I for lecithin:cholesterol acyltransferase, or en-

plasma. Levels are low in the fully fed condition and

zyme inhibitors, eg, apo A-II and apo C-III for lipopro-

rise to 0.7-0.8 µeq/mL in the starved state. In uncon-

tein lipase, apo C-I for cholesteryl ester transfer protein;

trolled diabetes mellitus, the level may rise to as much

and (3) they act as ligands for interaction with lipopro-

as 2 µeq/mL.

LIPID TRANSPORT & STORAGE

207

Peripheral apoprotein

are also to be found in chyle; however, most of the

(eg, apo C)

plasma VLDL are of hepatic origin. They are the vehi-

cles of transport of triacylglycerol from the liver to

the extrahepatic tissues.

Free

Phospholipid

cholesterol

There are striking similarities in the mechanisms of

Cholesteryl

formation of chylomicrons by intestinal cells and of

ester

VLDL by hepatic parenchymal cells (Figure 25-2), per-

haps because—apart from the mammary gland—the

Triacylglycerol

intestine and liver are the only tissues from which par-

ticulate lipid is secreted. Newly secreted or “nascent”

chylomicrons and VLDL contain only a small amount

of apolipoproteins C and E, and the full complement is

acquired from HDL in the circulation (Figures 25-3

Core of mainly

and 25-4). Apo B is essential for chylomicron and

nonpolar lipids

VLDL formation. In abetalipoproteinemia (a rare dis-

ease), lipoproteins containing apo B are not formed and

Integral

apoprotein

Monolayer of mainly

lipid droplets accumulate in the intestine and liver.

(eg, apo B)

amphipathic lipids

A more detailed account of the factors controlling

hepatic VLDL secretion is given below.

Figure 25-1. Generalized structure of a plasma

lipoprotein. The similarities with the structure of the

plasma membrane are to be noted. Small amounts of

CHYLOMICRONS & VERY LOW

cholesteryl ester and triacylglycerol are to be found in

DENSITY LIPOPROTEINS ARE

the surface layer and a little free cholesterol in the core.

RAPIDLY CATABOLIZED

The clearance of labeled chylomicrons from the blood

Free fatty acids are removed from the blood ex-

is rapid, the half-time of disappearance being under 1

tremely rapidly and oxidized (fulfilling 25-50% of en-

hour in humans. Larger particles are catabolized more

ergy requirements in starvation) or esterified to form

quickly than smaller ones. Fatty acids originating from

triacylglycerol in the tissues. In starvation, esterified

chylomicron triacylglycerol are delivered mainly to adi-

lipids from the circulation or in the tissues are oxidized

pose tissue, heart, and muscle (80%), while about 20%

as well, particularly in heart and skeletal muscle cells,

goes to the liver. However, the liver does not metabo-

where considerable stores of lipid are to be found.

lize native chylomicrons or VLDL significantly; thus,

The free fatty acid uptake by tissues is related di-

the fatty acids in the liver must be secondary to their

rectly to the plasma free fatty acid concentration, which

metabolism in extrahepatic tissues.

in turn is determined by the rate of lipolysis in adipose

tissue. After dissociation of the fatty acid-albumin com-

plex at the plasma membrane, fatty acids bind to a

Triacylglycerols of Chylomicrons & VLDL

membrane fatty acid transport protein that acts as a

Are Hydrolyzed by Lipoprotein Lipase

transmembrane cotransporter with Na⁺. On entering

Lipoprotein lipase is located on the walls of blood cap-

the cytosol, free fatty acids are bound by intracellular

illaries, anchored to the endothelium by negatively

fatty acid-binding proteins. The role of these proteins

charged proteoglycan chains of heparan sulfate. It has

in intracellular transport is thought to be similar to that

been found in heart, adipose tissue, spleen, lung, renal

of serum albumin in extracellular transport of long-

medulla, aorta, diaphragm, and lactating mammary

chain fatty acids.

gland, though it is not active in adult liver. It is not

normally found in blood; however, following injection

TRIACYLGLYCEROL IS TRANSPORTED

of heparin, lipoprotein lipase is released from its hep-

FROM THE INTESTINES IN

aran sulfate binding into the circulation. Hepatic li-

pase is bound to the sinusoidal surface of liver cells and

CHYLOMICRONS & FROM THE LIVER IN

is released by heparin. This enzyme, however, does not

VERY LOW DENSITY LIPOPROTEINS

react readily with chylomicrons or VLDL but is con-

By definition, chylomicrons are found in chyle formed

cerned with chylomicron remnant and HDL metabo-

only by the lymphatic system draining the intestine.

lism.

They are responsible for the transport of all dietary

Both phospholipids and apo C-II are required as

lipids into the circulation. Small quantities of VLDL

cofactors for lipoprotein lipase activity, while apo A-II

208

CHAPTER 25

Intestinal lumen

RER

•

RER

SER

Bile

canaliculus

VLDL

Fenestra

Endothelial

cell

Blood

Lymph vessel leading

capillary

to thoracic duct

Lumen of blood sinusoid

Figure 25-2. The formation and secretion of (A) chylomicrons by an intestinal cell and (B) very low density

lipoproteins by a hepatic cell. (RER, rough endoplasmic reticulum; SER, smooth endoplasmic reticulum; G, Golgi

apparatus; N, nucleus; C, chylomicrons; VLDL, very low density lipoproteins; E, endothelium; SD, space of Disse,

containing blood plasma.) Apolipoprotein B, synthesized in the RER, is incorporated into lipoproteins in the SER,

the main site of synthesis of triacylglycerol. After addition of carbohydrate residues in G, they are released from

the cell by reverse pinocytosis. Chylomicrons pass into the lymphatic system. VLDL are secreted into the space

of Disse and then into the hepatic sinusoids through fenestrae in the endothelial lining.

and apo C-III act as inhibitors. Hydrolysis takes place

The Action of Lipoprotein Lipase Forms

while the lipoproteins are attached to the enzyme on

Remnant Lipoproteins

the endothelium. Triacylglycerol is hydrolyzed progres-

Reaction with lipoprotein lipase results in the loss of

sively through a diacylglycerol to a monoacylglycerol

approximately 90% of the triacylglycerol of chylomi-

that is finally hydrolyzed to free fatty acid plus glycerol.

crons and in the loss of apo C (which returns to HDL)

Some of the released free fatty acids return to the circu-

but not apo E, which is retained. The resulting chy-

lation, attached to albumin, but the bulk is transported

lomicron remnant is about half the diameter of the

into the tissue (Figures 25-3 and 25-4). Heart lipopro-

parent chylomicron and is relatively enriched in choles-

tein lipase has a low K_m for triacylglycerol, about one-

terol and cholesteryl esters because of the loss of triacyl-

tenth of that for the enzyme in adipose tissue. This en-

glycerol

(Figure

25-3). Similar changes occur to

ables the delivery of fatty acids from triacylglycerol to

VLDL, with the formation of VLDL remnants or IDL

be redirected from adipose tissue to the heart in the

(intermediate-density lipoprotein) (Figure 25-4).

starved state when the plasma triacylglycerol decreases.

A similar redirection to the mammary gland occurs

during lactation, allowing uptake of lipoprotein triacyl-

The Liver Is Responsible for the Uptake

glycerol fatty acid for milk fat synthesis. The VLDL re-

of Remnant Lipoproteins

ceptor plays an important part in the delivery of fatty

acids from VLDL triacylglycerol to adipocytes by bind-

Chylomicron remnants are taken up by the liver by re-

ing VLDL and bringing it into close contact with

ceptor-mediated endocytosis, and the cholesteryl esters

lipoprotein lipase. In adipose tissue, insulin enhances

and triacylglycerols are hydrolyzed and metabolized.

lipoprotein lipase synthesis in adipocytes and its

Uptake is mediated by a receptor specific for apo E

translocation to the luminal surface of the capillary en-

(Figure 25-3), and both the LDL (apo B-100, E) re-

dothelium.

ceptor and the LRP (LDL receptor-related protein)

LIPID TRANSPORT & STORAGE

209

Dietary TG

Nascent

chylomicron

B-48

SMALL

Lymphatics

INTESTINE

Chylomicron

B-48

EXTRAHEPATIC

TISSUES

LDL

(apo B-100, E)

receptor

HDL

Cholesterol

LIPOPROTEIN LIPASE

Fatty acids

B-48

Fatty acids

LIVER

Chylomicron

Glycerol

LRP receptor

remnant

Figure 25-3. Metabolic fate of chylomicrons. (A, apolipoprotein A; B-48, apolipoprotein B-48; C ,

apolipoprotein C; E, apolipoprotein E; HDL, high-density lipoprotein; TG, triacylglycerol; C, cholesterol and

cholesteryl ester; P, phospholipid; HL, hepatic lipase; LRP, LDL receptor-related protein.) Only the predominant

lipids are shown.

are believed to take part. Hepatic lipase has a dual role:

graded in extrahepatic tissues and 70% in the liver. A

(1) in acting as a ligand to the lipoprotein and (2) in

positive correlation exists between the incidence of

hydrolyzing its triacylglycerol and phospholipid.

coronary atherosclerosis and the plasma concentra-

VLDL is the precursor of IDL, which is then con-

tion of LDL cholesterol. For further discussion of the

verted to LDL. Only one molecule of apo B-100 is

regulation of the LDL receptor, see Chapter 26.

present in each of these lipoprotein particles, and this is

conserved during the transformations. Thus, each LDL

HDL TAKES PART IN BOTH

particle is derived from only one VLDL particle (Figure

LIPOPROTEIN TRIACYLGLYCEROL

25-4). Two possible fates await IDL. It can be taken up

& CHOLESTEROL METABOLISM

by the liver directly via the LDL (apo B-100, E) recep-

tor, or it is converted to LDL. In humans, a relatively

HDL is synthesized and secreted from both liver and

large proportion forms LDL, accounting for the in-

intestine (Figure 25-5). However, apo C and apo E are

creased concentrations of LDL in humans compared

synthesized in the liver and transferred from liver HDL

with many other mammals.

to intestinal HDL when the latter enters the plasma. A

major function of HDL is to act as a repository for the

apo C and apo E required in the metabolism of chy-

LDL IS METABOLIZED VIA

lomicrons and VLDL. Nascent HDL consists of discoid

THE LDL RECEPTOR

phospholipid bilayers containing apo A and free choles-

The liver and many extrahepatic tissues express the

terol. These lipoproteins are similar to the particles

LDL (B-100, E) receptor. It is so designated because it

found in the plasma of patients with a deficiency of the

is specific for apo B-100 but not B-48, which lacks the

plasma enzyme lecithin:cholesterol acyltransferase

carboxyl terminal domain of B-100 containing the

(LCAT) and in the plasma of patients with obstructive

LDL receptor ligand, and it also takes up lipoproteins

jaundice. LCAT—and the LCAT activator apo A-I—

rich in apo E. This receptor is defective in familial hy-

bind to the disk, and the surface phospholipid and free

percholesterolemia. Approximately 30% of LDL is de-

cholesterol are converted into cholesteryl esters and

210

CHAPTER 25

Nascent

VLDL

B-100

VLDL

B-100

EXTRAHEPATIC

TISSUES

LDL

(apo B-100, E)

PC C

receptor

HDL

Fatty acids

LIPOPROTEIN LIPASE

B-100

Cholesterol

B-100

Fatty acids

IDL

LIVER

(VLDL remnant)

LDL

(apo B-100, E)

Glycerol

receptor

Final destruction in

liver, extrahepatic

tissues (eg, lympho-

EXTRAHEPATIC

cytes, fibroblasts)

TISSUES

via endocytosis

Figure 25-4. Metabolic fate of very low density lipoproteins (VLDL) and production of low-density

lipoproteins (LDL). (A, apolipoprotein A; B-100, apolipoprotein B-100; C , apolipoprotein C; E, apolipoprotein

E; HDL, high-density lipoprotein; TG, triacylglycerol; IDL, intermediate-density lipoprotein; C, cholesterol and

cholesteryl ester; P, phospholipid.) Only the predominant lipids are shown. It is possible that some IDL is also

metabolized via the LRP.

lysolecithin (Chapter 24). The nonpolar cholesteryl es-

then esterified by LCAT, increasing the size of the par-

ters move into the hydrophobic interior of the bilayer,

ticles to form the less dense HDL₂. The cycle is com-

whereas lysolecithin is transferred to plasma albumin.

pleted by the re-formation of HDL₃, either after selec-

Thus, a nonpolar core is generated, forming a spherical,

tive delivery of cholesteryl ester to the liver via the

pseudomicellar HDL covered by a surface film of polar

SR-B1 or by hydrolysis of HDL₂ phospholipid and tri-

lipids and apolipoproteins. In this way, the LCAT sys-

acylglycerol by hepatic lipase. In addition, free apo A-I

tem is involved in the removal of excess unesterified

is released by these processes and forms pre

-HDL

cholesterol from lipoproteins and tissues. The class B

after associating with a minimum amount of phospho-

scavenger receptor B1

(SR-B1) has recently been

lipid and cholesterol. Preβ-HDL is the most potent

identified as an HDL receptor in the liver and in

form of HDL in inducing cholesterol efflux from the

steroidogenic tissues. HDL binds to the receptor via

tissues to form discoidal HDL. Surplus apo A-I is de-

apo A-I and cholesteryl ester is selectively delivered to

stroyed in the kidney.

the cells, but the particle itself, including apo A-I, is not

HDL concentrations vary reciprocally with plasma

taken up. The transport of cholesterol from the tissues

triacylglycerol concentrations and directly with the ac-

to the liver is known as reverse cholesterol transport

tivity of lipoprotein lipase. This may be due to surplus

and is mediated by an HDL cycle (Figure 25-5). The

surface constituents, eg, phospholipid and apo A-I

smaller HDL₃ accepts cholesterol from the tissues via

being released during hydrolysis of chylomicrons and

the ATP-binding cassette transporter-1

(ABC-1).

VLDL and contributing toward the formation of preβ-

ABC-1 is a member of a family of transporter proteins

HDL and discoidal HDL. HDL₂ concentrations are in-

that couple the hydrolysis of ATP to the binding of a

versely related to the incidence of coronary athero-

substrate, enabling it to be transported across the mem-

sclerosis, possibly because they reflect the efficiency of

brane. After being accepted by HDL₃, the cholesterol is

reverse cholesterol transport. HDL_c (HDL₁) is found in

LIPID TRANSPORT & STORAGE

211

Bile C and

bile acids

A-1

SMALL INTESTINE

LIVER

Synthesis

LCAT

Synthesis

Kidney

C CE PL

Discoidal

HDL

SR-B1

HEPATIC

A-1

LIPASE

PL C

A-1

Preβ-HDL

Phospholipid

bilayer

A-1

TISSUES

LCAT

ABC-1

HDL₂

HDL₃

Figure 25-5. Metabolism of high-density lipoprotein (HDL) in reverse cholesterol transport.

(LCAT, lecithin:cholesterol acyltransferase; C, cholesterol; CE, cholesteryl ester; PL, phospholipid;

A-I, apolipoprotein A-I; SR-B1, scavenger receptor B1; ABC-1, ATP binding cassette transporter 1.)

Preβ-HDL, HDL₂, HDL₃—see Table 25-1. Surplus surface constituents from the action of lipopro-

tein lipase on chylomicrons and VLDL are another source of preβ-HDL. Hepatic lipase activity is

increased by androgens and decreased by estrogens, which may account for higher concentra-

tions of plasma HDL₂ in women.

the blood of diet-induced hypercholesterolemic ani-

Hepatic VLDL Secretion Is Related

mals. It is rich in cholesterol, and its sole apolipopro-

to Dietary & Hormonal Status

tein is apo E. It appears that all plasma lipoproteins are

The cellular events involved in VLDL formation and

interrelated components of one or more metabolic cy-

secretion have been described above. Hepatic triacyl-

cles that together are responsible for the complex

glycerol synthesis provides the immediate stimulus for

process of plasma lipid transport.

the formation and secretion of VLDL. The fatty acids

used are derived from two possible sources: (1) synthe-

sis within the liver from acetyl-CoA derived mainly

THE LIVER PLAYS A CENTRAL ROLE IN

from carbohydrate (perhaps not so important in hu-

LIPID TRANSPORT & METABOLISM

mans) and (2) uptake of free fatty acids from the circu-

The liver carries out the following major functions in

lation. The first source is predominant in the well-fed

lipid metabolism: (1) It facilitates the digestion and ab-

condition, when fatty acid synthesis is high and the

sorption of lipids by the production of bile, which con-

level of circulating free fatty acids is low. As triacylglyc-

tains cholesterol and bile salts synthesized within the

erol does not normally accumulate in the liver under

liver de novo or from uptake of lipoprotein cholesterol

this condition, it must be inferred that it is transported

(Chapter 26). (2) The liver has active enzyme systems

from the liver in VLDL as rapidly as it is synthesized

for synthesizing and oxidizing fatty acids (Chapters 21

and that the synthesis of apo B-100 is not rate-limiting.

and 22) and for synthesizing triacylglycerols and phos-

Free fatty acids from the circulation are the main source

pholipids (Chapter 24). (3) It converts fatty acids to ke-

during starvation, the feeding of high-fat diets, or in di-

tone bodies (ketogenesis) (Chapter 22). (4) It plays an

abetes mellitus, when hepatic lipogenesis is inhibited.

integral part in the synthesis and metabolism of plasma

Factors that enhance both the synthesis of triacylglyc-

lipoproteins (this chapter).

erol and the secretion of VLDL by the liver include (1)

212

CHAPTER 25

the fed state rather than the starved state; (2) the feed-

causing lipid peroxidation. Some protection against this

ing of diets high in carbohydrate (particularly if they

is provided by the antioxidant action of vitamin E-sup-

contain sucrose or fructose), leading to high rates of li-

plemented diets. The action of ethionine is thought to

pogenesis and esterification of fatty acids; (3) high lev-

be due to a reduction in availability of ATP due to its

els of circulating free fatty acids;

(4) ingestion of

replacing methionine in S-adenosylmethionine, trap-

ethanol; and (5) the presence of high concentrations of

ping available adenine and preventing synthesis of

insulin and low concentrations of glucagon, which en-

ATP. Orotic acid also causes fatty liver; it is believed to

hance fatty acid synthesis and esterification and inhibit

interfere with glycosylation of the lipoprotein, thus in-

their oxidation (Figure 25-6).

hibiting release, and may also impair the recruitment of

triacylglycerol to the particles. A deficiency of vitamin

E enhances the hepatic necrosis of the choline defi-

CLINICAL ASPECTS

ciency type of fatty liver. Added vitamin E or a source

of selenium has a protective effect by combating lipid

Imbalance in the Rate of Triacylglycerol

peroxidation. In addition to protein deficiency, essen-

Formation & Export Causes Fatty Liver

tial fatty acid and vitamin deficiencies (eg, linoleic acid,

For a variety of reasons, lipid—mainly as triacylglyc-

pyridoxine, and pantothenic acid) can cause fatty infil-

erol—can accumulate in the liver (Figure 25-6). Exten-

tration of the liver.

sive accumulation is regarded as a pathologic condition.

When accumulation of lipid in the liver becomes

Ethanol Also Causes Fatty Liver

chronic, fibrotic changes occur in the cells that progress

to cirrhosis and impaired liver function.

Alcoholism leads to fat accumulation in the liver, hy-

Fatty livers fall into two main categories. The first

perlipidemia, and ultimately cirrhosis. The exact

type is associated with raised levels of plasma free

mechanism of action of ethanol in the long term is still

fatty acids resulting from mobilization of fat from adi-

uncertain. Ethanol consumption over a long period

pose tissue or from the hydrolysis of lipoprotein triacyl-

leads to the accumulation of fatty acids in the liver that

glycerol by lipoprotein lipase in extrahepatic tissues.

are derived from endogenous synthesis rather than from

The production of VLDL does not keep pace with the

increased mobilization from adipose tissue. There is no

increasing influx and esterification of free fatty acids, al-

impairment of hepatic synthesis of protein after ethanol

lowing triacylglycerol to accumulate, causing a fatty

ingestion. Oxidation of ethanol by alcohol dehydrogen-

liver. This occurs during starvation and the feeding of

ase leads to excess production of NADH.

high-fat diets. The ability to secrete VLDL may also be

impaired (eg, in starvation). In uncontrolled diabetes

ALCOHOL

DEHYDROGENASE

mellitus, twin lamb disease, and ketosis in cattle,

fatty infiltration is sufficiently severe to cause visible

CH₃

CH₂

CH₃

CHO

pallor (fatty appearance) and enlargement of the liver

NAD⁺ NADH + H⁺

with possible liver dysfunction.

Ethanol

Acetaldehyde

The second type of fatty liver is usually due to a

metabolic block in the production of plasma lipo-

The NADH generated competes with reducing

proteins, thus allowing triacylglycerol to accumulate.

equivalents from other substrates, including fatty acids,

Theoretically, the lesion may be due to (1) a block in

for the respiratory chain, inhibiting their oxidation, and

apolipoprotein synthesis, (2) a block in the synthesis of

decreasing activity of the citric acid cycle. The net effect

the lipoprotein from lipid and apolipoprotein, (3) a

of inhibiting fatty acid oxidation is to cause increased

failure in provision of phospholipids that are found in

esterification of fatty acids in triacylglycerol, resulting

lipoproteins, or (4) a failure in the secretory mechanism

in the fatty liver. Oxidation of ethanol leads to the for-

itself.

mation of acetaldehyde, which is oxidized by aldehyde

One type of fatty liver that has been studied exten-

dehydrogenase, producing acetate. Other effects of

sively in rats is due to a deficiency of choline, which

ethanol may include increased lipogenesis and choles-

has therefore been called a lipotropic factor. The an-

terol synthesis from acetyl-CoA, and lipid peroxidation.

tibiotic puromycin, ethionine (α-amino-γ-mercaptobu-

The increased [NADH]/[NAD⁺] ratio also causes in-

tyric acid), carbon tetrachloride, chloroform, phospho-

creased

[lactate]/[pyruvate], resulting in hyperlactic-

rus, lead, and arsenic all cause fatty liver and a marked

acidemia, which decreases excretion of uric acid, aggra-

reduction in concentration of VLDL in rats. Choline

vating gout. Some metabolism of ethanol takes place

will not protect the organism against these agents but

via a cytochrome P450-dependent microsomal ethanol

appears to aid in recovery. The action of carbon tetra-

oxidizing system (MEOS) involving NADPH and O₂.

chloride probably involves formation of free radicals

This system increases in activity in chronic alcoholism

LIPID TRANSPORT & STORAGE

213

VLDL

Apo C

HDL

Nascent

Apo E

BLOOD

VLDL

Nascent

LIVER

VLDL

HEPATOCYTE

Glycosyl

Golgi complex

residues

Smooth

Orotic acid

Carbon tetrachloride

endoplasmic

Destruction

Puromycin

of surplus apo B-100

Amino

reticulum

Ethionine

Carbon

acids

tetrachloride

Cholesterol

Apo B-100

Protein

Cholesteryl

Apo C

synthesis

ester

Apo E

Polyribosomes

Rough

Membrane

endoplasmic

synthesis

reticulum

Nascent

polypeptide

chains of

Triacylglycerol*

Phospholipid

apo B-100

Cholesterol feeding

EFA deficiency

Lipid

EFA

Choline

TRIACYLGLYCEROL

deficiency

1,2-Diacylglycerol

CDP-choline

Phosphocholine

Choline

Glucagon

Insulin

Ethanol

Acyl-CoA

Oxidation

–

Insulin

FFA

Lipogenesis from

carbohydrate

Figure 25-6. The synthesis of very low density lipoprotein (VLDL) in the liver and the possible loci of action of

factors causing accumulation of triacylglycerol and a fatty liver. (EFA, essential fatty acids; FFA, free fatty acids;

HDL, high-density lipoproteins; Apo, apolipoprotein; M, microsomal triacylglycerol transfer protein.) The pathways

indicated form a basis for events depicted in Figure 25-2. The main

triacylglycerol

pool in liver is not on the direct

pathway of VLDL synthesis from acyl-CoA. Thus, FFA, insulin, and glucagon have immediate effects on VLDL secre-

tion as their effects impinge directly on the small triacylglycerol* precursor pool. In the fully fed state, apo B-100 is

synthesized in excess of requirements for VLDL secretion and the surplus is destroyed in the liver. During transla-

tion of apo B-100, microsomal transfer protein-mediated lipid transport enables lipid to become associated with

the nascent polypeptide chain. After release from the ribosomes, these particles fuse with more lipids from the

smooth endoplasmic reticulum, producing nascent VLDL.

214

CHAPTER 25

and may account for the increased metabolic clearance

Glucose

in this condition. Ethanol will also inhibit the metabo-

Insulin

lism of some drugs, eg, barbiturates, by competing for

BLOOD

cytochrome P450-dependent enzymes.

ADIPOSE TISSUE

Glucose 6-phosphate

MEOS

CH₃

CH₂

+ NADPH + H⁺ + O₂

Ethanol

CH₃

CHO

+ NADP⁺ + 2H₂O

Glycolysis

Acetaldehyde

CO2

PPP

Acetyl-CoA

In some Asian populations and Native Americans,

NADPH + H+

alcohol consumption results in increased adverse reac-

CO2

tions to acetaldehyde owing to a genetic defect of mito-

chondrial aldehyde dehydrogenase.

Glycerol

Acyl-CoA

3-phosphate

Esterification

ADIPOSE TISSUE IS THE MAIN STORE

OF TRIACYLGLYCEROL IN THE BODY

ATP

CoA

The triacylglycerol stores in adipose tissue are continu-

ACYL-CoA

ally undergoing lipolysis (hydrolysis) and reesterifica-

SYNTHETASE

HORMONE-

tion (Figure 25-7). These two processes are entirely dif-

SENSITIVE

ferent pathways involving different reactants and

LIPASE

enzymes. This allows the processes of esterification or

lipolysis to be regulated separately by many nutritional,

Lipolysis

metabolic, and hormonal factors. The resultant of these

two processes determines the magnitude of the free

FFA

Glycerol

(pool 2)

(pool 1)

fatty acid pool in adipose tissue, which in turn deter-

mines the level of free fatty acids circulating in the

plasma. Since the latter has most profound effects upon

LIPOPROTEIN

the metabolism of other tissues, particularly liver and

LIPASE

muscle, the factors operating in adipose tissue that reg-

ulate the outflow of free fatty acids exert an influence

FFA

Glycerol

far beyond the tissue itself.

BLOOD

(chylomicrons, VLDL)

The Provision of Glycerol 3-Phosphate

Regulates Esterification: Lipolysis Is

Controlled by Hormone-Sensitive Lipase

FFA

Glycerol

(Figure 25-7)

Figure 25-7. Metabolism of adipose tissue. Hor-

Triacylglycerol is synthesized from acyl-CoA and glyc-

mone-sensitive lipase is activated by ACTH, TSH,

erol 3-phosphate (Figure 24-2). Because the enzyme

glucagon, epinephrine, norepinephrine, and vaso-

glycerol kinase is not expressed in adipose tissue, glyc-

pressin and inhibited by insulin, prostaglandin E₁, and

erol cannot be utilized for the provision of glycerol

nicotinic acid. Details of the formation of glycerol

3-phosphate, which must be supplied by glucose via

3-phosphate from intermediates of glycolysis are

glycolysis.

shown in Figure 24-2. (PPP, pentose phosphate path-

Triacylglycerol undergoes hydrolysis by a hormone-

way; TG, triacylglycerol; FFA, free fatty acids; VLDL, very

sensitive lipase to form free fatty acids and glycerol.

low density lipoprotein.)

This lipase is distinct from lipoprotein lipase that cat-

alyzes lipoprotein triacylglycerol hydrolysis before its

uptake into extrahepatic tissues (see above). Since glyc-

erol cannot be utilized, it diffuses into the blood,

whence it is utilized by tissues such as those of the liver

and kidney, which possess an active glycerol kinase.

LIPID TRANSPORT & STORAGE

215

The free fatty acids formed by lipolysis can be recon-

regulated in a coordinate manner by phosphorylation-

verted in the tissue to acyl-CoA by acyl-CoA syn-

dephosphorylation mechanisms.

thetase and reesterified with glycerol 3-phosphate to

A principal action of insulin in adipose tissue is to

form triacylglycerol. Thus, there is a continuous cycle

inhibit the activity of hormone-sensitive lipase, reduc-

of lipolysis and reesterification within the tissue.

ing the release not only of free fatty acids but of glycerol

However, when the rate of reesterification is not suffi-

as well. Adipose tissue is much more sensitive to insulin

cient to match the rate of lipolysis, free fatty acids accu-

than are many other tissues, which points to adipose

mulate and diffuse into the plasma, where they bind to

tissue as a major site of insulin action in vivo.

albumin and raise the concentration of plasma free fatty

acids.

Several Hormones Promote Lipolysis

Increased Glucose Metabolism Reduces

Other hormones accelerate the release of free fatty acids

the Output of Free Fatty Acids

from adipose tissue and raise the plasma free fatty acid

concentration by increasing the rate of lipolysis of the

When the utilization of glucose by adipose tissue is in-

triacylglycerol stores (Figure 25-8). These include epi-

creased, the free fatty acid outflow decreases. However,

nephrine, norepinephrine, glucagon, adrenocorticotro-

the release of glycerol continues, demonstrating that the

pic hormone (ACTH), α- and β-melanocyte-stimulat-

effect of glucose is not mediated by reducing the rate of

ing hormones (MSH), thyroid-stimulating hormone

lipolysis. The effect is due to the provision of glycerol

(TSH), growth hormone (GH), and vasopressin. Many

3-phosphate, which enhances esterification of free fatty

of these activate the hormone-sensitive lipase. For an

acids. Glucose can take several pathways in adipose tis-

optimal effect, most of these lipolytic processes require

sue, including oxidation to CO₂ via the citric acid

the presence of glucocorticoids and thyroid hor-

cycle, oxidation in the pentose phosphate pathway,

mones. These hormones act in a facilitatory or per-

conversion to long-chain fatty acids, and formation of

missive capacity with respect to other lipolytic en-

acylglycerol via glycerol

3-phosphate (Figure

25-7).

docrine factors.

When glucose utilization is high, a larger proportion of

The hormones that act rapidly in promoting lipoly-

the uptake is oxidized to CO₂ and converted to fatty

sis, ie, catecholamines, do so by stimulating the activity

acids. However, as total glucose utilization decreases,

of adenylyl cyclase, the enzyme that converts ATP to

the greater proportion of the glucose is directed to the

cAMP. The mechanism is analogous to that responsible

formation of glycerol 3-phosphate for the esterification

for hormonal stimulation of glycogenolysis

(Chap-

of acyl-CoA, which helps to minimize the efflux of free

ter 18). cAMP, by stimulating cAMP-dependent pro-

fatty acids.

tein kinase, activates hormone-sensitive lipase. Thus,

processes which destroy or preserve cAMP influence

HORMONES REGULATE

lipolysis. cAMP is degraded to 5′-AMP by the enzyme

FAT MOBILIZATION

cyclic 3 ,5 -nucleotide phosphodiesterase. This en-

zyme is inhibited by methylxanthines such as caffeine

Insulin Reduces the Output

and theophylline. Insulin antagonizes the effect of the

of Free Fatty Acids

lipolytic hormones. Lipolysis appears to be more sensi-

The rate of release of free fatty acids from adipose tissue

tive to changes in concentration of insulin than are glu-

is affected by many hormones that influence either the

cose utilization and esterification. The antilipolytic ef-

rate of esterification or the rate of lipolysis. Insulin in-

fects of insulin, nicotinic acid, and prostaglandin E₁ are

hibits the release of free fatty acids from adipose tissue,

accounted for by inhibition of the synthesis of cAMP at

which is followed by a fall in circulating plasma free

the adenylyl cyclase site, acting through a G_i protein.

fatty acids. It enhances lipogenesis and the synthesis of

Insulin also stimulates phosphodiesterase and the lipase

acylglycerol and increases the oxidation of glucose to

phosphatase that inactivates hormone-sensitive lipase.

CO₂ via the pentose phosphate pathway. All of these ef-

The effect of growth hormone in promoting lipolysis is

fects are dependent on the presence of glucose and can

dependent on synthesis of proteins involved in the for-

be explained, to a large extent, on the basis of the abil-

mation of cAMP. Glucocorticoids promote lipolysis via

ity of insulin to enhance the uptake of glucose into adi-

synthesis of new lipase protein by a cAMP-independent

pose cells via the GLUT 4 transporter. Insulin also in-

pathway, which may be inhibited by insulin, and also

creases the activity of pyruvate dehydrogenase, acetyl-

by promoting transcription of genes involved in the

CoA carboxylase, and glycerol phosphate acyltrans-

cAMP signal cascade. These findings help to explain

ferase, reinforcing the effects of increased glucose up-

the role of the pituitary gland and the adrenal cortex in

take on the enhancement of fatty acid and acylglycerol

enhancing fat mobilization. The recently discovered

synthesis. These three enzymes are now known to be

body weight regulatory hormone, leptin, stimulates

216

CHAPTER 25

Epinephrine,

ACTH,

Insulin, prostaglandin E₁,

norepinephrine

TSH,

nicotinic acid

(

)

glucagon

β-Adrenergic

blockers

ATP

Thyroid hormone

Hormone-sensitive

ADENYLYL

lipase b

Insulin

GTP

FFA

CYCLASE

(inactive)

ATP

Growth hormone

cAMP-

dependent

Lipase

Inhibitors of

cAMP

Mg²

protein

phosphatase

protein synthesis

Adenosine

kinase

TRIACYL-

Methyl-

ADP

GLYCEROL

xanthines

–

PHOSPHODI-

Hormone-sensitive

–

(eg, caffeine)

ESTERASE

lipase a

(active)

FFA +

Diacylglycerol

Thyroid hormone

Hormone-sensitive

lipase

5′ AMP

Insulin

FFA +

Insulin

2-Monoacylglycerol

lipase

Inhibitors of

Glucocorticoids

protein synthesis

FFA + glycerol

Figure 25-8.

Control of adipose tissue lipolysis. (TSH, thyroid-stimulating hormone; FFA, free fatty acids.)

Note the cascade sequence of reactions affording amplification at each step. The lipolytic stimulus is “switched

off” by removal of the stimulating hormone; the action of lipase phosphatase; the inhibition of the lipase and

adenylyl cyclase by high concentrations of FFA; the inhibition of adenylyl cyclase by adenosine; and the removal

of cAMP by the action of phosphodiesterase. ACTH, TSH, and glucagon may not activate adenylyl cyclase in vivo,

since the concentration of each hormone required in vitro is much higher than is found in the circulation. Posi-

tive ( + ) and negative ( − ) regulatory effects are represented by broken lines and substrate flow by solid lines.

lipolysis and inhibits lipogenesis by influencing the ac-

citrate lyase, a key enzyme in lipogenesis, does not ap-

tivity of the enzymes in the pathways for the break-

pear to be present, and other lipogenic enzymes—eg,

down and synthesis of fatty acids.

glucose-6-phosphate dehydrogenase and the malic en-

The sympathetic nervous system, through liberation

zyme—do not undergo adaptive changes. Indeed, it has

of norepinephrine in adipose tissue, plays a central role

been suggested that in humans there is a “carbohydrate

in the mobilization of free fatty acids. Thus, the in-

excess syndrome” due to a unique limitation in ability

creased lipolysis caused by many of the factors de-

to dispose of excess carbohydrate by lipogenesis. In

scribed above can be reduced or abolished by denerva-

birds, lipogenesis is confined to the liver, where it is

tion of adipose tissue or by ganglionic blockade.

particularly important in providing lipids for egg for-

mation, stimulated by estrogens. Human adipose tissue

is unresponsive to most of the lipolytic hormones apart

A Variety of Mechanisms Have Evolved for

from the catecholamines.

Fine Control of Adipose Tissue Metabolism

On consideration of the profound derangement of

Human adipose tissue may not be an important site of

metabolism in diabetes mellitus (due in large part to

lipogenesis. There is no significant incorporation of

increased release of free fatty acids from the depots) and

glucose or pyruvate into long-chain fatty acids; ATP-

the fact that insulin to a large extent corrects the condi-

LIPID TRANSPORT & STORAGE

217

INNER

tion, it must be concluded that insulin plays a promi-

OUTSIDE

MITOCHONDRIAL

INSIDE

nent role in the regulation of adipose tissue metabolism.

MEMBRANE

BROWN ADIPOSE TISSUE

Norepinephine

PROMOTES THERMOGENESIS

F₀

Brown adipose tissue is involved in metabolism particu-

ATP

H⁺

larly at times when heat generation is necessary. Thus,

synthase

cAMP

the tissue is extremely active in some species in arousal

F₀

from hibernation, in animals exposed to cold (nonshiv-

ering thermogenesis), and in heat production in the

Heat

newborn animal. Though not a prominent tissue in hu-

H⁺

Hormone-

mans, it is present in normal individuals, where it could

sensitive

lipase

be responsible for “diet-induced thermogenesis.” It is

noteworthy that brown adipose tissue is reduced or ab-

Respiratory

sent in obese persons. The tissue is characterized by a

chain

well-developed blood supply and a high content of mi-

Triacyl-

tochondria and cytochromes but low activity of ATP

glycerol

synthase. Metabolic emphasis is placed on oxidation of

H⁺

both glucose and fatty acids. Norepinephrine liberated

FFA

from sympathetic nerve endings is important in increas-

ing lipolysis in the tissue and increasing synthesis of

Thermogenin

lipoprotein lipase to enhance utilization of triacylglyc-

erol-rich lipoproteins from the circulation. Oxidation

Acyl-CoA

Reducing

and phosphorylation are not coupled in mitochondria

equivalents

of this tissue, and the phosphorylation that does occur

is at the substrate level, eg, at the succinate thiokinase

Heat

step and in glycolysis. Thus, oxidation produces much

heat, and little free energy is trapped in ATP. A ther-

Purine

mogenic uncoupling protein, thermogenin, acts as a

nucleotides

proton conductance pathway dissipating the electro-

chemical potential across the mitochondrial membrane

(Figure 25-9).

Carnitine

transporter

SUMMARY

• Since nonpolar lipids are insoluble in water, for

transport between the tissues in the aqueous blood

plasma they are combined with amphipathic lipids

Figure 25-9. Thermogenesis in brown adipose tis-

and proteins to make water-miscible lipoproteins.

sue. Activity of the respiratory chain produces heat in

• Four major groups of lipoproteins are recognized:

addition to translocating protons (Chapter 12). These

Chylomicrons transport lipids resulting from diges-

protons dissipate more heat when returned to the

tion and absorption. Very low density lipoproteins

inner mitochondrial compartment via thermogenin in-

(VLDL) transport triacylglycerol from the liver. Low-

stead of generating ATP when returning via the F₁ ATP

density lipoproteins (LDL) deliver cholesterol to the

synthase. The passage of H⁺ via thermogenin is inhib-

tissues, and high-density lipoproteins (HDL) remove

ited by purine nucleotides when brown adipose tissue

cholesterol from the tissues in the process known as

is unstimulated. Under the influence of norepinephrine,

reverse cholesterol transport.

the inhibition is removed by the production of free

• Chylomicrons and VLDL are metabolized by hydrol-

fatty acids (FFA) and acyl-CoA. Note the dual role of

ysis of their triacylglycerol, and lipoprotein remnants

acyl-CoA in both facilitating the action of thermogenin

are left in the circulation. These are taken up by liver,

and supplying reducing equivalents for the respiratory

but some of the remnants

(IDL) resulting from

chain.

and − signify positive or negative regulatory

VLDL form LDL which is taken up by the liver and

effects.

other tissues via the LDL receptor.

218

CHAPTER 25

• Apolipoproteins constitute the protein moiety of

REFERENCES

lipoproteins. They act as enzyme activators (eg, apo

Chappell DA, Medh JD: Receptor-mediated mechanisms of

C-II and apo A-I) or as ligands for cell receptors (eg,

lipoprotein remnant catabolism. Prog Lipid Res 1998;37:

apo A-I, apo E, and apo B-100).

393.

• Triacylglycerol is the main storage lipid in adipose

Eaton S et al: Multiple biochemical effects in the pathogenesis of

tissue. Upon mobilization, free fatty acids and glyc-

fatty liver. Eur J Clin Invest 1997;27:719.

erol are released. Free fatty acids are an important

Goldberg IJ, Merkel M: Lipoprotein lipase: physiology, biochem-

fuel source.

istry and molecular biology. Front Biosci 2001;6:D388.

• Brown adipose tissue is the site of

“nonshivering

Holm C et al: Molecular mechanisms regulating hormone sensitive

lipase and lipolysis. Annu Rev Nutr 2000;20:365.

thermogenesis.” It is found in hibernating and new-

Kaikans RM, Bass NM, Ockner RK: Functions of fatty acid bind-

born animals and is present in small quantity in hu-

ing proteins. Experientia 1990;46:617.

mans. Thermogenesis results from the presence of an

Lardy H, Shrago E: Biochemical aspects of obesity. Annu Rev

uncoupling protein, thermogenin, in the inner mito-

Biochem 1990;59:689.

chondrial membrane.

Rye K-A et al: Overview of plasma lipid transport. In: Plasma

Lipids and Their Role in Disease. Barter PJ, Rye K-A (editors).

Harwood Academic Publishers, 1999.

Shelness GS, Sellers JA: Very-low-density lipoprotein assembly and

secretion. Curr Opin Lipidol 2001;12:151.

Various authors: Biochemistry of Lipids, Lipoproteins and Mem-

branes. Vance DE, Vance JE (editors). Elsevier, 1996.

Various authors: Brown adipose tissue—role in nutritional energet-

ics. (Symposium.) Proc Nutr Soc 1989;48:165.

Cholesterol Synthesis,Transport,

& Excretion

Peter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DSc

BIOMEDICAL IMPORTANCE

from mevalonate by loss of CO₂ (Figure 26-2). (3) Six

isoprenoid units condense to form squalene. (4) Squa-

Cholesterol is present in tissues and in plasma either as

lene cyclizes to give rise to the parent steroid, lanos-

free cholesterol or as a storage form, combined with a

terol. (5) Cholesterol is formed from lanosterol (Figure

long-chain fatty acid as cholesteryl ester. In plasma,

26-3).

both forms are transported in lipoproteins (Chapter

25). Cholesterol is an amphipathic lipid and as such is

Step 1—Biosynthesis of Mevalonate: HMG-CoA

an essential structural component of membranes and of

(3-hydroxy-3-methylglutaryl-CoA) is formed by the re-

the outer layer of plasma lipoproteins. It is synthesized

actions used in mitochondria to synthesize ketone bod-

in many tissues from acetyl-CoA and is the precursor of

ies (Figure 22-7). However, since cholesterol synthesis

all other steroids in the body such as corticosteroids, sex

is extramitochondrial, the two pathways are distinct.

hormones, bile acids, and vitamin D. As a typical prod-

Initially, two molecules of acetyl-CoA condense to

uct of animal metabolism, cholesterol occurs in foods

form acetoacetyl-CoA catalyzed by cytosolic thiolase.

of animal origin such as egg yolk, meat, liver, and

Acetoacetyl-CoA condenses with a further molecule of

brain. Plasma low-density lipoprotein (LDL) is the ve-

acetyl-CoA catalyzed by HMG-CoA synthase to form

hicle of uptake of cholesterol and cholesteryl ester into

HMG-CoA, which is reduced to mevalonate by

many tissues. Free cholesterol is removed from tissues

NADPH catalyzed by HMG-CoA reductase. This is

by plasma high-density lipoprotein (HDL) and trans-

the principal regulatory step in the pathway of choles-

ported to the liver, where it is eliminated from the body

terol synthesis and is the site of action of the most effec-

either unchanged or after conversion to bile acids in the

tive class of cholesterol-lowering drugs, the HMG-CoA

process known as reverse cholesterol transport. Cho-

reductase inhibitors (statins) (Figure 26-1).

lesterol is a major constituent of gallstones. However,

Step 2—Formation of Isoprenoid Units: Meval-

its chief role in pathologic processes is as a factor in the

onate is phosphorylated sequentially by ATP by three

genesis of atherosclerosis of vital arteries, causing cere-

kinases, and after decarboxylation (Figure 26-2) the ac-

brovascular, coronary, and peripheral vascular disease.

tive isoprenoid unit, isopentenyl diphosphate, is

formed.

CHOLESTEROL IS DERIVED

Step 3—Six Isoprenoid Units Form Squalene:

ABOUT EQUALLY FROM THE DIET

Isopentenyl diphosphate is isomerized by a shift of the

& FROM BIOSYNTHESIS

double bond to form dimethylallyl diphosphate, then

condensed with another molecule of isopentenyl

A little more than half the cholesterol of the body arises

diphosphate to form the ten-carbon intermediate ger-

by synthesis (about 700 mg/d), and the remainder is

anyl diphosphate (Figure 26-2). A further condensa-

provided by the average diet. The liver and intestine ac-

tion with isopentenyl diphosphate forms farnesyl

count for approximately 10% each of total synthesis in

diphosphate. Two molecules of farnesyl diphosphate

humans. Virtually all tissues containing nucleated cells

condense at the diphosphate end to form squalene. Ini-

are capable of cholesterol synthesis, which occurs in the

tially, inorganic pyrophosphate is eliminated, forming

endoplasmic reticulum and the cytosol.

presqualene diphosphate, which is then reduced by

NADPH with elimination of a further inorganic py-

Acetyl-CoA Is the Source of All Carbon

rophosphate molecule.

Atoms in Cholesterol

Step 4—Formation of Lanosterol: Squalene can

The biosynthesis of cholesterol may be divided into five

fold into a structure that closely resembles the steroid

steps: (1) Synthesis of mevalonate occurs from acetyl-

nucleus (Figure 26-3). Before ring closure occurs, squa-

CoA (Figure 26-1). (2) Isoprenoid units are formed

lene is converted to squalene 2,3-epoxide by a mixed-

219

220

CHAPTER 26

Farnesyl Diphosphate Gives Rise

CH₃

C S CoA

to Dolichol & Ubiquinone

2 Acetyl-CoA

The polyisoprenoids dolichol

(Figure

14-20 and

Chapter 47) and ubiquinone (Figure 12-5) are formed

THIOLASE

from farnesyl diphosphate by the further addition of up

(dolichol) or

3-7

(ubiquinone) isopentenyl

CoA SH

diphosphate residues, respectively. Some GTP-binding

CH₃

proteins in the cell membrane are prenylated with far-

CH₂

CoA

nesyl or geranylgeranyl (20 carbon) residues. Protein

Acetoacetyl-CoA

prenylation is believed to facilitate the anchoring of

proteins into lipoid membranes and may also be in-

H₂O

CH₃

C S CoA

volved in protein-protein interactions and membrane-

Acetyl-CoA

HMG-CoA SYNTHASE

associated protein trafficking.

CoA SH

CH₃

CHOLESTEROL SYNTHESIS IS

CONTROLLED BY REGULATION

^-OOC

CH₂

S CoA

OF HMG-CoA REDUCTASE

3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA)

Regulation of cholesterol synthesis is exerted near the

Bile acid, cholesterol

beginning of the pathway, at the HMG-CoA reductase

2NADPH + 2H⁺

step. The reduced synthesis of cholesterol in starving

Statins, eg,

animals is accompanied by a decrease in the activity of

HMG-CoA REDUCTASE

simvastatin

the enzyme. However, it is only hepatic synthesis that is

2NADP⁺ + CoA SH

inhibited by dietary cholesterol. HMG-CoA reductase

Mevalonate

CH₃

in liver is inhibited by mevalonate, the immediate prod-

uct of the pathway, and by cholesterol, the main prod-

OOC

C CH₂

CH₂

uct. Cholesterol (or a metabolite, eg, oxygenated sterol)

represses transcription of the HMG-CoA reductase

Mevalonate

gene and is also believed to influence translation. A di-

urnal variation occurs in both cholesterol synthesis

Figure 26-1. Biosynthesis of mevalonate. HMG-CoA

and reductase activity. In addition to these mechanisms

reductase is inhibited by atorvastatin, pravastatin, and

regulating the rate of protein synthesis, the enzyme ac-

simvastatin. The open and solid circles indicate the fate

tivity is also modulated more rapidly by posttransla-

of each of the carbons in the acetyl moiety of acetyl-

tional modification (Figure 26-4). Insulin or thyroid

CoA.

hormone increases HMG-CoA reductase activity,

whereas glucagon or glucocorticoids decrease it. Activ-

ity is reversibly modified by phosphorylation-dephos-

function oxidase in the endoplasmic reticulum, squa-

phorylation mechanisms, some of which may be

lene epoxidase. The methyl group on C₁₄ is transferred

cAMP-dependent and therefore immediately responsive

to C₁₃ and that on C₈ to C₁₄ as cyclization occurs, cat-

to glucagon. Attempts to lower plasma cholesterol in

alyzed by oxidosqualene:lanosterol cyclase.

humans by reducing the amount of cholesterol in the

Step 5—Formation of Cholesterol: The forma-

diet produce variable results. Generally, a decrease of

tion of cholesterol from lanosterol takes place in the

100 mg in dietary cholesterol causes a decrease of ap-

membranes of the endoplasmic reticulum and involves

proximately 0.13 mmol/L of serum.

changes in the steroid nucleus and side chain (Figure

26-3). The methyl groups on C₁₄ and C₄ are removed

MANY FACTORS INFLUENCE THE

to form 14-desmethyl lanosterol and then zymosterol.

CHOLESTEROL BALANCE IN TISSUES

The double bond at C₈-C₉ is subsequently moved to

C₅-C₆ in two steps, forming desmosterol. Finally, the

In tissues, cholesterol balance is regulated as follows (Fig-

double bond of the side chain is reduced, producing

ure 26-5): Cell cholesterol increase is due to uptake of

cholesterol. The exact order in which the steps de-

cholesterol-containing lipoproteins by receptors, eg, the

scribed actually take place is not known with cer-

LDL receptor or the scavenger receptor; uptake of free

tainty.

cholesterol from cholesterol-rich lipoproteins to the cell

ATP

ADP

CH₃

Mg2+

^-OOC

CH₂

^–OOC

CH₂

MEVALONATE

CH₂

O P

KINASE

Mevalonate

Mevalonate 5-phosphate

ATP

PHOSPHOMEVALONATE

Mg2+

KINASE

ADP

ATP

O P

CH₃

Mg2+

^-OOC

CH₂

^-OOC

CH₂

DIPHOSPHOMEVALONATE

CH₂

O P P

CH₂

O P

KINASE

Mevalonate 3-phospho-5-diphosphate

Mevalonate 5-diphosphate

CO₂ + P_i

HMG-CoA

DIPHOSPHO-

MEVALONATE

trans -Methyl-

CH₃

DECARBOXYLASE

CH₃

glutaconate

shunt

CH₂

ISOPENTENYL-

CH₃

O P P

CH₂

O P P

DIPHOSPHATE

3,3-Dimethylallyl

ISOMERASE

Isopentenyl

diphosphate

Isopentenyl tRNA

CIS-PRENYL

TRANSFERASE

CH₃

CH₂

Prenylated proteins

CH₃

CH₂

O P P

Geranyl diphosphate

CIS-PRENYL

TRANSFERASE

PP_i

TRANS-PRENYL

CIS-PRENYL

TRANSFERASE

Side chain of

CH₂

Dolichol

ubiquinone

O P P

Farnesyl diphosphate

Heme a

NADPH + H⁺

SQUALENE SYNTHETASE

Mg2+, Mn2+

2PP_i

NADP⁺

CH₂

*CH₂

Squalene

Figure 26-2. Biosynthesis of squalene, ubiquinone, dolichol, and other polyisoprene derivatives. (HMG,

3-hydroxy-3-methylglutaryl;

⋅⋅, cytokinin.) A farnesyl residue is present in heme a of cytochrome oxidase.

⋅

The carbon marked with asterisk becomes C₁₁ or C₁₂ in squalene. Squalene synthetase is a microsomal en-

zyme; all other enzymes indicated are soluble cytosolic proteins, and some are found in peroxisomes.

221

222

CHAPTER 26

CH₃

CoA

^-OOC

CH₂

CH₂OH

CH C

CH CH₂-

CO₂

H₂O

Acetyl-CoA

Mevalonate

Isoprenoid unit

CH₃

CH₂

CH₃

CH₂

CH₃

CH₂

HC C

CH₂

HC C

Squalene

CH₃

epoxide

CH₂

CH₃

CH₂

SQUALENE

CH₃

EPOXIDASE

CH₃

NADPH

CH₂

¹/2 O₂

CH₂

FAD

CH₂

Squalene

OXIDOSQUALENE:

CH₃

LANOSTEROL

CH₃

CYCLASE

COOH

2CO₂

NADPH

O₂, NADPH

O₂

NAD⁺

Lanosterol

14-Desmethyl

Zymosterol

lanosterol

ISOMERASE

NADPH

1112

O₂

∆²⁴-REDUCTASE

Cholesterol

Desmosterol

∆^7,24-Cholestadienol

(24-dehydrocholesterol)

Triparanol

Figure 26-3. Biosynthesis of cholesterol. The numbered positions are those of the steroid nucleus and the

open and solid circles indicate the fate of each of the carbons in the acetyl moiety of acetyl-CoA. Asterisks: Refer

to labeling of squalene in Figure 26-2.

CHOLESTEROL SYNTHESIS, TRANSPORT, & EXCRETION

223

REDUCTASE

ATP

KINASE

(inactive)

REDUCTASE

Insulin

PROTEIN

KINASE

PHOSPHATASES

KINASE

REDUCTASE

Glucagon

ADP

KINASE

H₂O

(active)

ATP

ADP

Inhibitor-1-

cAMP

phosphate*

HMG-CoA

LDL-cholesterol

REDUCTASE

(active)

(inactive)

Cholesterol

H₂O

Insulin

PROTEIN

Oxysterols

PHOSPHATASES

Enzyme synthesis

Figure 26-4.

Possible mechanisms in the regulation of cholesterol synthesis by HMG-CoA reductase. Insulin

has a dominant role compared with glucagon. Asterisk: See Figure 18-6.

membrane; cholesterol synthesis; and hydrolysis of cho-

ity; and down-regulates synthesis of the LDL receptor.

lesteryl esters by the enzyme cholesteryl ester hydrolase.

Thus, the number of LDL receptors on the cell surface

Decrease is due to efflux of cholesterol from the mem-

is regulated by the cholesterol requirement for mem-

brane to HDL, promoted by LCAT (lecithin:cholesterol

branes, steroid hormones, or bile acid synthesis (Figure

acyltransferase) (Chapter 25); esterification of cholesterol

26-5). The apo B-100, E receptor is a “high-affinity”

by ACAT (acyl-CoA:cholesterol acyltransferase); and uti-

LDL receptor, which may be saturated under most cir-

lization of cholesterol for synthesis of other steroids, such

cumstances. Other “low-affinity” LDL receptors also

as hormones, or bile acids in the liver.

appear to be present in addition to a scavenger path-

way, which is not regulated.

The LDL Receptor Is Highly Regulated

LDL (apo B-100, E) receptors occur on the cell surface

CHOLESTEROL IS TRANSPORTED

in pits that are coated on the cytosolic side of the cell

BETWEEN TISSUES IN PLASMA

membrane with a protein called clathrin. The glycopro-

LIPOPROTEINS

tein receptor spans the membrane, the B-100 binding

(Figure 26-6)

region being at the exposed amino terminal end. After

binding, LDL is taken up intact by endocytosis. The

In Western countries, the total plasma cholesterol in

apoprotein and cholesteryl ester are then hydrolyzed in

humans is about 5.2 mmol/L, rising with age, though

the lysosomes, and cholesterol is translocated into the

there are wide variations between individuals. The

cell. The receptors are recycled to the cell surface. This

greater part is found in the esterified form. It is trans-

influx of cholesterol inhibits in a coordinated man-

ported in lipoproteins of the plasma, and the highest

ner HMG-CoA synthase, HMG-CoA reductase, and,

proportion of cholesterol is found in the LDL. Dietary

therefore, cholesterol synthesis; stimulates ACAT activ-

cholesterol equilibrates with plasma cholesterol in days

224

CHAPTER 26

CELL MEMBRANE

Recycling

Receptor

–

vesicle

LDL (apo B -100, E)

synthesis

receptors

Cholesterol

(in coated pits)

Lysosome

synthesis

Endosome

ACAT

LDL

Unesterified

cholesterol

Coated

pool

vesicle

(mainly in membranes)

Scavenger receptor or

LDL

nonregulated pathway

Lysosome

HYDROLASE

LDL

Synthesis

VLDL

of steroids

ABC-1

CE A-1

A-1

LCAT

PL C

Preβ-HDL

HDL3

Figure 26-5. Factors affecting cholesterol balance at the cellular level. Reverse cholesterol transport may

be initiated by preβ HDL binding to the ABC-1 transporter protein via apo A-I. Cholesterol is then moved out

of the cell via the transporter, lipidating the HDL, and the larger particles then dissociate from the ABC-1 mol-

ecule. (C, cholesterol; CE, cholesteryl ester; PL, phospholipid; ACAT, acyl-CoA:cholesterol acyltransferase; LCAT,

lecithin:cholesterol acyltransferase; A-I, apolipoprotein A-I; LDL, low-density lipoprotein; VLDL, very low den-

sity lipoprotein.) LDL and HDL are not shown to scale.

and with tissue cholesterol in weeks. Cholesteryl ester

ates a concentration gradient and draws in cholesterol

in the diet is hydrolyzed to cholesterol, which is then

from tissues and from other lipoproteins (Figures 26-5

absorbed by the intestine together with dietary unesteri-

and 26-6), thus enabling HDL to function in reverse

fied cholesterol and other lipids. With cholesterol syn-

cholesterol transport (Figure 25-5).

thesized in the intestines, it is then incorporated into

chylomicrons. Of the cholesterol absorbed, 80-90% is

esterified with long-chain fatty acids in the intestinal

Cholesteryl Ester Transfer Protein

mucosa. Ninety-five percent of the chylomicron choles-

Facilitates Transfer of Cholesteryl Ester

terol is delivered to the liver in chylomicron remnants,

From HDL to Other Lipoproteins

and most of the cholesterol secreted by the liver in

This protein is found in plasma of humans and many

VLDL is retained during the formation of IDL and ul-

timately LDL, which is taken up by the LDL receptor

other species, associated with HDL. It facilitates transfer

of cholesteryl ester from HDL to VLDL, IDL, and LDL

in liver and extrahepatic tissues (Chapter 25).

in exchange for triacylglycerol, relieving product inhibi-

tion of LCAT activity in HDL. Thus, in humans, much

Plasma LCAT Is Responsible for Virtually

of the cholesteryl ester formed by LCAT finds its way to

All Plasma Cholesteryl Ester in Humans

the liver via VLDL remnants (IDL) or LDL (Figure

LCAT activity is associated with HDL containing apo

26-6). The triacylglycerol-enriched HDL₂ delivers its

A-I. As cholesterol in HDL becomes esterified, it cre-

cholesterol to the liver in the HDL cycle (Figure 25-5).

CHOLESTEROL SYNTHESIS, TRANSPORT, & EXCRETION

225

ENTEROHEPATIC CIRCULATION

HEPATIC PORTAL VEIN

Diet (0.4 g/d)

GALL

BLADDER

Synthesis

–

Bile acids

(total pool, 3-5 g)

BILE DUCT

Unesterified

cholesterol

pool

ACAT

Bile

acids

VLDL

Chylomicron

ILEUM

LDL

(apo B-100, E)

LIVER

receptor

LDL

LRP receptor

Bile acids

A-I

(0.6 g/d)

(0.4 g/d)

IDL

Feces

HDL

(VLDL remnant)

Chylomicron

remnant

LPL

LDL

(apo B-100, E)

receptor

EXTRAHEPATIC

Synthesis

TISSUES

Figure 26-6. Transport of cholesterol between the tissues in humans. (C, unesterified cholesterol; CE, cho-

lesteryl ester; TG, triacylglycerol; VLDL, very low density lipoprotein; IDL, intermediate-density lipoprotein; LDL,

low-density lipoprotein; HDL, high-density lipoprotein; ACAT, acyl-CoA:cholesterol acyltransferase; LCAT,

lecithin:cholesterol acyltransferase; A-I, apolipoprotein A-I; CETP, cholesteryl ester transfer protein; LPL, lipopro-

tein lipase; HL, hepatic lipase; LRP, LDL receptor-related protein.)

CHOLESTEROL IS EXCRETED FROM THE

feces; it is formed from cholesterol by the bacteria in

the lower intestine.

BODY IN THE BILE AS CHOLESTEROL OR

BILE ACIDS (SALTS)

Bile Acids Are Formed From Cholesterol

About 1 g of cholesterol is eliminated from the body

per day. Approximately half is excreted in the feces after

The primary bile acids are synthesized in the liver from

conversion to bile acids. The remainder is excreted as

cholesterol. These are cholic acid (found in the largest

cholesterol. Coprostanol is the principal sterol in the

amount) and chenodeoxycholic acid (Figure 26-7).

226

CHAPTER 26

Vitamin C

NADPH + H⁺

NADP⁺

O₂

7α-HYDROXYLASE

Cholesterol

7α-Hydroxycholesterol

12α-HYDROX-

Bile

YLASE

acids

Vitamin C

O₂

deficiency

NADPH + H⁺

(Several

2 CoA SH

steps)

2 CoA SH

Propionyl-CoA

S CoA

(CH₂)

SO₃H

CoA SH

Taurine

S CoA

Chenodeoxycholyl-CoA

Taurocholic acid

Glycine

(primary bile acid)

CoA SH

Tauro- and glyco-

Cholyl-CoA

chenodeoxycholic acid

(primary bile acids)

Deconjugation

CH₂COOH

+ 7α-dehydroxylation

COOH

Glycocholic acid

(primary bile acid)

Deconjugation

+ 7α-dehydroxylation

Deoxycholic acid

Lithocholic acid

(secondary bile acid)

Figure 26-7. Biosynthesis and degradation of bile acids. A second pathway in mitochondria involves hy-

droxylation of cholesterol by sterol 27-hydroxylase. Asterisk: Catalyzed by microbial enzymes.

The 7α-hydroxylation of cholesterol is the first and

chenodeoxycholyl-CoA (Figure 26-7). A second path-

principal regulatory step in the biosynthesis of bile acids

way in mitochondria involving the 27-hydroxylation of

catalyzed by 7

-hydroxylase, a microsomal enzyme. A

cholesterol by sterol 27-hydroxylase as the first step is

typical monooxygenase, it requires oxygen, NADPH,

responsible for a significant proportion of the primary

and cytochrome P450. Subsequent hydroxylation steps

bile acids synthesized. The primary bile acids (Figure

are also catalyzed by monooxygenases. The pathway of

26-7) enter the bile as glycine or taurine conjugates.

bile acid biosynthesis divides early into one subpathway

Conjugation takes place in peroxisomes. In humans, the

leading to cholyl-CoA, characterized by an extra α-OH

ratio of the glycine to the taurine conjugates is normally

group on position 12, and another pathway leading to

3:1. In the alkaline bile, the bile acids and their conju-

CHOLESTEROL SYNTHESIS, TRANSPORT, & EXCRETION

227

gates are assumed to be in a salt form—hence the term

ized by the deposition of cholesterol and cholesteryl

“bile salts.”

ester from the plasma lipoproteins into the artery wall.

A portion of the primary bile acids in the intestine is

Diseases in which prolonged elevated levels of VLDL,

subjected to further changes by the activity of the in-

IDL, chylomicron remnants, or LDL occur in the

testinal bacteria. These include deconjugation and 7α-

blood (eg, diabetes mellitus, lipid nephrosis, hypothy-

dehydroxylation, which produce the secondary bile

roidism, and other conditions of hyperlipidemia) are

acids, deoxycholic acid and lithocholic acid.

often accompanied by premature or more severe ather-

osclerosis. There is also an inverse relationship between

HDL (HDL₂) concentrations and coronary heart dis-

Most Bile Acids Return to the Liver

ease, and some consider that the most predictive rela-

in the Enterohepatic Circulation

tionship is the LDL:HDL cholesterol ratio. This is

Although products of fat digestion, including choles-

consistent with the function of HDL in reverse choles-

terol, are absorbed in the first 100 cm of small intestine,

terol transport. Susceptibility to atherosclerosis varies

the primary and secondary bile acids are absorbed al-

widely among species, and humans are one of the few

most exclusively in the ileum, and 98-99% are re-

in which the disease can be induced by diets high in

turned to the liver via the portal circulation. This is

cholesterol.

known as the enterohepatic circulation (Figure 26-6).

However, lithocholic acid, because of its insolubility, is

Diet Can Play an Important Role in

not reabsorbed to any significant extent. Only a small

Reducing Serum Cholesterol

fraction of the bile salts escapes absorption and is there-

fore eliminated in the feces. Nonetheless, this represents

Hereditary factors play the greatest role in determining

a major pathway for the elimination of cholesterol.

individual serum cholesterol concentrations; however,

Each day the small pool of bile acids (about 3-5 g) is

dietary and environmental factors also play a part, and

cycled through the intestine six to ten times and an

the most beneficial of these is the substitution in the

amount of bile acid equivalent to that lost in the feces is

diet of polyunsaturated and monounsaturated fatty

synthesized from cholesterol, so that a pool of bile acids

acids for saturated fatty acids. Plant oils such as corn oil

of constant size is maintained. This is accomplished by

and sunflower seed oil contain a high proportion of

a system of feedback controls.

polyunsaturated fatty acids, while olive oil contains a

high concentration of monounsaturated fatty acids. On

the other hand, butterfat, beef fat, and palm oil contain

Bile Acid Synthesis Is Regulated

a high proportion of saturated fatty acids. Sucrose and

at the 7

-Hydroxylase Step

fructose have a greater effect in raising blood lipids, par-

The principal rate-limiting step in the biosynthesis of

ticularly triacylglycerols, than do other carbohydrates.

bile acids is at the cholesterol 7

-hydroxylase reac-

The reason for the cholesterol-lowering effect of

tion (Figure 26-7). The activity of the enzyme is feed-

polyunsaturated fatty acids is still not fully understood.

back-regulated via the nuclear bile acid-binding recep-

It is clear, however, that one of the mechanisms in-

tor farnesoid X receptor (FXR). When the size of the

volved is the up-regulation of LDL receptors by poly-

bile acid pool in the enterohepatic circulation increases,

and monounsaturated as compared with saturated fatty

FXR is activated and transcription of the cholesterol

acids, causing an increase in the catabolic rate of LDL,

7α-hydroxylase gene is suppressed. Chenodeoxycholic

the main atherogenic lipoprotein. In addition, saturated

acid is particularly important in activating FXR. Cho-

fatty acids cause the formation of smaller VLDL parti-

lesterol

7α-hydroxylase activity is also enhanced by

cles that contain relatively more cholesterol, and they

cholesterol of endogenous and dietary origin and regu-

are utilized by extrahepatic tissues at a slower rate than

lated by insulin, glucagon, glucocorticoids, and thyroid

are larger particles—tendencies that may be regarded as

hormone.

atherogenic.

Lifestyle Affects the Serum

CLINICAL ASPECTS

Cholesterol Level

The Serum Cholesterol Is Correlated With

Additional factors considered to play a part in coronary

the Incidence of Atherosclerosis &

heart disease include high blood pressure, smoking,

Coronary Heart Disease

male gender, obesity (particularly abdominal obesity),

While cholesterol is believed to be chiefly concerned in

lack of exercise, and drinking soft as opposed to hard

the relationship, other serum lipids such as triacylglyc-

water. Factors associated with elevation of plasma FFA

erols may also play a role. Atherosclerosis is character-

followed by increased output of triacylglycerol and cho-

228

CHAPTER 26

lesterol into the circulation in VLDL include emotional

of coronary heart disease. This may be due to elevation

stress and coffee drinking. Premenopausal women ap-

of HDL concentrations resulting from increased syn-

pear to be protected against many of these deleterious

thesis of apo A-I and changes in activity of cholesteryl

factors, and this is thought to be related to the benefi-

ester transfer protein. It has been claimed that red wine

cial effects of estrogen. There is an association between

is particularly beneficial, perhaps because of its content

moderate alcohol consumption and a lower incidence

of antioxidants. Regular exercise lowers plasma LDL

Table 26-1. Primary disorders of plasma lipoproteins (dyslipoproteinemias).

Name

Defect

Remarks

Hypolipoproteinemias

No chylomicrons, VLDL, or LDL are

Rare; blood acylglycerols low; intestine and liver

Abetalipoproteinemia

formed because of defect in the

accumulate acylglycerols. Intestinal malabsorp-

loading of apo B with lipid.

tion. Early death avoidable by administration of

large doses of fat-soluble vitamins, particularly

vitamin E.

Familial alpha-lipoprotein deficiency

All have low or near absence of HDL.

Tendency toward hypertriacylglycerolemia as a

Tangier disease

result of absence of apo C-II, causing inactive

Fish-eye disease

LPL. Low LDL levels. Atherosclerosis in the el-

Apo-A-I deficiencies

derly.

Hyperlipoproteinemias

Hypertriacylglycerolemia due to de-

Slow clearance of chylomicrons and VLDL. Low

Familial lipoprotein lipase

ficiency of LPL, abnormal LPL, or apo

levels of LDL and HDL. No increased risk of coro-

deficiency (type I)

C-II deficiency causing inactive LPL.

nary disease.

Familial hypercholesterolemia

Defective LDL receptors or mutation

Elevated LDL levels and hypercholesterolemia,

(type IIa)

in ligand region of apo B-100.

resulting in atherosclerosis and coronary disease.

Familial type III hyperlipoprotein-

Deficiency in remnant clearance by

Increase in chylomicron and VLDL remnants of

emia (broad beta disease, rem-

the liver is due to abnormality in apo

density < 1.019 (β-VLDL). Causes hypercholes-

nant removal disease, familial

E. Patients lack isoforms E3 and E4

terolemia, xanthomas, and atherosclerosis.

dysbetalipoproteinemia)

and have only E2, which does not

react with the E receptor.¹

Familial hypertriacylglycerolemia

Overproduction of VLDL often

Cholesterol levels rise with the VLDL concentra-

(type IV)

associated with glucose intolerance

tion. LDL and HDL tend to be subnormal. This

and hyperinsulinemia.

type of pattern is commonly associated with

coronary heart disease, type II diabetes mellitus,

obesity, alcoholism, and administration of

progestational hormones.

Familial hyperalphalipoproteinemia

Increased concentrations of HDL.

A rare condition apparently beneficial to health

and longevity.

Hepatic lipase deficiency

Deficiency of the enzyme leads to

Patients have xanthomas and coronary heart

accumulation of large triacylgly-

disease.

cerol-rich HDL and VLDL remnants.

Familial lecithin:cholesterol

Absence of LCAT leads to block in

Plasma concentrations of cholesteryl esters and

acyltransferase (LCAT) deficiency

reverse cholesterol transport. HDL

lysolecithin are low. Present is an abnormal LDL

remains as nascent disks incapable

fraction, lipoprotein X, found also in patients

of taking up and esterifying choles-

with cholestasis. VLDL is abnormal (β-VLDL).

terol.

Familial lipoprotein(a) excess

Lp(a) consists of 1 mol of LDL

Premature coronary heart disease due to athero-

attached to 1 mol of apo(a). Apo(a)

sclerosis, plus thrombosis due to inhibition of

shows structural homologies to plas-

fibrinolysis.

minogen.

¹There is an association between patients possessing the apo E4 allele and the incidence of Alzheimer’s disease. Apparently, apo E4 binds

more avidly to β-amyloid found in neuritic plaques.

CHOLESTEROL SYNTHESIS, TRANSPORT, & EXCRETION

229

but raises HDL. Triacylglycerol concentrations are also

acids, and vitamin D. It also plays an important

reduced, due most likely to increased insulin sensitivity,

structural role in membranes and in the outer layer of

which enhances expression of lipoprotein lipase.

lipoproteins.

•

Cholesterol is synthesized in the body entirely from

When Diet Changes Fail, Hypolipidemic

acetyl-CoA. Three molecules of acetyl-CoA form

mevalonate via the important regulatory reaction for

Drugs Will Reduce Serum Cholesterol

the pathway, catalyzed by HMG-CoA reductase.

& Triacylglycerol

Next, a five-carbon isoprenoid unit is formed, and

Significant reductions of plasma cholesterol can be ef-

six of these condense to form squalene. Squalene un-

fected medically by the use of cholestyramine resin or

dergoes cyclization to form the parent steroid lanos-

surgically by the ileal exclusion operations. Both proce-

terol, which, after the loss of three methyl groups,

dures block the reabsorption of bile acids, causing in-

forms cholesterol.

creased bile acid synthesis in the liver. This increases

•

Cholesterol synthesis in the liver is regulated partly

cholesterol excretion and up-regulates LDL receptors,

by cholesterol in the diet. In tissues, cholesterol bal-

lowering plasma cholesterol. Sitosterol is a hypocholes-

ance is maintained between the factors causing gain

terolemic agent that acts by blocking the absorption of

of cholesterol (eg, synthesis, uptake via the LDL or

cholesterol from the gastrointestinal tract.

scavenger receptors) and the factors causing loss of

Several drugs are known to block the formation of

cholesterol (eg, steroid synthesis, cholesteryl ester for-

cholesterol at various stages in the biosynthetic path-

mation, excretion). The activity of the LDL receptor

way. The statins inhibit HMG-CoA reductase, thus

is modulated by cellular cholesterol levels to achieve

up-regulating LDL receptors. Statins currently in use

this balance. In reverse cholesterol transport, HDL

include atorvastatin, simvastatin, and pravastatin. Fi-

(preβ-HDL, discoidal, or HDL₃) takes up cholesterol

brates such as clofibrate and gemfibrozil act mainly to

from the tissues and LCAT esterifies it and deposits

lower plasma triacylglycerols by decreasing the secretion

it in the core of HDL, which is converted to HDL₂.

of triacylglycerol and cholesterol-containing VLDL by

The cholesteryl ester in HDL₂ is taken up by the

the liver. In addition, they stimulate hydrolysis of

liver, either directly or after transfer to VLDL, IDL,

VLDL triacylglycerols by lipoprotein lipase. Probucol

or LDL via the cholesteryl ester transfer protein.

appears to increase LDL catabolism via receptor-

•

Excess cholesterol is excreted from the liver in the

independent pathways, but its antioxidant properties

bile as cholesterol or bile salts. A large proportion of

may be more important in preventing accumulation of

bile salts is absorbed into the portal circulation and

oxidized LDL, which has enhanced atherogenic proper-

returned to the liver as part of the enterohepatic cir-

ties, in arterial walls. Nicotinic acid reduces the flux of

culation.

FFA by inhibiting adipose tissue lipolysis, thereby in-

•

Elevated levels of cholesterol present in VLDL, IDL,

hibiting VLDL production by the liver.

or LDL are associated with atherosclerosis, whereas

high levels of HDL have a protective effect.

Primary Disorders of the Plasma

•

Inherited defects in lipoprotein metabolism lead to a

Lipoproteins (Dyslipoproteinemias)

primary condition of hypo- or hyperlipoproteinemia.

Are Inherited

Conditions such as diabetes mellitus, hypothy-

Inherited defects in lipoprotein metabolism lead to the

roidism, kidney disease, and atherosclerosis exhibit

secondary abnormal lipoprotein patterns that resem-

primary condition of either hypo- or hyperlipopro-

teinemia (Table 26-1). In addition, diseases such as

ble certain primary conditions.

diabetes mellitus, hypothyroidism, kidney disease

(nephrotic syndrome), and atherosclerosis are associ-

ated with secondary abnormal lipoprotein patterns that

REFERENCES

are very similar to one or another of the primary inher-

Illingworth DR: Management of hypercholesterolemia. Med Clin

ited conditions. Virtually all of the primary conditions

North Am 2000;84:23.

are due to a defect at a stage in lipoprotein formation,

Ness GC, Chambers CM: Feedback and hormonal regulation of

transport, or destruction (see Figures 25-4, 26-5, and

hepatic

3-hydroxy-3-methylglutaryl coenzyme A reductase:

26-6). Not all of the abnormalities are harmful.

the concept of cholesterol buffering capacity. Proc Soc Exp

Biol Med 2000;224:8.

SUMMARY

Parks DJ et al: Bile acids: natural ligands for a nuclear orphan re-

ceptor. Science 1999;284:1365.

• Cholesterol is the precursor of all other steroids in

Princen HMG: Regulation of bile acid synthesis. Curr Pharm De-

the body, eg, corticosteroids, sex hormones, bile

sign 1997;3:59.

Integration of Metabolism—

The Provision of Metabolic Fuels

David A Bender, PhD, & Peter A. Mayes, PhD, DSc

BIOMEDICAL IMPORTANCE

MANY METABOLIC FUELS

ARE INTERCONVERTIBLE

An adult human weighing 70 kg requires about 10-12

MJ (2400-2900 kcal) from metabolic fuels each day.

Carbohydrate in excess of immediate requirements as

This requirement is met from carbohydrates (40-60%),

fuel or for synthesis of glycogen in muscle and liver may

lipids

(mainly triacylglycerol, 30-40%), protein (10-

be used for lipogenesis (Chapter 21) and hence triacyl-

15%), and alcohol if consumed. The mix being oxi-

glycerol synthesis in both adipose tissue and liver

dized varies depending on whether the subject is in the

(whence it is exported in very low density lipoprotein).

fed or starving state and on the intensity of physical

The importance of lipogenesis in human beings is un-

work. The requirement for metabolic fuels is relatively

clear; in Western countries, dietary fat provides

constant throughout the day, since average physical ac-

35-45% of energy intake, while in less developed coun-

tivity only increases metabolic rate by about 40-50%

tries where carbohydrate may provide 60-75% of en-

over the basal metabolic rate. However, most people

ergy intake the total intake of food may be so low that

consume their daily intake of metabolic fuels in two or

there is little surplus for lipogenesis. A high intake of fat

three meals, so there is a need to form reserves of carbo-

inhibits lipogenesis.

hydrate (glycogen in liver and muscle) and lipid (tri-

Fatty acids (and ketone bodies formed from them)

acylglycerol in adipose tissue) for use between meals.

cannot be used for the synthesis of glucose. The reac-

If the intake of fuels is consistently greater than en-

tion of pyruvate dehydrogenase, forming acetyl-CoA, is

ergy expenditure, the surplus is stored, largely as fat,

irreversible, and for every two-carbon unit from acetyl-

leading to the development of obesity and its associated

CoA that enters the citric acid cycle there is a loss of

health hazards. If the intake of fuels is consistently

two carbon atoms as carbon dioxide before only one

lower than energy expenditure, there will be negligible

molecule of oxaloacetate is re-formed—ie, there is no

fat and carbohydrate reserves, and amino acids arising

net increase. This means that acetyl-CoA (and therefore

from protein turnover will be used for energy rather

any substrates that yield acetyl-CoA) can never be used

than replacement protein synthesis, leading to emacia-

for gluconeogenesis (Chapter 19). The (relatively rare)

tion and eventually death.

fatty acids with an odd number of carbon atoms yield

After a normal meal there is an ample supply of car-

propionyl-CoA as the product of the final cycle of β-

bohydrate, and the fuel for most tissues is glucose. In

oxidation (Chapter 22), and this can be a substrate for

the starving state, glucose must be spared for use by the

gluconeogenesis, as can the glycerol released by lipolysis

central nervous system (which is largely dependent on

of adipose tissue triacylglycerol reserves. Most of the

glucose) and the erythrocytes (which are wholly reliant

amino acids in excess of requirements for protein syn-

on glucose). Other tissues can utilize alternative fuels

thesis

(arising from the diet or from tissue protein

such as fatty acids and ketone bodies. As glycogen re-

turnover) yield pyruvate, or five- and four-carbon in-

serves become depleted, so amino acids arising from

termediates of the citric acid cycle. Pyruvate can be

protein turnover and glycerol arising from lipolysis are

carboxylated to oxaloacetate, which is the primary

used for gluconeogenesis. These events are largely con-

substrate for gluconeogenesis, and the five- and

trolled by the hormones insulin and glucagon. In dia-

four-carbon intermediates also result in a net increase in

betes mellitus there is either impaired synthesis and

the formation of oxaloacetate, which is then available

secretion of insulin (type 1 diabetes mellitus) or im-

for gluconeogenesis. These amino acids are classified as

paired sensitivity of tissues to insulin action (type 2 di-

glucogenic. Lysine and leucine yield only acetyl-CoA

abetes mellitus), leading to severe metabolic derange-

on oxidation and thus cannot be used for gluconeogen-

ment. In cattle the demands of heavy lactation can lead

esis, while phenylalanine, tyrosine, tryptophan, and

to ketosis, as can the demands of twin pregnancy in

isoleucine give rise to both acetyl-CoA and to interme-

sheep.

diates of the citric acid cycle that can be used for gluco-

231

232

CHAPTER 27

neogenesis. Those amino acids that give rise to acetyl-

rate of synthesis of glucose 6-phosphate. This is in ex-

CoA are classified as ketogenic because in the starving

cess of the liver’s requirement for energy and is used

state much of the acetyl-CoA will be used for synthesis

mainly for synthesis of glycogen. In both liver and

of ketone bodies in the liver.

skeletal muscle, insulin acts to stimulate glycogen syn-

thase and inhibit glycogen phosphorylase. Some of the

glucose entering the liver may also be used for lipogene-

sis and synthesis of triacylglycerol. In adipose tissue, in-

A SUPPLY OF METABOLIC FUELS

sulin stimulates glucose uptake, its conversion to fatty

IS PROVIDED IN BOTH THE FED

acids, and their esterification; and inhibits intracellular

& STARVING STATES

lipolysis and the release of free fatty acids.

(Figure 27-1)

The products of lipid digestion enter the circulation

as triacylglycerol-rich chylomicrons (Chapter 25). In

Glucose Is Always Required by the Central

adipose tissue and skeletal muscle, lipoprotein lipase is

Nervous System & Erythrocytes

activated in response to insulin; the resultant free fatty

Erythrocytes lack mitochondria and hence are wholly

acids are largely taken up to form triacylglycerol re-

reliant on glycolysis and the pentose phosphate path-

serves, while the glycerol remains in the blood stream

way. The brain can metabolize ketone bodies to meet

and is taken up by the liver and used for glycogen syn-

about 20% of its energy requirements; the remainder

thesis or lipogenesis. Free fatty acids remaining in the

must be supplied by glucose. The metabolic changes

blood stream are taken up by the liver and reesterified.

that occur in starvation are the consequences of the

The lipid-depleted chylomicron remnants are also

need to preserve glucose and the limited reserves of

cleared by the liver, and surplus liver triacylglycerol—

glycogen in liver for use by the brain and erythrocytes

including that from lipogenesis—is exported in very

and to ensure the provision of alternative fuels for other

low density lipoprotein.

tissues. The fetus and synthesis of lactose in milk also

Under normal feeding patterns the rate of tissue

require a significant amount of glucose.

protein catabolism is more or less constant throughout

the day; it is only in cachexia that there is an increased

rate of protein catabolism. There is net protein catabo-

lism in the postabsorptive phase of the feeding cycle

In the Fed State, Metabolic Fuel

and net protein synthesis in the absorptive phase, when

Reserves Are Laid Down

the rate of synthesis increases by about 20-25%. The

For several hours after a meal, while the products of di-

increased rate of protein synthesis is, again, a response

gestion are being absorbed, there is an abundant supply

to insulin action. Protein synthesis is an energy-expen-

of metabolic fuels. Under these conditions, glucose is

sive process, accounting for up to almost 20% of energy

the major fuel for oxidation in most tissues; this is ob-

expenditure in the fed state, when there is an ample

served as an increase in the respiratory quotient (the

supply of amino acids from the diet, but under 9% in

ratio of carbon dioxide produced to oxygen consumed)

the starved state.

from about 0.8 in the starved state to near 1 (Table

27-1).

Metabolic Fuel Reserves Are Mobilized

Glucose uptake into muscle and adipose tissue is

in the Starving State

controlled by insulin, which is secreted by the B islet

cells of the pancreas in response to an increased concen-

There is a small fall in plasma glucose upon starvation,

tration of glucose in the portal blood. An early response

then little change as starvation progresses (Table 27-2;

to insulin in muscle and adipose tissue is the migration

Figure

27-2). Plasma free fatty acids increase with

of glucose transporter vesicles to the cell surface, expos-

onset of starvation but then plateau. There is an initial

ing active glucose transporters (GLUT 4). These in-

delay in ketone body production, but as starvation pro-

sulin-sensitive tissues will only take up glucose from the

gresses the plasma concentration of ketone bodies in-

blood stream to any significant extent in the presence of

creases markedly.

the hormone. As insulin secretion falls in the starved

In the postabsorptive state, as the concentration of

state, so the transporters are internalized again, reduc-

glucose in the portal blood falls, so insulin secretion de-

ing glucose uptake.

creases, resulting in skeletal muscle and adipose tissue

The uptake of glucose into the liver is independent

taking up less glucose. The increase in secretion of

of insulin, but liver has an isoenzyme of hexokinase

glucagon from the A cells of the pancreas inhibits

(glucokinase) with a high K_m, so that as the concentra-

glycogen synthase and activates glycogen phosphorylase

tion of glucose entering the liver increases, so does the

in liver. The resulting glucose 6-phosphate in liver is

234

CHAPTER 27

Table 27-1. Energy yields, oxygen consumption, and carbon

dioxide production in the oxidation of metabolic fuels.

Energy Yield

O₂ Consumed

CO₂ Produced

Oxygen

(kJ/g)

(L/g)

(kJ/L)

Carbohydrate

0.829

1.00

Protein

0.966

0.782

0.81

Fat

2.016

1.427

0.71

hydrolyzed by glucose-6-phosphatase, and glucose is re-

Although muscle takes up and preferentially oxidizes

leased into the blood stream for use by other tissues,

free fatty acids in the starving state, it cannot meet all of

particularly the brain and erythrocytes.

its energy requirements by β-oxidation. By contrast, the

Muscle glycogen cannot contribute directly to

liver has a greater capacity for β-oxidation than it re-

plasma glucose, since muscle lacks glucose-6-phos-

quires to meet its own energy needs and forms more

phatase, and the primary purpose of muscle glycogen is

acetyl-CoA than can be oxidized. This acetyl-CoA is

to provide a source of glucose 6-phosphate for energy-

used to synthesize ketone bodies (Chapter 22), which

yielding metabolism in the muscle itself. However,

are major metabolic fuels for skeletal and heart muscle

acetyl-CoA formed by oxidation of fatty acids in muscle

and can meet some of the brain’s energy needs. In pro-

inhibits pyruvate dehydrogenase and leads to citrate ac-

longed starvation, glucose may represent less than 10%

cumulation, which in turn inhibits phosphofructoki-

of whole body energy-yielding metabolism. Further-

nase and therefore glycolysis, thus sparing glucose. Any

more, as a result of protein catabolism, an increasing

accumulated pyruvate is transaminated to alanine at the

number of amino acids are released and utilized in the

expense of amino acids arising from breakdown of pro-

liver and kidneys for gluconeogenesis.

tein reserves. The alanine—and much of the keto acids

resulting from this transamination—are exported from

muscle and taken up by the liver, where the alanine is

Plasma glucagon

transaminated to yield pyruvate. The resultant amino

acids are largely exported back to muscle to provide

amino groups for formation of more alanine, while the

pyruvate is a major substrate for gluconeogenesis in the

liver.

In adipose tissue, the effect of the decrease in insulin

and increase in glucagon results in inhibition of lipo-

genesis, inactivation of lipoprotein lipase, and activa-

tion of hormone-sensitive lipase

(Chapter 25). This

leads to release of increased amounts of glycerol (a sub-

strate for gluconeogenesis in the liver) and free fatty

acids, which are used by skeletal muscle and liver as

their preferred metabolic fuels, so sparing glucose.

Blood glucose

Table 27-2. Plasma concentrations of metabolic

fuels (mmol/L) in the fed and starving states.

40 Hours

7 Days

Fed

Starvation

12-24

Glucose

5.5

3.6

3.5

Hours of starvation

Free fatty acids

0.30

1.15

1.19

Figure 27-2. Relative changes in metabolic parame-

Ketone bodies

Negligible

2.9

4.5

ters during the onset of starvation.

236

CHAPTER 27

CLINICAL ASPECTS

A summary of the major and unique metabolic fea-

tures of the principal tissues is presented in Table 27-3.

In prolonged starvation, as adipose tissue reserves are

depleted there is a very considerable increase in the net

rate of protein catabolism to provide amino acids not

SUMMARY

only as substrates for gluconeogenesis but also as the

• The body can interconvert the majority of foodstuffs.

main metabolic fuel of the tissues. Death results when

essential tissue proteins are catabolized beyond the

However, there is no net conversion of most fatty

acids

(or other acetyl-CoA-forming substances) to

point at which they can sustain this metabolic drain. In

patients with cachexia as a result of release of cytokines

glucose. Most amino acids, arising from the diet or

from tissue protein, can be used for gluconeogenesis,

in response to tumors and a number of other patho-

logic conditions, there is an increase in the rate of tissue

as can the glycerol from triacylglycerol.

protein catabolism as well as a considerably increased

• In starvation, glucose must be provided for the brain

metabolic rate, resulting in a state of advanced starva-

and erythrocytes; initially, this is supplied from liver

tion. Again, death results when essential tissue proteins

glycogen reserves. To spare glucose, muscle and other

have been catabolized.

tissues reduce glucose uptake in response to lowered

The high demand for glucose by the fetus and for

insulin secretion; they also oxidize fatty acids and ke-

synthesis of lactose in lactation can lead to ketosis. This

tone bodies preferentially to glucose.

may be seen as mild ketosis with hypoglycemia in

• Adipose tissue releases free fatty acids in starvation,

women, but in lactating cattle and in ewes carrying

and these are used by many tissues as fuel. Further-

twins there may be very pronounced ketosis and pro-

more, in the liver they are the substrate for synthesis

found hypoglycemia.

of ketone bodies.

In poorly controlled type 1 diabetes mellitus, pa-

• Ketosis, a metabolic adaptation to starvation, is exac-

tients may become hyperglycemic, partly as a result of

erbated in pathologic conditions such as diabetes

lack of insulin to stimulate uptake and utilization of

mellitus and ruminant ketosis.

glucose and partly because of increased gluconeogenesis

from amino acids in the liver. At the same time, the

lack of insulin results in increased lipolysis in adipose

REFERENCES

tissue, and the resultant free fatty acids are substrates

for ketogenesis in the liver. It is possible that in very se-

Bender DA: Introduction to Nutrition and Metabolism, 3rd edition.

Taylor & Francis, 2002.

vere diabetes utilization of ketone bodies in muscle

Caprio S et al: Oxidative fuel metabolism during mild hypo-

(and other tissues) is impaired because of lack of ox-

glycemia: critical role of free fatty acids. Am J Physiol

aloacetate (most tissues have a requirement for some

1989;256:E413.

glucose metabolism to maintain an adequate amount of

Fell D: Understanding the Control of Metabolism. Portland Press,

oxaloacetate for citric acid cycle activity). In uncon-

1997.

trolled diabetes, the magnitude of ketosis may be such

Frayn KN: Metabolic Regulation—A Human Perspective. Portland

as to result in severe acidosis (ketoacidosis) since ace-

Press, 1996.

toacetic acid and 3-hydroxybutyric acid are relatively

McNamara JP: Role and regulation of metabolism in adipose tissue

strong acids. Coma results from both the acidosis and

during lactation. J Nutr Biochem 1995;6:120.

the considerably increased osmolarity of extracellular

Randle PJ: The glucose-fatty acid cycle—biochemical aspects. Ath-

fluid (mainly due to the hyperglycemia).

erosclerosis Rev 1991;22:183.

SECTION III

Metabolism of Proteins & Amino Acids

Biosynthesis of the Nutritionally

Nonessential Amino Acids

Victor W. Rodwell, PhD

BIOMEDICAL IMPORTANCE

those three enzymes is to transform ammonium ion

into the α-amino nitrogen of various amino acids.

All 20 of the amino acids present in proteins are essential

Glutamate and Glutamine. Reductive amination of

for health. While comparatively rare in the Western

α-ketoglutarate is catalyzed by glutamate dehydrogenase

world, amino acid deficiency states are endemic in cer-

(Figure 28-1). Amination of glutamate to glutamine is

tain regions of West Africa where the diet relies heavily

catalyzed by glutamine synthetase (Figure 28-2).

on grains that are poor sources of amino acids such as

Alanine. Transamination of pyruvate forms alanine

tryptophan and lysine. These disorders include kwash-

(Figure 28-3).

iorkor, which results when a child is weaned onto a

Aspartate and Asparagine. Transamination of

starchy diet poor in protein; and marasmus, in which

oxaloacetate forms aspartate. The conversion of aspartate

both caloric intake and specific amino acids are deficient.

Humans can synthesize 12 of the 20 common amino

acids from the amphibolic intermediates of glycolysis and

of the citric acid cycle (Table 28-1). While nutritionally

Table 28-1. Amino acid requirements

nonessential, these 12 amino acids are not “nonessential.”

of humans.

All 20 amino acids are biologically essential. Of the 12 nu-

tritionally nonessential amino acids, nine are formed from

Nutritionally Essential

Nutritionally Nonessential

amphibolic intermediates and three (cysteine, tyrosine

Arginine¹

Alanine

and hydroxylysine) from nutritionally essential amino

Histidine

Asparagine

acids. Identification of the twelve amino acids that hu-

Isoleucine

Aspartate

mans can synthesize rested primarily on data derived from

Leucine

Cysteine

feeding diets in which purified amino acids replaced pro-

Lysine

Glutamate

tein. This chapter considers only the biosynthesis of the

Methionine

Glutamine

twelve amino acids that are synthesized in human tissues,

Phenylalanine

Glycine

not the other eight that are synthesized by plants.

Threonine

Hydroxyproline²

Tryptophan

Hydroxylysine²

NUTRITIONALLY NONESSENTIAL

Valine

Proline

Serine

AMINO ACIDS HAVE SHORT

Tyrosine

BIOSYNTHETIC PATHWAYS

¹“Nutritionally semiessential.” Synthesized at rates inadequate

The enzymes glutamate dehydrogenase, glutamine syn-

to support growth of children.

thetase, and aminotransferases occupy central positions

²Not necessary for protein synthesis but formed during post-

in amino acid biosynthesis. The combined effect of

translational processing of collagen.

237

238

CHAPTER 28

NH₃

^-O

^–O

O^-

Pyruvate

Alanine

α-Ketoglutarate

L-Glutamate

NH4+

H₂O

Glu or Asp

α-Ketoglutarate or oxaloacetate

NAD(P)H + H⁺

NAD(P)

Figure 28-3. Formation of alanine by transamina-

tion of pyruvate. The amino donor may be glutamate or

Figure 28-1. The glutamate dehydrogenase

aspartate. The other product thus is α-ketoglutarate or

reaction.

oxaloacetate.

to asparagine is catalyzed by asparagine synthetase (Fig-

ure 28-4), which resembles glutamine synthetase (Fig-

ure 28-2) except that glutamine, not ammonium ion,

provides the nitrogen. Bacterial asparagine synthetases

O NH3+

O NH

can, however, also use ammonium ion. Coupled hy-

O^-

H₂N

drolysis of PP_i to P_i by pyrophosphatase ensures that

the reaction is strongly favored.

Serine. Oxidation of the α-hydroxyl group of the

L-Aspartate

L-Asparagine

glycolytic intermediate 3-phosphoglycerate converts it

to an oxo acid, whose subsequent transamination and

Gln

Glu

dephosphorylation leads to serine (Figure 28-5).

Glycine. Glycine aminotransferases can catalyze the

synthesis of glycine from glyoxylate and glutamate or

Mg-ATP

Mg-AMP + PP_i

alanine. Unlike most aminotransferase reactions, these

strongly favor glycine synthesis. Additional important

Figure 28-4. The asparagine synthetase reaction.

mammalian routes for glycine formation are from

Note similarities to and differences from the glutamine

choline (Figure 28-6) and from serine (Figure 28-7).

synthetase reaction (Figure 28-2).

Proline. Proline is formed from glutamate by rever-

sal of the reactions of proline catabolism (Figure 28-8).

Cysteine. Cysteine, while not nutritionally essen-

tial, is formed from methionine, which is nutritionally

essential. Following conversion of methionine to ho-

O⁻

NADH

O⁻

D-3-Phosphoglycerate

Phosphohydroxy

pyruvate

α-AA

NH₃

^-O

O^-

H₂N

O^-

α-KA

L-Glutamate

L-Glutamine

NH₃

H₂O

O⁻

NH₄

L-Serine

Phospho-L-serine

Mg-ATP

Mg-ADP + P_i

Figure 28-5. Serine biosynthesis. (α-AA, α-amino

Figure 28-2. The glutamine synthetase reaction.

acids; α-KA, α-keto acids.)

BIOSYNTHESIS OF THE NUTRITIONALLY NONESSENTIAL AMINO ACIDS

239

CH₃

H₃C

N⁺ CH₃

H₃C

N⁺ CH₃

NADH

O⁻

-O

NH3+

Choline

Betaine aldehyde

H₂O

L-Glutamate

L-Glutamate-

γ-semialdehyde

NAD⁺

H₂O

H₃C

CH₃

CH3

[CH₃]

H₃C

N⁺ CH₃

O⁻

Dimethylglycine O

Betaine

O⁻

NADH

O⁻

NH2+

NH⁺

[CH₂O]

L-Proline

∆²-Pyrrolidine-

5-carboxylate

CH₃

Figure 28-8. Biosynthesis of proline from glutamate

[CH₂O]

NH3+

by reversal of reactions of proline catabolism.

O⁻

Sarcosine

Glycine

Figure 28-6. Formation of glycine from choline.

NH3+

O⁻

mocysteine (see Chapter 30), homocysteine and serine

form cysteine and homoserine (Figure 28-9).

L-Serine

Tyrosine. Phenylalanine hydroxylase converts

H₃N⁺

phenylalanine to tyrosine

(Figure

28-10). Provided

H₂O

that the diet contains adequate nutritionally essential

NH3+

phenylalanine, tyrosine is nutritionally nonessential.

O⁻

But since the reaction is irreversible, dietary tyrosine

L-Homocysteine

cannot replace phenylalanine. Catalysis by this mixed-

H₃N⁺

function oxygenase incorporates one atom of O₂ into

H₂O

phenylalanine and reduces the other atom to water. Re-

ducing power, provided as tetrahydrobiopterin, derives

Cystathionine

ultimately from NADPH.

NH3+

O⁻

Methylene

H₄ folate

H₃N⁺

NH3+

L-Cysteine

NH3+

O^-

L-Homoserine

Serine

Glycine

Figure 28-9. Conversion of homocysteine and ser-

Figure 28-7. The serine hydroxymethyltransferase

ine to homoserine and cysteine. The sulfur of cysteine

reaction. The reaction is freely reversible. (H₄ folate,

derives from methionine and the carbon skeleton from

tetrahydrofolate.)

serine.

240

CHAPTER 28

NADP⁺ NADPH + H⁺

α-Ketoglutarate

[¹⁸O] Succinate

Fe2+

¹⁸O₂

Tetrahydro-

Dihydro-

Ascorbate

¹⁸OH

biopterin

Pro

O₂

H₂O

Figure 28-11. The prolyl hydroxylase reaction. The

CH₂

COO-

CH₂

COO⁻

substrate is a proline-rich peptide. During the course of

the reaction, molecular oxygen is incorporated into

NH3+

both succinate and proline. Lysyl hydroxylase catalyzes

an analogous reaction.

L-Phenylalanine

L-Tyrosine

H₂N

amino acids, tissue aminotransferases reversibly inter-

CH₃

convert all three amino acids and their corresponding

α-keto acids. These α-keto acids thus can replace their

OH OH

amino acids in the diet.

Tetrahydrobiopterin

Selenocysteine. While not normally considered

an amino acid present in proteins, selenocysteine oc-

Figure 28-10. The phenylalanine hydroxylase reac-

curs at the active sites of several enzymes. Examples in-

tion. Two distinct enzymatic activities are involved. Ac-

clude the human enzymes thioredoxin reductase, glu-

tivity II catalyzes reduction of dihydrobiopterin by

tathione peroxidase, and the deiodinase that converts

NADPH, and activity I the reduction of O₂ to H₂O and of

thyroxine to triiodothyronine. Unlike hydroxyproline

phenylalanine to tyrosine. This reaction is associated

or hydroxylysine, selenocysteine arises co-translation-

with several defects of phenylalanine metabolism dis-

ally during its incorporation into peptides. The UGA

cussed in Chapter 30.

anticodon of the unusual tRNA designated tRNA^Sec

normally signals STOP. The ability of the protein syn-

Hydroxyproline and Hydroxylysine. Hydroxy-

thetic apparatus to identify a selenocysteine-specific

proline and hydroxylysine are present principally in

UGA codon involves the selenocysteine insertion ele-

collagen. Since there is no tRNA for either hydroxy-

ment, a stem-loop structure in the untranslated region

lated amino acid, neither dietary hydroxyproline nor

of the mRNA. Selenocysteine-tRNA^Sec is first charged

hydroxylysine is incorporated into protein. Both are

with serine by the ligase that charges tRNA^Ser. Subse-

completely degraded (see Chapter 30). Hydroxyproline

quent replacement of the serine oxygen by selenium

and hydroxylysine arise from proline and lysine, but

involves selenophosphate formed by selenophosphate

only after these amino acids have been incorporated

synthase (Figure 28-12).

into peptides. Hydroxylation of peptide-bound prolyl

and lysyl residues is catalyzed by prolyl hydroxylase and

lysyl hydroxylase of tissues, including skin and skeletal

muscle, and of granulating wounds (Figure 28-11).

The hydroxylases are mixed-function oxygenases that

H Se

CH₂

COO^-

require substrate, molecular O₂, ascorbate, Fe2+, and

α-ketoglutarate. For every mole of proline or lysine hy-

NH3+

droxylated, one mole of α-ketoglutarate is decarboxy-

lated to succinate. One atom of O₂ is incorporated into

proline or lysine, the other into succinate

(Figure

Se + ATP

AMP + P_i

+ H Se

O^-

28-11). A deficiency of the vitamin C required for

O^-

these hydroxylases results in scurvy.

Valine, Leucine, and Isoleucine. While leucine,

Figure 28-12. Selenocysteine (top) and the reaction

valine, and isoleucine are all nutritionally essential

catalyzed by selenophosphate synthetase (bottom).

BIOSYNTHESIS OF THE NUTRITIONALLY NONESSENTIAL AMINO ACIDS

241

SUMMARY

• Selenocysteine, an essential active site residue in sev-

eral mammalian enzymes, arises by co-translational

• All vertebrates can form certain amino acids from

insertion of a previously modified tRNA.

amphibolic intermediates or from other dietary

amino acids. The intermediates and the amino acids

to which they give rise are α-ketoglutarate (Glu, Gln,

Pro, Hyp), oxaloacetate (Asp, Asn) and 3-phospho-

REFERENCES

glycerate (Ser, Gly).

Brown KM, Arthur JR: Selenium, selenoproteins and human

• Cysteine, tyrosine, and hydroxylysine are formed

health: a review. Public Health Nutr 2001;4:593.

from nutritionally essential amino acids. Serine pro-

Combs GF, Gray WP: Chemopreventive agents—selenium. Phar-

vides the carbon skeleton and homocysteine the sul-

macol Ther 1998;79:179.

fur for cysteine biosynthesis. Phenylalanine hydroxy-

Mercer LP, Dodds SJ, Smith DI: Dispensable, indispensable, and

lase converts phenylalanine to tyrosine.

conditionally indispensable amino acid ratios in the diet. In:

• Neither dietary hydroxyproline nor hydroxylysine is

Absorption and Utilization of Amino Acids. Friedman M (edi-

incorporated into proteins because no codon or

tor). CRC Press, 1989.

tRNA dictates their insertion into peptides.

Nordberg J et al: Mammalian thioredoxin reductase is irreversibly

inhibited by dinitrohalobenzenes by alkylation of both the

• Peptidyl hydroxyproline and hydroxylysine are

redox active selenocysteine and its neighboring cysteine

formed by hydroxylation of peptidyl proline or lysine

residue. J Biol Chem 1998;273:10835.

in reactions catalyzed by mixed-function oxidases

Scriver CR et al (editors): The Metabolic and Molecular Bases of In-

that require vitamin C as cofactor. The nutritional

herited Disease, 8th ed. McGraw-Hill, 2001.

disease scurvy reflects impaired hydroxylation due to

St Germain DL, Galton VA: The deiodinase family of selenopro-

a deficiency of vitamin C.

teins. Thyroid 1997;7:655.

Catabolism of Proteins

& of Amino Acid Nitrogen

Victor W. Rodwell, PhD

BIOMEDICAL IMPORTANCE

zymes have a t_1/2 of 0.5-2 hours. PEST sequences, re-

gions rich in proline (P), glutamate (E), serine (S), and

This chapter describes how the nitrogen of amino acids is

threonine (T), target some proteins for rapid degrada-

converted to urea and the rare disorders that accompany

tion. Intracellular proteases hydrolyze internal peptide

defects in urea biosynthesis. In normal adults, nitrogen

bonds. The resulting peptides are then degraded to

intake matches nitrogen excreted. Positive nitrogen bal-

amino acids by endopeptidases that cleave internal

ance, an excess of ingested over excreted nitrogen, ac-

bonds and by aminopeptidases and carboxypeptidases

companies growth and pregnancy. Negative nitrogen

that remove amino acids sequentially from the amino

balance, where output exceeds intake, may follow

and carboxyl terminals, respectively. Degradation of

surgery, advanced cancer, and kwashiorkor or marasmus.

circulating peptides such as hormones follows loss of a

While ammonia, derived mainly from the α-amino

sialic acid moiety from the nonreducing ends of their

nitrogen of amino acids, is highly toxic, tissues convert

oligosaccharide chains. Asialoglycoproteins are internal-

ammonia to the amide nitrogen of nontoxic glutamine.

ized by liver cell asialoglycoprotein receptors and de-

Subsequent deamination of glutamine in the liver re-

graded by lysosomal proteases termed cathepsins.

leases ammonia, which is then converted to nontoxic

Extracellular, membrane-associated, and long-lived

urea. If liver function is compromised, as in cirrhosis or

intracellular proteins are degraded in lysosomes by

hepatitis, elevated blood ammonia levels generate clini-

ATP-independent processes. By contrast, degradation

cal signs and symptoms. Rare metabolic disorders in-

of abnormal and other short-lived proteins occurs in

volve each of the five urea cycle enzymes.

the cytosol and requires ATP and ubiquitin. Ubiquitin,

so named because it is present in all eukaryotic cells, is a

PROTEIN TURNOVER OCCURS

small (8.5 kDa) protein that targets many intracellular

proteins for degradation. The primary structure of

IN ALL FORMS OF LIFE

ubiquitin is highly conserved. Only 3 of 76 residues

The continuous degradation and synthesis of cellular

differ between yeast and human ubiquitin. Several mol-

proteins occur in all forms of life. Each day humans

ecules of ubiquitin are attached by non-α-peptide

turn over 1-2% of their total body protein, principally

bonds formed between the carboxyl terminal of ubiqui-

muscle protein. High rates of protein degradation occur

tin and the ε-amino groups of lysyl residues in the tar-

in tissues undergoing structural rearrangement—eg,

get protein (Figure 29-1). The residue present at its

uterine tissue during pregnancy, tadpole tail tissue dur-

amino terminal affects whether a protein is ubiquiti-

ing metamorphosis, or skeletal muscle in starvation. Of

nated. Amino terminal Met or Ser retards whereas Asp

the liberated amino acids, approximately 75% are reuti-

or Arg accelerates ubiquitination. Degradation occurs

lized. The excess nitrogen forms urea. Since excess

in a multicatalytic complex of proteases known as the

amino acids are not stored, those not immediately in-

proteasome.

corporated into new protein are rapidly degraded.

ANIMALS CONVERT

-AMINO NITROGEN

PROTEASES & PEPTIDASES DEGRADE

TO VARIED END PRODUCTS

PROTEINS TO AMINO ACIDS

Different animals excrete excess nitrogen as ammonia,

The susceptibility of a protein to degradation is ex-

uric acid, or urea. The aqueous environment of

pressed as its half-life (t_1/2), the time required to lower

teleostean fish, which are ammonotelic (excrete ammo-

its concentration to half the initial value. Half-lives of

nia), compels them to excrete water continuously, facil-

liver proteins range from under 30 minutes to over 150

itating excretion of highly toxic ammonia. Birds, which

hours. Typical “housekeeping” enzymes have t_1/2 values

must conserve water and maintain low weight, are uri-

of over 100 hours. By contrast, many key regulatory en-

cotelic and excrete uric acid as semisolid guano. Many

242

CATABOLISM OF PROTEINS & OF AMINO ACID NITROGEN

243

UB C O^- + E₁ SH + ATP

AMP + PP_i + UB C S E₁

UB C S E₁ + E₂ SH

E₁ SH + UB C S E₂

E₃

3. UB C S E₂ + H₂N ε Protein

E₂ SH + UB C N ε Protein

Figure 29-1. Partial reactions in the attachment of ubiquitin (UB) to

proteins. (1) The terminal COOH of ubiquitin forms a thioester bond with

an -SH of E₁ in a reaction driven by conversion of ATP to AMP and PP_i. Sub-

sequent hydrolysis of PP_i by pyrophosphatase ensures that reaction 1 will

proceed readily. (2) A thioester exchange reaction transfers activated ubiq-

uitin to E₂. (3) E₃ catalyzes transfer of ubiquitin to ε-amino groups of lysyl

residues of target proteins.

land animals, including humans, are ureotelic and ex-

acids except lysine, threonine, proline, and hydroxypro-

crete nontoxic, water-soluble urea. High blood urea lev-

line participate in transamination. Transamination is

els in renal disease are a consequence—not a cause—of

readily reversible, and aminotransferases also function

impaired renal function.

in amino acid biosynthesis. The coenzyme pyridoxal

phosphate

(PLP) is present at the catalytic site of

aminotransferases and of many other enzymes that act

BIOSYNTHESIS OF UREA

on amino acids. PLP, a derivative of vitamin B₆, forms

Urea biosynthesis occurs in four stages: (1) transamina-

an enzyme-bound Schiff base intermediate that can re-

tion, (2) oxidative deamination of glutamate, (3) am-

arrange in various ways. During transamination, bound

monia transport, and (4) reactions of the urea cycle

PLP serves as a carrier of amino groups. Rearrangement

(Figure 29-2).

forms an α-keto acid and enzyme-bound pyridoxamine

phosphate, which forms a Schiff base with a second

keto acid. Following removal of α-amino nitrogen by

Transamination Transfers α-Amino

transamination, the remaining carbon “skeleton” is de-

Nitrogen to α-Ketoglutarate,

graded by pathways discussed in Chapter 30.

Forming Glutamate

Alanine-pyruvate aminotransferase (alanine amino-

Transamination interconverts pairs of α-amino acids

transferase) and glutamate-α-ketoglutarate aminotrans-

and α-keto acids (Figure 29-3). All the protein amino

ferase (glutamate aminotransferase) catalyze the transfer

α-Amino acid

α-Keto acid

TRANSAMINATION

NH₊

α-Ketoglutarate

L-Glutamate

O^-

R₁

OXIDATIVE

DEAMINATION

NH₃

CO₂

NH₊

UREA CYCLE

R₂

C O^-

Urea

Figure 29-2. Overall flow of nitrogen in amino acid

Figure 29-3. Transamination. The reaction is freely

catabolism.

reversible with an equilibrium constant close to unity.

244

CHAPTER 29

of amino groups to pyruvate (forming alanine) or to α-

NAD(P)⁺

NAD(P)H + H⁺

ketoglutarate (forming glutamate) (Figure 29-4). Each

aminotransferase is specific for one pair of substrates

NH₃

but nonspecific for the other pair. Since alanine is also a

substrate for glutamate aminotransferase, all the amino

L-Glutamate

α-Ketoglutarate

nitrogen from amino acids that undergo transamina-

tion can be concentrated in glutamate. This is impor-

Figure 29-5. The L-glutamate dehydrogenase reac-

tant because L-glutamate is the only amino acid that

tion. NAD(P)⁺ means that either NAD⁺ or NADP⁺ can

undergoes oxidative deamination at an appreciable rate

serve as co-substrate. The reaction is reversible but fa-

in mammalian tissues. The formation of ammonia

vors glutamate formation.

from α-amino groups thus occurs mainly via the α-

amino nitrogen of L-glutamate.

Transamination is not restricted to α-amino groups.

drogen peroxide (H₂O₂), which then is split to O₂ and

The δ-amino group of ornithine—but not the ε-amino

H₂O by catalase.

group of lysine—readily undergoes transamination.

Serum levels of aminotransferases are elevated in some

Ammonia Intoxication Is Life-Threatening

disease states (see Figure 7-11).

The ammonia produced by enteric bacteria and ab-

sorbed into portal venous blood and the ammonia pro-

L-GLUTAMATE DEHYDROGENASE

duced by tissues are rapidly removed from circulation

OCCUPIES A CENTRAL POSITION

by the liver and converted to urea. Only traces (10-20

µg/dL) thus normally are present in peripheral blood.

IN NITROGEN METABOLISM

This is essential, since ammonia is toxic to the central

Transfer of amino nitrogen to α-ketoglutarate forms L-

nervous system. Should portal blood bypass the liver,

glutamate. Release of this nitrogen as ammonia is then

systemic blood ammonia levels may rise to toxic levels.

catalyzed by hepatic L-glutamate dehydrogenase

This occurs in severely impaired hepatic function or the

(GDH), which can use either NAD⁺ or NADP⁺ (Fig-

development of collateral links between the portal and

ure 29-5). Conversion of α-amino nitrogen to ammo-

systemic veins in cirrhosis. Symptoms of ammonia in-

nia by the concerted action of glutamate aminotrans-

toxication include tremor, slurred speech, blurred vi-

ferase and GDH is often termed “transdeamination.”

sion, coma, and ultimately death. Ammonia may be

Liver GDH activity is allosterically inhibited by ATP,

toxic to the brain in part because it reacts with α-keto-

GTP, and NADH and activated by ADP. The reaction

glutarate to form glutamate. The resulting depleted lev-

catalyzed by GDH is freely reversible and functions also

els of α-ketoglutarate then impair function of the tri-

in amino acid biosynthesis (see Figure 28-1).

carboxylic acid (TCA) cycle in neurons.

Amino Acid Oxidases Also Remove

NH₃

NH₂

Nitrogen as Ammonia

AMINO ACID

O^-

OXIDASE

O^-

While their physiologic role is uncertain, L-amino acid

oxidases of liver and kidney convert amino acids to an

α-imino acid that decomposes to an α-keto acid with

α-Amino acid

Flavin

Flavin-H₂

α-Imino acid

release of ammonium ion (Figure 29-6). The reduced

H₂O

flavin is reoxidized by molecular oxygen, forming hy-

NH4+

H₂O₂

O₂

Pyruvate

α-Amino acid

O^-

CATALASE

L-Alanine

α-Keto acid

¹/2O₂

α-Keto acid

α-Ketoglutarate

α-Amino acid

Figure 29-6. Oxidative deamination catalyzed by

L-Glutamate

α-Keto acid

L-amino acid oxidase (L-α-amino acid:O₂ oxidoreduc-

Figure 29-4. Alanine aminotransferase (top) and

tase). The α-imino acid, shown in brackets, is not a

glutamate aminotransferase (bottom).

stable intermediate.

CATABOLISM OF PROTEINS & OF AMINO ACID NITROGEN

245

Glutamine Synthase Fixes Ammonia

NH₊

as Glutamine

H₂N

CH₂

O^-

CH₂

Formation of glutamine is catalyzed by mitochondrial

glutamine synthase (Figure 29-7). Since amide bond

synthesis is coupled to the hydrolysis of ATP to ADP

L-Glutamine

and P_i, the reaction strongly favors glutamine synthesis.

H₂O

One function of glutamine is to sequester ammonia in

a nontoxic form.

GLUTAMINASE

NH₊

Glutaminase & Asparaginase Deamidate

NH₊

Glutamine & Asparagine

CH₂

O^-

Hydrolytic release of the amide nitrogen of glutamine

CH₂

as ammonia, catalyzed by glutaminase (Figure 29-8),

strongly favors glutamate formation. The concerted ac-

L-Glutamate

tion of glutamine synthase and glutaminase thus cat-

alyzes the interconversion of free ammonium ion and

Figure 29-8. The glutaminase reaction proceeds es-

glutamine. An analogous reaction is catalyzed by L-as-

sentially irreversibly in the direction of glutamate and

paraginase.

NH4+ formation. Note that the amide nitrogen, not the

α-amino nitrogen, is removed.

Formation & Secretion of Ammonia

Maintains Acid-Base Balance

Excretion into urine of ammonia produced by renal tubu-

reactions of Figure

29-9. Of the six participating

lar cells facilitates cation conservation and regulation of

amino acids, N-acetylglutamate functions solely as an

acid-base balance. Ammonia production from intracellu-

enzyme activator. The others serve as carriers of the

lar renal amino acids, especially glutamine, increases in

atoms that ultimately become urea. The major meta-

metabolic acidosis and decreases in metabolic alkalosis.

bolic role of ornithine, citrulline, and argininosucci-

nate in mammals is urea synthesis. Urea synthesis is a

cyclic process. Since the ornithine consumed in reac-

UREA IS THE MAJOR END PRODUCT OF

tion 2 is regenerated in reaction 5, there is no net loss

NITROGEN CATABOLISM IN HUMANS

or gain of ornithine, citrulline, argininosuccinate, or

Synthesis of 1 mol of urea requires 3 mol of ATP plus

arginine. Ammonium ion, CO₂, ATP, and aspartate

1 mol each of ammonium ion and of the α-amino nitro-

are, however, consumed. Some reactions of urea syn-

gen of aspartate. Five enzymes catalyze the numbered

thesis occur in the matrix of the mitochondrion, other

reactions in the cytosol (Figure 29-9).

NH₊

Carbamoyl Phosphate Synthase I

CH₂

O^-

CH₂

Initiates Urea Biosynthesis

Condensation of CO₂, ammonia, and ATP to form

L-Glutamate

carbamoyl phosphate is catalyzed by mitochondrial

Mg-ATP

NH₊

carbamoyl phosphate synthase I (reaction 1, Figure

29-9). A cytosolic form of this enzyme, carbamoyl

GLUTAMINE

phosphate synthase II, uses glutamine rather than am-

SYNTHASE

monia as the nitrogen donor and functions in pyrimi-

Mg-ADP

H₂O

dine biosynthesis (see Chapter 34). Carbamoyl phos-

+ P_i

NH₊

phate synthase I, the rate-limiting enzyme of the urea

cycle, is active only in the presence of its allosteric acti-

H₂

CH₂

O^-

CH₂

vator N-acetylglutamate, which enhances the affinity

of the synthase for ATP. Formation of carbamoyl phos-

phate requires 2 mol of ATP, one of which serves as a

L-Glutamine

phosphate donor. Conversion of the second ATP to

Figure 29-7. The glutamine synthase reaction

AMP and pyrophosphate, coupled to the hydrolysis of

strongly favors glutamine synthesis.

pyrophosphate to orthophosphate, provides the driving

246

CHAPTER 29

CO₂

NH4+

CO₂ + NH4+

NH₂

Urea

C O

CARBAMOYL

NH₂

H₂O

2Mg-ATP

NH3+

PHOSPHATE

SYNTHASE I

N-Acetyl-

glutamate

CH₂NH₃

ARGINASE

CH₂ NH

CH₂

2Mg-ADP + Pi

CH₂

NH₃

COO⁻

L-Ornithine

L-Arginine

HC COO⁻

−

OOC

H2N C

O⁻

ORNITHINE

TRANSCARBAMOYLASE

Fumarate

O⁻

Carbamoyl

phosphate

ARGININOSUCCINASE

NH₂

COO⁻

NH CH

CH₂

CH₂ NH

CH₂

COO⁻

CH₂

Argininosuccinate

NH₃

ARGININOSUCCINIC ACID

COO⁻

SYNTHASE

COO⁻

L-Citrulline

Mg-ATP

AMP + Mg-PP_i

COO⁻

H₂N C

CH₂

COO⁻

L-Aspartate

Figure 29-9. Reactions and intermediates of urea biosynthesis. The nitrogen-containing groups that

contribute to the formation of urea are shaded. Reactions 1 and 2

occur in the matrix of liver mitochon-

dria and reactions

4 , and

in liver cytosol. CO₂ (as bicarbonate), ammonium ion, ornithine, and cit-

rulline enter the mitochondrial matrix via specific carriers (see heavy dots) present in the inner membrane of

liver mitochondria.

force for synthesis of the amide bond and the mixed

Carbamoyl Phosphate Plus Ornithine

acid anhydride bond of carbamoyl phosphate. The con-

Forms Citrulline

certed action of GDH and carbamoyl phosphate syn-

thase I thus shuttles nitrogen into carbamoyl phos-

L-Ornithine transcarbamoylase catalyzes transfer

phate, a compound with high group transfer potential.

the carbamoyl group of carbamoyl phosphate to or-

The reaction proceeds stepwise. Reaction of bicarbo-

nithine, forming citrulline and orthophosphate (reac-

nate with ATP forms carbonyl phosphate and ADP.

tion 2, Figure 29-9). While the reaction occurs in the

Ammonia then displaces ADP, forming carbamate and

mitochondrial matrix, both the formation of ornithine

orthophosphate. Phosphorylation of carbamate by the

and the subsequent metabolism of citrulline take place

second ATP then forms carbamoyl phosphate.

in the cytosol. Entry of ornithine into mitochondria

CATABOLISM OF PROTEINS & OF AMINO ACID NITROGEN

247

and exodus of citrulline from mitochondria therefore

of ammonia that accompanies enhanced protein degra-

involve mitochondrial inner membrane transport sys-

dation.

tems (Figure 29-9).

METABOLIC DISORDERS ARE

Citrulline Plus Aspartate

ASSOCIATED WITH EACH REACTION

Forms Argininosuccinate

OF THE UREA CYCLE

Argininosuccinate synthase links aspartate and cit-

Metabolic disorders of urea biosynthesis, while ex-

rulline via the amino group of aspartate (reaction 3,

tremely rare, illustrate four important principles: (1)

Figure 29-9) and provides the second nitrogen of urea.

Defects in any of several enzymes of a metabolic path-

The reaction requires ATP and involves intermediate

way enzyme can result in similar clinical signs and

formation of citrullyl-AMP. Subsequent displacement

symptoms. (2) The identification of intermediates and

of AMP by aspartate then forms citrulline.

of ancillary products that accumulate prior to a meta-

bolic block provides insight into the reaction that is im-

Cleavage of Argininosuccinate

paired. (3) Precise diagnosis requires quantitative assay

Forms Arginine & Fumarate

of the activity of the enzyme thought to be defective.

Cleavage of argininosuccinate, catalyzed by argini-

(4) Rational therapy must be based on an understand-

nosuccinase, proceeds with retention of nitrogen in

ing of the underlying biochemical reactions in normal

arginine and release of the aspartate skeleton as fu-

and impaired individuals.

marate (reaction 4, Figure 29-9). Addition of water to

All defects in urea synthesis result in ammonia in-

fumarate forms L-malate, and subsequent NAD⁺-

toxication. Intoxication is more severe when the meta-

dependent oxidation of malate forms oxaloacetate.

bolic block occurs at reactions 1 or 2 since some cova-

These two reactions are analogous to reactions of the

lent linking of ammonia to carbon has already occurred

citric acid cycle (see Figure 16-3) but are catalyzed by

if citrulline can be synthesized. Clinical symptoms

cytosolic fumarase and malate dehydrogenase. Transami-

common to all urea cycle disorders include vomiting,

nation of oxaloacetate by glutamate aminotransferase

avoidance of high-protein foods, intermittent ataxia, ir-

then re-forms aspartate. The carbon skeleton of aspartate-

ritability, lethargy, and mental retardation. The clinical

fumarate thus acts as a carrier of the nitrogen of gluta-

features and treatment of all five disorders discussed

mate into a precursor of urea.

below are similar. Significant improvement and mini-

mization of brain damage accompany a low-protein

Cleavage of Arginine Releases Urea

diet ingested as frequent small meals to avoid sudden

& Re-forms Ornithine

increases in blood ammonia levels.

Hyperammonemia Type 1. A consequence of

Hydrolytic cleavage of the guanidino group of arginine,

carbamoyl phosphate synthase I deficiency

(reac-

catalyzed by liver arginase, releases urea (reaction 5,

tion 1, Figure 29-9), this relatively infrequent condition

Figure 29-9). The other product, ornithine, reenters

(estimated frequency 1:62,000) probably is familial.

liver mitochondria for additional rounds of urea syn-

Hyperammonemia Type 2. A deficiency of or-

thesis. Ornithine and lysine are potent inhibitors of

nithine transcarbamoylase (reaction 2, Figure 29-9)

arginase, competitive with arginine. Arginine also serves

produces this X chromosome-linked deficiency. The

as the precursor of the potent muscle relaxant nitric

mothers also exhibit hyperammonemia and an aversion

oxide (NO) in a Ca2+-dependent reaction catalyzed by

to high-protein foods. Levels of glutamine are elevated

NO synthase (see Figure 49-15).

in blood, cerebrospinal fluid, and urine, probably due

to enhanced glutamine synthesis in response to elevated

Carbamoyl Phosphate Synthase I Is the

levels of tissue ammonia.

Pacemaker Enzyme of the Urea Cycle

Citrullinemia. In this rare disorder, plasma and

The activity of carbamoyl phosphate synthase I is deter-

cerebrospinal fluid citrulline levels are elevated and

mined by N-acetylglutamate, whose steady-state level is

1-2 g of citrulline are excreted daily. One patient lacked

dictated by its rate of synthesis from acetyl-CoA and

detectable argininosuccinate synthase activity

(reac-

glutamate and its rate of hydrolysis to acetate and glu-

tion 3, Figure 29-9). In another, the K_m for citrulline

tamate. These reactions are catalyzed by N-acetylglu-

was 25 times higher than normal. Citrulline and argini-

tamate synthase and N-acetylglutamate hydrolase, re-

nosuccinate, which contain nitrogen destined for urea

spectively. Major changes in diet can increase the

synthesis, serve as alternative carriers of excess nitrogen.

concentrations of individual urea cycle enzymes 10-fold

Feeding arginine enhanced excretion of citrulline in these

to 20-fold. Starvation, for example, elevates enzyme lev-

patients. Similarly, feeding benzoate diverts ammonia

els, presumably to cope with the increased production

nitrogen to hippurate via glycine (see Figure 31-1).

248

CHAPTER 29

Argininosuccinicaciduria. A rare disease charac-

• Transamination channels α-amino acid nitrogen into

terized by elevated levels of argininosuccinate in blood,

glutamate. L-Glutamate dehydrogenase (GDH) oc-

cerebrospinal fluid, and urine is associated with friable,

cupies a central position in nitrogen metabolism.

tufted hair (trichorrhexis nodosa). Both early-onset and

• Glutamine synthase converts NH₃ to nontoxic gluta-

late-onset types are known. The metabolic defect is

mine. Glutaminase releases NH₃ for use in urea syn-

the absence of argininosuccinase (reaction 4, Figure

thesis.

29-9). Diagnosis by measurement of erythrocyte argini-

• NH₃, CO₂, and the amide nitrogen of aspartate pro-

nosuccinase activity can be performed on umbilical

vide the atoms of urea.

cord blood or amniotic fluid cells. As for citrullinemia,

• Hepatic urea synthesis takes place in part in the mi-

feeding arginine and benzoate promotes nitrogen excre-

tochondrial matrix and in part in the cytosol. Inborn

tion.

errors of metabolism are associated with each reac-

Hyperargininemia. This defect is characterized by

tion of the urea cycle.

elevated blood and cerebrospinal fluid arginine levels,

low erythrocyte levels of arginase (reaction 5, Figure

• Changes in enzyme levels and allosteric regulation of

carbamoyl phosphate synthase by N-acetylglutamate

29-9), and a urinary amino acid pattern resembling

that of lysine-cystinuria. This pattern may reflect com-

regulate urea biosynthesis.

petition by arginine with lysine and cystine for reab-

sorption in the renal tubule. A low-protein diet lowers

REFERENCES

plasma ammonia levels and abolishes lysine-cystinuria.

Brooks P et al: Subcellular localization of proteasomes and their

regulatory complexes in mammalian cells. Biochem J 2000;

Gene Therapy Offers Promise for

346:155.

Curthoys NP, Watford M: Regulation of glutaminase activity and

Correcting Defects in Urea Biosynthesis

glutamine metabolism. Annu Rev Nutr 1995;15:133.

Gene therapy for rectification of defects in the enzymes

Hershko A, Ciechanover A: The ubiquitin system. Annu Rev

of the urea cycle is an area of active investigation. En-

Biochem 1998;67:425.

couraging preliminary results have been obtained, for

Iyer R et al: The human arginases and arginase deficiency. J Inherit

example, in animal models using an adenoviral vector

Metab Dis 1998;21:86.

to treat citrullinemia.

Lim CB et al: Reduction in the rates of protein and amino acid ca-

tabolism to slow down the accumulation of endogenous am-

monia: a strategy potentially adopted by mudskippers during

SUMMARY

aerial exposure in constant darkness. J Exp Biol

2001;

204:1605.

• Human subjects degrade 1-2% of their body protein

Patejunas G et al: Evaluation of gene therapy for citrullinaemia

daily at rates that vary widely between proteins and

using murine and bovine models. J Inherit Metab Dis

with physiologic state. Key regulatory enzymes often

1998;21:138.

have short half-lives.

Pickart CM. Mechanisms underlying ubiquitination. Annu Rev

Biochem 2001;70:503.

• Proteins are degraded by both ATP-dependent and

ATP-independent pathways. Ubiquitin targets many

Scriver CR et al (editors): The Metabolic and Molecular Bases of In-

herited Disease, 8th ed. McGraw-Hill, 2001.

intracellular proteins for degradation. Liver cell sur-

Tuchman M et al: The biochemical and molecular spectrum of or-

face receptors bind and internalize circulating asialo-

nithine transcarbamoylase deficiency. J Inherit Metab Dis

glycoproteins destined for lysosomal degradation.

1998;21:40.

• Ammonia is highly toxic. Fish excrete NH₃ directly;

Turner MA et al: Human argininosuccinate lyase: a structural basis

birds convert NH₃ to uric acid. Higher vertebrates

for intragenic complementation. Proc Natl Acad Sci U S A

convert NH₃ to urea.

1997;94:9063.

Catabolism of the Carbon Skeletons

of Amino Acids

Victor W. Rodwell, PhD

BIOMEDICAL IMPORTANCE

TRANSAMINATION TYPICALLY INITIATES

AMINO ACID CATABOLISM

This chapter considers conversion of the carbon skele-

tons of the common L-amino acids to amphibolic inter-

Removal of α-amino nitrogen by transamination (see

mediates and the metabolic diseases or “inborn errors of

Figure 28-3) is the first catabolic reaction of amino

metabolism” associated with these processes. Left un-

acids except in the case of proline, hydroxyproline,

treated, they can result in irreversible brain damage and

threonine, and lysine. The residual hydrocarbon skele-

early mortality. Prenatal or early postnatal detection

ton is then degraded to amphibolic intermediates as

and timely initiation of treatment thus are essential.

outlined in Figure 30-1.

Many of the enzymes concerned can be detected in cul-

Asparagine, Aspartate, Glutamine, and Gluta-

tured amniotic fluid cells, which facilitates early diag-

mate. All four carbons of asparagine and aspartate

nosis by amniocentesis. Treatment consists primarily of

form oxaloacetate (Figure 30-2, top). Analogous reac-

feeding diets low in the amino acids whose catabolism

tions convert glutamine and glutamate to

-ketoglu-

is impaired. While many changes in the primary struc-

tarate (Figure 30-2, bottom). Since the enzymes also

ture of enzymes have no adverse effects, others modify

fulfill anabolic functions, no metabolic defects are asso-

the three-dimensional structure of catalytic or regula-

ciated with the catabolism of these four amino acids.

tory sites, lower catalytic efficiency (lower V_max or ele-

Proline. Proline forms dehydroproline, glutamate-

vate K_m), or alter the affinity for an allosteric regulator

γ-semialdehyde, glutamate, and, ultimately,

-ketoglu-

of activity. A variety of mutations thus may give rise to

tarate (Figure 30-3, top). The metabolic block in type

the same clinical signs and symptoms.

I hyperprolinemia is at proline dehydrogenase.

Ala

Cys

Arg

Gly

His

α-Ketoglutarate

Glutamate

Hyp

Gln

Ser

Pro

Thr

lle

Leu

lle

Citrate

Succinyl-CoA

Met

Trp

Val

Pyruvate

Citrate

cycle

Acetyl-CoA

Acetoacetyl-CoA

Tyr

Oxaloacetate

Fumarate

Phe

Leu, Lys,

Phe, Trp,

Tyr

Aspartate

Asn

Figure 30-1. Amphibolic intermediates formed from the carbon skeletons

of amino acids.

249

250

CHAPTER 30

H₂O

NH4+

PYR

ALA

NH₂

O^-

CH₂

C NH₂

ASPARAGINASE

C NH3+

TRANSAMINASE

COO^-

COO

COO^-

L-Asparagine

L-Aspartate

Oxaloacetate

H₂O

NH₄

PYR

ALA

NH₂

O^-

Figure 30-2. Catabolism of L-as-

CH₂

paragine (top) and of L-glutamine

CH₂

(bottom) to amphibolic intermedi-

GLUTAMINASE

TRANSAMINASE

ates. (PYR, pyruvate; ALA, L-alanine.)

H C NH

C NH₃

In this and subsequent figures, color

COO^-

highlights portions of the molecules

L-Glutamine

L-Glutamate

α-Ketoglutarate

undergoing chemical change.

There is no associated impairment of hydroxyproline

Glycine. The glycine synthase complex of liver mi-

catabolism. The metabolic block in type II hyperpro-

tochondria splits glycine to CO₂ and NH4+ and forms

linemia is at glutamate-

-semialdehyde dehydroge-

N⁵,N¹⁰-methylene tetrahydrofolate (Figure 30-5).

nase, which also functions in hydroxyproline catabo-

Glycinuria results from a defect in renal tubular re-

lism. Both proline and hydroxyproline catabolism thus

absorption. The defect in primary hyperoxaluria is the

are affected and ∆¹-pyrroline-3-hydroxy-5-carboxylate

failure to catabolize glyoxylate formed by deamination

(see Figure 30-10) is excreted.

of glycine. Subsequent oxidation of glyoxylate to ox-

Arginine and Ornithine. Arginine is converted to

alate results in urolithiasis, nephrocalcinosis, and early

ornithine, glutamate γ-semialdehyde, and then

-ke-

mortality from renal failure or hypertension.

toglutarate (Figure 30-3, bottom). Mutations in or-

Serine. Following conversion to glycine, catalyzed

nithine

-aminotransferase elevate plasma and urinary

by serine hydroxymethyltransferase (Figure 30-5),

ornithine and cause gyrate atrophy of the retina.

serine catabolism merges with that of glycine (Figure

Treatment involves restricting dietary arginine. In hy-

30-6).

perornithinemia-hyperammonemia syndrome, a de-

Alanine. Transamination of alanine forms pyru-

fective mitochondrial ornithine-citrulline antiporter

vate. Perhaps for the reason advanced under glutamate

(see Figure 29-9) impairs transport of ornithine into

and aspartate catabolism, there is no known metabolic

mitochondria for use in urea synthesis.

defect of alanine catabolism. Cysteine. Cystine is first

Histidine. Catabolism of histidine proceeds via

reduced to cysteine by cystine reductase

(Figure

urocanate,

4-imidazolone-5-propionate, and N-for-

30-7). Two different pathways then convert cysteine to

miminoglutamate (Figlu). Formimino group transfer to

pyruvate (Figure 30-8).

tetrahydrofolate forms glutamate, then

-ketoglu-

There are numerous abnormalities of cysteine me-

tarate (Figure 30-4). In folic acid deficiency, group

tabolism. Cystine, lysine, arginine, and ornithine are

transfer is impaired and Figlu is excreted. Excretion of

excreted in cystine-lysinuria (cystinuria), a defect in

Figlu following a dose of histidine thus has been used

renal reabsorption. Apart from cystine calculi, cystin-

to detect folic acid deficiency. Benign disorders of histi-

uria is benign. The mixed disulfide of L-cysteine and

dine catabolism include histidinemia and urocanic

L-homocysteine (Figure 30-9) excreted by cystinuric

aciduria associated with impaired histidase.

patients is more soluble than cystine and reduces for-

mation of cystine calculi. Several metabolic defects

result in vitamin B₆-responsive or -unresponsive ho-

SIX AMINO ACIDS FORM PYRUVATE

mocystinurias. Defective carrier-mediated transport

All of the carbons of glycine, serine, alanine, and cys-

of cystine results in cystinosis (cystine storage dis-

teine and two carbons of threonine form pyruvate and

ease) with deposition of cystine crystals in tissues

subsequently acetyl-CoA.

and early mortality from acute renal failure. Despite

Figure 30-3. Top: Catabolism of proline. Numerals indicate

sites of the metabolic defects in 1

type I and 2

type II hyper-

prolinemias. Bottom: Catabolism of arginine. Glutamate-γ-

O⁻

semialdehyde forms α-ketoglutarate as shown above. 3 , site of

the metabolic defect in hyperargininemia.

L-Proline

NAD⁺

PROLINE

H₃N⁺

DEHYDROGENASE

NADH + H⁺

^-O

NH⁺

O⁻

L-Histidine

H₂O

HISTIDASE

NH4+

NH3+

O⁻

^-O

CH₂

L-Glutamate-γ-semialdehyde

Urocanate

H₂O

NAD⁺

GLUTAMATE SEMIALDEHYDE

UROCANASE

DEHYDROGENASE

NADH + H⁺

L-Glutamate

^-O

CH₂

α-Ketoglutarate

4-Imidazolone-5-propionate

NH3+

H₂O

−

H₂N

IMIDAZOLONE PROPIONATE

CH₂

HYDROLASE

L-Arginine

NH₂

H₂O

^–O

O^-

ARGINASE

CH₂

Urea

NH3+

N -Formiminoglutamate (Figlu)

O⁻

H₄ folate

CH₂

GLUTAMATE FORMIMINO

TRANSFERASE

NH3+

⁵-Formimino

H₄ folate

L-Ornithine

L-Glutamate

α-KG

α-Ketoglutarate

Glu

Figure 30-4. Catabolism of L-histidine to α-ketoglu-

tarate. (H₄ folate, tetrahydrofolate.) Histidase is the

L-Glutamate-γ-semialdehyde

probable site of the metabolic defect in histidinemia.

251

252

CHAPTER 30

Methylene

NH₃

H₄ folate

O⁻

NH3+

Cysteine

H₂C

O^-

CH₂

O^-

H₂C

[O]

L-Serine

Glycine

CYSTEINE

DIOXYGENASE

Figure 30-5. Interconversion of serine and glycine

catalyzed by serine hydroxymethyltransferase. (H₄ fo-

NH₃

late, tetrahydrofolate.)

O⁻

Cysteine sulfinate

⁻O₂S

NH3+

α-Keto acid

CH₂

O^- + NAD⁺

TRANSAMINASE

α-Amino acid

Glycine

H₄ folate

O⁻

Sulfinylpyruvate

H₂C

N⁵,N¹⁰-CH₂-H₄ folate

^–O₂S

⁺ + NADH + H⁺

CO₂ + NH₄

DESULFINASE

SO32−

Figure 30-6. Reversible cleavage of glycine by the

mitochondrial glycine synthase complex. (PLP, pyri-

Pyruvate

doxal phosphate.)

CYSTEINE

α-KA

NH3+

TRANSAMINASE

α-AA

O⁻

H₂C

3-Mercaptopyruvate

−

(thiolpyruvate)

H₂C

CH₂

HS O

⁻O

NADH

NH₃

L-Cystine

+ H⁺

NADH + H⁺

H₂S NAD

CYSTINE

Pyruvate

−

REDUCTASE

NAD⁺

H₂C

NH3+

3-Mercaptolactate

O⁻

CH₂

Figure 30-8. Catabolism of L-cysteine via the cys-

teine sulfinate pathway (top) and by the 3-mercaptopy-

L-Cysteine

ruvate pathway (bottom).

Figure 30-7. The cystine reductase reaction.

CATABOLISM OF THE CARBON SKELETONS OF AMINO ACIDS

253

CH₂

S S

CH₂

H C

NH3+

CH₂

O^-

COO^-

H C

NH3+

COO^-

(Cysteine)

(Homocysteine)

4-Hydroxy-L-proline

Figure 30-9.

Mixed disulfide of cysteine and homo-

HYDROXYPROLINE

cysteine.

DEHYDROGENASE

NH3+

NH⁺

H₃C

O⁻

O^-

L-∆¹-Pyrroline-3-hydroxy-5-carboxylate

L-Threonine

H₂

THREONINE

NONENZYMATIC

ALDOLASE

Glycine

NH3+

H₃C

O^-

CH₂

Acetaldehyde

γ-Hydroxy-L-glutamate-γ-semialdehyde

NAD

H₂O

ALDEHYDE

DEHYDROGENASE

NADH+H

NADH + H⁺

H₃C

O⁻

NH₃

^–O

O^-

CH₂

Acetate

CoASH

Mg-ATP

Erythro-γ-hydroxy-L-glutamate

ACETATE

α-KA

THIOKINASE

TRANSAMINASE

H₂O

Mg-ADP

α-AA

^-O

O^-

H₃C

S CoA

CH₂

α-Keto-γ-hydroxyglutarate

Acetyl-CoA

AN ALDOLASE

Figure 30-10. Conversion of threonine to glycine

(see Figure 30-6) and acetyl-CoA.

^–O

O^-

H₃C

Figure 30-11. Intermediates in L-hydroxyproline catabolism. (α-KA,

α-keto acid; α-AA, α-amino acid.) Numerals identify sites of metabolic

Glyoxylate

Pyruvate

defects in 1

hyperhydroxyprolinemia and 2

type II hyperprolinemia.

NH3+

O^-

α-KG

Glu

[O]

Ascorbate

¹CO₂

CH₂

PLP

Cu2+

CH₂

O^-

TYROSINE

p-HYDROXYPHENYLPYRUVATE

TRANSAMINASE

HYDROXYLASE

L-Tyrosine

p-Hydroxyphenylpyruvate

Homogentisate

[O]

CH₂

O^-

Fe2+

Glutathione

O^-

CH₂

O^-

CH₂

O^-

MALEYLACETOACETATE

HOMOGENTISATE

CIS, TRANS ISOMERASE

OXIDASE

Maleylacetoacetate

Fumarylacetoacetate

(rewritten)

O^-

H₂O

Fumarate

FUMARYLACETOACETATE

CoASH

HYDROLASE

H₃C

CH₂

O^-

H₃C

S CoA

H₃C

O^-

β-KETOTHIOLASE

Acetoacetate

Acetyl-CoA

Acetate

Figure 30-12. Intermediates in tyrosine catabolism. Carbons are numbered to emphasize their ultimate fate. (α-KG, α-ketoglutarate;

Glu, glutamate; PLP, pyridoxal phosphate.) Circled numerals represent the probable sites of the metabolic defects in 1

type II tyrosinemia;

neonatal tyrosinemia; 3

alkaptonuria; and 4

type I tyrosinemia, or tyrosinosis.

CATABOLISM OF THE CARBON SKELETONS OF AMINO ACIDS

255

epidemiologic data suggesting a relationship between

those of tyrosine

(Figure

30-12). Hyperphenylala-

plasma homocysteine and cardiovascular disease,

ninemias arise from defects in phenylalanine hydroxy-

whether homocysteine represents a causal cardiovas-

lase itself (type I, classic phenylketonuria or PKU), in

cular risk factor remains controversial.

dihydrobiopterin reductase (types II and III), or in di-

Threonine. Threonine is cleaved to acetaldehyde

hydrobiopterin biosynthesis (types IV and V) (Figure

and glycine. Oxidation of acetaldehyde to acetate is fol-

28-10). Alternative catabolites are excreted

(Figure

lowed by formation of acetyl-CoA (Figure 30-10). Ca-

30-13). DNA probes facilitate prenatal diagnosis of de-

tabolism of glycine is discussed above.

fects in phenylalanine hydroxylase or dihydrobiopterin

4-Hydroxyproline. Catabolism of 4-hydroxy-L-pro-

reductase. A diet low in phenylalanine can prevent the

line forms, successively, L-∆¹-pyrroline-3-hydroxy-5-car-

mental retardation of PKU

(frequency

1:10,000

boxylate, γ-hydroxy-L-glutamate-γ-semialdehyde, erythro-

γ-hydroxy-L-glutamate, and α-keto-γ-hydroxyglutarate.

An aldol-type cleavage then forms glyoxylate plus pyru-

CH₂

COO^-

vate (Figure 30-11). A defect in 4-hydroxyproline de-

hydrogenase results in hyperhydroxyprolinemia,

which is benign. There is no associated impairment of

NH₃

proline catabolism.

L-Phenylalanine

α-Ketoglutarate

TWELVE AMINO ACIDS FORM

TRANSAMINASE

ACETYL-CoA

L-Glutamate

Tyrosine. Figure 30-12 diagrams the conversion of

CH₂

COO^-

tyrosine to amphibolic intermediates. Since ascorbate is

the reductant for conversion of p-hydroxyphenylpyru-

vate to homogentisate, scorbutic patients excrete in-

Phenylpyruvate

completely oxidized products of tyrosine catabolism.

Subsequent catabolism forms maleylacetoacetate, fu-

NAD⁺

NADH + H⁺

marylacetoacetate, fumarate, acetoacetate, and ulti-

mately acetyl-CoA.

H₂O

The probable metabolic defect in type I tyrosine-

NADH + H⁺

NAD⁺

mia (tyrosinosis) is at fumarylacetoacetate hydrolase

CO₂

(reaction 4, Figure 30-12). Therapy employs a diet low

in tyrosine and phenylalanine. Untreated acute and

CH₂

COO^-

chronic tyrosinosis leads to death from liver failure. Al-

COO^-

ternate metabolites of tyrosine are also excreted in type

II tyrosinemia (Richner-Hanhart syndrome), a de-

fect in tyrosine aminotransferase (reaction 1, Figure

Phenylacetate

Phenyllactate

30-12), and in neonatal tyrosinemia, due to lowered

L-Glutamine

p-hydroxyphenylpyruvate hydroxylase activity (reaction

2, Figure

30-12). Therapy employs a diet low in

protein.

H₂O

Alkaptonuria was first described in the 16th cen-

COO^-

tury. Characterized in 1859, it provided the basis for

CH₂

N C H

Garrod’s classic ideas concerning heritable metabolic

disorders. The defect is lack of homogentisate oxidase

CH₂

(reaction 3, Figure 30-12). The urine darkens on expo-

CH₂

sure to air due to oxidation of excreted homogentisate.

Phenylacetylglutamine

Late in the disease, there is arthritis and connective tis-

CONH₂

sue pigmentation (ochronosis) due to oxidation of ho-

mogentisate to benzoquinone acetate, which polymer-

Figure 30-13. Alternative pathways of phenylala-

izes and binds to connective tissue.

nine catabolism in phenylketonuria. The reactions also

Phenylalanine. Phenylalanine is first converted to

occur in normal liver tissue but are of minor signifi-

tyrosine (see Figure 28-10). Subsequent reactions are

cance.

258

CHAPTER 30

of newborn infants is compulsory in the United States

and many other countries.

Lysine. Figure 30-14 summarizes the catabolism of

CH₂

lysine. Lysine first forms a Schiff base with α-ketoglu-

tarate, which is reduced to saccharopine. In one form

O^-

of periodic hyperlysinemia, elevated lysine competi-

H₂

H3+

tively inhibits liver arginase (see Figure 29-9), causing

hyperammonemia. Restricting dietary lysine relieves the

3-Hydroxykynurenine

ammonemia, whereas ingestion of a lysine load precipi-

tates severe crises and coma. In a different periodic hy-

perlysinemia, lysine catabolites accumulate, but even a

NH4+

lysine load does not trigger hyperammonemia. In addi-

tion to impaired synthesis of saccharopine, some pa-

tients cannot cleave saccharopine.

Tryptophan. Tryptophan is degraded to amphi-

bolic intermediates via the kynurenine-anthranilate

pathway

(Figure

30-15). Tryptophan oxygenase

O^-

(tryptophan pyrrolase) opens the indole ring, incor-

porates molecular oxygen, and forms N-formylkynure-

nine. An iron porphyrin metalloprotein that is in-

Xanthurenate

ducible in liver by adrenal corticosteroids and by

tryptophan, tryptophan oxygenase is feedback-

Figure 30-16. Formation of xanthurenate in vitamin

inhibited by nicotinic acid derivatives, including

B₆ deficiency. Conversion of the tryptophan metabolite

NADPH. Hydrolytic removal of the formyl group of

3-hydroxykynurenine to 3-hydroxyanthranilate is im-

N-formylkynurenine, catalyzed by kynurenine formy-

paired (see Figure 30-15). A large portion is therefore

lase, produces kynurenine. Since kynureninase re-

converted to xanthurenate.

quires pyridoxal phosphate, excretion of xanthurenate

(Figure 30-16) in response to a tryptophan load is di-

agnostic of vitamin B₆ deficiency. Hartnup disease re-

births). Elevated blood phenylalanine may not be de-

flects impaired intestinal and renal transport of trypto-

tectable until 3-4 days postpartum. False-positives in

phan and other neutral amino acids. Indole derivatives

premature infants may reflect delayed maturation of en-

of unabsorbed tryptophan formed by intestinal bacteria

zymes of phenylalanine catabolism. A less reliable

are excreted. The defect limits tryptophan availability

screening test employs FeCl₃

to detect urinary

for niacin biosynthesis and accounts for the pellagra-

phenylpyruvate. FeCl₃ screening for PKU of the urine

like signs and symptoms.

COO^-

⁺H₃N

C H

⁺H₃N

CH₂

H₂O

+ PP

CH₂

P P P

CH₂

Adenine

⁺S

Adenine

CH₃

L-METHIONINE

CH₃

ADENOSYLTRANSFERASE

Ribose

HO OH

L-Methionine

ATP

S-Adenosyl-L-methionine

(“active methionine”)

Figure 30-17. Formation of S-adenosylmethionine. ~CH₃ represents the high

group transfer potential of “active methionine.”

CATABOLISM OF THE CARBON SKELETONS OF AMINO ACIDS

259

NH3+

Methionine. Methionine reacts with ATP forming

S-adenosylmethionine,

“active methionine”

(Figure

H₃C

O^-

CH₂

30-17). Subsequent reactions form propionyl-CoA

(Figure 30-18) and ultimately succinyl-CoA (see Fig-

L-Methionine

ure 19-2).

ATP

THE INITIAL REACTIONS ARE COMMON

P_i + PP_i

TO ALL THREE BRANCHED-CHAIN

S-Adenosyl-L-methionine

AMINO ACIDS

Acceptor

Reactions 1-3 of Figure 30-19 are analogous to those

of fatty acid catabolism. Following transamination, all

CH₃-Acceptor

three α-keto acids undergo oxidative decarboxylation

catalyzed by mitochondrial branched-chain

-keto

S-Adenosyl-L-homocysteine

acid dehydrogenase. This multimeric enzyme complex

H₂O

of a decarboxylase, a transacylase, and a dihydrolipoyl

dehydrogenase closely resembles pyruvate dehydroge-

Adenosine

nase (see Figure 17-5). Its regulation also parallels that

of pyruvate dehydrogenase, being inactivated by phos-

NH3+

phorylation and reactivated by dephosphorylation (see

O^-

Figure 17-6).

CH₂

Reaction 3 is analogous to the dehydrogenation of

fatty acyl-CoA thioesters (see Figure 22-3). In isova-

L-Homocysteine

leric acidemia, ingestion of protein-rich foods ele-

CH₂

CYSTATHIONINE

vates isovalerate, the deacylation product of isovaleryl-

–

β-SYNTHASE

CoA. Figures 30-20, 30-21, and 30-22 illustrate the

NH₃

subsequent reactions unique to each amino acid skele-

H₂O

L-Serine

ton.

NH3+

METABOLIC DISORDERS OF BRANCHED-

CH₂

CHAIN AMINO ACID CATABOLISM

CH₂

–

As the name implies, the odor of urine in maple syrup

Cystathionine

urine disease

(branched-chain ketonuria) suggests

NH₃

maple syrup or burnt sugar. The biochemical defect in-

H₂O

volves the

-keto acid decarboxylase complex (reac-

tion 2, Figure 30-19). Plasma and urinary levels of

leucine, isoleucine, valine, α-keto acids, and α-hydroxy

CH₂

H₃C

O^-

acids (reduced α-keto acids) are elevated. The mecha-

^-O

CH₂

nism of toxicity is unknown. Early diagnosis, especially

NH₄

prior to 1 week of age, employs enzymatic analysis.

L-Cysteine

α-Ketobutyrate

Prompt replacement of dietary protein by an amino

acid mixture that lacks leucine, isoleucine, and valine

CoASH

NAD

averts brain damage and early mortality.

Mutation of the dihydrolipoate reductase compo-

CO₂

NADH + H

nent impairs decarboxylation of branched-chain α-

keto acids, of pyruvate, and of α-ketoglutarate. In in-

termittent branched-chain ketonuria, the α-keto

H₃C

acid decarboxylase retains some activity, and symp-

CH₂

CoA

toms occur later in life. The impaired enzyme in iso-

Propionyl-CoA

valeric acidemia is isovaleryl-CoA dehydrogenase

Figure 30-18. Conversion of methionine to propi-

(reaction 3, Figure 30-19). Vomiting, acidosis, and

onyl-CoA.

coma follow ingestion of excess protein. Accumulated

262

CHAPTER 30

isovaleryl-CoA is hydrolyzed to isovalerate and ex-

creted.

H₂C

S CoA

CH₃

SUMMARY

Methacrylyl-CoA

•

Excess amino acids are catabolized to amphibolic in-

H₂O

termediates used as sources of energy or for carbohy-

drate and lipid biosynthesis.

•

Transamination is the most common initial reaction

H₂C

of amino acid catabolism. Subsequent reactions re-

S CoA

move any additional nitrogen and restructure the hy-

CH₃

drocarbon skeleton for conversion to oxaloacetate,

β-Hydroxyisobutyryl-CoA

α-ketoglutarate, pyruvate, and acetyl-CoA.

H₂O

•

Metabolic diseases associated with glycine catabolism

include glycinuria and primary hyperoxaluria.

CoASH

•

Two distinct pathways convert cysteine to pyruvate.

Metabolic disorders of cysteine catabolism include

H₂C

cystine-lysinuria, cystine storage disease, and the ho-

mocystinurias.

CH₃

•

Threonine catabolism merges with that of glycine

β-Hydroxyisobutyrate

after threonine aldolase cleaves threonine to glycine

NAD⁺

and acetaldehyde.

NADH + H

•

Following transamination, the carbon skeleton of ty-

rosine is degraded to fumarate and acetoacetate.

Metabolic diseases of tyrosine catabolism include ty-

rosinosis, Richner-Hanhart syndrome, neonatal ty-

–

rosinemia, and alkaptonuria.

CH₃

•

Metabolic disorders of phenylalanine catabolism in-

Methylmalonate semialdehyde

clude phenylketonuria

(PKU) and several hyper-

α-AA

phenylalaninemias.

CoASH

NAD⁺

•

Neither nitrogen of lysine undergoes transamination.

α-KA

Metabolic diseases of lysine catabolism include peri-

NADH + H⁺

odic and persistent forms of hyperlysinemia-

ammonemia.

H₂C

•

The catabolism of leucine, valine, and isoleucine pre-

S CoA

sents many analogies to fatty acid catabolism. Meta-

CH₃

bolic disorders of branched-chain amino acid catabo-

Methylmalonyl-CoA

β-Aminoisobutyrate

lism include hypervalinemia, maple syrup urine

disease, intermittent branched-chain ketonuria, iso-

B₁₂ COENZYME

valeric acidemia, and methylmalonic aciduria.

REFERENCES

CH₂

Blacher J, Safar ME: Homocysteine, folic acid, B vitamins and car-

H₂ C

S CoA

diovascular risk. J Nutr Health Aging 2001;5:196.

Cooper AJL: Biochemistry of the sulfur-containing amino acids.

Annu Rev Biochem 1983;52:187.

Succinyl-CoA

Gjetting T et al: A phenylalanine hydroxylase amino acid polymor-

phism with implications for molecular diagnostics. Mol

Figure 30-22. Subsequent catabolism of the

Genet Metab 2001;73:280.

methacrylyl-CoA formed from L-valine (see Figure

Harris RA et al: Molecular cloning of the branched-chain α-ke-

30-19). (α-KA, α-keto acid; α-AA, α-amino acid.)

toacid dehydrogenase kinase and the CoA-dependent methyl-

Conversion of Amino Acids

to Specialized Products

Victor W. Rodwell, PhD

BIOMEDICAL IMPORTANCE

β-aminoisobutyrate are elevated in the rare metabolic

disorder hyperbeta-alaninemia.

Important products derived from amino acids include

heme, purines, pyrimidines, hormones, neurotransmit-

-Alanyl Dipeptides

ters, and biologically active peptides. In addition, many

proteins contain amino acids that have been modified

The β-alanyl dipeptides carnosine and anserine

for a specific function such as binding calcium or as in-

(N-methylcarnosine)

(Figure

31-2) activate myosin

termediates that serve to stabilize proteins—generally

ATPase, chelate copper, and enhance copper uptake.

structural proteins—by subsequent covalent cross-link-

β-Alanyl-imidazole buffers the pH of anaerobically

ing. The amino acid residues in those proteins serve as

contracting skeletal muscle. Biosynthesis of carnosine is

precursors for these modified residues. Small peptides

catalyzed by carnosine synthetase in a two-stage reac-

or peptide-like molecules not synthesized on ribosomes

tion that involves initial formation of an enzyme-bound

fulfill specific functions in cells. Histamine plays a cen-

acyl-adenylate of β-alanine and subsequent transfer of

tral role in many allergic reactions. Neurotransmitters

the β-alanyl moiety to L-histidine.

derived from amino acids include γ-aminobutyrate,

5-hydroxytryptamine

(serotonin), dopamine, norepi-

ATP+

-Alanine

→

-Alanyl−AMP →+PP

nephrine, and epinephrine. Many drugs used to treat

Alanyl−AMP

Histidine

Carnosine+AMP

β-

→

neurologic and psychiatric conditions affect the metab-

olism of these neurotransmitters.

Hydrolysis of carnosine to β-alanine and L-histidine is

catalyzed by carnosinase. The heritable disorder

carnosinase deficiency is characterized by carnosinuria.

Glycine

Homocarnosine (Figure 31-2), present in human

Metabolites and pharmaceuticals excreted as water-

brain at higher levels than carnosine, is synthesized in

soluble glycine conjugates include glycocholic acid

brain tissue by carnosine synthetase. Serum carnosinase

(Chapter 24) and hippuric acid formed from the food

does not hydrolyze homocarnosine. Homocarnosinosis,

additive benzoate

(Figure

31-1). Many drugs, drug

a rare genetic disorder, is associated with progressive

metabolites, and other compounds with carboxyl

spastic paraplegia and mental retardation.

groups are excreted in the urine as glycine conjugates.

Glycine is incorporated into creatine (see Figure 31-6),

Phosphorylated Serine, Threonine,

the nitrogen and α-carbon of glycine are incorporated

& Tyrosine

into the pyrrole rings and the methylene bridge carbons

of heme (Chapter 32), and the entire glycine molecule

The phosphorylation and dephosphorylation of seryl,

becomes atoms 4, 5, and 7 of purines (Figure 34-1).

threonyl, and tyrosyl residues regulate the activity of

certain enzymes of lipid and carbohydrate metabolism

and the properties of proteins that participate in signal

transduction cascades.

-Alanine

β-Alanine, a metabolite of cysteine (Figure 34-9), is

Methionine

present in coenzyme A and as β-alanyl dipeptides, prin-

cipally carnosine (see below). Mammalian tissues form

S-Adenosylmethionine, the principal source of methyl

β-alanine from cytosine (Figure 34-9), carnosine, and

groups in the body, also contributes its carbon skeleton

anserine (Figure 31-2). Mammalian tissues transami-

for the biosynthesis of the 3-diaminopropane portions

nate β-alanine, forming malonate semialdehyde. Body

of the polyamines spermine and spermidine (Figure

fluid and tissue levels of β-alanine, taurine, and

31-4).

264

CONVERSION OF AMINO ACIDS TO SPECIALIZED PRODUCTS

265

(CH₃)₃

NH2+

O^-

CH₂

Benzoate

Ergothioneine

ATP

CoASH

CH₂

NH3+

AMP + PP_i

NH₂

O^-

S CoA

CH₂

Benzoyl-CoA

Carnosine

Glycine

CH₂

NH3+

CH₂

CoASH

CH₃

CH₂

O^-

CH₂

Anserine

Hippurate

Figure 31-1. Biosynthesis of hippurate. Analogous

CH₂

reactions occur with many acidic drugs and catabolites.

CH₂

NH2+

Cysteine

O^-

CH₂

L-Cysteine is a precursor of the thioethanolamine por-

tion of coenzyme A and of the taurine that conjugates

with bile acids such as taurocholic acid (Chapter 26).

Homocarnosine

Figure 31-2.

Compounds related to histidine. The

Histidine

boxes surround the components not derived from histi-

Decarboxylation of histidine to histamine is catalyzed by

dine. The SH group of ergothioneine derives from cys-

a broad-specificity aromatic L-amino acid decarboxylase

teine.

that also catalyzes the decarboxylation of dopa, 5-hy-

droxytryptophan, phenylalanine, tyrosine, and trypto-

phan. α-Methyl amino acids, which inhibit decarboxy-

tric oxide

(NO) that serves as a neurotransmitter,

lase activity, find application as antihypertensive agents.

smooth muscle relaxant, and vasodilator. Synthesis of

Histidine compounds present in the human body in-

NO, catalyzed by NO synthase, involves the NADPH-

clude ergothioneine, carnosine, and dietary anserine

dependent reaction of L-arginine with O₂ to yield L-cit-

(Figure 31-2). Urinary levels of 3-methylhistidine are

rulline and NO.

unusually low in patients with Wilson’s disease.

Polyamines

Ornithine & Arginine

The polyamines spermidine and spermine

(Figure

Arginine is the formamidine donor for creatine synthe-

31-4) function in cell proliferation and growth, are

sis (Figure 31-6) and via ornithine to putrescine, sper-

growth factors for cultured mammalian cells, and stabi-

mine, and spermidine (Figure 31-3) Arginine is also

lize intact cells, subcellular organelles, and membranes.

the precursor of the intercellular signaling molecule ni-

Pharmacologic doses of polyamines are hypothermic

266

CHAPTER 31

PROTEINS

NITRIC OXIDE

UREA

PROTEINS

CREATINE

ARGININE

PHOSPHATE,

CREATININE

PROLINE

ORNITHINE

ARGININE

PHOSPHATE

Glutamate-γ-

PUTRESCINE,

semialdehyde

SPERMIDINE,

SPERMINE

GLUTAMATE

Figure 31-3. Arginine, ornithine, and proline metabolism. Reactions with solid ar-

rows all occur in mammalian tissues. Putrescine and spermine synthesis occurs in

both mammals and bacteria. Arginine phosphate of invertebrate muscle functions

as a phosphagen analogous to creatine phosphate of mammalian muscle (see

Figure 31-6).

and hypotensive. Since they bear multiple positive

mine), a potent vasoconstrictor and stimulator of

charges, polyamines associate readily with DNA and

smooth muscle contraction. Catabolism of serotonin is

RNA. Figure 31-4 summarizes polyamine biosynthesis.

initiated by monoamine oxidase-catalyzed oxidative

deamination to 5-hydroxyindoleacetate. The psychic

stimulation that follows administration of iproniazid

Tryptophan

results from its ability to prolong the action of sero-

Following hydroxylation of tryptophan to 5-hydroxy-

tonin by inhibiting monoamine oxidase. In carcinoid

tryptophan by liver tyrosine hydroxylase, subsequent

(argentaffinoma), tumor cells overproduce serotonin.

decarboxylation forms serotonin

(5-hydroxytrypta-

Urinary metabolites of serotonin in patients with carci-

H₂

⁺H₃N

⁺N

NH3+

Spermidine

Decarboxylated

S -adenosylmethionine

SPERMINE

SYNTHASE

Methylthio-

adenosine

Figure 31-4. Conversion of spermidine to spermine.

H₂

Spermidine formed from putrescine (decarboxylated

⁺H₃N

⁺N

L-ornithine) by transfer of a propylamine moiety from

NH₃

H₂

decarboxylated S-adenosylmethionine accepts a

Spermine

second propylamine moiety to form spermidine.

CONVERSION OF AMINO ACIDS TO SPECIALIZED PRODUCTS

267

noid include N-acetylserotonin glucuronide and the

NH₃

glycine conjugate of 5-hydroxyindoleacetate. Serotonin

O^-

and 5-methoxytryptamine are metabolized to the corre-

CH₂

sponding acids by monoamine oxidase. N-Acetylation

of serotonin, followed by O-methylation in the pineal

L-Tyrosine

body, forms melatonin. Circulating melatonin is taken

H₄ • biopterin

up by all tissues, including brain, but is rapidly metabo-

TYROSINE

lized by hydroxylation followed by conjugation with

HYDROXYLASE

sulfate or with glucuronic acid.

H₂ • biopterin

Kidney tissue, liver tissue, and fecal bacteria all con-

vert tryptophan to tryptamine, then to indole 3-acetate.

NH₃

The principal normal urinary catabolites of tryptophan

O^-

are 5-hydroxyindoleacetate and indole 3-acetate.

CH₂

Tyrosine

Dopa

Neural cells convert tyrosine to epinephrine and norepi-

PLP

nephrine (Figure 31-5). While dopa is also an interme-

DOPA

DECARBOXYLASE

diate in the formation of melanin, different enzymes

CO₂

hydroxylate tyrosine in melanocytes. Dopa decarboxy-

lase, a pyridoxal phosphate-dependent enzyme, forms

dopamine. Subsequent hydroxylation by dopamine

β-oxidase then forms norepinephrine. In the adrenal

CH₂

medulla, phenylethanolamine-N-methyltransferase uti-

CH₂

NH₃

lizes S-adenosylmethionine to methylate the primary

Dopamine

amine of norepinephrine, forming epinephrine (Figure

31-5). Tyrosine is also a precursor of triiodothyronine

O₂

DOPAMINE

and thyroxine (Chapter 42).

β-OXIDASE

Cu2+

Vitamin C

Creatinine

Creatinine is formed in muscle from creatine phosphate

by irreversible, nonenzymatic dehydration and loss of

CH₂

phosphate (Figure 31-6). The 24-hour urinary excre-

NH3+

tion of creatinine is proportionate to muscle mass.

Glycine, arginine, and methionine all participate in cre-

Norepinephrine

atine biosynthesis. Synthesis of creatine is completed by

methylation of guanidoacetate by S-adenosylmethio-

S-Adenosylmethionine

PHENYLETHANOL-

nine (Figure 31-6).

AMINE N-METHYL-

TRANSFERASE

S-Adenosylhomocysteine

-Aminobutyrate

γ-Aminobutyrate (GABA) functions in brain tissue as

an inhibitory neurotransmitter by altering transmem-

brane potential differences. It is formed by decarboxyla-

CH₂

CH₃

tion of L-glutamate, a reaction catalyzed by L-glutamate

H₂

decarboxylase

(Figure

31-7). Transamination of γ-

aminobutyrate forms succinate semialdehyde (Figure

Epinephrine

31-7), which may then undergo reduction to γ-hydroxy-

Figure 31-5. Conversion of tyrosine to epinephrine

butyrate, a reaction catalyzed by L-lactate dehydro-

genase, or oxidation to succinate and thence via the cit-

and norepinephrine in neuronal and adrenal cells. (PLP,

ric acid cycle to CO₂ and H₂O. A rare genetic disorder

pyridoxal phosphate.)

of GABA metabolism involves a defective GABA amino-

transferase, an enzyme that participates in the catabo-

lism of GABA subsequent to its postsynaptic release in

brain tissue.

NH₂

H₂N

(Kidney)

ARGININE-GLYCINE

TRANSAMIDINASE

NH₂

CH₂

H₂N

CH₂

HN CH₂

COO^-

CH₂

⁺H₃N

COO^-

Ornithine

Glycocyamine

(guanidoacetate)

C NH

Glycine

(Liver)

COO^-

S-Adenosyl-

ATP

-Arginine

methionine

GUANIDOACETATE

METHYLTRANSFERASE

S-Adenosyl-

ADP

homocysteine

NONENZYMATIC

IN MUSCLE

HN C

CH₂

N CH₂

COO^-

CH₃

P_i + H₂O

CH₃

Creatinine

Creatine phosphate

Figure 31-6. Biosynthesis and metabolism of creatine and creatinine.

COO^-

C NH3+

α-KA

CH₂

L-GLUTAMATE

DECARBOXYLASE

TRANSAMINASE

CH2

α-AA

COO^-

PLP

L-Glutamate

CO₂

CH₂OH

COO^-

C O

⁺H

CH₂

COO^-

CH₂

γ-Aminobutyrate

COO^-

γ-Hydroxybutyrate

[O]

COO

α-Ketoglutarate

NAD⁺

PLP

LACTATE

DEHYDROGENASE

[NH4+]

NADH + H⁺

SUCCINIC

SEMIALDEHYDE

C H

COO

DEHYDROGENASE

CH₂

NAD⁺

NADH + H

COO^-

Succinate semialdehyde

Succinate

Figure 31-7. Metabolism of γ-aminobutyrate. (α-KA, α-keto acids; α-AA, α-amino acids; PLP, pyri-

doxal phosphate.)

268

CONVERSION OF AMINO ACIDS TO SPECIALIZED PRODUCTS

269

SUMMARY

• Decarboxylation of histidine forms histamine, and

several dipeptides are derived from histidine and

• In addition to their roles in proteins and polypep-

β-alanine.

tides, amino acids participate in a wide variety of ad-

• Arginine serves as the formamidine donor for crea-

ditional biosynthetic processes.

tine biosynthesis, participates in polyamine biosyn-

• Glycine participates in the biosynthesis of heme,

thesis, and provides the nitrogen of nitric oxide

purines, and creatine and is conjugated to bile acids

(NO).

and to the urinary metabolites of many drugs.

• Important tryptophan metabolites include serotonin,

• In addition to its roles in phospholipid and sphingo-

melanin, and melatonin.

sine biosynthesis, serine provides carbons 2 and 8 of

• Tyrosine forms both epinephrine and norepineph-

purines and the methyl group of thymine.

rine, and its iodination forms thyroid hormone.

• S-Adenosylmethionine, the methyl group donor for

many biosynthetic processes, also participates directly

in spermine and spermidine biosynthesis.

• Glutamate and ornithine form the neurotransmitter

γ-aminobutyrate (GABA).

REFERENCE

• The thioethanolamine of coenzyme A and the tau-

Scriver CR et al (editors): The Metabolic and Molecular Bases of

rine of taurocholic acid arise from cysteine.

Inherited Disease, 8th ed. McGraw-Hill, 2001.

Porphyrins & Bile Pigments

Robert K. Murray, MD, PhD

BIOMEDICAL IMPORTANCE

substituent positions numbered as shown in Figure

32-2. Various porphyrins are represented in Figures

The biochemistry of the porphyrins and of the bile pig-

32-2, 32-3, and 32-4.

ments is presented in this chapter. These topics are

The arrangement of the acetate (A) and propionate

closely related, because heme is synthesized from por-

(P) substituents in the uroporphyrin shown in Figure

phyrins and iron, and the products of degradation of

32-2 is asymmetric (in ring IV, the expected order of

heme are the bile pigments and iron.

the A and P substituents is reversed). A porphyrin with

Knowledge of the biochemistry of the porphyrins

this type of asymmetric substitution is classified as a

and of heme is basic to understanding the varied func-

type III porphyrin. A porphyrin with a completely sym-

tions of hemoproteins (see below) in the body. The

metric arrangement of the substituents is classified as a

porphyrias are a group of diseases caused by abnormal-

type I porphyrin. Only types I and III are found in na-

ities in the pathway of biosynthesis of the various por-

ture, and the type III series is far more abundant (Figure

phyrins. Although porphyrias are not very prevalent,

32-3)—and more important because it includes heme.

physicians must be aware of them. A much more preva-

Heme and its immediate precursor, protoporphyrin

lent clinical condition is jaundice, due to elevation of

IX (Figure 32-4), are both type III porphyrins (ie, the

bilirubin in the plasma. This elevation is due to over-

methyl groups are asymmetrically distributed, as in type

production of bilirubin or to failure of its excretion and

III coproporphyrin). However, they are sometimes

is seen in numerous diseases ranging from hemolytic

identified as belonging to series IX, because they were

anemias to viral hepatitis and to cancer of the pancreas.

designated ninth in a series of isomers postulated by

Hans Fischer, the pioneer worker in the field of por-

phyrin chemistry.

METALLOPORPHYRINS

& HEMOPROTEINS ARE

IMPORTANT IN NATURE

HEME IS SYNTHESIZED FROM

Porphyrins are cyclic compounds formed by the linkage

SUCCINYL-COA & GLYCINE

of four pyrrole rings through

HC  methenyl

Heme is synthesized in living cells by a pathway that has

bridges (Figure 32-1). A characteristic property of the

been much studied. The two starting materials are suc-

porphyrins is the formation of complexes with metal

cinyl-CoA, derived from the citric acid cycle in mito-

ions bound to the nitrogen atom of the pyrrole rings.

chondria, and the amino acid glycine. Pyridoxal phos-

Examples are the iron porphyrins such as heme of he-

phate is also necessary in this reaction to

“activate”

moglobin and the magnesium-containing porphyrin

glycine. The product of the condensation reaction be-

chlorophyll, the photosynthetic pigment of plants.

tween succinyl-CoA and glycine is α-amino-β-ketoadipic

Proteins that contain heme

(hemoproteins) are

acid, which is rapidly decarboxylated to form α-amino-

widely distributed in nature. Examples of their impor-

levulinate (ALA) (Figure 32-5). This reaction sequence

tance in humans and animals are listed in Table 32-1.

is catalyzed by ALA synthase, the rate-controlling en-

zyme in porphyrin biosynthesis in mammalian liver.

Synthesis of ALA occurs in mitochondria. In the cy-

Natural Porphyrins Have Substituent Side

tosol, two molecules of ALA are condensed by the en-

Chains on the Porphin Nucleus

zyme ALA dehydratase to form two molecules of water

The porphyrins found in nature are compounds in

and one of porphobilinogen (PBG) (Figure 32-5). ALA

which various side chains are substituted for the eight

dehydratase is a zinc-containing enzyme and is sensitive

hydrogen atoms numbered in the porphin nucleus

to inhibition by lead, as can occur in lead poisoning.

shown in Figure 32-1. As a simple means of showing

The formation of a cyclic tetrapyrrole—ie, a por-

these substitutions, Fischer proposed a shorthand for-

phyrin—occurs by condensation of four molecules of

mula in which the methenyl bridges are omitted and

PBG (Figure 32-6). These four molecules condense in a

each pyrrole ring is shown as indicated with the eight

head-to-tail manner to form a linear tetrapyrrole, hy-

270

PORPHYRINS & BILE PIGMENTS

271

Pyrrole

III

Figure 32-2. Uroporphyrin III. A (acetate) =

C CH

CH₂COOH; P (propionate) = CH₂CH₂COOH.

8 HC

IV NH

(CH₂), which do not form a conjugated ring sys-

tem. Thus, these compounds are colorless (as are all

porphyrinogens). However, the porphyrinogens are

III

readily auto-oxidized to their respective colored por-

phyrins. These oxidations are catalyzed by light and by

the porphyrins that are formed.

Uroporphyrinogen III is converted to copropor-

Porphin

phyrinogen III by decarboxylation of all of the acetate

(C₂₀H₁₄N₄)

(A) groups, which changes them to methyl (M) sub-

Figure 32-1. The porphin molecule. Rings are la-

stituents. The reaction is catalyzed by uroporphyrino-

gen decarboxylase, which is also capable of converting

beled I, II, III, and IV. Substituent positions on the rings

uroporphyrinogen I to coproporphyrinogen I (Figure

are labeled 1, 2, 3, 4, 5, 6, 7, and 8. The methenyl

32-7). Coproporphyrinogen III then enters the mito-

bridges ( HC) are labeled α, β, γ, and δ.

chondria, where it is converted to protoporphyrinogen

III and then to protoporphyrin III. Several steps are

droxymethylbilane (HMB). The reaction is catalyzed by

involved in this conversion. The mitochondrial enzyme

uroporphyrinogen I synthase, also named PBG deami-

coproporphyrinogen oxidase catalyzes the decarboxy-

nase or HMB synthase. HMB cyclizes spontaneously to

lation and oxidation of two propionic side chains to

form uroporphyrinogen I (left-hand side of Figure

form protoporphyrinogen. This enzyme is able to act

32-6) or is converted to uroporphyrinogen III by the

only on type III coproporphyrinogen, which would ex-

action of uroporphyrinogen III synthase (right-hand side

plain why type I protoporphyrins do not generally occur

of Figure 32-6). Under normal conditions, the uropor-

in nature. The oxidation of protoporphyrinogen to pro-

phyrinogen formed is almost exclusively the III isomer,

toporphyrin is catalyzed by another mitochondrial en-

but in certain of the porphyrias (discussed below), the

zyme, protoporphyrinogen oxidase. In mammalian

type I isomers of porphyrinogens are formed in excess.

liver, the conversion of coproporphyrinogen to proto-

Note that both of these uroporphyrinogens have

porphyrin requires molecular oxygen.

the pyrrole rings connected by methylene bridges

Formation of Heme Involves Incorporation

of Iron Into Protoporphyrin

Table 32-1. Examples of some important human

The final step in heme synthesis involves the incorpora-

and animal hemoproteins.¹

tion of ferrous iron into protoporphyrin in a reaction

catalyzed by ferrochelatase (heme synthase), another

Protein

Function

mitochondrial enzyme (Figure 32-4).

Hemoglobin

Transport of oxygen in blood

A summary of the steps in the biosynthesis of the

Myoglobin

Storage of oxygen in muscle

porphyrin derivatives from PBG is given in Figure

Cytochrome c

Involvement in electron transport chain

32-8. The last three enzymes in the pathway and ALA

Cytochrome P450

Hydroxylation of xenobiotics

synthase are located in the mitochondrion, whereas the

Catalase

Degradation of hydrogen peroxide

other enzymes are cytosolic. Both erythroid and non-

Tryptophan

Oxidation of trypotophan

erythroid (“housekeeping”) forms of the first four en-

pyrrolase

zymes are found. Heme biosynthesis occurs in most

¹The functions of the above proteins are described in various

mammalian cells with the exception of mature erythro-

chapters of this text.

cytes, which do not contain mitochondria. However,

272

CHAPTER 32

Uroporphyrins were first

found in the urine, but they

are not restricted to urine.

Uroporphyrin I

Uroporphyrin III

Coproporphyrins were first

isolated from feces, but they

are also found in urine.

Coproporphyrin I

Coproporphyrin III

Figure 32-3. Uroporphyrins and coproporphyrins. A (acetate); P (propionate); M

(methyl) = CH₃; V (vinyl) = CHCH₂.

approximately 85% of heme synthesis occurs in eryth-

ALAS1. This repression-derepression mechanism is de-

roid precursor cells in the bone marrow and the major-

picted diagrammatically in Figure 32-9. Thus, the rate

ity of the remainder in hepatocytes.

of synthesis of ALAS1 increases greatly in the absence

The porphyrinogens described above are colorless,

of heme and is diminished in its presence. The turnover

containing six extra hydrogen atoms as compared with

rate of ALAS1 in rat liver is normally rapid (half-life

the corresponding colored porphyrins. These reduced

about 1 hour), a common feature of an enzyme catalyz-

porphyrins (the porphyrinogens) and not the corre-

ing a rate-limiting reaction. Heme also affects transla-

sponding porphyrins are the actual intermediates in the

tion of the enzyme and its transfer from the cytosol to

biosynthesis of protoporphyrin and of heme.

the mitochondrion.

Many drugs when administered to humans can re-

sult in a marked increase in ALAS1. Most of these

ALA Synthase Is the Key Regulatory

drugs are metabolized by a system in the liver that uti-

Enzyme in Hepatic Biosynthesis of Heme

lizes a specific hemoprotein, cytochrome P450 (see

ALA synthase occurs in both hepatic (ALAS1) and ery-

Chapter 53). During their metabolism, the utilization

throid (ALAS2) forms. The rate-limiting reaction in the

of heme by cytochrome P450 is greatly increased,

synthesis of heme in liver is that catalyzed by ALAS1

which in turn diminishes the intracellular heme con-

(Figure

32-5), a regulatory enzyme. It appears that

centration. This latter event effects a derepression of

heme, probably acting through an aporepressor mole-

ALAS1 with a corresponding increased rate of heme

cule, acts as a negative regulator of the synthesis of

synthesis to meet the needs of the cells.

Fe2+

FERROCHELATASE

Protoporphyrin III (IX)

Heme

(parent porphyrin of heme)

(prosthetic group of hemoglobin)

Figure 32-4. Addition of iron to protoporphyrin to form heme.

PORPHYRINS & BILE PIGMENTS

273

COOH

CH₂

ALA

CH₂

ALA

CH₂

SYNTHASE

Succinyl-CoA

CH₂

(“active”

CoA • SH

CO₂

succinate)

C O

S CoA

C NH₂

Pyridoxal

phosphate

COOH

Glycine

C NH₂

α-Amino-β-ketoadipate

δ-Aminolevulinate (ALA)

COOH

CH₂

COOH

CH₂

2H₂O

CH₂

ALA

C O

DEHYDRATASE

CH₂

NH₂

Two molecules of

Porphobilinogen

δ-aminolevulinate

(first precursor pyrrole)

Figure 32-5. Biosynthesis of porphobilinogen. ALA synthase occurs in the mitochon-

dria, whereas ALA dehydratase is present in the cytosol.

Several factors affect drug-mediated derepression of

side chains present. This band is termed the Soret band

ALAS1 in liver—eg, the administration of glucose can

after its discoverer, the French physicist Charles Soret.

prevent it, as can the administration of hematin (an ox-

When porphyrins dissolved in strong mineral acids

idized form of heme).

or in organic solvents are illuminated by ultraviolet

The importance of some of these regulatory mecha-

light, they emit a strong red fluorescence. This fluores-

nisms is further discussed below when the porphyrias

cence is so characteristic that it is often used to detect

are described.

small amounts of free porphyrins. The double bonds

Regulation of the erythroid form of ALAS (ALAS2)

joining the pyrrole rings in the porphyrins are responsi-

differs from that of ALAS1. For instance, it is not in-

ble for the characteristic absorption and fluorescence of

duced by the drugs that affect ALAS1, and it does not

these compounds; these double bonds are absent in the

undergo feedback regulation by heme.

porphyrinogens.

An interesting application of the photodynamic

properties of porphyrins is their possible use in the

PORPHYRINS ARE COLORED

treatment of certain types of cancer, a procedure called

& FLUORESCE

cancer phototherapy. Tumors often take up more por-

phyrins than do normal tissues. Thus, hematopor-

The various porphyrinogens are colorless, whereas the

phyrin or other related compounds are administered to

various porphyrins are all colored. In the study of por-

a patient with an appropriate tumor. The tumor is then

phyrins or porphyrin derivatives, the characteristic ab-

exposed to an argon laser, which excites the porphyrins,

sorption spectrum that each exhibits—in both the visible

producing cytotoxic effects.

and the ultraviolet regions of the spectrum—is of great

value. An example is the absorption curve for a solution

Spectrophotometry Is Used to Test

of porphyrin in 5% hydrochloric acid (Figure 32-10).

for Porphyrins & Their Precursors

Note particularly the sharp absorption band near 400

nm. This is a distinguishing feature of the porphin ring

Coproporphyrins and uroporphyrins are of clinical in-

and is characteristic of all porphyrins regardless of the

terest because they are excreted in increased amounts in

274

CHAPTER 32

HOOC

COOH

pain and of a variety of neuropsychiatric findings); oth-

H₂C

CH₂

erwise, patients will be subjected to inappropriate treat-

CH₂

ments. It has been speculated that King George III had

a type of porphyria, which may account for his periodic

confinements in Windsor Castle and perhaps for some

of his views regarding American colonists. Also, the

H₂C

photosensitivity (favoring nocturnal activities) and se-

NH₂

vere disfigurement exhibited by some victims of con-

genital erythropoietic porphyria have led to the sugges-

Four molecules of

porphobilinogen

tion that these individuals may have been the

prototypes of so-called werewolves. No evidence to sup-

UROPORPHYRINOGEN I

4NH3

SYNTHASE

port this notion has been adduced.

Hydroxymethylbilane

(linear tetrapyrrole)

Biochemistry Underlies the

SPONTANEOUS

UROPORPHYRINOGEN III

Causes, Diagnoses, & Treatments

CYCLIZATION

SYNTHASE

of the Porphyrias

Six major types of porphyria have been described, re-

sulting from depressions in the activities of enzymes 3

through 8 shown in Figure 32-9 (see also Table 32-2).

H₂

Assay of the activity of one or more of these enzymes

using an appropriate source (eg, red blood cells) is thus

important in making a definitive diagnosis in a sus-

CH₂

pected case of porphyria. Individuals with low activities

of enzyme 1 (ALAS2) develop anemia, not porphyria

(see Table 32-2). Patients with low activities of enzyme

III

2 (ALA dehydratase) have been reported, but very

H₂

rarely; the resulting condition is called ALA dehy-

dratase-deficient porphyria.

Type I

Type III

In general, the porphyrias described are inherited in

uroporphyrinogen

an autosomal dominant manner, with the exception of

congenital erythropoietic porphyria, which is inherited

Figure 32-6. Conversion of porphobilinogen to uro-

in a recessive mode. The precise abnormalities in the

porphyrinogens. Uroporphyrinogen synthase I is also

genes directing synthesis of the enzymes involved in

called porphobilinogen (PBG) deaminase or hydroxy-

heme biosynthesis have been determined in some in-

methylbilane (HMB) synthase.

stances. Thus, the use of appropriate gene probes has

made possible the prenatal diagnosis of some of the

porphyrias.

the porphyrias. These compounds, when present in

As is true of most inborn errors, the signs and symp-

urine or feces, can be separated from each other by ex-

toms of porphyria result from either a deficiency of

traction with appropriate solvent mixtures. They can

metabolic products beyond the enzymatic block or

then be identified and quantified using spectrophoto-

from an accumulation of metabolites behind the block.

metric methods.

If the enzyme lesion occurs early in the pathway

ALA and PBG can also be measured in urine by ap-

prior to the formation of porphyrinogens (eg, enzyme 3

propriate colorimetric tests.

of Figure 32-9, which is affected in acute intermittent

porphyria), ALA and PBG will accumulate in body tis-

sues and fluids

(Figure

32-11). Clinically, patients

THE PORPHYRIAS ARE GENETIC

complain of abdominal pain and neuropsychiatric

DISORDERS OF HEME METABOLISM

symptoms. The precise biochemical cause of these

The porphyrias are a group of disorders due to abnor-

symptoms has not been determined but may relate to

malities in the pathway of biosynthesis of heme; they

elevated levels of ALA or PBG or to a deficiency of

can be genetic or acquired. They are not prevalent, but

heme.

it is important to consider them in certain circum-

On the other hand, enzyme blocks later in the path-

stances (eg, in the differential diagnosis of abdominal

way result in the accumulation of the porphyrinogens

PORPHYRINS & BILE PIGMENTS

275

4CO₂

III

P A

P M

Uroporphyrinogen I

Coproporphyrinogen I

UROPORPHYRINOGEN

DECARBOXYLASE

III

Figure 32-7. Decarboxylation of uropor-

4CO₂

P A

P M

phyrinogens to coproporphyrinogens in cy-

Uroporphyrinogen III

Coproporphyrinogen III

tosol. (A, acetyl; M, methyl; P, propionyl.)

Porphobilinogen

UROPORPHYRINOGEN I

SYNTHASE

Hydroxymethylbilane

UROPORPHYRINOGEN III

SYNTHASE

SPONTANEOUS

Uroporphyrin

Uroporphyrinogen

Uroporphyrin

III

Light

III

Light

UROPORPHYRINOGEN

DECARBOXYLASE

4CO₂

Coproporphyrin

Coproporphyrinogen

Coproporphyrin

III

Light

III

Light

COPROPORPHYRINOGEN

OXIDASE

Protoporphyrinogen III

Or light in vitro

PROTOPORPHYRINOGEN

OXIDASE

Protoporphyrin III

Fe2+

FERROCHELATASE

Heme

Figure 32-8. Steps in the biosynthesis of the porphyrin derivatives from porphobilinogen. Uropor-

phyrinogen I synthase is also called porphobilinogen deaminase or hydroxymethylbilane synthase.

276

CHAPTER 32

Hemoproteins

Proteins

Heme

Aporepressor

8. FERROCHELATASE

Fe2+

Protoporphyrin III

7. PROTOPORPHYRINOGEN

OXIDASE

Protoporphyrinogen III

6. COPROPORPHYRINOGEN

OXIDASE

Coproporphyrinogen III

5. UROPORPHYRINOGEN

DECARBOXYLASE

Uroporphyrinogen III

4. UROPORPHYRINOGEN III

SYNTHASE

Hydroxymethylbilane

3. UROPORPHYRINOGEN I

SYNTHASE

Porphobilinogen

2. ALA

DEHYDRATASE

ALA

1. ALA

SYNTHASE

Succinyl-CoA + Glycine

Figure 32-9. Intermediates, enzymes, and regulation of heme syn-

thesis. The enzyme numbers are those referred to in column 1 of Table

32-2. Enzymes 1, 6, 7, and 8 are located in mitochondria, the others in

the cytosol. Mutations in the gene encoding enzyme 1 causes X-linked

sideroblastic anemia. Mutations in the genes encoding enzymes 2-8

cause the porphyrias, though only a few cases due to deficiency of en-

zyme 2 have been reported. Regulation of hepatic heme synthesis oc-

curs at ALA synthase (ALAS1) by a repression-derepression mecha-

nism mediated by heme and its hypothetical aporepressor. The

dotted lines indicate the negative ( − ) regulation by repression. En-

zyme 3 is also called porphobilinogen deaminase or hydroxymethyl-

bilane synthase.

PORPHYRINS & BILE PIGMENTS

277

Mutations in DNA

Abnormalities of the

enzymes of heme synthesis

Accumulation of

ALA and PBG and/or

porphyrinogens in skin

decrease in heme in

and tissues

cells and body fluids

300

400

500

600

700

Spontaneous oxidation

Wavelength (nm)

Neuropsychiatric signs

of porphyrinogens to

and symptoms

porphyrins

Figure 32-10. Absorption spectrum of hematopor-

phyrin (0.01% solution in 5% HCl).

Photosensitivity

Figure 32-11. Biochemical causes of the major

signs and symptoms of the porphyrias.

Table 32-2. Summary of major findings in the porphyrias.¹

Enzyme Involved²

Type, Class, and MIM Number

Major Signs and Symptoms

Results of Laboratory Tests

1. ALA synthase

X-linked sideroblastic anemia³

Anemia

Red cell counts and hemoglobin

(erythroid form)

(erythropoietic) (MIM

decreased

201300)

2. ALA dehydratase

ALA dehydratase deficiency

Abdominal pain, neuropsychiatric

Urinary δ-aminolevulinic acid

(hepatic) (MIM 125270)

symptoms

3. Uroporphyrinogen I

Acute intermittent porphyria

Abdominal pain, neuropsychiatric

Urinary porphobilinogen positive,

synthase⁴

(hepatic) (MIM 176000)

symptoms

uroporphyrin positive

4. Uroporphyrinogen III

Congenital erythropoietic

No photosensitivity

Uroporphyrin positive, porpho-

synthase

(erythropoietic) (MIM

bilinogen negative

263700)

5. Uroporphyrinogen

Porphyria cutanea tarda (he-

Photosensitivity

Uroporphyrin positive, porpho-

decarboxylase

patic) (MIM 176100)

bilinogen negative

6. Coproporphyrinogen

Hereditary coproporphyria

Photosensitivity, abdominal pain,

Urinary porphobilinogen posi-

oxidase

(hepatic) (MIM 121300)

neuropsychiatric symptoms

tive, urinary uroporphyrin

positive, fecal protopor-

phyrin positive

7. Protoporphyrinogen

Variegate porphyria (hepatic)

Photosensitivity, abdominal pain,

Urinary porphobilinogen posi-

oxidase

(MIM 176200)

neuropsychiatric symptoms

tive, fecal protoporphyrin

positive

8. Ferrochelatase

Protoporphyria (erythropoietic)

Photosensitivity

Fecal protoporphyrin posi-

(MIM 177000)

tive, red cell protoporphyrin

positive

¹Only the biochemical findings in the active stages of these diseases are listed. Certain biochemical abnormalities are detectable in the la-

tent stages of some of the above conditions. Conditions 3, 5, and 8 are generally the most prevalent porphyrias.

²The numbering of the enzymes in this table corresponds to that used in Figure 32-9.

³X-linked sideroblastic anemia is not a porphyria but is included here because δ−aminolevulinic acid synthase is involved.

⁴This enzyme is also called porphobilinogen deaminase or hydroxymethylbilane synthase.

278

CHAPTER 32

indicated in Figures 32-9 and 32-11. Their oxidation

chrome P450. Ingestion of large amounts of carbohy-

products, the corresponding porphyrin derivatives,

drates (glucose loading) or administration of hematin (a

cause photosensitivity, a reaction to visible light of

hydroxide of heme) may repress ALAS1, resulting in di-

about 400 nm. The porphyrins, when exposed to light

minished production of harmful heme precursors. Pa-

of this wavelength, are thought to become “excited”

tients exhibiting photosensitivity may benefit from ad-

and then react with molecular oxygen to form oxygen

ministration of β-carotene; this compound appears to

radicals. These latter species injure lysosomes and other

lessen production of free radicals, thus diminishing

organelles. Damaged lysosomes release their degradative

photosensitivity. Sunscreens that filter out visible light

enzymes, causing variable degrees of skin damage, in-

can also be helpful to such patients.

cluding scarring.

The porphyrias can be classified on the basis of the

organs or cells that are most affected. These are gener-

CATABOLISM OF HEME

ally organs or cells in which synthesis of heme is partic-

PRODUCES BILIRUBIN

ularly active. The bone marrow synthesizes considerable

hemoglobin, and the liver is active in the synthesis of

Under physiologic conditions in the human adult, 1-2

another hemoprotein, cytochrome P450. Thus, one

× 10⁸ erythrocytes are destroyed per hour. Thus, in 1

classification of the porphyrias is to designate them as

day, a 70-kg human turns over approximately 6 g of he-

predominantly either erythropoietic or hepatic; the

moglobin. When hemoglobin is destroyed in the body,

types of porphyrias that fall into these two classes are so

globin is degraded to its constituent amino acids,

characterized in Table 32-2. Porphyrias can also be

which are reused, and the iron of heme enters the iron

classified as acute or cutaneous on the basis of their

pool, also for reuse. The iron-free porphyrin portion of

clinical features. Why do specific types of porphyria af-

heme is also degraded, mainly in the reticuloendothelial

fect certain organs more markedly than others? A par-

cells of the liver, spleen, and bone marrow.

tial answer is that the levels of metabolites that cause

The catabolism of heme from all of the heme pro-

damage (eg, ALA, PBG, specific porphyrins, or lack of

teins appears to be carried out in the microsomal frac-

heme) can vary markedly in different organs or cells de-

tions of cells by a complex enzyme system called heme

pending upon the differing activities of their heme-

oxygenase. By the time the heme derived from heme

forming enzymes.

proteins reaches the oxygenase system, the iron has usu-

As described above, ALAS1 is the key regulatory en-

ally been oxidized to the ferric form, constituting

zyme of the heme biosynthetic pathway in liver. A large

hemin. The heme oxygenase system is substrate-in-

number of drugs (eg, barbiturates, griseofulvin) induce

ducible. As depicted in Figure 32-12, the hemin is re-

the enzyme. Most of these drugs do so by inducing cy-

duced to heme with NADPH, and, with the aid of

tochrome P450 (see Chapter 53), which uses up heme

more NADPH, oxygen is added to the α-methenyl

and thus derepresses (induces) ALAS1. In patients with

bridge between pyrroles I and II of the porphyrin. The

porphyria, increased activities of ALAS1 result in in-

ferrous iron is again oxidized to the ferric form. With

creased levels of potentially harmful heme precursors

the further addition of oxygen, ferric ion is released,

prior to the metabolic block. Thus, taking drugs that

carbon monoxide is produced, and an equimolar

cause induction of cytochrome P450 (so-called micro-

quantity of biliverdin results from the splitting of the

somal inducers) can precipitate attacks of porphyria.

tetrapyrrole ring.

The diagnosis of a specific type of porphyria can

In birds and amphibia, the green biliverdin IX is ex-

generally be established by consideration of the clinical

creted; in mammals, a soluble enzyme called biliverdin

and family history, the physical examination, and ap-

reductase reduces the methenyl bridge between pyrrole

propriate laboratory tests. The major findings in the six

III and pyrrole IV to a methylene group to produce

principal types of porphyria are listed in Table 32-2.

bilirubin, a yellow pigment (Figure 32-12).

High levels of lead can affect heme metabolism by

It is estimated that 1 g of hemoglobin yields 35 mg

combining with SH groups in enzymes such as fer-

of bilirubin. The daily bilirubin formation in human

rochelatase and ALA dehydratase. This affects por-

adults is approximately 250-350 mg, deriving mainly

phyrin metabolism. Elevated levels of protoporphyrin

from hemoglobin but also from ineffective erythro-

are found in red blood cells, and elevated levels of ALA

poiesis and from various other heme proteins such as

and of coproporphyrin are found in urine.

cytochrome P450.

It is hoped that treatment of the porphyrias at the

The chemical conversion of heme to bilirubin by

gene level will become possible. In the meantime, treat-

reticuloendothelial cells can be observed in vivo as the

ment is essentially symptomatic. It is important for pa-

purple color of the heme in a hematoma is slowly con-

tients to avoid drugs that cause induction of cyto-

verted to the yellow pigment of bilirubin.

280

CHAPTER 32

Bilirubin formed in peripheral tissues is transported

converted to water-soluble derivatives by conjugation in

to the liver by plasma albumin. The further metabolism

preparation for excretion (see Chapter 53).

of bilirubin occurs primarily in the liver. It can be di-

The conjugation of bilirubin is catalyzed by a spe-

vided into three processes: (1) uptake of bilirubin by

cific glucuronosyltransferase. The enzyme is mainly

liver parenchymal cells,

(2) conjugation of bilirubin

located in the endoplasmic reticulum, uses UDP-

with glucuronate in the endoplasmic reticulum, and (3)

glucuronic acid as the glucuronosyl donor, and is re-

secretion of conjugated bilirubin into the bile. Each of

ferred to as bilirubin-UGT. Bilirubin monoglucuronide

these processes will be considered separately.

is an intermediate and is subsequently converted to the

diglucuronide (Figures 32-13 and 32-14). Most of the

bilirubin excreted in the bile of mammals is in the form

THE LIVER TAKES UP BILIRUBIN

of bilirubin diglucuronide. However, when bilirubin

conjugates exist abnormally in human plasma (eg, in

Bilirubin is only sparingly soluble in water, but its solu-

obstructive jaundice), they are predominantly mono-

bility in plasma is increased by noncovalent binding to

glucuronides. Bilirubin-UGT activity can be induced

albumin. Each molecule of albumin appears to have

by a number of clinically useful drugs, including phe-

one high-affinity site and one low-affinity site for

nobarbital. More information about glucuronosylation

bilirubin. In 100 mL of plasma, approximately 25 mg

is presented below in the discussion of inherited disor-

of bilirubin can be tightly bound to albumin at its high-

ders of bilirubin conjugation.

affinity site. Bilirubin in excess of this quantity can be

bound only loosely and thus can easily be detached and

diffuse into tissues. A number of compounds such as

Bilirubin Is Secreted Into Bile

antibiotics and other drugs compete with bilirubin for

Secretion of conjugated bilirubin into the bile occurs by

the high-affinity binding site on albumin. Thus, these

an active transport mechanism, which is probably rate-

compounds can displace bilirubin from albumin and

limiting for the entire process of hepatic bilirubin me-

have significant clinical effects.

tabolism. The protein involved is MRP-2 (multidrug

In the liver, the bilirubin is removed from albumin

resistance-like protein 2), also called multispecific or-

and taken up at the sinusoidal surface of the hepato-

ganic anion transporter (MOAT). It is located in the

cytes by a carrier-mediated saturable system. This facil-

plasma membrane of the bile canalicular membrane

itated transport system has a very large capacity, so

and handles a number of organic anions. It is a member

that even under pathologic conditions the system does

of the family of ATP-binding cassette (ABC) trans-

not appear to be rate-limiting in the metabolism of

porters. The hepatic transport of conjugated bilirubin

bilirubin.

into the bile is inducible by those same drugs that are

Since this facilitated transport system allows the

capable of inducing the conjugation of bilirubin. Thus,

equilibrium of bilirubin across the sinusoidal mem-

the conjugation and excretion systems for bilirubin be-

brane of the hepatocyte, the net uptake of bilirubin will

have as a coordinated functional unit.

be dependent upon the removal of bilirubin via subse-

Figure 32-15 summarizes the three major processes

quent metabolic pathways.

involved in the transfer of bilirubin from blood to bile.

Once bilirubin enters the hepatocytes, it can bind to

Sites that are affected in a number of conditions caus-

certain cytosolic proteins, which help to keep it solubi-

ing jaundice (see below) are also indicated.

lized prior to conjugation. Ligandin (a family of glu-

tathione S-transferases) and protein Y are the involved

proteins. They may also help to prevent efflux of biliru-

bin back into the blood stream.

O O

^-OOC(CH₂O)₄C

C(CH₂O)₄COO^-

Conjugation of Bilirubin With Glucuronic

H₂C

CH₂

Acid Occurs in the Liver

H₂C

M V M

Bilirubin is nonpolar and would persist in cells (eg,

III

bound to lipids) if not rendered water-soluble. Hepato-

cytes convert bilirubin to a polar form, which is readily

excreted in the bile, by adding glucuronic acid mole-

Figure 32-13. Structure of bilirubin diglucuronide

cules to it. This process is called conjugation and can

(conjugated, “direct-reacting” bilirubin). Glucuronic

employ polar molecules other than glucuronic acid (eg,

acid is attached via ester linkage to the two propionic

sulfate). Many steroid hormones and drugs are also

acid groups of bilirubin to form an acylglucuronide.

PORPHYRINS & BILE PIGMENTS

281

UDP-GLUCOSE

DEHYDROGENASE

UDP-Glucose

UDP-Glucuronic acid

2NAD⁺

2NADH + 2H⁺

UDP-GLUCURONOSYL-

TRANSFERASE

UDP-Glucuronic acid

Bilirubin monoglucuronide

Bilirubin

UDP

Figure 32-14. Conjugation of bilirubin

with glucuronic acid. The glucuronate donor,

UDP-GLUCURONOSYL-

UDP-glucuronic acid, is formed from UDP-

TRANSFERASE

UDP-Glucuronic acid

Bilirubin diglucuronide

glucose as depicted. The UDP-glucuronosyl-

Bilirubin monoglucuronide

UDP

transferase is also called bilirubin-UGT.

Conjugated Bilirubin Is Reduced to

Urobilinogen by Intestinal Bacteria

As the conjugated bilirubin reaches the terminal ileum

and the large intestine, the glucuronides are removed by

BLOOD

specific bacterial enzymes (

-glucuronidases), and the

Bilirubin • Albumin

pigment is subsequently reduced by the fecal flora to a

group of colorless tetrapyrrolic compounds called uro-

1. UPTAKE

bilinogens (Figure 32-16). In the terminal ileum and

large intestine, a small fraction of the urobilinogens is re-

absorbed and reexcreted through the liver to constitute

HEPATOCYTE

the enterohepatic urobilinogen cycle. Under abnormal

Bilirubin

conditions, particularly when excessive bile pigment is

2. CONJUGATION

formed or liver disease interferes with this intrahepatic

Neonatal jaundice

cycle, urobilinogen may also be excreted in the urine.

UDP-GlcUA

“Toxic” jaundice

Normally, most of the colorless urobilinogens

UDP-GlcUA

Crigler-Najjar syndrome

formed in the colon by the fecal flora are oxidized there

Gilbert syndrome

to urobilins (colored compounds) and are excreted in

the feces

(Figure

32-16). Darkening of feces upon

Bilirubin diglucuronide

standing in air is due to the oxidation of residual uro-

bilinogens to urobilins.

3. SECRETION

Dubin-Johnson syndrome

HYPERBILIRUBINEMIA CAUSES

JAUNDICE

BILE DUCTULE

When bilirubin in the blood exceeds 1 mg/dL (17.1

Bilirubin diglucuronide

µmol/L), hyperbilirubinemia exists. Hyperbilirubine-

mia may be due to the production of more bilirubin

Figure 32-15.

Diagrammatic representation of the

than the normal liver can excrete, or it may result from

three major processes (uptake, conjugation, and secre-

the failure of a damaged liver to excrete bilirubin pro-

tion) involved in the transfer of bilirubin from blood to

duced in normal amounts. In the absence of hepatic

bile. Certain proteins of hepatocytes, such as ligandin (a

damage, obstruction of the excretory ducts of the

family of glutathione S-transferase) and Y protein, bind

liver—by preventing the excretion of bilirubin—will

intracellular bilirubin and may prevent its efflux into the

also cause hyperbilirubinemia. In all these situations,

blood stream. The process affected in a number of con-

bilirubin accumulates in the blood, and when it reaches

ditions causing jaundice is also shown.

a certain concentration (approximately 2-2.5 mg/dL),

282

CHAPTER 32

H₂C

CH₂

H₂C

CH₂

Mesobilirubinogen

Stercobilinogen

Stercobilin

(C₃₃H₄₄O₆N₄)

(L-Urobilinogen)

(L-Urobilin)

Figure 32-16. Structure of some bile pigments.

it diffuses into the tissues, which then become yellow.

soluble, can react directly with the diazo reagent, so

That condition is called jaundice or icterus.

that the “direct bilirubin” of van den Bergh is actually a

In clinical studies of jaundice, measurement of

bilirubin conjugate (bilirubin glucuronide).

bilirubin in the serum is of great value. A method for

Depending on the type of bilirubin present in

quantitatively assaying the bilirubin content of the

plasma—ie, unconjugated or conjugated—hyperbiliru-

serum was first devised by van den Bergh by application

binemia may be classified as retention hyperbiliru-

of Ehrlich’s test for bilirubin in urine. The Ehrlich reac-

binemia, due to overproduction, or regurgitation hy-

tion is based on the coupling of diazotized sulfanilic

perbilirubinemia, due to reflux into the bloodstream

acid (Ehrlich’s diazo reagent) and bilirubin to produce

because of biliary obstruction.

a reddish-purple azo compound. In the original proce-

Because of its hydrophobicity, only unconjugated

dure as described by Ehrlich, methanol was used to

bilirubin can cross the blood-brain barrier into the cen-

provide a solution in which both bilirubin and the

tral nervous system; thus, encephalopathy due to hyper-

diazo regent were soluble. Van den Bergh inadvertently

bilirubinemia (kernicterus) can occur only in connec-

omitted the methanol on an occasion when assay of bile

tion with unconjugated bilirubin, as found in retention

pigment in human bile was being attempted. To his

hyperbilirubinemia. On the other hand, because of its

surprise, normal development of the color occurred “di-

water-solubility, only conjugated bilirubin can appear

rectly.” This form of bilirubin that would react without

in urine. Accordingly, choluric jaundice (choluria is

the addition of methanol was thus termed “direct-

the presence of bile pigments in the urine) occurs only

reacting.” It was then found that this same direct reac-

in regurgitation hyperbilirubinemia, and acholuric

tion would also occur in serum from cases of jaundice

jaundice occurs only in the presence of an excess of un-

due to biliary obstruction. However, it was still neces-

conjugated bilirubin.

sary to add methanol to detect bilirubin in normal

serum or that which was present in excess in serum

Elevated Amounts of Unconjugated

from cases of hemolytic jaundice where no evidence of

Bilirubin in Blood Occur in a Number

obstruction was to be found. To that form of bilirubin

of Conditions

which could be measured only after the addition of

methanol, the term “indirect-reacting” was applied.

A. HEMOLYTIC ANEMIAS

It was subsequently discovered that the indirect

Hemolytic anemias are important causes of unconju-

bilirubin is “free” (unconjugated) bilirubin en route to

gated hyperbilirubinemia, though unconjugated hyper-

the liver from the reticuloendothelial tissues, where the

bilirubinemia is usually only slight (< 4 mg/dL; < 68.4

bilirubin was originally produced by the breakdown of

µmol/L) even in the event of extensive hemolysis be-

heme porphyrins. Since this bilirubin is not water-solu-

cause of the healthy liver’s large capacity for handling

ble, it requires methanol to initiate coupling with the

bilirubin.

diazo reagent. In the liver, the free bilirubin becomes

conjugated with glucuronic acid, and the conjugate,

B. NEONATAL “PHYSIOLOGIC JAUNDICE”

bilirubin glucuronide, can then be excreted into the

This transient condition is the most common cause of

bile. Furthermore, conjugated bilirubin, being water-

unconjugated hyperbilirubinemia. It results from an ac-

PORPHYRINS & BILE PIGMENTS

283

celerated hemolysis around the time of birth and an im-

mushroom poisoning. These acquired disorders are due

mature hepatic system for the uptake, conjugation, and

to hepatic parenchymal cell damage, which impairs

secretion of bilirubin. Not only is the bilirubin-UGT

conjugation.

activity reduced, but there probably is reduced synthesis

of the substrate for that enzyme, UDP-glucuronic acid.

Obstruction in the Biliary Tree Is the

Since the increased amount of bilirubin is unconju-

Commonest Cause of Conjugated

gated, it is capable of penetrating the blood-brain bar-

rier when its concentration in plasma exceeds that

Hyperbilirubinemia

which can be tightly bound by albumin

(20-25

A. OBSTRUCTION OF THE BILIARY TREE

mg/dL). This can result in a hyperbilirubinemic toxic

Conjugated hyperbilirubinemia commonly results from

encephalopathy, or kernicterus, which can cause men-

blockage of the hepatic or common bile ducts, most

tal retardation. Because of the recognized inducibility

often due to a gallstone or to cancer of the head of the

of this bilirubin-metabolizing system, phenobarbital

pancreas. Because of the obstruction, bilirubin diglu-

has been administered to jaundiced neonates and is ef-

curonide cannot be excreted. It thus regurgitates into

fective in this disorder. In addition, exposure to blue

the hepatic veins and lymphatics, and conjugated

light (phototherapy) promotes the hepatic excretion of

bilirubin appears in the blood and urine (choluric jaun-

unconjugated bilirubin by converting some of the

dice).

bilirubin to other derivatives such as maleimide frag-

The term cholestatic jaundice is used to include all

ments and geometric isomers that are excreted in the

cases of extrahepatic obstructive jaundice. It also covers

bile.

those cases of jaundice that exhibit conjugated hyper-

C. CRIGLER-NAJJAR SYNDROME, TYPE I;

bilirubinemia due to micro-obstruction of intrahepatic

CONGENITAL NONHEMOLYTIC JAUNDICE

biliary ductules by swollen, damaged hepatocytes (eg, as

Type I Crigler-Najjar syndrome is a rare autosomal re-

may occur in infectious hepatitis).

cessive disorder. It is characterized by severe congenital

jaundice (serum bilirubin usually exceeds 20 mg/dL)

B. DUBIN-JOHNSON SYNDROME

due to mutations in the gene encoding bilirubin-UGT

This benign autosomal recessive disorder consists of

activity in hepatic tissues. The disease is often fatal

conjugated hyperbilirubinemia in childhood or during

within the first 15 months of life. Children with this

adult life. The hyperbilirubinemia is caused by muta-

condition have been treated with phototherapy, result-

tions in the gene encoding MRP-2 (see above), the pro-

ing in some reduction in plasma bilirubin levels. Phe-

tein involved in the secretion of conjugated bilirubin

nobarbital has no effect on the formation of bilirubin

into bile. The centrilobular hepatocytes contain an ab-

glucuronides in patients with type I Crigler-Najjar syn-

normal black pigment that may be derived from epi-

drome. A liver transplant may be curative.

nephrine.

D. CRIGLER-NAJJAR SYNDROME, TYPE II

C. ROTOR SYNDROME

This rare inherited disorder also results from mutations

This is a rare benign condition characterized by chronic

in the gene encoding bilirubin-UGT, but some activity

conjugated hyperbilirubinemia and normal liver histol-

of the enzyme is retained and the condition has a more

ogy. Its precise cause has not been identified, but it is

benign course than type I. Serum bilirubin concentra-

thought to be due to an abnormality in hepatic storage.

tions usually do not exceed 20 mg/dL. Patients with

this condition can respond to treatment with large

doses of phenobarbital.

Some Conjugated Bilirubin Can Bind

Covalently to Albumin

E. GILBERT SYNDROME

Again, this is caused by mutations in the gene encoding

When levels of conjugated bilirubin remain high in

bilirubin-UGT, but approximately

30% of the en-

plasma, a fraction can bind covalently to albumin (delta

zyme’s activity is preserved and the condition is entirely

bilirubin). Because it is bound covalently to albumin,

harmless.

this fraction has a longer half-life in plasma than does

conventional conjugated bilirubin. Thus, it remains ele-

F. TOXIC HYPERBILIRUBINEMIA

vated during the recovery phase of obstructive jaundice

Unconjugated hyperbilirubinemia can result from

after the remainder of the conjugated bilirubin has de-

toxin-induced liver dysfunction such as that caused by

clined to normal levels; this explains why some patients

chloroform, arsphenamines, carbon tetrachloride, ace-

continue to appear jaundiced after conjugated bilirubin

taminophen, hepatitis virus, cirrhosis, and Amanita

levels have returned to normal.

284

CHAPTER 32

Table 32-3. Laboratory results in normal patients and patients with three different causes of jaundice.

Condition

Serum Bilirubin

Urine Urobilinogen

Urine Bilirubin

Fecal Urobilinogen

Normal

Direct: 0.1-0.4 mg/dL

0-4 mg/24 h

Absent

40-280 mg/24 h

Indirect: 0.2-0.7 mg/dL

Hemolytic anemia

↑ Indirect

Increased

Absent

Increased

Hepatitis

↑ Direct and indirect

Decreased if micro-

Present if micro-

Decreased

obstruction is

obstruction occurs

present

Obstructive jaundice¹

↑ Direct

Absent

Present

Trace to absent

¹The commonest causes of obstructive (posthepatic) jaundice are cancer of the head of the pancreas and a gallstone lodged in the com-

mon bile duct. The presence of bilirubin in the urine is sometimes referred to as choluria—therefore, hepatitis and obstruction of the

common bile duct cause choluric jaundice, whereas the jaundice of hemolytic anemia is referred to as acholuric. The laboratory results in

patients with hepatitis are variable, depending on the extent of damage to parenchymal cells and the extent of micro-obstruction to bile

ductules. Serum levels of ALT and AST are usually markedly elevated in hepatitis, whereas serum levels of alkaline phosphatase are ele-

vated in obstructive liver disease.

Urobilinogen & Bilirubin in Urine

which four pyrrole rings are joined by methenyl

Are Clinical Indicators

bridges. The eight side groups (methyl, vinyl, and

propionyl substituents) on the four pyrrole rings of

Normally, there are mere traces of urobilinogen in the

heme are arranged in a specific sequence.

urine. In complete obstruction of the bile duct, no

•

Biosynthesis of the heme ring occurs in mitochondria

urobilinogen is found in the urine, since bilirubin has

and cytosol via eight enzymatic steps. It commences

no access to the intestine, where it can be converted to

with formation of δ-aminolevulinate

(ALA) from

urobilinogen. In this case, the presence of bilirubin

succinyl-CoA and glycine in a reaction catalyzed by

(conjugated) in the urine without urobilinogen suggests

ALA synthase, the regulatory enzyme of the pathway.

obstructive jaundice, either intrahepatic or posthepatic.

•

Genetically determined abnormalities of seven of the

In jaundice secondary to hemolysis, the increased

eight enzymes involved in heme biosynthesis result in

production of bilirubin leads to increased production of

the inherited porphyrias. Red blood cells and liver

urobilinogen, which appears in the urine in large

are the major sites of metabolic expression of the por-

amounts. Bilirubin is not usually found in the urine in

phyrias. Photosensitivity and neurologic problems

hemolytic jaundice

(because unconjugated bilirubin

are common complaints. Intake of certain com-

does not pass into the urine), so that the combination

pounds (such as lead) can cause acquired porphyrias.

of increased urobilinogen and absence of bilirubin is

Increased amounts of porphyrins or their precursors

suggestive of hemolytic jaundice. Increased blood de-

can be detected in blood and urine, facilitating diag-

struction from any cause brings about an increase in

nosis.

urine urobilinogen.

Table 32-3 summarizes laboratory results obtained

•

Catabolism of the heme ring is initiated by the en-

on patients with three different causes of jaundice—he-

zyme heme oxygenase, producing a linear tetrapyr-

molytic anemia (a prehepatic cause), hepatitis (a hepatic

role.

cause), and obstruction of the common bile duct (a

•

Biliverdin is an early product of catabolism and on

posthepatic cause). Laboratory tests on blood (evalua-

reduction yields bilirubin. The latter is transported

tion of the possibility of a hemolytic anemia and mea-

by albumin from peripheral tissues to the liver, where

surement of prothrombin time) and on serum (eg, elec-

it is taken up by hepatocytes. The iron of heme and

trophoresis of proteins; activities of the enzymes ALT,

the amino acids of globin are conserved and reuti-

AST, and alkaline phosphatase) are also important in

lized.

helping to distinguish between prehepatic, hepatic, and

•

In the liver, bilirubin is made water-soluble by conju-

posthepatic causes of jaundice.

gation with two molecules of glucuronic acid and is

secreted into the bile. The action of bacterial en-

SUMMARY

zymes in the gut produces urobilinogen and urobilin,

which are excreted in the feces and urine.

• Hemoproteins, such as hemoglobin and the cy-

tochromes, contain heme. Heme is an iron-por-

•

Jaundice is due to elevation of the level of bilirubin

phyrin compound

(Fe2+-protoporphyrin IX) in

in the blood. The causes of jaundice can be classified

PORPHYRINS & BILE PIGMENTS

285

as prehepatic (eg, hemolytic anemias), hepatic (eg,

Berk PD, Wolkoff AW: Bilirubin metabolism and the hyperbiliru-

binemias. In: Harrison’s Principles of Internal Medicine, 15th

hepatitis), and posthepatic

(eg, obstruction of the

ed. Braunwald E et al (editors). McGraw-Hill, 2001.

common bile duct). Measurements of plasma total

Chowdhury JR et al: Hereditary jaundice and disorders of bilirubin

and nonconjugated bilirubin, of urinary urobilino-

metabolism. In: The Metabolic and Molecular Bases of Inher-

gen and bilirubin, and of certain serum enzymes as

ited Disease, 8th ed. Scriver CR et al (editors). McGraw-Hill,

well as inspection of stool samples help distinguish

2001.

between these causes.

Desnick RJ: The porphyrias. In: Harrison’s Principles of Internal

Medicine, 15th ed. Braunwald E et al (editors). McGraw-Hill,

2001.

REFERENCES

Elder GH: Haem synthesis and the porphyrias. In: Scientific Foun-

Anderson KE et al: Disorders of heme biosynthesis: X-linked sid-

dations of Biochemistry in Clinical Practice, 2nd ed. Williams

eroblastic anemia and the porphyrias. In: The Metabolic and

DL, Marks V (editors). Butterworth-Heinemann, 1994.

Molecular Bases of Inherited Disease, 8th ed. Scriver CR et al

(editors). McGraw-Hill, 2001.

SECTION IV

Structure, Function, & Replication

of Informational Macromolecules

Nucleotides

Victor W. Rodwell, PhD

BIOMEDICAL IMPORTANCE

Nucleotides—the monomer units or building blocks of

nucleic acids—serve multiple additional functions. They

²C

form a part of many coenzymes and serve as donors of

N₉

phosphoryl groups (eg, ATP or GTP), of sugars (eg,

UDP- or GDP-sugars), or of lipid (eg, CDP-acylglyc-

Purine

Pyrimidine

erol). Regulatory nucleotides include the second mes-

Figure 33-1. Purine and pyrimidine. The atoms are

sengers cAMP and cGMP, the control by ADP of ox-

numbered according to the international system.

idative phosphorylation, and allosteric regulation of

enzyme activity by ATP, AMP, and CTP. Synthetic

purine and pyrimidine analogs that contain halogens,

thiols, or additional nitrogen are employed for chemo-

Nucleosides & Nucleotides

therapy of cancer and AIDS and as suppressors of the

immune response during organ transplantation.

Nucleosides are derivatives of purines and pyrimidines

that have a sugar linked to a ring nitrogen. Numerals

PURINES, PYRIMIDINES, NUCLEOSIDES,

with a prime (eg, 2′ or 3′) distinguish atoms of the

sugar from those of the heterocyclic base. The sugar in

& NUCLEOTIDES

ribonucleosides is D-ribose, and in deoxyribonucleo-

Purines and pyrimidines are nitrogen-containing hete-

sides it is 2-deoxy-D-ribose. The sugar is linked to the

rocycles, cyclic compounds whose rings contain both

heterocyclic base via a

-N-glycosidic bond, almost al-

carbon and other elements (hetero atoms). Note that

ways to N-1 of a pyrimidine or to N-9 of a purine (Fig-

the smaller pyrimidine has the longer name and the

ure 33-3).

larger purine the shorter name and that their six-atom

rings are numbered in opposite directions

(Figure

33-1). The planar character of purines and pyrimidines

facilitates their close association, or “stacking,” which

stabilizes double-stranded DNA (Chapter 36). The oxo

and amino groups of purines and pyrimidines exhibit

keto-enol and amine-imine tautomerism (Figure 33-2),

but physiologic conditions strongly favor the amino

Figure 33-2. Tautomerism of the oxo and amino

and oxo forms.

functional groups of purines and pyrimidines.

286

NUCLEOTIDES

287

NH₂

H₂N

Adenosine

Cytidine

Guanosine

Uridine

Figure 33-3. Ribonucleosides, drawn as the syn conformers.

Mononucleotides are nucleosides with a phosphoryl

cleotides. Both therefore exist as syn or anti conformers

group esterified to a hydroxyl group of the sugar. The

(Figure 33-5). While both conformers occur in nature,

3′- and 5′-nucleotides are nucleosides with a phospho-

anti conformers predominate. Table

33-1 lists the

ryl group on the 3′- or 5′-hydroxyl group of the sugar,

major purines and pyrimidines and their nucleoside

respectively. Since most nucleotides are 5′-, the prefix

and nucleotide derivatives. Single-letter abbreviations

“5′-” is usually omitted when naming them. UMP and

are used to identify adenine (A), guanine (G), cytosine

dAMP thus represent nucleotides with a phosphoryl

(C), thymine (T), and uracil (U), whether free or pre-

group on C-5 of the pentose. Additional phosphoryl

sent in nucleosides or nucleotides. The prefix

“d”

groups linked by acid anhydride bonds to the phos-

(deoxy) indicates that the sugar is

2′-deoxy-D-ribose

phoryl group of a mononucleotide form nucleoside

(eg, dGTP) (Figure 33-6).

diphosphates and triphosphates (Figure 33-4).

Steric hindrance by the base restricts rotation about

Nucleic Acids Also Contain

the β-N-glycosidic bond of nucleosides and nu-

Additional Bases

Small quantities of additional purines and pyrimidines

occur in DNA and RNAs. Examples include 5-methyl-

cytosine of bacterial and human DNA,

5-hydroxy-

Adenine

methylcytosine of bacterial and viral nucleic acids, and

mono- and di-N-methylated adenine and guanine of

CH₂

NH₂

Ribose

O^-

P O

HO OH

O^-

Adenosine 5′-monophosphate (AMP)

Adenosine 5′-diphosphate (ADP)

Syn

Anti

Adenosine 5′-triphosphate (ATP)

Figure 33-5.

The syn and anti conformers of adeno-

Figure 33-4. ATP, its diphosphate, and its

sine differ with respect to orientation about the N-gly-

monophosphate.

cosidic bond.

NUCLEOTIDES

289

NH₂

CH₃

CH₂OH

H₂N

CH₂

5-Methylcytosine

5-Hydroxymethylcytosine

^-O

H₃C

CH₃

Figure 33-9. cAMP, 3′,5′-cyclic AMP, and cGMP.

H₂N

Nucleotides Serve Diverse

Dimethylaminoadenine

7-Methylguanine

Physiologic Functions

Figure 33-7. Four uncommon naturally occurring

Nucleotides participate in reactions that fulfill physio-

pyrimidines and purines.

logic functions as diverse as protein synthesis, nucleic

acid synthesis, regulatory cascades, and signal transduc-

tion pathways.

mammalian messenger RNAs (Figure 33-7). These

atypical bases function in oligonucleotide recognition

Nucleoside Triphosphates Have High

and in regulating the half-lives of RNAs. Free nu-

Group Transfer Potential

cleotides include hypoxanthine, xanthine, and uric acid

(see Figure 34-8), intermediates in the catabolism of

Acid anhydrides, unlike phosphate esters, have high

adenine and guanine. Methylated heterocyclic bases of

group transfer potential. ∆⁰′ for the hydrolysis of each

plants include the xanthine derivatives caffeine of cof-

of the terminal phosphates of nucleoside triphosphates

fee, theophylline of tea, and theobromine of cocoa (Fig-

is about −7 kcal/mol (−30 kJ/mol). The high group

ure 33-8).

transfer potential of purine and pyrimidine nucleoside

Posttranslational modification of preformed polynu-

triphosphates permits them to function as group trans-

cleotides can generate additional bases such as

fer reagents. Cleavage of an acid anhydride bond typi-

pseudouridine, in which D-ribose is linked to C-5 of

cally is coupled with a highly endergonic process such

uracil by a carbon-to-carbon bond rather than by a

as covalent bond synthesis—eg, polymerization of nu-

β-N-glycosidic bond. The nucleotide pseudouridylic

cleoside triphosphates to form a nucleic acid.

acid Ψ arises by rearrangement of UMP of a preformed

In addition to their roles as precursors of nucleic

tRNA. Similarly, methylation by S-adenosylmethionine

acids, ATP, GTP, UTP, CTP, and their derivatives

of a UMP of preformed tRNA forms TMP (thymidine

each serve unique physiologic functions discussed in

monophosphate), which contains ribose rather than de-

other chapters. Selected examples include the role of

oxyribose.

ATP as the principal biologic transducer of free energy;

the second messenger cAMP (Figure 33-9); adenosine

3′-phosphate-5′-phosphosulfate

(Figure

33-10), the

sulfate donor for sulfated proteoglycans (Chapter 48)

and for sulfate conjugates of drugs; and the methyl

group donor S-adenosylmethionine

(Figure

33-11).

CH₃

Figure 33-8. Caffeine, a trimethylxanthine. The di-

Adenine Ribose

O SO₃

methylxanthines theobromine and theophylline are

similar but lack the methyl group at N-1 and at N-7, re-

Figure 33-10. Adenosine 3′-phosphate-5′-phos-

spectively.

phosulfate.

290

CHAPTER 33

NH₂

Table 33-2. Many coenzymes and related

compounds are derivatives of adenosine

monophosphate.

NH₂

COO^-

CH₃

CH₂

Adenine

CH₂

N N

NH3+

P O

CH₂

HO OH

O^-

Adenosine

Methionine

R'' O OR'

Figure 33-11. S-Adenosylmethionine.

D-Ribose

Coenzyme

GTP serves as an allosteric regulator and as an energy

Active methionine

Methionine*

source for protein synthesis, and cGMP (Figure 33-9)

Amino acid adenylates

Amino acid

serves as a second messenger in response to nitric oxide

Active sulfate

SO32−

PO32−

(NO) during relaxation of smooth muscle (Chapter

3′,5′-Cyclic AMP

PO32−

†

48). UDP-sugar derivatives participate in sugar epimer-

NAD*

†

izations and in biosynthesis of glycogen, glucosyl disac-

NADP*

PO32−

†

charides, and the oligosaccharides of glycoproteins and

FAD

†

proteoglycans (Chapters 47 and 48). UDP-glucuronic

CoASH

PO32−

acid forms the urinary glucuronide conjugates of biliru-

*Replaces phosphoryl group.

bin (Chapter 32) and of drugs such as aspirin. CTP

^†R is a B vitamin derivative.

participates in biosynthesis of phosphoglycerides,

sphingomyelin, and other substituted sphingosines

(Chapter 24). Finally, many coenzymes incorporate nu-

cleotides as well as structures similar to purine and

nucleic acids thus often is expressed in terms of “ab-

pyrimidine nucleotides (see Table 33-2).

sorbance at 260 nm.”

Nucleotides Are Polyfunctional Acids

SYNTHETIC NUCLEOTIDE ANALOGS

ARE USED IN CHEMOTHERAPY

Nucleosides or free purine or pyrimidine bases are un-

charged at physiologic pH. By contrast, the primary

Synthetic analogs of purines, pyrimidines, nucleosides,

phosphoryl groups (pK about 1.0) and secondary phos-

and nucleotides altered in either the heterocyclic ring or

phoryl groups (pK about 6.2) of nucleotides ensure that

the sugar moiety have numerous applications in clinical

they bear a negative charge at physiologic pH. Nu-

medicine. Their toxic effects reflect either inhibition of

cleotides can, however, act as proton donors or accep-

enzymes essential for nucleic acid synthesis or their in-

tors at pH values two or more units above or below

corporation into nucleic acids with resulting disruption

neutrality.

of base-pairing. Oncologists employ

5-fluoro- or

iodouracil, 3-deoxyuridine, 6-thioguanine and 6-mer-

captopurine, 5- or 6-azauridine, 5- or 6-azacytidine,

Nucleotides Absorb Ultraviolet Light

and 8-azaguanine (Figure 33-12), which are incorpo-

The conjugated double bonds of purine and pyrimidine

rated into DNA prior to cell division. The purine ana-

derivatives absorb ultraviolet light. The mutagenic ef-

log allopurinol, used in treatment of hyperuricemia and

fect of ultraviolet light results from its absorption by

gout, inhibits purine biosynthesis and xanthine oxidase

nucleotides in DNA with accompanying chemical

activity. Cytarabine is used in chemotherapy of cancer.

changes. While spectra are pH-dependent, at pH 7.0 all

Finally, azathioprine, which is catabolized to 6-mercap-

the common nucleotides absorb light at a wavelength

topurine, is employed during organ transplantation to

close to 260 nm. The concentration of nucleotides and

suppress immunologic rejection.

NUCLEOTIDES

291

⁶N

⁸N

H₂N

2′

HO OH

HO H

5-Iodo-2′-deoxyuridine

5-Fluorouracil

6-Azauridine

8-Azaguanine

H₂N

6-Mercaptopurine

6-Thioguanine

Alloburinol

Figure 33-12. Selected synthetic pyrimidine and purine analogs.

Nonhydrolyzable Nucleoside

(absent from DNA) functions as a nucleophile during

Triphosphate Analogs Serve as

hydrolysis of the 3′,5′-phosphodiester bond.

Research Tools

Synthetic nonhydrolyzable analogs of nucleoside

Polynucleotides Are Directional

triphosphates (Figure 33-13) allow investigators to dis-

Macromolecules

tinguish the effects of nucleotides due to phosphoryl

Phosphodiester bonds link the 3′- and 5′-carbons of ad-

transfer from effects mediated by occupancy of al-

jacent monomers. Each end of a nucleotide polymer

losteric nucleotide-binding sites on regulated enzymes.

thus is distinct. We therefore refer to the “5′- end” or

the “3′- end” of polynucleotides, the 5′- end being the

POLYNUCLEOTIDES

one with a free or phosphorylated 5′-hydroxyl.

The 5′-phosphoryl group of a mononucleotide can es-

terify a second OH group, forming a phosphodi-

Polynucleotides Have Primary Structure

ester. Most commonly, this second OH group is the

3′-OH of the pentose of a second nucleotide. This

The base sequence or primary structure of a polynu-

forms a dinucleotide in which the pentose moieties are

cleotide can be represented as shown below. The phos-

linked by a 3′ → 5′ phosphodiester bond to form the

phodiester bond is represented by P or p, bases by a sin-

“backbone” of RNA and DNA.

gle letter, and pentoses by a vertical line.

While formation of a dinucleotide may be repre-

sented as the elimination of water between two

monomers, the reaction in fact strongly favors phos-

phodiester hydrolysis. Phosphodiesterases rapidly cat-

alyze the hydrolysis of phosphodiester bonds whose

spontaneous hydrolysis is an extremely slow process.

Consequently, DNA persists for considerable periods

and has been detected even in fossils. RNAs are far less

stable than DNA since the 2′-hydroxyl group of RNA

292

CHAPTER 33

SUMMARY

Pu /Py R O

O^-

•

Under physiologic conditions, the amino and oxo

tautomers of purines, pyrimidines, and their deriva-

O^-

tives predominate.

Parent nucleoside triphosphate

•

Nucleic acids contain, in addition to A, G, C, T, and

U, traces of 5-methylcytosine, 5-hydroxymethylcyto-

sine, pseudouridine (Ψ), or N-methylated bases.

Pu /Py R O

CH₂

O^-

•

Most nucleosides contain D-ribose or

2-deoxy-D-

O^-

ribose linked to N-1 of a pyrimidine or to N-9 of a

β,γ-Methylene derivative

purine by a β-glycosidic bond whose syn conformers

predominate.

•

A primed numeral locates the position of the phos-

Pu /Py R O

O^-

phate on the sugars of mononucleotides

(eg,

3′-

GMP, 5′-dCMP). Additional phosphoryl groups

O^-

linked to the first by acid anhydride bonds form nu-

β,γ-Imino derivative

cleoside diphosphates and triphosphates.

Figure 33-13. Synthetic derivatives of nucleoside

•

Nucleoside triphosphates have high group transfer

triphosphates incapable of undergoing hydrolytic re-

potential and participate in covalent bond syntheses.

lease of the terminal phosphoryl group. (Pu/Py, a

The cyclic phosphodiesters cAMP and cGMP func-

purine or pyrimidine base; R, ribose or deoxyribose.)

tion as intracellular second messengers.

Shown are the parent (hydrolyzable) nucleoside

•

Mononucleotides linked by 3′ → 5′-phosphodiester

triphosphate (top) and the unhydrolyzable β-methyl-

bonds form polynucleotides, directional macromole-

ene (center) and γ-imino derivatives (bottom).

cules with distinct 3′- and 5′- ends. For pTpGpTp or

TGCATCA, the 5′- end is at the left, and all phos-

phodiester bonds are 3′ → 5′.

Where all the phosphodiester bonds are 5′ → 3′, a

•

Synthetic analogs of purine and pyrimidine bases and

more compact notation is possible:

their derivatives serve as anticancer drugs either by

inhibiting an enzyme of nucleotide biosynthesis or

pGpGpApTpCpA

by being incorporated into DNA or RNA.

This representation indicates that the 5′-hydroxyl—

REFERENCES

but not the 3′-hydroxyl—is phosphorylated.

The most compact representation shows only the

Adams RLP, Knowler JT, Leader DP: The Biochemistry of the Nu-

base sequence with the 5′- end on the left and the 3′-

cleic Acids, 11th ed. Chapman & Hall, 1992.

end on the right. The phosphoryl groups are assumed

Blackburn GM, Gait MJ: Nucleic Acids in Chemistry & Biology. IRL

but not shown:

Press, 1990.

Bugg CE, Carson WM, Montgomery JA: Drugs by design. Sci Am

GGATCA

1992;269(6):92.

Metabolism of Purine &

Pyrimidine Nucleotides

Victor W. Rodwell, PhD

BIOMEDICAL IMPORTANCE

(synthesis de novo), (2) phosphoribosylation of purines,

and (3) phosphorylation of purine nucleosides.

The biosynthesis of purines and pyrimidines is strin-

gently regulated and coordinated by feedback mecha-

nisms that ensure their production in quantities and at

INOSINE MONOPHOSPHATE (IMP)

times appropriate to varying physiologic demand. Ge-

IS SYNTHESIZED FROM AMPHIBOLIC

netic diseases of purine metabolism include gout,

Lesch-Nyhan syndrome, adenosine deaminase defi-

INTERMEDIATES

ciency, and purine nucleoside phosphorylase deficiency.

Figure 34-2 illustrates the intermediates and reactions

By contrast, apart from the orotic acidurias, there are

for conversion of α-D-ribose 5-phosphate to inosine

few clinically significant disorders of pyrimidine catab-

monophosphate (IMP). Separate branches then lead to

olism.

AMP and GMP (Figure 34-3). Subsequent phosphoryl

transfer from ATP converts AMP and GMP to ADP

and GDP. Conversion of GDP to GTP involves a sec-

PURINES & PYRIMIDINES ARE

ond phosphoryl transfer from ATP, whereas conversion

DIETARILY NONESSENTIAL

of ADP to ATP is achieved primarily by oxidative

Human tissues can synthesize purines and pyrimidines

phosphorylation (see Chapter 12).

from amphibolic intermediates. Ingested nucleic acids

and nucleotides, which therefore are dietarily nonessen-

tial, are degraded in the intestinal tract to mononu-

Multifunctional Catalysts Participate in

cleotides, which may be absorbed or converted to

Purine Nucleotide Biosynthesis

purine and pyrimidine bases. The purine bases are then

In prokaryotes, each reaction of Figure 34-2 is cat-

oxidized to uric acid, which may be absorbed and ex-

alyzed by a different polypeptide. By contrast, in eu-

creted in the urine. While little or no dietary purine or

karyotes, the enzymes are polypeptides with multiple

pyrimidine is incorporated into tissue nucleic acids, in-

catalytic activities whose adjacent catalytic sites facili-

jected compounds are incorporated. The incorporation

tate channeling of intermediates between sites. Three

of injected [³H]thymidine into newly synthesized DNA

distinct multifunctional enzymes catalyze reactions 3,

thus is used to measure the rate of DNA synthesis.

4, and 6, reactions 7 and 8, and reactions 10 and 11 of

Figure 34-2.

BIOSYNTHESIS OF PURINE NUCLEOTIDES

Purine and pyrimidine nucleotides are synthesized in

Antifolate Drugs or Glutamine Analogs

vivo at rates consistent with physiologic need. Intracel-

Block Purine Nucleotide Biosynthesis

lular mechanisms sense and regulate the pool sizes of

nucleotide triphosphates

(NTPs), which rise during

The carbons added in reactions 4 and 5 of Figure 34-2

growth or tissue regeneration when cells are rapidly di-

are contributed by derivatives of tetrahydrofolate.

viding. Early investigations of nucleotide biosynthesis

Purine deficiency states, which are rare in humans, gen-

employed birds, and later ones used Escherichia coli.

erally reflect a deficiency of folic acid. Compounds that

Isotopic precursors fed to pigeons established the source

inhibit formation of tetrahydrofolates and therefore

of each atom of a purine base (Figure 34-1) and initi-

block purine synthesis have been used in cancer

ated study of the intermediates of purine biosynthesis.

chemotherapy. Inhibitory compounds and the reactions

Three processes contribute to purine nucleotide

they inhibit include azaserine (reaction 5, Figure 34-2),

biosynthesis. These are, in order of decreasing impor-

diazanorleucine (reaction 2), 6-mercaptopurine (reac-

tance:

(1) synthesis from amphibolic intermediates

tions 13 and 14), and mycophenolic acid (reaction 14).

293

294

CHAPTER 34

Respiratory CO₂

and therefore utilize exogenous purines to form nu-

Glycine

cleotides.

Aspartate

AMP & GMP Feedback-Regulate PRPP

Glutamyl Amidotransferase

C²

N⁵,N10 -Methenyl-

Since biosynthesis of IMP consumes glycine, gluta-

tetrahydrofolate

N¹⁰ -Formyl-

mine, tetrahydrofolate derivatives, aspartate, and ATP,

tetrahydro-

it is advantageous to regulate purine biosynthesis. The

folate

major determinant of the rate of de novo purine nu-

cleotide biosynthesis is the concentration of PRPP,

whose pool size depends on its rates of synthesis, uti-

Amide nitrogen of glutamine

lization, and degradation. The rate of PRPP synthesis

depends on the availability of ribose 5-phosphate and

Figure 34-1. Sources of the nitrogen and carbon

on the activity of PRPP synthase, an enzyme sensitive

atoms of the purine ring. Atoms 4, 5, and 7 (shaded) de-

to feedback inhibition by AMP, ADP, GMP, and

rive from glycine.

GDP.

AMP & GMP Feedback-Regulate

“SALVAGE REACTIONS” CONVERT

Their Formation From IMP

PURINES & THEIR NUCLEOSIDES TO

Two mechanisms regulate conversion of IMP to GMP

MONONUCLEOTIDES

and AMP. AMP and GMP feedback-inhibit adenylo-

Conversion of purines, their ribonucleosides, and their

succinate synthase and IMP dehydrogenase (reactions

deoxyribonucleosides to mononucleotides involves so-

12 and 14, Figure 34-3), respectively. Furthermore,

called “salvage reactions” that require far less energy

conversion of IMP to adenylosuccinate en route to

than de novo synthesis. The more important mecha-

AMP requires GTP, and conversion of xanthinylate

nism involves phosphoribosylation by PRPP (structure

(XMP) to GMP requires ATP. This cross-regulation

II, Figure 34-2) of a free purine (Pu) to form a purine

between the pathways of IMP metabolism thus serves

5′-mononucleotide (Pu-RP).

to decrease synthesis of one purine nucleotide when

there is a deficiency of the other nucleotide. AMP and

GMP also inhibit hypoxanthine-guanine phosphoribo-

Pu+PR−PP→PRP+PP

syltransferase, which converts hypoxanthine and gua-

nine to IMP and GMP (Figure 34-4), and GMP feed-

back-inhibits PRPP glutamyl amidotransferase (reaction

Two phosphoribosyl transferases then convert adenine

2, Figure 34-2).

to AMP and hypoxanthine and guanine to IMP or

GMP (Figure 34-4). A second salvage mechanism in-

volves phosphoryl transfer from ATP to a purine ri-

REDUCTION OF RIBONUCLEOSIDE

bonucleoside (PuR):

DIPHOSPHATES FORMS

DEOXYRIBONUCLEOSIDE

PuR + ATP → PuR − P + ADP

DIPHOSPHATES

Reduction of the 2′-hydroxyl of purine and pyrimidine

Adenosine kinase catalyzes phosphorylation of adeno-

ribonucleotides, catalyzed by the ribonucleotide re-

sine and deoxyadenosine to AMP and dAMP, and de-

ductase complex (Figure 34-5), forms deoxyribonu-

oxycytidine kinase phosphorylates deoxycytidine and

cleoside diphosphates (dNDPs). The enzyme complex

2′-deoxyguanosine to dCMP and dGMP.

is active only when cells are actively synthesizing DNA.

Liver, the major site of purine nucleotide biosynthe-

Reduction requires thioredoxin, thioredoxin reductase,

sis, provides purines and purine nucleosides for salvage

and NADPH. The immediate reductant, reduced

and utilization by tissues incapable of their biosynthe-

thioredoxin, is produced by NADPH:thioredoxin re-

sis. For example, human brain has a low level of PRPP

ductase

(Figure

34-5). Reduction of ribonucleoside

amidotransferase (reaction 2, Figure 34-2) and hence

diphosphates (NDPs) to deoxyribonucleoside diphos-

depends in part on exogenous purines. Erythrocytes

phates (dNDPs) is subject to complex regulatory con-

and polymorphonuclear leukocytes cannot synthesize

trols that achieve balanced production of deoxyribonu-

5-phosphoribosylamine

(structure III, Figure

34-2)

cleotides for synthesis of DNA (Figure 34-6).

296

CHAPTER 34

OOC C C COO

OOC

C COO-

H₂

H₂O

OOC

C COO

NH₂

GTP, Mg

ADENYLOSUCCINASE

ADENYLOSUCCINATE

R-5- P

SYNTHASE

R-5-

R-5- P

Inosine monophosphate

Adenylosuccinate

Adenosine monophosphate

(IMP)

(AMPS)

(AMP)

NAD

H₂O

IMP DEHYDROGENASE

NADH

+ H

Glutamine

Glutamate

ATP

H₂ N

TRANSAMIDINASE

R-5-

Xanthosine monophosphate

Guanosine monophosphate

(XMP)

(GMP)

Figure 34-3. Conversion of IMP to AMP and GMP.

BIOSYNTHESIS OF PYRIMIDINE

THE DEOXYRIBONUCLEOSIDES OF

NUCLEOTIDES

URACIL & CYTOSINE ARE SALVAGED

Figure 34-7 summarizes the roles of the intermediates

While mammalian cells reutilize few free pyrimidines,

and enzymes of pyrimidine nucleotide biosynthesis.

“salvage reactions” convert the ribonucleosides uridine

The catalyst for the initial reaction is cytosolic carbamoyl

and cytidine and the deoxyribonucleosides thymidine

phosphate synthase II, a different enzyme from the mi-

and deoxycytidine to their respective nucleotides. ATP-

tochondrial carbamoyl phosphate synthase I of urea syn-

dependent phosphoryltransferases (kinases) catalyze the

thesis (Figure 29-9). Compartmentation thus provides

phosphorylation of the nucleoside diphosphates 2′-de-

two independent pools of carbamoyl phosphate. PRPP,

oxycytidine, 2′-deoxyguanosine, and 2′-deoxyadenosine

an early participant in purine nucleotide synthesis (Fig-

to their corresponding nucleoside triphosphates. In ad-

ure 34-2), is a much later participant in pyrimidine

dition, orotate phosphoribosyltransferase

(reaction

biosynthesis.

Figure 34-7), an enzyme of pyrimidine nucleotide syn-

thesis, salvages orotic acid by converting it to orotidine

Multifunctional Proteins

monophosphate (OMP).

Catalyze the Early Reactions

of Pyrimidine Biosynthesis

Methotrexate Blocks Reduction

of Dihydrofolate

Five of the first six enzyme activities of pyrimidine

biosynthesis reside on multifunctional polypeptides.

Reaction 12 of Figure 34-7 is the only reaction of pyrimi-

One such polypeptide catalyzes the first three reactions

dine nucleotide biosynthesis that requires a tetrahydrofo-

of Figure 34-2 and ensures efficient channeling of car-

late derivative. The methylene group of N⁵,N¹⁰-methyl-

bamoyl phosphate to pyrimidine biosynthesis. A second

ene-tetrahydrofolate is reduced to the methyl group that

bifunctional enzyme catalyzes reactions 5 and 6.

is transferred, and tetrahydrofolate is oxidized to dihydro-

METABOLISM OF PURINE & PYRIMIDINE NUCLEOTIDES

297

NH₂

RIBONUCLEOTIDE

PRPP

PP_i

REDUCTASE

Ribonucleoside

2 ′-Deoxyribonucleoside

diphosphate

Adenine

Reduced

Oxidized

ADENINE

PHOSPHORIBOSYL

thioredoxin

TRANSFERASE

THIOREDOXIN

REDUCTASE

AMP

NADP⁺

NADPH + H⁺

Figure 34-5. Reduction of ribonucleoside diphos-

PRPP

PP_i

phates to 2′-deoxyribonucleoside diphosphates.

a nucleotide in which the ribosyl phosphate is attached

to N-1 of the pyrimidine ring. The anticancer drug

Hypoxanthine

5-fluorouracil (Figure 33-12) is also phosphoribosy-

lated by orotate phosphoribosyl transferase.

HYPOXANTHINE-GUANINE

PHOSPHORIBOSYLTRANSFERASE

REGULATION OF PYRIMIDINE

IMP

NUCLEOTIDE BIOSYNTHESIS

Gene Expression & Enzyme Activity

Both Are Regulated

The activities of the first and second enzymes of pyrim-

H₂N

idine nucleotide biosynthesis are controlled by allosteric

Guanine

PRPP

PP_i

CDP

2′dCDP

2′dCTP

H H

ATP

OH OH

GMP

UDP

2′dUDP

2′dTTP

Figure 34-4. Phosphoribosylation of adenine, hy-

poxanthine, and guanine to form AMP, IMP, and GMP,

respectively.

folate. For further pyrimidine synthesis to occur, dihydro-

folate must be reduced back to tetrahydrofolate, a reac-

GDP

2′dGDP

2′dGTP

tion catalyzed by dihydrofolate reductase. Dividing cells,

–

which must generate TMP and dihydrofolate, thus are es-

pecially sensitive to inhibitors of dihydrofolate reductase

such as the anticancer drug methotrexate.

ADP

2′dADP

2′dATP

Certain Pyrimidine Analogs Are

Figure 34-6. Regulation of the reduction of purine

Substrates for Enzymes of Pyrimidine

and pyrimidine ribonucleotides to their respective

Nucleotide Biosynthesis

2′-deoxyribonucleotides. Solid lines represent chemical

Orotate phosphoribosyltransferase (reaction 5, Figure

flow. Broken lines show negative ( - ) or positive ( + )

34-7) converts the drug allopurinol (Figure 33-12) to

feedback regulation.

METABOLISM OF PURINE & PYRIMIDINE NUCLEOTIDES

299

regulation. Carbamoyl phosphate synthase II (reaction

NH₂

1, Figure 34-7) is inhibited by UTP and purine nu-

cleotides but activated by PRPP. Aspartate transcar-

bamoylase (reaction 2, Figure 34-7) is inhibited by

N N

CTP but activated by ATP. In addition, the first three

and the last two enzymes of the pathway are regulated

by coordinate repression and derepression.

Purine & Pyrimidine Nucleotide

Biosynthesis Are Coordinately Regulated

Adenosine

Purine and pyrimidine biosynthesis parallel one an-

H₂O

other mole for mole, suggesting coordinated control of

their biosynthesis. Several sites of cross-regulation char-

NH+

acterize purine and pyrimidine nucleotide biosynthesis.

The PRPP synthase reaction (reaction 1, Figure 34-2),

which forms a precursor essential for both processes, is

feedback-inhibited by both purine and pyrimidine nu-

cleotides.

H₂N

N N

HUMANS CATABOLIZE PURINES

TO URIC ACID

Humans convert adenosine and guanosine to uric acid

(Figure 34-8). Adenosine is first converted to inosine

Inosine

Guanosine

by adenosine deaminase. In mammals other than

higher primates, uricase converts uric acid to the water-

P_i

soluble product allantoin. However, since humans lack

uricase, the end product of purine catabolism in hu-

Ribose 1-phosphate

mans is uric acid.

GOUT IS A METABOLIC DISORDER

OF PURINE CATABOLISM

H₂N

N NH

Hypoxanthine

Guanine

Various genetic defects in PRPP synthetase (reaction 1,

Figure 34-2) present clinically as gout. Each defect—

H₂O + O₂

eg, an elevated V_max, increased affinity for ribose 5-

phosphate, or resistance to feedback inhibition—results

HN₃

in overproduction and overexcretion of purine catabo-

H₂O

lites. When serum urate levels exceed the solubility

limit, sodium urate crystalizes in soft tissues and joints

and causes an inflammatory reaction, gouty arthritis.

However, most cases of gout reflect abnormalities in

Xanthine

renal handling of uric acid.

H₂O + O₂

H₂O₂

Figure 34-8. Formation of uric acid from purine nucleosides

by way of the purine bases hypoxanthine, xanthine, and gua-

nine. Purine deoxyribonucleosides are degraded by the same

catabolic pathway and enzymes, all of which exist in the mucosa

of the mammalian gastrointestinal tract.

Uric acid

300

CHAPTER 34

OTHER DISORDERS OF

CATABOLISM OF PYRIMIDINES

PURINE CATABOLISM

PRODUCES WATER-SOLUBLE

METABOLITES

While purine deficiency states are rare in human sub-

jects, there are numerous genetic disorders of purine ca-

Unlike the end products of purine catabolism, those

tabolism. Hyperuricemias may be differentiated based

of pyrimidine catabolism are highly water-soluble:

on whether patients excrete normal or excessive quanti-

CO₂, NH₃, β-alanine, and β-aminoisobutyrate (Figure

ties of total urates. Some hyperuricemias reflect specific

34-9). Excretion of β-aminoisobutyrate increases in

enzyme defects. Others are secondary to diseases such

leukemia and severe x-ray radiation exposure due to in-

as cancer or psoriasis that enhance tissue turnover.

creased destruction of DNA. However, many persons

of Chinese or Japanese ancestry routinely excrete

β-aminoisobutyrate. Humans probably transaminate

Lesch-Nyhan Syndrome

β-aminoisobutyrate to methylmalonate semialdehyde,

Lesch-Nyhan syndrome, an overproduction hyper-

which then forms succinyl-CoA (Figure 19-2).

uricemia characterized by frequent episodes of uric acid

lithiasis and a bizarre syndrome of self-mutilation, re-

Pseudouridine Is Excreted Unchanged

flects a defect in hypoxanthine-guanine phosphoribo-

Since no human enzyme catalyzes hydrolysis or phos-

syl transferase, an enzyme of purine salvage (Figure

phorolysis of pseudouridine, this unusual nucleoside is

34-4). The accompanying rise in intracellular PRPP re-

excreted unchanged in the urine of normal subjects.

sults in purine overproduction. Mutations that decrease

or abolish hypoxanthine-guanine phosphoribosyltrans-

ferase activity include deletions, frameshift mutations,

OVERPRODUCTION OF PYRIMIDINE

base substitutions, and aberrant mRNA splicing.

CATABOLITES IS ONLY RARELY

ASSOCIATED WITH CLINICALLY

Von Gierke’s Disease

SIGNIFICANT ABNORMALITIES

Purine overproduction and hyperuricemia in von

Since the end products of pyrimidine catabolism are

Gierke’s disease

(glucose-6-phosphatase deficiency)

highly water-soluble, pyrimidine overproduction results

occurs secondary to enhanced generation of the PRPP

in few clinical signs or symptoms. In hyperuricemia as-

precursor ribose 5-phosphate. An associated lactic aci-

sociated with severe overproduction of PRPP, there is

dosis elevates the renal threshold for urate, elevating

overproduction of pyrimidine nucleotides and in-

total body urates.

creased excretion of β-alanine. Since N⁵,N¹⁰-methyl-

ene-tetrahydrofolate is required for thymidylate synthe-

sis, disorders of folate and vitamin B₁₂

metabolism

Hypouricemia

result in deficiencies of TMP.

Hypouricemia and increased excretion of hypoxanthine

and xanthine are associated with xanthine oxidase de-

Orotic Acidurias

ficiency due to a genetic defect or to severe liver dam-

The orotic aciduria that accompanies Reye’s syndrome

age. Patients with a severe enzyme deficiency may ex-

probably is a consequence of the inability of severely

hibit xanthinuria and xanthine lithiasis.

damaged mitochondria to utilize carbamoyl phosphate,

which then becomes available for cytosolic overproduc-

Adenosine Deaminase & Purine

tion of orotic acid. Type I orotic aciduria reflects a de-

ficiency of both orotate phosphoribosyltransferase and

Nucleoside Phosphorylase Deficiency

orotidylate decarboxylase

(reactions

5 and 6, Figure

Adenosine deaminase deficiency is associated with an

34-7); the rarer type II orotic aciduria is due to a defi-

immunodeficiency disease in which both thymus-

ciency only of orotidylate decarboxylase (reaction 6,

derived lymphocytes (T cells) and bone marrow-de-

Figure 34-7).

rived lymphocytes (B cells) are sparse and dysfunc-

tional. Purine nucleoside phosphorylase deficiency is

Deficiency of a Urea Cycle Enzyme Results

associated with a severe deficiency of T cells but appar-

in Excretion of Pyrimidine Precursors

ently normal B cell function. Immune dysfunctions ap-

pear to result from accumulation of dGTP and dATP,

Increased excretion of orotic acid, uracil, and uridine

which inhibit ribonucleotide reductase and thereby de-

accompanies a deficiency in liver mitochondrial or-

plete cells of DNA precursors.

nithine transcarbamoylase (reaction 2, Figure 29-9).

METABOLISM OF PURINE & PYRIMIDINE NUCLEOTIDES

301

Excess carbamoyl phosphate exits to the cytosol, where

NH₂

it stimulates pyrimidine nucleotide biosynthesis. The

resulting mild orotic aciduria is increased by high-

nitrogen foods.

Cytosine

Drugs May Precipitate Orotic Aciduria

1/2

O₂

Allopurinol (Figure 33-12), an alternative substrate for

orotate phosphoribosyltransferase

(reaction

5, Figure

34-7), competes with orotic acid. The resulting nu-

cleotide product also inhibits orotidylate decarboxylase

(reaction 6, Figure 34-7), resulting in orotic aciduria

CH₃

and orotidinuria. 6-Azauridine, following conversion

to 6-azauridylate, also competitively inhibits orotidylate

O N

decarboxylase (reaction 6, Figure 34-7), enhancing ex-

cretion of orotic acid and orotidine.

Thymine

Uracil

NADPH + H⁺

SUMMARY

•

Ingested nucleic acids are degraded to purines and

pyrimidines. New purines and pyrimidines are

NADP⁺

formed from amphibolic intermediates and thus are

dietarily nonessential.

CH₃

•

Several reactions of IMP biosynthesis require folate

derivatives and glutamine. Consequently, antifolate

drugs and glutamine analogs inhibit purine biosyn-

O N

thesis.

Dihydrouracil

Dihydrothymine

•

Oxidation and amination of IMP forms AMP and

GMP, and subsequent phosphoryl transfer from

H₂O

ATP forms ADP and GDP. Further phosphoryl

transfer from ATP to GDP forms GTP. ADP is con-

verted to ATP by oxidative phosphorylation. Reduc-

tion of NDPs forms dNDPs.

COO⁻

CH₃

H₂N

CH₂

•

Hepatic purine nucleotide biosynthesis is stringently

H₂N

CH₂

regulated by the pool size of PRPP and by feedback

CH₂

inhibition of PRPP-glutamyl amidotransferase by

AMP and GMP.

β -Ureidopropionate

β -Ureidoisobutyrate

•

Coordinated regulation of purine and pyrimidine

(N -carbamoyl-β -alanine)

(N -carbamoyl-β -amino-

isobutyrate)

nucleotide biosynthesis ensures their presence in pro-

portions appropriate for nucleic acid biosynthesis

and other metabolic needs.

•

Humans catabolize purines to uric acid (pK_a 5.8),

CO₂

+ NH₃

present as the relatively insoluble acid at acidic pH or

as its more soluble sodium urate salt at a pH near

3N⁺

CH₂

COO⁻

H₃N+ CH2

CH COO⁻

neutrality. Urate crystals are diagnostic of gout.

β-Alanine

CH₃

Other disorders of purine catabolism include Lesch-

-Aminoisobutyrate

Nyhan syndrome, von Gierke’s disease, and hypo-

uricemias.

Figure 34-9. Catabolism of pyrimidines.

•

Since pyrimidine catabolites are water-soluble, their

overproduction does not result in clinical abnormali-

ties. Excretion of pyrimidine precursors can, how-

ever, result from a deficiency of ornithine transcar-

bamoylase because excess carbamoyl phosphate is

available for pyrimidine biosynthesis.

302

CHAPTER 34

Martinez J et al: Human genetic disorders, a phylogenetic perspec-

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syndrome. Hum Genet 1998;103:311.

Harris MD, Siegel LB, Alloway JA: Gout and hyperuricemia. Am

Zalkin H, Dixon JE: De novo purine nucleotide synthesis. Prog

Family Physician 1999;59:925.

Nucleic Acid Res Mol Biol 1992;42:259.

Lipkowitz MS et al: Functional reconstitution, membrane target-

ing, genomic structure, and chromosomal localization of a

human urate transporter. J Clin Invest 2001;107:1103.

Nucleic Acid Structure & Function

Daryl K. Granner, MD

BIOMEDICAL IMPORTANCE

The informational content of DNA (the genetic code)

resides in the sequence in which these monomers—

The discovery that genetic information is coded along

purine and pyrimidine deoxyribonucleotides—are or-

the length of a polymeric molecule composed of only

dered. The polymer as depicted possesses a polarity;

four types of monomeric units was one of the major sci-

one end has a 5′-hydroxyl or phosphate terminal while

entific achievements of the twentieth century. This

the other has a 3′-phosphate or hydroxyl terminal. The

polymeric molecule, DNA, is the chemical basis of

importance of this polarity will become evident. Since

heredity and is organized into genes, the fundamental

the genetic information resides in the order of the

units of genetic information. The basic information

monomeric units within the polymers, there must exist

pathway—ie, DNA directs the synthesis of RNA,

a mechanism of reproducing or replicating this specific

which in turn directs protein synthesis—has been eluci-

information with a high degree of fidelity. That re-

dated. Genes do not function autonomously; their

quirement, together with x-ray diffraction data from

replication and function are controlled by various gene

the DNA molecule and the observation of Chargaff

products, often in collaboration with components of

that in DNA molecules the concentration of de-

various signal transduction pathways. Knowledge of the

oxyadenosine (A) nucleotides equals that of thymidine

structure and function of nucleic acids is essential in

(T) nucleotides (A = T), while the concentration of de-

understanding genetics and many aspects of pathophys-

oxyguanosine (G) nucleotides equals that of deoxycyti-

iology as well as the genetic basis of disease.

dine (C) nucleotides (G = C), led Watson, Crick, and

Wilkins to propose in the early 1950s a model of a dou-

ble-stranded DNA molecule. The model they proposed

DNA CONTAINS THE

is depicted in Figure 35-2. The two strands of this

GENETIC INFORMATION

double-stranded helix are held in register by hydrogen

The demonstration that DNA contained the genetic in-

bonds between the purine and pyrimidine bases of the

formation was first made in 1944 in a series of experi-

respective linear molecules. The pairings between the

ments by Avery, MacLeod, and McCarty. They showed

purine and pyrimidine nucleotides on the opposite

that the genetic determination of the character (type) of

strands are very specific and are dependent upon hydro-

the capsule of a specific pneumococcus could be trans-

gen bonding of A with T and G with C (Figure 35-3).

mitted to another of a different capsular type by intro-

This common form of DNA is said to be right-

ducing purified DNA from the former coccus into the

handed because as one looks down the double helix the

latter. These authors referred to the agent (later shown

base residues form a spiral in a clockwise direction. In

to be DNA) accomplishing the change as “transforming

the double-stranded molecule, restrictions imposed by

factor.” Subsequently, this type of genetic manipulation

the rotation about the phosphodiester bond, the fa-

has become commonplace. Similar experiments have

vored anti configuration of the glycosidic bond (Figure

recently been performed utilizing yeast, cultured mam-

33-8), and the predominant tautomers

(see Figure

malian cells, and insect and mammalian embryos as re-

33-3) of the four bases (A, G, T, and C) allow A to pair

cipients and cloned DNA as the donor of genetic infor-

only with T and G only with C, as depicted in Figure

mation.

35-3. This base-pairing restriction explains the earlier

observation that in a double-stranded DNA molecule

the content of A equals that of T and the content of G

DNA Contains Four Deoxynucleotides

equals that of C. The two strands of the double-helical

The chemical nature of the monomeric deoxynucleo-

molecule, each of which possesses a polarity, are an-

tide units of DNA—deoxyadenylate, deoxyguanylate,

tiparallel; ie, one strand runs in the 5′ to 3′ direction

deoxycytidylate, and thymidylate—is described in

and the other in the 3′ to 5′ direction. This is analogous

Chapter 33. These monomeric units of DNA are held

to two parallel streets, each running one way but carry-

in polymeric form by 3′,5′-phosphodiester bridges con-

ing traffic in opposite directions. In the double-

stituting a single strand, as depicted in Figure 35-1.

stranded DNA molecules, the genetic information re-

303

304

CHAPTER 35

5′

CH₂

NH₂

CH₂

H₃C

CH₂

NH₂

H H

CH₂

H H

3′

Figure 35-1.

A segment of one strand of a DNA molecule in which the purine and pyrimidine bases guanine

(G), cytosine (C), thymine (T), and adenine (A) are held together by a phosphodiester backbone between 2′-de-

oxyribosyl moieties attached to the nucleobases by an N-glycosidic bond. Note that the backbone has a polarity

(ie, a direction). Convention dictates that a single-stranded DNA sequence is written in the 5′ to 3′ direction (ie,

pGpCpTpA, where G, C, T, and A represent the four bases and p represents the interconnecting phosphates).

sides in the sequence of nucleotides on one strand, the

dine nucleotide, whereas the other pair, the A-T pair, is

template strand. This is the strand of DNA that is

held together by two hydrogen bonds. Thus, the G-C

copied during nucleic acid synthesis. It is sometimes re-

bonds are much more resistant to denaturation, or

ferred to as the noncoding strand. The opposite strand

“melting,” than A-T-rich regions.

is considered the coding strand because it matches the

RNA transcript that encodes the protein.

The Denaturation (Melting) of DNA

The two strands, in which opposing bases are held

Is Used to Analyze Its Structure

together by hydrogen bonds, wind around a central axis

in the form of a double helix. Double-stranded DNA

The double-stranded structure of DNA can be sepa-

exists in at least six forms (A-E and Z). The B form is

rated into two component strands (melted) in solution

usually found under physiologic conditions (low salt,

by increasing the temperature or decreasing the salt

high degree of hydration). A single turn of B-DNA

concentration. Not only do the two stacks of bases pull

about the axis of the molecule contains ten base pairs.

apart but the bases themselves unstack while still con-

The distance spanned by one turn of B-DNA is 3.4

nected in the polymer by the phosphodiester backbone.

nm. The width (helical diameter) of the double helix in

Concomitant with this denaturation of the DNA mole-

B-DNA is 2 nm.

cule is an increase in the optical absorbance of the

As depicted in Figure 35-3, three hydrogen bonds

purine and pyrimidine bases—a phenomenon referred

hold the deoxyguanosine nucleotide to the deoxycyti-

to as hyperchromicity of denaturation. Because of the

NUCLEIC ACID STRUCTURE & FUNCTION

305

CH₃

Thymidine

Minor groove

Adenosine

34 A

G C

Major groove

Cytosine

H Guanosine

20 A^o

Figure 35-3. Base pairing between deoxyadenosine

Figure 35-2.

A diagrammatic representation of the

and thymidine involves the formation of two hydrogen

Watson and Crick model of the double-helical structure

bonds. Three such bonds form between deoxycytidine

of the B form of DNA. The horizontal arrow indicates

and deoxyguanosine. The broken lines represent hy-

the width of the double helix (20 Å), and the vertical

drogen bonds.

arrow indicates the distance spanned by one complete

turn of the double helix (34 Å). One turn of B-DNA in-

cludes ten base pairs (bp), so the rise is 3.4 Å per bp.

or DNA-RNA hybrids to be separated at much lower

temperatures and minimizes the phosphodiester bond

The central axis of the double helix is indicated by the

breakage that occurs at high temperatures.

vertical rod. The short arrows designate the polarity of

the antiparallel strands. The major and minor grooves

are depicted. (A, adenine; C, cytosine; G, guanine;

Renaturation of DNA Requires

T, thymine; P, phosphate; S, sugar [deoxyribose].)

Base Pair Matching

Separated strands of DNA will renature or reassociate

when appropriate physiologic temperature and salt con-

stacking of the bases and the hydrogen bonding be-

ditions are achieved. The rate of reassociation depends

tween the stacks, the double-stranded DNA molecule

upon the concentration of the complementary strands.

exhibits properties of a rigid rod and in solution is a vis-

Reassociation of the two complementary DNA strands

cous material that loses its viscosity upon denaturation.

of a chromosome after DNA replication is a physiologic

The strands of a given molecule of DNA separate

example of renaturation (see below). At a given temper-

over a temperature range. The midpoint is called the

ature and salt concentration, a particular nucleic acid

melting temperature, or T_m. The T_m is influenced by

strand will associate tightly only with a complementary

the base composition of the DNA and by the salt con-

strand. Hybrid molecules will also form under appro-

centration of the solution. DNA rich in G-C pairs,

priate conditions. For example, DNA will form a hy-

which have three hydrogen bonds, melts at a higher tem-

brid with a complementary DNA (cDNA) or with a

perature than that rich in A-T pairs, which have two hy-

cognate messenger RNA (mRNA; see below). When

drogen bonds. A tenfold increase of monovalent cation

combined with gel electrophoresis techniques that sepa-

concentration increases the T_m by 16.6 °C. Formamide,

rate hybrid molecules by size and radioactive labeling to

which is commonly used in recombinant DNA experi-

provide a detectable signal, the resulting analytic tech-

ments, destabilizes hydrogen bonding between bases,

niques are called Southern (DNA/cDNA) and North-

thereby lowering the T_m. This allows the strands of DNA

ern blotting (DNA/RNA), respectively. These proce-

306

CHAPTER 35

dures allow for very specific identification of hybrids

the cell and organism, and it provides the information

from mixtures of DNA or RNA (see Chapter 40).

inherited by daughter cells or offspring. Both of these

functions require that the DNA molecule serve as a

There Are Grooves in the DNA Molecule

template—in the first case for the transcription of the

information into RNA and in the second case for the

Careful examination of the model depicted in Figure

replication of the information into daughter DNA mol-

35-2 reveals a major groove and a minor groove wind-

ecules.

ing along the molecule parallel to the phosphodiester

The complementarity of the Watson and Crick dou-

backbones. In these grooves, proteins can interact specif-

ble-stranded model of DNA strongly suggests that

ically with exposed atoms of the nucleotides (usually by

replication of the DNA molecule occurs in a semicon-

H bonding) and thereby recognize and bind to specific

servative manner. Thus, when each strand of the dou-

nucleotide sequences without disrupting the base pair-

ble-stranded parental DNA molecule separates from its

ing of the double-helical DNA molecule. As discussed in

complement during replication, each serves as a tem-

Chapters 37 and 39, regulatory proteins control the ex-

plate on which a new complementary strand is synthe-

pression of specific genes via such interactions.

sized (Figure 35-4). The two newly formed double-

stranded daughter DNA molecules, each containing

DNA Exists in Relaxed

one strand (but complementary rather than identical)

& Supercoiled Forms

from the parent double-stranded DNA molecule, are

then sorted between the two daughter cells (Figure

In some organisms such as bacteria, bacteriophages, and

35-5). Each daughter cell contains DNA molecules

many DNA-containing animal viruses, the ends of the

with information identical to that which the parent

DNA molecules are joined to create a closed circle with

possessed; yet in each daughter cell the DNA molecule

no covalently free ends. This of course does not destroy

of the parent cell has been only semiconserved.

the polarity of the molecules, but it eliminates all free 3′

and 5′ hydroxyl and phosphoryl groups. Closed circles

exist in relaxed or supercoiled forms. Supercoils are intro-

THE CHEMICAL NATURE OF RNA DIFFERS

duced when a closed circle is twisted around its own axis

FROM THAT OF DNA

or when a linear piece of duplex DNA, whose ends are

fixed, is twisted. This energy-requiring process puts the

Ribonucleic acid (RNA) is a polymer of purine and

molecule under stress, and the greater the number of su-

pyrimidine ribonucleotides linked together by

3′,5′-

percoils, the greater the stress or torsion (test this by

phosphodiester bridges analogous to those in DNA

twisting a rubber band). Negative supercoils are formed

(Figure 35-6). Although sharing many features with

when the molecule is twisted in the direction opposite

DNA, RNA possesses several specific differences:

from the clockwise turns of the right-handed double

helix found in B-DNA. Such DNA is said to be under-

(1) In RNA, the sugar moiety to which the phos-

wound. The energy required to achieve this state is, in a

phates and purine and pyrimidine bases are attached is

sense, stored in the supercoils. The transition to another

ribose rather than the 2′-deoxyribose of DNA.

form that requires energy is thereby facilitated by the un-

(2) The pyrimidine components of RNA differ from

derwinding. One such transition is strand separation,

those of DNA. Although RNA contains the ribonu-

which is a prerequisite for DNA replication and tran-

cleotides of adenine, guanine, and cytosine, it does not

scription. Supercoiled DNA is therefore a preferred form

possess thymine except in the rare case mentioned

in biologic systems. Enzymes that catalyze topologic

below. Instead of thymine, RNA contains the ribonu-

changes of DNA are called topoisomerases. Topoisom-

cleotide of uracil.

erases can relax or insert supercoils. The best-character-

(3) RNA exists as a single strand, whereas DNA ex-

ized example is bacterial gyrase, which induces negative

ists as a double-stranded helical molecule. However,

supercoiling in DNA using ATP as energy source. Ho-

given the proper complementary base sequence with

mologs of this enzyme exist in all organisms and are im-

opposite polarity, the single strand of RNA—as

portant targets for cancer chemotherapy.

demonstrated in Figure 35-7—is capable of folding

back on itself like a hairpin and thus acquiring double-

stranded characteristics.

DNA PROVIDES A TEMPLATE FOR

(4) Since the RNA molecule is a single strand com-

REPLICATION & TRANSCRIPTION

plementary to only one of the two strands of a gene, its

The genetic information stored in the nucleotide se-

guanine content does not necessarily equal its cytosine

quence of DNA serves two purposes. It is the source of

content, nor does its adenine content necessarily equal

information for the synthesis of all protein molecules of

its uracil content.

NUCLEIC ACID STRUCTURE & FUNCTION

307

OLD

5′

3′

Original

parent molecule

First-generation

daughter molecules

3′

5′

Second-generation

daughter molecules

G C

Figure 35-5. DNA replication is semiconservative.

During a round of replication, each of the two strands

of DNA is used as a template for synthesis of a new,

complementary strand.

3′

5′

3′

5′

OLD

NEW

OLD

Figure 35-4.

The double-stranded structure of DNA

and the template function of each old strand (dark

complementarity, an RNA molecule can bind specifi-

shading) on which a new (light shading) complemen-

cally via the base-pairing rules to its template DNA

tary strand is synthesized.

strand; it will not bind (“hybridize”) with the other

(coding) strand of its gene. The sequence of the RNA

molecule (except for U replacing T) is the same as that

of the coding strand of the gene (Figure 35-8).

(5) RNA can be hydrolyzed by alkali to 2′,3′ cyclic

diesters of the mononucleotides, compounds that can-

Nearly All of the Several Species of RNA

not be formed from alkali-treated DNA because of the

Are Involved in Some Aspect of Protein

absence of a 2′-hydroxyl group. The alkali lability of

Synthesis

RNA is useful both diagnostically and analytically.

Those cytoplasmic RNA molecules that serve as tem-

Information within the single strand of RNA is con-

plates for protein synthesis (ie, that transfer genetic in-

tained in its sequence (“primary structure”) of purine

formation from DNA to the protein-synthesizing ma-

and pyrimidine nucleotides within the polymer. The

chinery) are designated messenger RNAs, or mRNAs.

sequence is complementary to the template strand of

Many other cytoplasmic RNA molecules (ribosomal

the gene from which it was transcribed. Because of this

RNAs; rRNAs) have structural roles wherein they con-

308

CHAPTER 35

5′

NH₂

CH₂

NH₂

CH₂

H H

NH₂

H H

CH₂

H H

3′

Figure 35-6.

A segment of a ribonucleic acid (RNA) molecule in which the purine and pyrimidine bases—

guanine (G), cytosine (C), uracil (U), and adenine (A)—are held together by phosphodiester bonds between ribo-

syl moieties attached to the nucleobases by N-glycosidic bonds. Note that the polymer has a polarity as indi-

cated by the labeled 3′- and 5′-attached phosphates.

tribute to the formation and function of ribosomes (the

The genetic material for some animal and plant

organellar machinery for protein synthesis) or serve as

viruses is RNA rather than DNA. Although some RNA

adapter molecules

(transfer RNAs; tRNAs) for the

viruses never have their information transcribed into a

translation of RNA information into specific sequences

DNA molecule, many animal RNA viruses—specifi-

of polymerized amino acids.

cally, the retroviruses (the HIV virus, for example)—are

Some RNA molecules have intrinsic catalytic activ-

transcribed by an RNA-dependent DNA polymerase,

ity. The activity of these ribozymes often involves the

the so-called reverse transcriptase, to produce a dou-

cleavage of a nucleic acid. An example is the role of

ble-stranded DNA copy of their RNA genome. In

RNA in catalyzing the processing of the primary tran-

many cases, the resulting double-stranded DNA tran-

script of a gene into mature messenger RNA.

script is integrated into the host genome and subse-

Much of the RNA synthesized from DNA templates

quently serves as a template for gene expression and

in eukaryotic cells, including mammalian cells, is de-

from which new viral RNA genomes can be tran-

graded within the nucleus, and it never serves as either a

scribed.

structural or an informational entity within the cellular

cytoplasm.

RNA Is Organized in Several

In all eukaryotic cells there are small nuclear RNA

Unique Structures

(snRNA) species that are not directly involved in pro-

tein synthesis but play pivotal roles in RNA processing.

In all prokaryotic and eukaryotic organisms, three main

These relatively small molecules vary in size from 90 to

classes of RNA molecules exist: messenger RNA

about 300 nucleotides (Table 35-1).

(mRNA), transfer RNA (tRNA), and ribosomal RNA

NUCLEIC ACID STRUCTURE & FUNCTION

309

Table 35-1. Some of the species of small stable

RNAs found in mammalian cells.

Loop

Length

Molecules

Name

(nucleotides)

per Cell

Localization

165

1 × 10⁶

Nucleoplasm/hnRNA

188

5 × 10⁵

Nucleoplasm

216

3 × 10⁵

Nucleolus

139

1 × 10⁵

Nucleoplasm

118

2 × 10⁵

Nucleoplasm

106

3 × 10⁵

Perichromatin granules

Stem

4.5S

91-95

3 x 10⁵

Nucleus and cytoplasm

280

5 × 10⁵

Nucleus and cytoplasm

7-2

290

1 × 10⁵

Nucleus and cytoplasm

7-3

300

2 × 10⁵

Nucleus

Messenger RNAs, particularly in eukaryotes, have

some unique chemical characteristics. The 5′ terminal

of mRNA is “capped” by a 7-methylguanosine triphos-

phate that is linked to an adjacent 2′-O-methyl ribonu-

cleoside at its 5′-hydroxyl through the three phosphates

5′

3′

(Figure 35-10). The mRNA molecules frequently con-

tain internal 6-methyladenylates and other 2′-O-ribose

Figure 35-7.

Diagrammatic representation of the

methylated nucleotides. The cap is involved in the

secondary structure of a single-stranded RNA molecule

recognition of mRNA by the translating machinery,

in which a stem loop, or “hairpin,” has been formed and

and it probably helps stabilize the mRNA by preventing

is dependent upon the intramolecular base pairing.

the attack of 5′-exonucleases. The protein-synthesizing

Note that A forms hydrogen bonds with U in RNA.

machinery begins translating the mRNA into proteins

beginning downstream of the 5′ or capped terminal.

The other end of most mRNA molecules, the 3′-hy-

(rRNA). Each differs from the others by size, function,

droxyl terminal, has an attached polymer of adenylate

and general stability.

residues

20-250 nucleotides in length. The specific

function of the poly(A) “tail” at the 3′-hydroxyl termi-

nal of mRNAs is not fully understood, but it seems that

A. MESSENGER RNA (MRNA)

it maintains the intracellular stability of the specific

This class is the most heterogeneous in size and stabil-

mRNA by preventing the attack of 3′-exonucleases.

ity. All members of the class function as messengers

Some mRNAs, including those for some histones, do

conveying the information in a gene to the protein-

not contain poly(A). The poly(A) tail, because it will

synthesizing machinery, where each serves as a template

form a base pair with oligodeoxythymidine polymers

on which a specific sequence of amino acids is polymer-

attached to a solid substrate like cellulose, can be used

ized to form a specific protein molecule, the ultimate

to separate mRNA from other species of RNA, includ-

gene product (Figure 35-9).

ing mRNA molecules that lack this tail.

DNA strands:

Coding

5′ —

TGGAAT TGTGAGCGGATAACAAT T TCACACAGGAAACAGCTATGACCATG

— 3′

Template

3′ —

ACCT TAACACTCGCCTAT TGT TAAAGTGTGTCCT T TGTCGATACTGGTAC

— 5′

RNA

5′

p AUUGUGAGCGGAUAACAAUUUCACACAGGAAACAGCUAUGACCAUG

3′

transcript

Figure 35-8. The relationship between the sequences of an RNA transcript and its gene, in which the cod-

ing and template strands are shown with their polarities. The RNA transcript with a 5′ to 3′ polarity is comple-

mentary to the template strand with its 3′ to 5′ polarity. Note that the sequence in the RNA transcript and its

polarity is the same as that in the coding strand, except that the U of the transcript replaces the T of the gene.

310

CHAPTER 35

DNA

5′

3′

5′

mRNA

5′

3′

Protein synthesis on mRNA template

3′

5′

Figure 35-9. The expression of genetic in-

Completed

Ribosome

protein

formation in DNA into the form of an mRNA

molecule

transcript. This is subsequently translated by

ribosomes into a specific protein molecule.

In mammalian cells, including cells of humans, the

The D, T C, and extra arms help define a specific

mRNA molecules present in the cytoplasm are not the

tRNA.

RNA products immediately synthesized from the DNA

Although tRNAs are quite stable in prokaryotes, they

template but must be formed by processing from a pre-

are somewhat less stable in eukaryotes. The opposite is

cursor molecule before entering the cytoplasm. Thus,

true for mRNAs, which are quite unstable in prokary-

in mammalian nuclei, the immediate products of gene

otes but generally stable in eukaryotic organisms.

transcription constitute a fourth class of RNA mole-

C. RIBOSOMAL RNA (RRNA)

cules. These nuclear RNA molecules are very heteroge-

neous in size and are quite large. The heterogeneous

A ribosome is a cytoplasmic nucleoprotein structure

nuclear RNA (hnRNA) molecules may have a molecu-

that acts as the machinery for the synthesis of proteins

lar weight in excess of

10⁷, whereas the molecular

from the mRNA templates. On the ribosomes, the

weight of mRNA molecules is generally less than 2 ×

mRNA and tRNA molecules interact to translate into a

10⁶. As discussed in Chapter 37, hnRNA molecules are

specific protein molecule information transcribed from

processed to generate the mRNA molecules which then

the gene. In active protein synthesis, many ribosomes

enter the cytoplasm to serve as templates for protein

are associated with an mRNA molecule in an assembly

synthesis.

called the polysome.

The components of the mammalian ribosome,

B. TRANSFER RNA (TRNA)

which has a molecular weight of about 4.2 × 10⁶ and a

tRNA molecules vary in length from 74 to 95 nu-

sedimentation velocity of

80S (Svedberg units), are

cleotides. They also are generated by nuclear processing

shown in Table 35-2. The mammalian ribosome con-

of a precursor molecule (Chapter 37). The tRNA mole-

tains two major nucleoprotein subunits—a larger one

cules serve as adapters for the translation of the infor-

with a molecular weight of 2.8 × 10⁶ (60S) and a

mation in the sequence of nucleotides of the mRNA

smaller subunit with a molecular weight of 1.4 × 10⁶

into specific amino acids. There are at least 20 species

(40S). The 60S subunit contains a 5S ribosomal RNA

of tRNA molecules in every cell, at least one (and often

(rRNA), a 5.8S rRNA, and a 28S rRNA; there are also

several) corresponding to each of the 20 amino acids re-

probably more than 50 specific polypeptides. The 40S

quired for protein synthesis. Although each specific

subunit is smaller and contains a single 18S rRNA and

tRNA differs from the others in its sequence of nu-

approximately 30 distinct polypeptide chains. All of the

cleotides, the tRNA molecules as a class have many fea-

ribosomal RNA molecules except the 5S rRNA are

tures in common. The primary structure—ie, the nu-

processed from a single 45S precursor RNA molecule in

cleotide sequence—of all tRNA molecules allows

the nucleolus (Chapter 37). 5S rRNA is independently

extensive folding and intrastrand complementarity to

transcribed. The highly methylated ribosomal RNA

generate a secondary structure that appears like a

molecules are packaged in the nucleolus with the spe-

cloverleaf (Figure 35-11).

cific ribosomal proteins. In the cytoplasm, the ribo-

All tRNA molecules contain four main arms. The

somes remain quite stable and capable of many transla-

acceptor arm terminates in the nucleotides CpCpAOH.

tion cycles. The functions of the ribosomal RNA

These three nucleotides are added posttranscription-

molecules in the ribosomal particle are not fully under-

ally. The tRNA-appropriate amino acid is attached to

stood, but they are necessary for ribosomal assembly

the 3′-OH group of the A moiety of the acceptor arm.

and seem to play key roles in the binding of mRNA to

NUCLEIC ACID STRUCTURE & FUNCTION

311

OH OH

C C

H H

H₂N

H₂C

5′

O^-

NH₂

O^-

5′

CH₃

O^-

CAP

H H

2′

3′

C C

OCH₃

5′

CH₂

mRNA

O^-

H H

3′ C

O^-

Figure 35-10.

The cap structure attached to the 5′ terminal of most eukaryotic messen-

ger RNA molecules. A 7-methylguanosine triphosphate (black) is attached at the 5′ terminal

of the mRNA (shown in blue), which usually contains a 2′-O-methylpurine nucleotide.

These modifications (the cap and methyl group) are added after the mRNA is transcribed

from DNA.

ribosomes

and its translation. Recent studies suggest

size from 90 to 300 nucleotides and are

present

that an rRNA component performs the peptidyl trans-

100,000-1,000,000 copies per cell.

ferase activity and thus is an enzyme (a ribozyme).

Small nuclear RNAs (snRNAs), a subset of these

RNAs, are significantly involved in mRNA processing

and gene regulation. Of the several snRNAs, U1, U2,

D. SMALL STABLE RNA

U4, U5, and U6 are involved in intron removal and the

A large number of discrete, highly conserved, and small

processing of hnRNA into mRNA (Chapter 37). The

stable RNA species are found in eukaryotic cells. The

U7 snRNA may be involved in production of the cor-

majority of these molecules are complexed with pro-

rect 3′ ends of histone mRNA—which lacks a poly(A)

teins to form ribonucleoproteins and are distributed in

tail. The U4 and U6 snRNAs may also be required for

the nucleus, in the cytoplasm, or in both. They range in

poly(A) processing.

312

CHAPTER 35

SPECIFIC NUCLEASES DIGEST

NUCLEIC ACIDS

Enzymes capable of degrading nucleic acids have been

3′

recognized for many years. These nucleases can be clas-

Acceptor

sified in several ways. Those which exhibit specificity

arm

for deoxyribonucleic acid are referred to as deoxyri-

bonucleases. Those which specifically hydrolyze ri-

5′P

bonucleic acids are ribonucleases. Within both of

these classes are enzymes capable of cleaving internal

Region of hydrogen

phosphodiester bonds to produce either 3′-hydroxyl

bonding between

base pairs

and 5′-phosphoryl terminals or

5′-hydroxyl and

3′-

phosphoryl terminals. These are referred to as endonu-

TψC arm

cleases. Some are capable of hydrolyzing both strands

of a double-stranded molecule, whereas others can

only cleave single strands of nucleic acids. Some nucle-

ases can hydrolyze only unpaired single strands, while

others are capable of hydrolyzing single strands partici-

pating in the formation of a double-stranded molecule.

D arm

There exist classes of endonucleases that recognize spe-

Extra arm

cific sequences in DNA; the majority of these are the

restriction endonucleases, which have in recent years

become important tools in molecular genetics and med-

Alkylated purine

ical sciences. A list of some currently recognized restric-

tion endonucleases is presented in Table 40-2.

Anticodon arm

Some nucleases are capable of hydrolyzing a nu-

cleotide only when it is present at a terminal of a mole-

Figure 35-11. Typical aminoacyl tRNA in which the

cule; these are referred to as exonucleases. Exonucle-

amino acid (aa) is attached to the 3′ CCA terminal. The

ases act in one direction (3′ → 5′ or 5′ → 3′) only. In

anticodon, TΨC, and dihydrouracil (D) arms are indi-

bacteria, a 3′ → 5′ exonuclease is an integral part of the

cated, as are the positions of the intramolecular hydro-

DNA replication machinery and there serves to edit—

gen bonding between these base pairs. (From Watson

or proofread—the most recently added deoxynucleo-

tide for base-pairing errors.

1976, 1970, 1965, by W.A. Benjamin, Inc., Menlo Park, Cali-

fornia.)

Table 35-2. Components of mammalian ribosomes.¹

Mass

Protein

RNA

Component

(mw)

Number Mass

Size Mass Bases

40S subunit

1.4 × 10⁶

~35

7 × 10⁵

18S

7 × 10⁵

1900

60S subunit

2.8 × 10⁶

~50

1 × 10⁶

35,000

120

5.8S

45,000

160

28S

1.6 × 10⁶

4700

¹The ribosomal subunits are defined according to their sedimentation ve-

locity in Svedberg units (40S or 60S). This table illustrates the total mass

(MW) of each. The number of unique proteins and their total mass (MW) and

the RNA components of each subunit in size (Svedberg units), mass, and

number of bases are listed.

NUCLEIC ACID STRUCTURE & FUNCTION

313

SUMMARY

sis. The linear array of nucleotides in RNA consists

of A, G, C, and U, and the sugar moiety is ribose.

• DNA consists of four bases—A, G, C, and T—

• The major forms of RNA include messenger RNA

which are held in linear array by phosphodiester

(mRNA), ribosomal RNA

(rRNA), and transfer

bonds through the 3′ and 5′ positions of adjacent de-

RNA (tRNA). Certain RNA molecules act as cata-

oxyribose moieties.

lysts (ribozymes).

• DNA is organized into two strands by the pairing of

bases A to T and G to C on complementary strands.

These strands form a double helix around a central

axis.

REFERENCES

• The 3 × 10⁹ base pairs of DNA in humans are orga-

Green R, Noller HF: Ribosomes and translation. Annu Rev Bio-

nized into the haploid complement of 23 chromo-

chem 1997;66:689.

somes. The exact sequence of these 3 billion nu-

Guthrie C, Patterson B: Spliceosomal snRNAs. Ann Rev Genet

cleotides defines the uniqueness of each individual.

1988;22:387.

• DNA provides a template for its own replication and

Hunt T: DNA Makes RNA Makes Protein. Elsevier, 1983.

thus maintenance of the genotype and for the tran-

Watson JD, Crick FHC: Molecular structure of nucleic acids. Na-

scription of the 30,000-50,000 genes into a variety

ture 1953;171:737.

of RNA molecules.

Watson JD: The Double Helix. Atheneum, 1968.

• RNA exists in several different single-stranded struc-

Watson JD et al: Molecular Biology of the Gene, 5th ed. Benjamin-

tures, most of which are involved in protein synthe-

Cummings, 2000.

DNA Organization, Replication,

& Repair

Daryl K. Granner, MD, & P. Anthony Weil, PhD

BIOMEDICAL IMPORTANCE*

larger than histones) and a small quantity of RNA. The

nonhistone proteins include enzymes involved in DNA

The genetic information in the DNA of a chromosome

replication, such as DNA topoisomerases. Also in-

can be transmitted by exact replication or it can be ex-

cluded are proteins involved in transcription, such as

changed by a number of processes, including crossing

the RNA polymerase complex. The double-stranded

over, recombination, transposition, and conversion.

DNA helix in each chromosome has a length that is

These provide a means of ensuring adaptability and di-

thousands of times the diameter of the cell nucleus.

versity for the organism but, when these processes go

One purpose of the molecules that comprise chro-

awry, can also result in disease. A number of enzyme

matin, particularly the histones, is to condense the

systems are involved in DNA replication, alteration,

DNA. Electron microscopic studies of chromatin have

and repair. Mutations are due to a change in the base

demonstrated dense spherical particles called nucleo-

sequence of DNA and may result from the faulty repli-

somes, which are approximately 10 nm in diameter

cation, movement, or repair of DNA and occur with a

and connected by DNA filaments (Figure 36-1). Nu-

frequency of about one in every 10⁶ cell divisions. Ab-

cleosomes are composed of DNA wound around a col-

normalities in gene products (either in protein function

lection of histone molecules.

or amount) can be the result of mutations that occur in

coding or regulatory-region DNA. A mutation in a

Histones Are the Most Abundant

germ cell will be transmitted to offspring (so-called ver-

Chromatin Proteins

tical transmission of hereditary disease). A number of

factors, including viruses, chemicals, ultraviolet light,

The histones are a small family of closely related basic

and ionizing radiation, increase the rate of mutation.

proteins. H1 histones are the ones least tightly bound

Mutations often affect somatic cells and so are passed

to chromatin Figure 36-1) and are, therefore, easily re-

on to successive generations of cells, but only within an

moved with a salt solution, after which chromatin be-

organism. It is becoming apparent that a number of

comes soluble. The organizational unit of this soluble

diseases—and perhaps most cancers—are due to the

chromatin is the nucleosome. Nucleosomes contain

combined effects of vertical transmission of mutations

four classes of histones: H2A, H2B, H3, and H4. The

as well as horizontal transmission of induced mutations.

structures of all four histones—H2A, H2B, H3, and

H4, the so-called core histones forming the nucleo-

CHROMATIN IS THE CHROMOSOMAL

some—have been highly conserved between species.

This extreme conservation implies that the function of

MATERIAL EXTRACTED FROM NUCLEI

histones is identical in all eukaryotes and that the entire

OF CELLS OF EUKARYOTIC ORGANISMS

molecule is involved quite specifically in carrying out

Chromatin consists of very long double-stranded DNA

this function. The carboxyl terminal two-thirds of the

molecules and a nearly equal mass of rather small basic

molecules have a typical random amino acid composi-

proteins termed histones as well as a smaller amount of

tion, while their amino terminal thirds are particularly

nonhistone proteins (most of which are acidic and

rich in basic amino acids. These four core histones are

subject to at least five types of covalent modifica-

tion: acetylation, methylation, phosphorylation, ADP-

ribosylation, and covalent linkage (H2A only) to ubiq-

*So far as is possible, the discussion in this chapter and in Chapters

uitin. These histone modifications probably play an

37, 38, and 39 will pertain to mammalian organisms, which are, of

important role in chromatin structure and function as

course, among the higher eukaryotes. At times it will be necessary

illustrated in Table 36-1.

to refer to observations in prokaryotic organisms such as bacteria

and viruses, but in such cases the information will be of a kind that

The histones interact with each other in very specific

can be extrapolated to mammalian organisms.

ways. H3 and H4 form a tetramer containing two mol-

314

DNA ORGANIZATION, REPLICATION, & REPAIR

315

In the nucleosome, the DNA is supercoiled in a left-

handed helix over the surface of the disk-shaped histone

octamer (Figure 36-2). The majority of core histone

proteins interact with the DNA on the inside of the su-

percoil without protruding, though the amino terminal

tails of all the histones probably protrude outside of this

structure and are available for regulatory covalent mod-

ifications (see Table 36-1).

The (H3/H4)₂ tetramer itself can confer nucleo-

some-like properties on DNA and thus has a central

role in the formation of the nucleosome. The addition

of two H2A-H2B dimers stabilizes the primary particle

and firmly binds two additional half-turns of DNA pre-

viously bound only loosely to the (H3/H4)₂. Thus,

1.75 superhelical turns of DNA are wrapped around

the surface of the histone octamer, protecting 146 base

pairs of DNA and forming the nucleosome core particle

Figure 36-1. Electron micrograph of nucleosomes

(Figure 36-2). The core particles are separated by an

attached by strands of nucleic acid. (The bar represents

about 30-bp linker region of DNA. Most of the DNA

2.5 µm.) (Reproduced, with permission, from Oudet P,

is in a repeating series of these structures, giving the so-

Gross-Bellard M, Chambon P: Electron microscopic and

called “beads-on-a-string” appearance when examined

biochemical evidence that chromatin structure is a re-

by electron microscopy (see Figure 36-1).

peating unit. Cell 1975;4:281.)

The assembly of nucleosomes is mediated by one of

several chromatin assembly factors facilitated by histone

chaperones, proteins such as the anionic nuclear protein

ecules of each (H3/H4)₂, while H2A and H2B form

nucleoplasmin. As the nucleosome is assembled, his-

dimers

(H2A-H2B). Under physiologic conditions,

tones are released from the histone chaperones. Nucleo-

these histone oligomers associate to form the histone oc-

somes appear to exhibit preference for certain regions on

tamer of the composition (H3/H4)₂-(H2A-H2B)₂.

specific DNA molecules, but the basis for this nonran-

dom distribution, termed phasing, is not completely

The Nucleosome Contains Histone & DNA

When the histone octamer is mixed with purified, dou-

ble-stranded DNA, the same x-ray diffraction pattern is

formed as that observed in freshly isolated chromatin.

Electron microscopic studies confirm the existence of

Histone octamer

reconstituted nucleosomes. Furthermore, the reconsti-

tution of nucleosomes from DNA and histones H2A,

H2B, H3, and H4 is independent of the organismal or

cellular origin of the various components. The histone

H1 and the nonhistone proteins are not necessary for

the reconstitution of the nucleosome core.

Histone

DNA

Table 36-1. Possible roles of modified histones.

Figure 36-2. Model for the structure of the nucleo-

1. Acetylation of histones H3 and H4 is associated with the ac-

some, in which DNA is wrapped around the surface of a

tivation or inactivation of gene transcription (Chapter 37).

flat protein cylinder consisting of two each of histones

2. Acetylation of core histones is associated with chromoso-

H2A, H2B, H3, and H4 that form the histone octamer.

mal assembly during DNA replication.

3. Phosphorylation of histone H1 is associated with the con-

The 146 base pairs of DNA, consisting of 1.75 superheli-

densation of chromosomes during the replication cycle.

cal turns, are in contact with the histone octamer. This

4. ADP-ribosylation of histones is associated with DNA repair.

protects the DNA from digestion by a nuclease. The po-

5. Methylation of histones is correlated with activation and

sition of histone H1, when it is present, is indicated by

repression of gene transcription.

the dashed outline at the bottom of the figure.

316

CHAPTER 36

understood. It is probably related to the relative physical

tion by a nuclease such as DNase I. DNase I makes sin-

flexibility of certain nucleotide sequences that are able to

gle-strand cuts in any segment of DNA (no sequence

accommodate the regions of kinking within the super-

specificity). It will digest DNA not protected by protein

coil as well as the presence of other DNA-bound factors

into its component deoxynucleotides. The sensitivity to

that limit the sites of nucleosome deposition.

DNase I of chromatin regions being actively transcribed

The super-packing of nucleosomes in nuclei is seem-

reflects only a potential for transcription rather than

ingly dependent upon the interaction of the H1 his-

transcription itself and in several systems can be corre-

tones with adjacent nucleosomes.

lated with a relative lack of 5-methyldeoxycytidine in the

DNA and particular histone covalent modifications

(phosphorylation, acetylation, etc; see Table 36-1).

HIGHER-ORDER STRUCTURES PROVIDE

Within the large regions of active chromatin there

FOR THE COMPACTION OF CHROMATIN

exist shorter stretches of 100-300 nucleotides that ex-

Electron microscopy of chromatin reveals two higher

hibit an even greater

(another tenfold) sensitivity to

orders of structure—the 10-nm fibril and the 30-nm

DNase I. These hypersensitive sites probably result

chromatin fiber—beyond that of the nucleosome itself.

from a structural conformation that favors access of the

The disk-like nucleosome structure has a 10-nm diame-

nuclease to the DNA. These regions are often located

ter and a height of 5 nm. The 10-nm fibril consists of

immediately upstream from the active gene and are the

nucleosomes arranged with their edges separated by a

location of interrupted nucleosomal structure caused by

small distance (30 bp of DNA) with their flat faces par-

the binding of nonhistone regulatory transcription factor

allel with the fibril axis (Figure 36-3). The 10-nm fibril

proteins. (See Chapters 37 and 39.) In many cases, it

is probably further supercoiled with six or seven nucleo-

seems that if a gene is capable of being transcribed, it

somes per turn to form the 30-nm chromatin fiber

very often has a DNase-hypersensitive site(s) in the chro-

(Figure 36-3). Each turn of the supercoil is relatively

matin immediately upstream. As noted above, nonhis-

flat, and the faces of the nucleosomes of successive

tone regulatory proteins involved in transcription control

turns would be nearly parallel to each other. H1 his-

and those involved in maintaining access to the template

tones appear to stabilize the 30-nm fiber, but their posi-

strand lead to the formation of hypersensitive sites. Hy-

tion and that of the variable length spacer DNA are not

persensitive sites often provide the first clue about the

clear. It is probable that nucleosomes can form a variety

presence and location of a transcription control element.

of packed structures. In order to form a mitotic chro-

Transcriptionally inactive chromatin is densely

mosome, the 30-nm fiber must be compacted in length

packed during interphase as observed by electron mi-

another 100-fold (see below).

croscopic studies and is referred to as heterochro-

In interphase chromosomes, chromatin fibers ap-

matin; transcriptionally active chromatin stains less

pear to be organized into 30,000-100,000 bp loops or

densely and is referred to as euchromatin. Generally,

domains anchored in a scaffolding (or supporting ma-

euchromatin is replicated earlier than heterochromatin

trix) within the nucleus. Within these domains, some

in the mammalian cell cycle (see below).

DNA sequences may be located nonrandomly. It has

There are two types of heterochromatin: constitutive

been suggested that each looped domain of chromatin

and facultative. Constitutive heterochromatin is al-

corresponds to one or more separate genetic functions,

ways condensed and thus inactive. It is found in the

containing both coding and noncoding regions of the

regions near the chromosomal centromere and at chro-

cognate gene or genes.

mosomal ends (telomeres). Facultative heterochro-

matin is at times condensed, but at other times it is ac-

tively transcribed and, thus, uncondensed and appears

SOME REGIONS OF CHROMATIN ARE

as euchromatin. Of the two members of the X chromo-

“ACTIVE” & OTHERS ARE “INACTIVE”

some pair in mammalian females, one X chromosome is

Generally, every cell of an individual metazoan organism

almost completely inactive transcriptionally and is hete-

contains the same genetic information. Thus, the differ-

rochromatic. However, the heterochromatic X chromo-

ences between cell types within an organism must be ex-

some decondenses during gametogenesis and becomes

plained by differential expression of the common genetic

transcriptionally active during early embryogenesis—

information. Chromatin containing active genes

(ie,

thus, it is facultative heterochromatin.

transcriptionally active chromatin) has been shown to

Certain cells of insects, eg, Chironomus, contain

differ in several ways from that of inactive regions. The

giant chromosomes that have been replicated for ten

nucleosome structure of active chromatin appears to be

cycles without separation of daughter chromatids.

altered, sometimes quite extensively, in highly active re-

These copies of DNA line up side by side in precise reg-

gions. DNA in active chromatin contains large regions

ister and produce a banded chromosome containing re-

(about 100,000 bases long) that are sensitive to diges-

gions of condensed chromatin and lighter bands of

318

CHAPTER 36

more extended chromatin. Transcriptionally active re-

sition of which is characteristic for a given chromosome

gions of these polytene chromosomes are especially

(Figure 36-5). The centromere is an adenine-thymine

decondensed into “puffs” that can be shown to contain

(A-T) rich region ranging in size from 10² (brewers’

the enzymes responsible for transcription and to be the

yeast) to 10⁶ (mammals) base pairs. It binds several pro-

sites of RNA synthesis (Figure 36-4).

teins with high affinity. This complex, called the kine-

tochore, provides the anchor for the mitotic spindle. It

thus is an essential structure for chromosomal segrega-

DNA IS ORGANIZED

tion during mitosis.

INTO CHROMOSOMES

The ends of each chromosome contain structures

At metaphase, mammalian chromosomes possess a

called telomeres. Telomeres consist of short, repeat

twofold symmetry, with the identical duplicated sister

TG-rich sequences. Human telomeres have a variable

chromatids connected at a centromere, the relative po-

number of repeats of the sequence 5′-TTAGGG-3′,

which can extend for several kilobases. Telomerase, a

multisubunit RNA-containing complex related to viral

RNA-dependent DNA polymerases (reverse transcrip-

tases), is the enzyme responsible for telomere synthesis

and thus for maintaining the length of the telomere.

Since telomere shortening has been associated with

both malignant transformation and aging, telomerase

has become an attractive target for cancer chemother-

apy and drug development. Each sister chromatid con-

tains one double-stranded DNA molecule. During in-

terphase, the packing of the DNA molecule is less dense

than it is in the condensed chromosome during

metaphase. Metaphase chromosomes are nearly com-

pletely transcriptionally inactive.

The human haploid genome consists of about

3 × 10⁹ bp and about 1.7 × 10⁷ nucleosomes. Thus, each

of the 23 chromatids in the human haploid genome

would contain on the average 1.3 × 10⁸ nucleotides in

one double-stranded DNA molecule. The length of

each DNA molecule must be compressed about 8000-

fold to generate the structure of a condensed metaphase

chromosome! In metaphase chromosomes, the 30-nm

BR3

chromatin fibers are also folded into a series of looped

domains, the proximal portions of which are anchored

to a nonhistone proteinaceous scaffolding within the

nucleus (Figure 36-3). The packing ratios of each of

Figure 36-4.

Illustration of the tight correlation be-

the orders of DNA structure are summarized in Table

tween the presence of RNA polymerase II and RNA syn-

36-2.

thesis. A number of genes are activated when Chirono-

The packaging of nucleoproteins within chromatids

mus tentans larvae are subjected to heat shock (39 °C

is not random, as evidenced by the characteristic pat-

for 30 minutes). A: Distribution of RNA polymerase II

terns observed when chromosomes are stained with spe-

(also called type B) in isolated chromosome IV from the

cific dyes such as quinacrine or Giemsa’s stain (Figure

salivary gland (at arrows). The enzyme was detected by

36-6).

immunofluorescence using an antibody directed

From individual to individual within a single

against the polymerase. The 5C and BR3 are specific

species, the pattern of staining (banding) of the entire

bands of chromosome IV, and the arrows indicate puffs.

chromosome complement is highly reproducible; none-

B: Autoradiogram of a chromosome IV that was incu-

theless, it differs significantly from other species, even

bated in ³H-uridine to label the RNA. Note the corre-

those closely related. Thus, the packaging of the nucleo-

spondence of the immunofluorescence and presence

proteins in chromosomes of higher eukaryotes must in

of the radioactive RNA (black dots). Bar = 7 µm. (Repro-

some way be dependent upon species-specific character-

duced, with permission, from Sass H: RNA polymerase B in

istics of the DNA molecules.

A combination of specialized staining techniques

1982 by the Massachusetts Institute of Technology.)

and high-resolution microscopy has allowed geneticists

DNA ORGANIZATION, REPLICATION, & REPAIR

319

Sister chromatid No. 2

Sister chromatid No. 1

Centromere

Figure 36-5. The two sister chromatids of

human chromosome 12 (× 27,850). The location

of the A+T-rich centromeric region connecting

sister chromatids is indicated, as are two of the

four telomeres residing at the very ends of the

chromatids that are attached one to the other at

the centromere. (Modified and reproduced, with

Telomeres

permission, from DuPraw EJ: DNA and Chromo-

(TTAGG)

somes. Holt, Rinehart, and Winston, 1970.)

to quite precisely map thousands of genes to specific re-

nonprotein coding DNA. Accordingly, the primary

gions of mouse and human chromosomes. With the re-

transcripts of DNA

(mRNA precursors, originally

cent elucidation of the human and mouse genome se-

termed hnRNA because this species of RNA was quite

quences, it has become clear that many of these visual

heterogeneous in size [length] and mostly restricted to

mapping methods were remarkably accurate.

the nucleus), contain noncoding intervening sequences

of RNA that must be removed in a process which also

joins together the appropriate coding segments to form

Coding Regions Are Often Interrupted

the mature mRNA. Most coding sequences for a single

by Intervening Sequences

mRNA are interrupted in the genome (and thus in the

primary transcript) by at least one—and in some cases

The protein coding regions of DNA, the transcripts

as many as 50—noncoding intervening sequences (in-

of which ultimately appear in the cytoplasm as single

trons). In most cases, the introns are much longer than

mRNA molecules, are usually interrupted in the eu-

the continuous coding regions (exons). The processing

karyotic genome by large intervening sequences of

of the primary transcript, which involves removal of in-

trons and splicing of adjacent exons, is described in de-

tail in Chapter 37.

The function of the intervening sequences, or in-

Table 36-2. The packing ratios of each of the

trons, is not clear. They may serve to separate func-

orders of DNA structure.

tional domains (exons) of coding information in a form

that permits genetic rearrangement by recombination

to occur more rapidly than if all coding regions for a

Chromatin Form

Packing Ratio

given genetic function were contiguous. Such an en-

Naked double-helical DNA

~1.0

hanced rate of genetic rearrangement of functional do-

10-nm fibril of nucleosomes

7-10

mains might allow more rapid evolution of biologic

25- to 30-nm chromatin fiber of superheli-

40-60

function. The relationships among chromosomal

cal nucleosomes

DNA, gene clusters on the chromosome, the exon-

Condensed metaphase chromosome of

8000

intron structure of genes, and the final mRNA product

loops

are illustrated in Figure 36-7.

320

CHAPTER 36

Figure 36-6. A human karyotype (of a man with a normal 46,XY constitution), in

which the metaphase chromosomes have been stained by the Giemsa method and

aligned according to the Paris Convention. (Courtesy of H Lawce and F Conte.)

MUCH OF THE MAMMALIAN GENOME

The DNA in a eukaryotic genome can be divided

into different

“sequence classes.” These are unique-

IS REDUNDANT & MUCH IS

sequence, or nonrepetitive, DNA and repetitive-

NOT TRANSCRIBED

sequence DNA. In the haploid genome, unique-se-

The haploid genome of each human cell consists of

quence DNA generally includes the single copy genes

3 × 10⁹ base pairs of DNA subdivided into 23 chromo-

that code for proteins. The repetitive DNA in the hap-

somes. The entire haploid genome contains sufficient

loid genome includes sequences that vary in copy num-

DNA to code for nearly 1.5 million average-sized

ber from two to as many as 10⁷ copies per cell.

genes. However, studies of mutation rates and of the

complexities of the genomes of higher organisms

strongly suggest that humans have < 100,000 proteins

More Than Half the DNA in Eukaryotic

encoded by the ~1.1% of the human genome that is

Organisms Is in Unique or

composed of exonic DNA. This implies that most of

Nonrepetitive Sequences

the DNA is noncoding—ie, its information is never

translated into an amino acid sequence of a protein

This estimation

(and the distribution of repetitive-

molecule. Certainly, some of the excess DNA sequences

sequence DNA) is based on a variety of DNA-RNA hy-

serve to regulate the expression of genes during devel-

bridization techniques and, more recently, on direct

opment, differentiation, and adaptation to the environ-

DNA sequencing. Similar techniques are used to esti-

ment. Some excess clearly makes up the intervening se-

mate the number of active genes in a population of

quences or introns (24% of the total human genome)

unique-sequence DNA. In brewers’ yeast

(Saccha-

that split the coding regions of genes, but much of the

romyces cerevisiae, a lower eukaryote), about two thirds

excess appears to be composed of many families of re-

of its 6200 genes are expressed. In typical tissues in a

peated sequences for which no functions have been

higher eukaryote (eg, mammalian liver and kidney), be-

clearly defined. A summary of the salient features of the

tween 10,000 and 15,000 genes are expressed. Differ-

human genome is presented in Chapter 40.

ent combinations of genes are expressed in each tissue,

DNA ORGANIZATION, REPLICATION, & REPAIR

321

Chromosome

1.5 × 10⁸ bp

(1-2 × 10³ genes)

Gene cluster

(~20 genes)

1.5 × 10⁶ bp

Gene

2 × 10⁴

Primary transcript

8 × 10³

mRNA

2 × 10³ nt

Figure 36-7. The relationship between chromosomal DNA and mRNA. The human

haploid DNA complement of 3 × 10⁹ base pairs (bp) is distributed between 23 chromo-

somes. Genes are clustered on these chromosomes. An average gene is 2 × 10⁴ bp in

length, including the regulatory region (hatched area), which is usually located at the 5′

end of the gene. The regulatory region is shown here as being adjacent to the transcrip-

tion initiation site (arrow). Most eukaryotic genes have alternating exons and introns. In

this example, there are nine exons (dark blue areas) and eight introns (light blue areas).

The introns are removed from the primary transcript by the processing reaction, and the

exons are ligated together in sequence to form the mature mRNA. (nt, nucleotides.)

of course, and how this is accomplished is one of the

Depending on their length, moderately repetitive

major unanswered questions in biology.

sequences are classified as long interspersed repeat

sequences

(LINEs) or short interspersed repeat

sequences

(SINEs). Both types appear to be

In Human DNA, at Least 30% of the

retroposons, ie, they arose from movement from one

Genome Consists of Repetitive Sequences

location to another (transposition) through an RNA

Repetitive-sequence DNA can be broadly classified as

intermediate by the action of reverse transcriptase that

moderately repetitive or as highly repetitive. The highly

transcribes an RNA template into DNA. Mammalian

repetitive sequences consist of 5-500 base pair lengths

genomes contain 20-50 thousand copies of the 6-7 kb

repeated many times in tandem. These sequences are

LINEs. These represent species-specific families of re-

usually clustered in centromeres and telomeres of the

peat elements. SINEs are shorter (70-300 bp), and

chromosome and are present in about 1-10 million

there may be more than 100,000 copies per genome.

copies per haploid genome. These sequences are tran-

Of the SINEs in the human genome, one family, the

scriptionally inactive and may play a structural role in

Alu family, is present in about 500,000 copies per hap-

the chromosome (see Chapter 40).

loid genome and accounts for at least 5-6% of the

The moderately repetitive sequences, which are de-

human genome. Members of the human Alu family

fined as being present in numbers of less than 10⁶

and their closely related analogs in other animals are

copies per haploid genome, are not clustered but are in-

transcribed as integral components of hnRNA or as dis-

terspersed with unique sequences. In many cases, these

crete RNA molecules, including the well-studied 4.5S

long interspersed repeats are transcribed by RNA poly-

RNA and 7S RNA. These particular family members

merase II and contain caps indistinguishable from those

are highly conserved within a species as well as between

on mRNA.

mammalian species. Components of the short inter-

322

CHAPTER 36

spersed repeats, including the members of the Alu fam-

with a gene in affected family members—and the lack

ily, may be mobile elements, capable of jumping into

of this association in unaffected members—may be the

and out of various sites within the genome (see below).

first clue about the genetic basis of a disease.

This can have disastrous results, as exemplified by the

Trinucleotide sequences that increase in number

insertion of Alu sequences into a gene, which, when so

(microsatellite instability) can cause disease. The unsta-

mutated, causes neurofibromatosis.

ble p(CGG)_n repeat sequence is associated with the

fragile X syndrome. Other trinucleotide repeats that

undergo dynamic mutation (usually an increase) are

Microsatellite Repeat Sequences

associated with Huntington’s chorea (CAG), myotonic

One category of repeat sequences exists as both dis-

dystrophy (CTG), spinobulbar muscular atrophy (CAG),

persed and grouped tandem arrays. The sequences con-

and Kennedy’s disease (CAG).

sist of

2-6 bp repeated up to 50 times. These mi-

crosatellite sequences most commonly are found as

ONE PERCENT OF CELLULAR DNA

dinucleotide repeats of AC on one strand and TG on

IS IN MITOCHONDRIA

the opposite strand, but several other forms occur, in-

cluding CG, AT, and CA. The AC repeat sequences are

The majority of the peptides in mitochondria (about

estimated to occur at 50,000-100,000 locations in the

54 out of 67) are coded by nuclear genes. The rest are

genome. At any locus, the number of these repeats may

coded by genes found in mitochondrial (mt) DNA.

vary on the two chromosomes, thus providing heterozy-

Human mitochondria contain two to ten copies of a

gosity of the number of copies of a particular mi-

small circular double-stranded DNA molecule that

crosatellite number in an individual. This is a heritable

makes up approximately 1% of total cellular DNA.

trait, and, because of their number and the ease of de-

This mtDNA codes for mt ribosomal and transfer

tecting them using the polymerase chain reaction

RNAs and for 13 proteins that play key roles in the res-

(PCR) (Chapter 40), AC repeats are very useful in con-

piratory chain. The linearized structural map of the

structing genetic linkage maps. Most genes are associ-

human mitochondrial genes is shown in Figure 36-8.

ated with one or more microsatellite markers, so the rel-

Some of the features of mtDNA are shown in Table

ative position of genes on chromosomes can be

36-3.

assessed, as can the association of a gene with a disease.

An important feature of human mitochondrial

Using PCR, a large number of family members can be

mtDNA is that—because all mitochondria are con-

rapidly screened for a certain microsatellite polymor-

tributed by the ovum during zygote formation—it is

phism. The association of a specific polymorphism

transmitted by maternal nonmendelian inheritance.

PH1

PH2

ATPase

12S

16S

ND1

ND2

CO1

CO2

CO3

ND4

ND5

CYT B

ND6

Figure 36-8. Maps of human mitochondrial genes. The maps represent the heavy (upper strand) and light

(lower map) strands of linearized mitochondrial (mt) DNA, showing the genes for the subunits of NADH-

coenzyme Q oxidoreductase (ND1 through ND6), cytochrome c oxidase (CO1 through CO3), cytochrome b

(CYT B), and ATP synthase (ATPase 8 and 6) and for the 12S and 16S ribosomal mt rRNAs. The transfer RNAs are de-

noted by small open boxes. The origin of heavy-strand (OH) and light-strand (OL) replication and the promoters

for the initiation of heavy-strand (PH1 and PH2) and light-strand (PL) transcription are indicated by arrows.

(Reproduced, with permission, from Moraes CT et al: Mitochondrial DNA deletions in progressive external ophthal-

moplegia and Kearns-Sayre syndrome. N Engl J Med 1989;320:1293.)

DNA ORGANIZATION, REPLICATION, & REPAIR

323

Table 36-3. Some major features of the structure

crossing over occurs as shown in Figure 36-9. This

and function of human mitochondrial DNA.¹

usually results in an equal and reciprocal exchange of

genetic information between homologous chromo-

somes. If the homologous chromosomes possess differ-

• Is circular, double-stranded, and composed of heavy (H)

ent alleles of the same genes, the crossover may produce

and a light (L) chains or strands.

noticeable and heritable genetic linkage differences. In

• Contains 16,569 bp.

the rare case where the alignment of homologous chro-

• Encodes 13 protein subunits of the respiratory chain (of a

mosomes is not exact, the crossing over or recombina-

total of about 67):

Seven subunits of NADH dehydrogenase (complex I)

tion event may result in an unequal exchange of infor-

Cytochrome b of complex III

mation. One chromosome may receive less genetic

Three subunits of cytochrome oxidase (complex IV)

material and thus a deletion, while the other partner of

Two subunits of ATP synthase

the chromosome pair receives more genetic material

• Encodes large (16s) and small (12s) mt ribosomal RNAs.

and thus an insertion or duplication (Figure 36-9).

• Encodes 22 mt tRNA molecules.

Unequal crossing over does occur in humans, as evi-

• Genetic code differs slightly from the standard code:

denced by the existence of hemoglobins designated

UGA (standard stop codon) is read as Trp.

Lepore and anti-Lepore (Figure 36-10). The farther

AGA and AGG (standard codons for Arg) are read as stop

apart two sequences are on an individual chromosome,

codons.

the greater the likelihood of a crossover recombination

• Contains very few untranslated sequences.

• High mutation rate (five to ten times that of nuclear DNA).

• Comparisons of mtDNA sequences provide evidence about

evolutionary origins of primates and other species.

¹Adapted from Harding AE: Neurological disease and mitochon-

drial genes. Trends Neurol Sci 1991;14:132.

Thus, in diseases resulting from mutations of mtDNA,

an affected mother would in theory pass the disease to

all of her children but only her daughters would trans-

mit the trait. However, in some cases, deletions in

mtDNA occur during oogenesis and thus are not inher-

ited from the mother. A number of diseases have now

been shown to be due to mutations of mtDNA. These

include a variety of myopathies, neurologic disorders,

and some cases of diabetes mellitus.

GENETIC MATERIAL CAN BE ALTERED

& REARRANGED

An alteration in the sequence of purine and pyrimidine

bases in a gene due to a change—a removal or an inser-

tion—of one or more bases may result in an altered

gene product. Such alteration in the genetic material re-

sults in a mutation whose consequences are discussed

in detail in Chapter 38.

Chromosomal Recombination Is One Way

of Rearranging Genetic Material

Genetic information can be exchanged between similar

or homologous chromosomes. The exchange or recom-

bination event occurs primarily during meiosis in

mammalian cells and requires alignment of homolo-

Figure 36-9.

The process of crossing-over between

gous metaphase chromosomes, an alignment that al-

homologous metaphase chromosomes to generate re-

most always occurs with great exactness. A process of

combinant chromosomes. See also Figure 36-12.

324

CHAPTER 36

Gγ

Aγ

βδ

Figure 36-10. The process of unequal cross-

Anti-

Lepore

over in the region of the mammalian genome

that harbors the structural genes encoding he-

Gγ

Aγ

moglobins and the generation of the unequal

recombinant products hemoglobin delta-beta

Lepore and beta-delta anti-Lepore. The exam-

Gγ

Aγ

ples given show the locations of the crossover

regions between amino acid residues. (Redrawn

and reproduced, with permission, from Clegg JB,

Gγ

Aγ

δβ

Weatherall DJ: β⁰ Thalassemia: Time for a reap-

Lepore

praisal? Lancet 1974;2:133.)

event. This is the basis for genetic mapping methods.

DNA polymerase, or reverse transcriptase—can be inte-

Unequal crossover affects tandem arrays of repeated

grated into chromosomes of the mammalian cell. The

DNAs whether they are related globin genes, as in Fig-

integration of the animal virus DNA into the animal

ure 36-10, or more abundant repetitive DNA. Un-

genome generally is not “site-specific” but does display

equal crossover through slippage in the pairing can re-

site preferences.

sult in expansion or contraction in the copy number of

the repeat family and may contribute to the expansion

Transposition Can Produce

and fixation of variant members throughout the array.

Processed Genes

In eukaryotic cells, small DNA elements that clearly are

Chromosomal Integration Occurs

not viruses are capable of transposing themselves in and

With Some Viruses

Some bacterial viruses (bacteriophages) are capable of

recombining with the DNA of a bacterial host in such a

way that the genetic information of the bacteriophage is

incorporated in a linear fashion into the genetic infor-

mation of the host. This integration, which is a form of

recombination, occurs by the mechanism illustrated in

Figure 36-11. The backbone of the circular bacterio-

phage genome is broken, as is that of the DNA mole-

cule of the host; the appropriate ends are resealed with

the proper polarity. The bacteriophage DNA is figura-

tively straightened out (“linearized”) as it is integrated

into the bacterial DNA molecule—frequently a closed

circle as well. The site at which the bacteriophage

genome integrates or recombines with the bacterial

genome is chosen by one of two mechanisms. If the

bacteriophage contains a DNA sequence homologous

to a sequence in the host DNA molecule, then a recom-

bination event analogous to that occurring between ho-

mologous chromosomes can occur. However, some

bacteriophages synthesize proteins that bind specific

sites on bacterial chromosomes to a nonhomologous

site characteristic of the bacteriophage DNA molecule.

Integration occurs at the site and is said to be “site-

specific.”

Many animal viruses, particularly the oncogenic

Figure 36-11.

The integration of a circular genome

viruses—either directly or, in the case of RNA viruses

from a virus (with genes A, B, and C) into the DNA mole-

such as HIV that causes AIDS, their DNA transcripts

cule of a host (with genes 1 and 2) and the consequent

generated by the action of the viral RNA-dependent

ordering of the genes.

DNA ORGANIZATION, REPLICATION, & REPAIR

325

out of the host genome in ways that affect the function

chromatids contains identical genetic information since

of neighboring DNA sequences. These mobile ele-

each is a product of the semiconservative replication of

ments, sometimes called “jumping DNA,” can carry

the original parent DNA molecule of that chromosome.

flanking regions of DNA and, therefore, profoundly af-

Crossing over occurs between these genetically identical

fect evolution. As mentioned above, the Alu family of

sister chromatids. Of course, these sister chromatid ex-

moderately repeated DNA sequences has structural

changes (Figure 36-12) have no genetic consequence as

characteristics similar to the termini of retroviruses,

long as the exchange is the result of an equal crossover.

which would account for the ability of the latter to

move into and out of the mammalian genome.

Immunoglobulin Genes Rearrange

Direct evidence for the transposition of other small

In mammalian cells, some interesting gene rearrange-

DNA elements into the human genome has been pro-

ments occur normally during development and differen-

vided by the discovery of “processed genes” for im-

munoglobulin molecules, α-globin molecules, and sev-

tiation. For example, in mice the V

L and CL genes for a

single immunoglobulin molecule (see Chapter 39) are

eral others. These processed genes consist of DNA

widely separated in the germ line DNA. In the DNA of a

sequences identical or nearly identical to those of the

differentiated immunoglobulin-producing (plasma) cell,

messenger RNA for the appropriate gene product. That

is, the

5′ nontranscribed region, the coding region

the same V

L and CL genes have been moved physically

closer together in the genome and into the same tran-

without intron representation, and the 3′ poly(A) tail

scription unit. However, even then, this rearrangement

are all present contiguously. This particular DNA se-

quence arrangement must have resulted from the re-

of DNA during differentiation does not bring the V

L and

verse transcription of an appropriately processed mes-

L genes into contiguity in the DNA. Instead, the DNA

senger RNA molecule from which the intron regions

had been removed and the poly(A) tail added. The only

recognized mechanism this reverse transcript could

have used to integrate into the genome would have

been a transposition event. In fact, these “processed

genes” have short terminal repeats at each end, as do

known transposed sequences in lower organisms. In the

absence of their transcription and thus genetic selection

for function, many of the processed genes have been

randomly altered through evolution so that they now

contain nonsense codons which preclude their ability to

encode a functional, intact protein (see Chapter 38).

Thus, they are referred to as “pseudogenes.”

Gene Conversion Produces

Rearrangements

Besides unequal crossover and transposition, a third

mechanism can effect rapid changes in the genetic ma-

terial. Similar sequences on homologous or nonhomol-

ogous chromosomes may occasionally pair up and elim-

inate any mismatched sequences between them. This

may lead to the accidental fixation of one variant or an-

other throughout a family of repeated sequences and

thereby homogenize the sequences of the members of

repetitive DNA families. This latter process is referred

to as gene conversion.

Figure 36-12.

Sister chromatid exchanges between

Sister Chromatids Exchange

human chromosomes. These are detectable by Giemsa

In diploid eukaryotic organisms such as humans, after

staining of the chromosomes of cells replicated for two

cells progress through the S phase they contain a

cycles in the presence of bromodeoxyuridine. The ar-

tetraploid content of DNA. This is in the form of sister

rows indicate some regions of exchange. (Courtesy of

chromatids of chromosome pairs. Each of these sister

S Wolff and J Bodycote.)

326

CHAPTER 36

contains an interspersed or interruption sequence of

Table 36-4. Steps involved in DNA replication

about 1200 base pairs at or near the junction of the V

in eukaryotes.

and C regions. The interspersed sequence is transcribed

genes, and the inter-

into RNA along with the V

L and CL

1. Identification of the origins of replication.

spersed information is removed from the RNA during its

2. Unwinding (denaturation) of dsDNA to provide an ssDNA

nuclear processing (Chapters 37 and 39).

template.

3. Formation of the replication fork.

DNA SYNTHESIS & REPLICATION

4. Initiation of DNA synthesis and elongation.

5. Formation of replication bubbles with ligation of the newly

ARE RIGIDLY CONTROLLED

synthesized DNA segments.

The primary function of DNA replication is under-

6. Reconstitution of chromatin structure.

stood to be the provision of progeny with the genetic

information possessed by the parent. Thus, the replica-

tion of DNA must be complete and carried out in such

a series of direct repeat DNA sequences. In bacterio-

a way as to maintain genetic stability within the organ-

phage λ, the oriλ is bound by the λ-encoded O protein

ism and the species. The process of DNA replication is

to four adjacent sites. In E coli, the oriC is bound by

complex and involves many cellular functions and sev-

the protein dnaA. In both cases, a complex is formed

eral verification procedures to ensure fidelity in replica-

consisting of 150-250 bp of DNA and multimers of

tion. About 30 proteins are involved in the replication

the DNA-binding protein. This leads to the local de-

of the E coli chromosome, and this process is almost

naturation and unwinding of an adjacent A+T-rich re-

certainly more complex in eukaryotic organisms. The

gion of DNA. Functionally similar autonomously

first enzymologic observations on DNA replication

replicating sequences (ARS) have been identified in

were made in E coli by Kornberg, who described in that

yeast cells. The ARS contains a somewhat degenerate

organism the existence of an enzyme now called DNA

11-bp sequence called the origin replication element

polymerase I. This enzyme has multiple catalytic activi-

(ORE). The ORE binds a set of proteins, analogous to

ties, a complex structure, and a requirement for the

the dnaA protein of E coli, which is collectively called

triphosphates of the four deoxyribonucleosides of ade-

the origin recognition complex (ORC). The ORE is

nine, guanine, cytosine, and thymine. The polymeriza-

located adjacent to an approximately 80-bp A+T-rich

tion reaction catalyzed by DNA polymerase I of E coli

sequence that is easy to unwind. This is called the DNA

has served as a prototype for all DNA polymerases of

unwinding element (DUE). The DUE is the origin of

both prokaryotes and eukaryotes, even though it is now

replication in yeast.

recognized that the major role of this polymerase is to

Consensus sequences similar to ori or ARS in struc-

complete replication on the lagging strand.

ture or function have not been precisely defined in

In all cells, replication can occur only from a single-

mammalian cells, though several of the proteins that

stranded DNA (ssDNA) template. Mechanisms must

participate in ori recognition and function have been

exist to target the site of initiation of replication and to

identified and appear quite similar to their yeast coun-

unwind the double-stranded DNA (dsDNA) in that re-

terparts in both amino acid sequence and function.

gion. The replication complex must then form. After

replication is complete in an area, the parent and daughter

Unwinding of DNA

strands must re-form dsDNA. In eukaryotic cells, an ad-

ditional step must occur. The dsDNA must precisely re-

The interaction of proteins with ori defines the start site

form the chromatin structure, including nucleosomes,

of replication and provides a short region of ssDNA es-

that existed prior to the onset of replication. Although this

sential for initiation of synthesis of the nascent DNA

entire process is not well understood in eukaryotic cells,

strand. This process requires the formation of a number

replication has been quite precisely described in prokary-

of protein-protein and protein-DNA interactions. A

otic cells, and the general principles are thought to be the

critical step is provided by a DNA helicase that allows

same in both. The major steps are listed in Table 36-4, il-

for processive unwinding of DNA. In uninfected E coli,

lustrated in Figure 36-13, and discussed, in sequence,

this function is provided by a complex of dnaB helicase

below. A number of proteins, most with specific enzy-

and the dnaC protein. Single-stranded DNA-binding

matic action, are involved in this process (Table 36-5).

proteins

(SSBs) stabilize this complex. In λ phage-

infected E coli, the phage protein P binds to dnaB and

the P/dnaB complex binds to oriλ by interacting with

The Origin of Replication

the O protein. dnaB is not an active helicase when in the

At the origin of replication (ori), there is an associa-

P/dnaB/O complex. Three E coli heat shock proteins

tion of sequence-specific dsDNA-binding proteins with

(dnaK, dnaJ, and GrpE) cooperate to remove the P

DNA ORGANIZATION, REPLICATION, & REPAIR

327

Ori

A + T region

A + T - rich

Ori-binding

Denaturation

region

protein

Binding

(

)

of SSB ( )

Binding of

factors,

formation of

3′

replication fork,

5′

initiation of

replication

Leading

strand

Polymerase

Helicase

3′

Primase

5′

SSB

5′

3′

Lagging

= Ori-binding protein

strand

5′

= Polymerase

= Nascent DNA

= RNA primer

Replication fork

= Helicase

= Primase

= SSB

Figure 36-13. Steps involved in DNA replication. This figure describes DNA replication in an E coli cell, but the

general steps are similar in eukaryotes. A specific interaction of a protein (the O protein) to the origin of replication

(ori) results in local unwinding of DNA at an adjacent A+T-rich region. The DNA in this area is maintained in the

single-strand conformation (ssDNA) by single-strand-binding proteins (SSBs). This allows a variety of proteins, includ-

ing helicase, primase, and DNA polymerase, to bind and to initiate DNA synthesis. The replication fork proceeds as

DNA synthesis occurs continuously (long arrow) on the leading strand and discontinuously (short arrows) on the lag-

ging strand. The nascent DNA is always synthesized in the 5′ to 3′ direction, as DNA polymerases can add a nucleotide

only to the 3′ end of a DNA strand.

protein and activate the dnaB helicase. In cooperation

(2) a primase initiates synthesis of an RNA molecule

with SSB, this leads to DNA unwinding and active

that is essential for priming DNA synthesis; (3) the

replication. In this way, the replication of the λ phage is

DNA polymerase initiates nascent, daughter strand

accomplished at the expense of replication of the host

synthesis; and (4) SSBs bind to ssDNA and prevent

E coli cell.

premature reannealing of ssDNA to dsDNA. These re-

actions are illustrated in Figure 36-13.

The polymerase III holoenzyme (the dnaE gene

Formation of the Replication Fork

product in E coli) binds to template DNA as part of a

A replication fork consists of four components that

multiprotein complex that consists of several polym-

form in the following sequence: (1) the DNA helicase

erase accessory factors (β, γ, δ, δ′, and τ). DNA polym-

unwinds a short segment of the parental duplex DNA;

erases only synthesize DNA in the 5′ to 3′ direction,

328

CHAPTER 36

Table 36-5. Classes of proteins involved

replication fork. Of all polymerases, it catalyzes the

in replication.

highest rate of chain elongation and is the most proces-

sive. It is capable of polymerizing 0.5 Mb of DNA dur-

ing one cycle on the leading strand. Pol III is a large

Protein

Function

(> 1 MDa), ten-subunit protein complex in E coli. The

DNA polymerases

Deoxynucleotide polymerization

two identical β subunits of pol III encircle the DNA

template in a sliding “clamp,” which accounts for the

Helicases

Processive unwinding of DNA

stability of the complex and for the high degree of pro-

Topoisomerases

Relieve torsional strain that results

cessivity the enzyme exhibits.

from helicase-induced unwinding

Polymerase II (pol II) is mostly involved in proof-

DNA primase

Initiates synthesis of RNA primers

reading and DNA repair. Polymerase I (pol I) com-

pletes chain synthesis between Okazaki fragments on

Single-strand binding

Prevent premature reannealing of

the lagging strand. Eukaryotic cells have counterparts

proteins

dsDNA

for each of these enzymes plus some additional ones. A

DNA ligase

Seals the single strand nick between

comparison is shown in Table 36-6.

the nascent chain and Okazaki frag-

In mammalian cells, the polymerase is capable of

ments on lagging strand

polymerizing about 100 nucleotides per second, a rate

at least tenfold slower than the rate of polymerization of

deoxynucleotides by the bacterial DNA polymerase

complex. This reduced rate may result from interfer-

and only one of the several different types of polym-

ence by nucleosomes. It is not known how the replica-

erases is involved at the replication fork. Because the

tion complex negotiates nucleosomes.

DNA strands are antiparallel (Chapter 35), the polym-

erase functions asymmetrically. On the leading (for-

ward) strand, the DNA is synthesized continuously.

Initiation & Elongation of DNA Synthesis

On the lagging (retrograde) strand, the DNA is syn-

The initiation of DNA synthesis (Figure 36-14) re-

thesized in short (1-5 kb; see Figure 36-16) fragments,

quires priming by a short length of RNA, about

the so-called Okazaki fragments. Several Okazaki frag-

10-200 nucleotides long. This priming process involves

ments (up to 250) must be synthesized, in sequence, for

the nucleophilic attack by the 3′-hydroxyl group of the

each replication fork. To ensure that this happens, the

RNA primer on the α phosphate of the first entering

helicase acts on the lagging strand to unwind dsDNA in

deoxynucleoside triphosphate

(N in Figure

36-14)

a 5′ to 3′ direction. The helicase associates with the pri-

with the splitting off of pyrophosphate. The 3′-hy-

mase to afford the latter proper access to the template.

droxyl group of the recently attached deoxyribonu-

This allows the RNA primer to be made and, in turn,

cleoside monophosphate is then free to carry out a

the polymerase to begin replicating the DNA. This is

nucleophilic attack on the next entering deoxyribonu-

an important reaction sequence since DNA poly-

cleoside triphosphate (N + 1 in Figure 36-14), again at

merases cannot initiate DNA synthesis de novo. The

its α phosphate moiety, with the splitting off of py-

mobile complex between helicase and primase has been

rophosphate. Of course, selection of the proper de-

called a primosome. As the synthesis of an Okazaki

oxyribonucleotide whose terminal 3′-hydroxyl group is

fragment is completed and the polymerase is released, a

to be attacked is dependent upon proper base pairing

new primer has been synthesized. The same polymerase

molecule remains associated with the replication fork

and proceeds to synthesize the next Okazaki fragment.

Table 36-6. A comparison of prokaryotic and

eukaryotic DNA polymerases.

The DNA Polymerase Complex

A number of different DNA polymerase molecules en-

E coli

Mammalian

Function

gage in DNA replication. These share three important

properties: (1) chain elongation, (2) processivity, and

Gap filling and synthesis of lagging

(3) proofreading. Chain elongation accounts for the

strand

rate (in nucleotides per second) at which polymeriza-

DNA proofreading and repair

tion occurs. Processivity is an expression of the number

DNA repair

of nucleotides added to the nascent chain before the

polymerase disengages from the template. The proof-

Mitochondrial DNA synthesis

reading function identifies copying errors and corrects

III

Processive, leading strand synthesis

them. In E coli, polymerase III (pol III) functions at the

330

CHAPTER 36

with the other strand of the DNA molecule according

of newly synthesized DNA by enzymes referred to as

to the rules proposed originally by Watson and Crick

DNA ligases.

(Figure 36-15). When an adenine deoxyribonucleoside

monophosphoryl moiety is in the template position, a

Replication Exhibits Polarity

thymidine triphosphate will enter and its α phosphate

will be attacked by the

3′-hydroxyl group of the

As has already been noted, DNA molecules are double-

deoxyribonucleoside monophosphoryl most recently

stranded and the two strands are antiparallel, ie, run-

added to the polymer. By this stepwise process, the

ning in opposite directions. The replication of DNA in

template dictates which deoxyribonucleoside triphos-

prokaryotes and eukaryotes occurs on both strands si-

phate is complementary and by hydrogen bonding

multaneously. However, an enzyme capable of poly-

holds it in place while the 3′-hydroxyl group of the

merizing DNA in the 3′ to 5′ direction does not exist in

growing strand attacks and incorporates the new nu-

any organism, so that both of the newly replicated

cleotide into the polymer. These segments of DNA

DNA strands cannot grow in the same direction simul-

attached to an RNA initiator component are the

taneously. Nevertheless, the same enzyme does replicate

Okazaki fragments (Figure 36-16). In mammals, after

both strands at the same time. The single enzyme repli-

many Okazaki fragments are generated, the replication

cates one strand

(“leading strand”) in a continuous

complex begins to remove the RNA primers, to fill in

manner in the 5′ to 3′ direction, with the same overall

the gaps left by their removal with the proper base-

forward direction. It replicates the other strand (“lag-

paired deoxynucleotide, and then to seal the fragments

ging strand”) discontinuously while polymerizing the

RNA primer

DNA template

Growing DNA polymer

Entering TTP

Figure 36-15. The RNA-primed synthesis of DNA demonstrating the template function of the

complementary strand of parental DNA.

DNA ORGANIZATION, REPLICATION, & REPAIR

331

DNA template

3′

5′

3′

Newly synthesized

RNA

10 bp

DNA strand

primer

100 bp

Okazaki fragments

Figure 36-16. The discontinuous polymerization of deoxyribonucleotides on the lagging

strand; formation of Okazaki fragments during lagging strand DNA synthesis is illustrated.

Okazaki fragments are 100-250 nt long in eukaryotes, 1000-2000 bp in prokaryotes.

nucleotides in short spurts of

150-250 nucleotides,

hours! Metazoan organisms get around this problem

again in the 5′ to 3′ direction, but at the same time it

using two strategies. First, replication is bidirectional.

faces toward the back end of the preceding RNA

Second, replication proceeds from multiple origins in

primer rather than toward the unreplicated portion.

each chromosome (a total of as many as 100 in hu-

This process of semidiscontinuous DNA synthesis is

mans). Thus, replication occurs in both directions

shown diagrammatically in Figures 36-13 and 36-16.

along all of the chromosomes, and both strands are

In the mammalian nuclear genome, most of the

replicated simultaneously. This replication process gen-

RNA primers are eventually removed as part of the

erates “replication bubbles” (Figure 36-17).

replication process, whereas after replication of the mi-

The multiple sites that serve as origins for DNA

tochondrial genome the small piece of RNA remains as

replication in eukaryotes are poorly defined except in a

an integral part of the closed circular DNA structure.

few animal viruses and in yeast. However, it is clear that

initiation is regulated both spatially and temporally,

since clusters of adjacent sites initiate replication syn-

Formation of Replication Bubbles

chronously. There are suggestions that functional do-

Replication proceeds from a single ori in the circular

mains of chromatin replicate as intact units, implying

bacterial chromosome, composed of roughly 6 × 10⁶ bp

that the origins of replication are specifically located

of DNA. This process is completed in about 30 min-

with respect to transcription units.

utes, a replication rate of 3 × 10⁵ bp/min. The entire

During the replication of DNA, there must be a sep-

mammalian genome replicates in approximately

aration of the two strands to allow each to serve as a

hours, the average period required for formation of a

template by hydrogen bonding its nucleotide bases to

tetraploid genome from a diploid genome in a replicat-

the incoming deoxynucleoside triphosphate. The separa-

ing cell. If a mammalian genome (3 × 10⁹ bp) repli-

tion of the DNA double helix is promoted by SSBs, spe-

cated at the same rate as bacteria (ie, 3 × 10⁵ bp/min)

cific protein molecules that stabilize the single-stranded

from but a single ori, replication would take over 150

structure as the replication fork progresses. These stabi-

Origin of replication

“Replication bubble”

3′

5′

3′

Unwinding proteins

at replication forks

Directions

of replication

Figure 36-17. The generation of “replication bubbles” during the process of DNA synthesis. The bidirectional

replication and the proposed positions of unwinding proteins at the replication forks are depicted.

332

CHAPTER 36

lizing proteins bind cooperatively and stoichiometrically

without requiring energy input, because of the forma-

to the single strands without interfering with the abili-

tion of a high-energy covalent bond between the nicked

ties of the nucleotides to serve as templates

(Figure

phosphodiester backbone and the nicking-sealing en-

36-13). In addition to separating the two strands of the

zyme. The nicking-resealing enzymes are called DNA

double helix, there must be an unwinding of the mole-

topoisomerases. This process is depicted diagrammati-

cule (once every 10 nucleotide pairs) to allow strand sep-

cally in Figure

36-18 and there compared with the

aration. This must happen in segments, given the time

ATP-dependent resealing carried out by the DNA li-

during which DNA replication occurs. There are multi-

gases. Topoisomerases are also capable of unwinding su-

ple “swivels” interspersed in the DNA molecules of all

percoiled DNA. Supercoiled DNA is a higher-ordered

organisms. The swivel function is provided by specific

structure occurring in circular DNA molecules wrapped

enzymes that introduce “nicks” in one strand of the

around a core, as depicted in Figure 36-19.

unwinding double helix, thereby allowing the unwind-

There exists in one species of animal viruses (retro-

ing process to proceed. The nicks are quickly resealed

viruses) a class of enzymes capable of synthesizing a sin-

Step 1

DNA topoisomerase I = E

DNA ligase = E

E + ATP

P R A

(AMP-Enzyme)

5′

P -E

Enzyme (E) -generated

Single-strand nick

single-strand nick

present

O 3′

3′

5′

3′

5′

E P R A

Step 2

5′

-E

P R A

Formation of high-

energy bond

Step 3

P R A

(AMP)

Nick repaired

Figure 36-18. Comparison of two types of nick-sealing reactions on DNA. The

series of reactions at left is catalyzed by DNA topoisomerase I, that at right by

DNA ligase; P = phosphate, R = ribose, A = ademine. (Slightly modified and repro-

duced, with permission, from Lehninger AL: Biochemistry, 2nd ed. Worth, 1975.)

DNA ORGANIZATION, REPLICATION, & REPAIR

333

preexisting and newly assembled histone octamers are

randomly distributed to each arm of the replication

fork.

DNA Synthesis Occurs During

the S Phase of the Cell Cycle

In animal cells, including human cells, the replication

of the DNA genome occurs only at a specified time

during the life span of the cell. This period is referred to

as the synthetic or S phase. This is usually temporally

separated from the mitotic phase by nonsynthetic peri-

ods referred to as gap 1 (G1) and gap 2 (G2), occurring

before and after the S phase, respectively

(Figure

36-20). Among other things, the cell prepares for DNA

synthesis in G1 and for mitosis in G2. The cell regu-

lates its DNA synthesis grossly by allowing it to occur

only at specific times and mostly in cells preparing to

divide by a mitotic process.

It appears that all eukaryotic cells have gene prod-

ucts that govern the transition from one phase of the

cell cycle to another. The cyclins are a family of pro-

teins whose concentration increases and decreases

throughout the cell cycle—thus their name. The cyclins

Figure 36-19.

Supercoiling of DNA. A left-handed

turn on, at the appropriate time, different cyclin-

toroidal (solenoidal) supercoil, at left, will convert to a

dependent protein kinases (CDKs) that phosphory-

right-handed interwound supercoil, at right, when the

late substrates essential for progression through the cell

cylindric core is removed. Such a transition is analogous

cycle (Figure 36-21). For example, cyclin D levels rise

to that which occurs when nucleosomes are disrupted

in late G1 phase and allow progression beyond the start

by the high salt extraction of histones from chromatin.

(yeast) or restriction point (mammals), the point be-

yond which cells irrevocably proceed into the S or

DNA synthesis phase.

The D cyclins activate CDK4 and CDK6. These

gle-stranded and then a double-stranded DNA mole-

two kinases are also synthesized during G1 in cells un-

cule from a single-stranded RNA template. This poly-

dergoing active division. The D cyclins and CDK4 and

merase, RNA-dependent DNA polymerase, or “reverse

CDK6 are nuclear proteins that assemble as a complex

transcriptase,” first synthesizes a DNA-RNA hybrid

in late G1 phase. The complex is an active serine-

molecule utilizing the RNA genome as a template. A

threonine protein kinase. One substrate for this kinase

specific nuclease, RNase H, degrades the RNA strand,

is the retinoblastoma (Rb) protein. Rb is a cell cycle

and the remaining DNA strand in turn serves as a tem-

regulator because it binds to and inactivates a transcrip-

plate to form a double-stranded DNA molecule con-

tion factor (E2F) necessary for the transcription of cer-

taining the information originally present in the RNA

tain genes (histone genes, DNA replication proteins,

genome of the animal virus.

etc) needed for progression from G1 to S phase. The

phosphorylation of Rb by CDK4 or CDK6 results in

the release of E2F from Rb-mediated transcription re-

Reconstitution of Chromatin Structure

pression—thus, gene activation ensues and cell cycle

There is evidence that nuclear organization and chro-

progression takes place.

matin structure are involved in determining the regu-

Other cyclins and CDKs are involved in different

lation and initiation of DNA synthesis. As noted

aspects of cell cycle progression (Table 36-7). Cyclin E

above, the rate of polymerization in eukaryotic cells,

and CDK2 form a complex in late G1. Cyclin E is

which have chromatin and nucleosomes, is tenfold

rapidly degraded, and the released CDK2 then forms a

slower than that in prokaryotic cells, which have

complex with cyclin A. This sequence is necessary for

naked DNA. It is also clear that chromatin structure

the initiation of DNA synthesis in S phase. A complex

must be re-formed after replication. Newly replicated

between cyclin B and CDK1 is rate-limiting for the

DNA is rapidly assembled into nucleosomes, and the

G2/M transition in eukaryotic cells.

334

CHAPTER 36

Improper spindle

detected

Figure 36-20. Mammalian cell cycle and cell

cycle checkpoints. DNA, chromosome, and chro-

mosome segregation integrity is continuously

G₂

monitored throughout the cell cycle. If DNA dam-

Damaged DNA

age is detected in either the G1 or the G2 phase of

detected

the cell cycle, if the genome is incompletely repli-

cated, or if normal chromosome segregation ma-

Damaged DNA

detected

chinery is incomplete (ie, a defective spindle), cells

will not progress through the phase of the cycle in

Incomplete

which defects are detected. In some cases, if the

replication

damage cannot be repaired, such cells undergo

detected

programmed cell death (apoptosis).

Many of the cancer-causing viruses (oncoviruses)

duced by several DNA viruses target the Rb transcrip-

and cancer-inducing genes (oncogenes) are capable of

tion repressor for inactivation, inducing cell division in-

alleviating or disrupting the apparent restriction that

appropriately.

normally controls the entry of mammalian cells from

During the S phase, mammalian cells contain

G1 into the S phase. From the foregoing, one might

greater quantities of DNA polymerase than during the

have surmised that excessive production of a cyclin—or

nonsynthetic phases of the cell cycle. Furthermore,

production at an inappropriate time—might result in

those enzymes responsible for formation of the sub-

abnormal or unrestrained cell division. In this context it

strates for DNA synthesis—ie, deoxyribonucleoside

is noteworthy that the bcl oncogene associated with B

triphosphates—are also increased in activity, and their

cell lymphoma appears to be the cyclin D1 gene. Simi-

activity will diminish following the synthetic phase

larly, the oncoproteins (or transforming proteins) pro-

until the reappearance of the signal for renewed DNA

Cdk1-cyclin B

Cdk1-cyclin A

G₂

Cdk4-cyclin D

Cdk6-cyclin D

G₁

Restriction

point

Cdk2-cyclin A

Figure 36-21. Schematic illustration of the

points during the mammalian cell cycle during

which the indicated cyclins and cyclin-dependent

kinases are activated. The thickness of the various

Cdk2-cyclin E

colored lines is indicative of the extent of activity.

DNA ORGANIZATION, REPLICATION, & REPAIR

335

Table 36-7. Cyclins and cyclin-dependent kinases

ring more frequently than once every 10⁸-10¹⁰ base

involved in cell cycle progression.

pairs of DNA synthesized. The mechanisms responsible

for this monitoring mechanism in E coli include the 3′

to 5′ exonuclease activities of one of the subunits of the

Cyclin

Kinase

Function

pol III complex and of the pol I molecule. The analo-

CDK4, CDK6

Progression past restriction point at

gous mammalian enzymes (δ and α) do not seem to

G1/S boundary

possess such a nuclease proofreading function. Other

E, A

CDK2

Initiation of DNA synthesis in early S

enzymes provide this repair function.

phase

Replication errors, even with a very efficient repair

system, lead to the accumulation of mutations. A

CDK1

Transition from G2 to M

human has 10¹⁴ nucleated cells each with 3 × 10⁹ base

pairs of DNA. If about 10¹⁶ cell divisions occur in a

lifetime and 10−10 mutations per base pair per cell gen-

eration escape repair, there may eventually be as many

synthesis. During the S phase, the nuclear DNA is

as one mutation per 10⁶ bp in the genome. Fortunately,

completely replicated once and only once. It seems

most of these will probably occur in DNA that does not

that once chromatin has been replicated, it is marked so

encode proteins or will not affect the function of en-

as to prevent its further replication until it again passes

coded proteins and so are of no consequence. In addi-

through mitosis. The molecular mechanisms for this

tion, spontaneous and chemically induced damage to

phenomenon have yet to be elucidated.

DNA must be repaired.

In general, a given pair of chromosomes will repli-

Damage to DNA by environmental, physical, and

cate simultaneously and within a fixed portion of the S

chemical agents may be classified into four types

phase upon every replication. On a chromosome, clus-

(Table 36-8). Abnormal regions of DNA, either from

ters of replication units replicate coordinately. The na-

copying errors or DNA damage, are replaced by four

ture of the signals that regulate DNA synthesis at these

mechanisms: (1) mismatch repair, (2) base excision-

levels is unknown, but the regulation does appear to be

repair, (3) nucleotide excision-repair, and (4) double-

an intrinsic property of each individual chromosome.

strand break repair (Table 36-9). These mechanisms

exploit the redundancy of information inherent in the

Enzymes Repair Damaged DNA

double helical DNA structure. The defective region in

one strand can be returned to its original form by rely-

The maintenance of the integrity of the information in

ing on the complementary information stored in the

DNA molecules is of utmost importance to the survival

unaffected strand.

of a particular organism as well as to survival of the

species. Thus, it can be concluded that surviving species

have evolved mechanisms for repairing DNA damage

occurring as a result of either replication errors or envi-

ronmental insults.

Table 36-8. Types of damage to DNA.

As described in Chapter 35, the major responsibility

for the fidelity of replication resides in the specific pair-

Single-base alteration

ing of nucleotide bases. Proper pairing is dependent

A. Depurination

upon the presence of the favored tautomers of the

B. Deamination of cytosine to uracil

purine and pyrimidine nucleotides, but the equilibrium

C. Deamination of adenine to hypoxanthine

whereby one tautomer is more stable than another is

D. Alkylation of base

only about 10⁴ or 10⁵ in favor of that with the greater

E. Insertion or deletion of nucleotide

stability. Although this is not favorable enough to en-

F. Base-analog incorporation

sure the high fidelity that is necessary, favoring of the

II.

Two-base alteration

preferred tautomers—and thus of the proper base pair-

A. UV light-induced thymine-thymine (pyrimidine) dimer

ing—could be ensured by monitoring the base pairing

B. Bifunctional alkylating agent cross-linkage

twice. Such double monitoring does appear to occur in

III. Chain breaks

both bacterial and mammalian systems: once at the

A. Ionizing radiation

time of insertion of the deoxyribonucleoside triphos-

B. Radioactive disintegration of backbone element

phates, and later by a follow-up energy-requiring mech-

C. Oxidative free radical formation

IV. Cross-linkage

anism that removes all improper bases which may occur

A. Between bases in same or opposite strands

in the newly formed strand. This “proofreading” pre-

B. Between DNA and protein molecules (eg, histones)

vents tautomer-induced misincorporation from occur-

336

CHAPTER 36

Table 36-9. Mechanism of DNA repair

CH₃

3′

5′

Mechanism

Problem

Solution

5′

3′

Mismatch

Copying errors (single

Methyl-directed

SINGLE-SITE STRAND CUT

repair

base or two- to five-

strand cutting, exo-

BY GATC ENDONUCLEASE

base unpaired loops)

nuclease digestion,

CH₃

and replacement

3′

5′

Base

Spontaneous, chem-

Base removal by N-

5′

3′

excision-

ical, or radiation dam-

glycosylase, abasic

repair

age to a single base

sugar removal, re-

DEFECT REMOVED

placement

BY EXONUCLEASE

Nucleotide

Spontaneous, chem-

Removal of an ap-

CH₃

excision-

ical, or radiation dam-

proximately 30-

3′

5′

repair

age to a DNA segment

nucleotide oligomer

5′

3′

and replacement

DEFECT REPAIRED

Double-

Ionizing radiation,

Synapsis, unwind-

BY POLYMERASE

strand

chemotherapy,

ing, alignment,

break repair

oxidative free

ligation

CH₃

radicals

3′

5′

RELIGATED

BY LIGASE

Mismatch Repair

CH₃

Mismatch repair corrects errors made when DNA is

3′

5′

copied. For example, a C could be inserted opposite an

A, or the polymerase could slip or stutter and insert two

5′

3′

to five extra unpaired bases. Specific proteins scan the

Figure 36-22.

Mismatch repair of DNA. This mecha-

newly synthesized DNA, using adenine methylation

nism corrects a single mismatch base pair (eg, C to A

within a GATC sequence as the point of reference (Fig-

rather than T to A) or a short region of unpaired DNA.

ure 36-22). The template strand is methylated, and the

The defective region is recognized by an endonuclease

newly synthesized strand is not. This difference allows

that makes a single-strand cut at an adjacent methy-

the repair enzymes to identify the strand that contains

lated GATC sequence. The DNA strand is removed

the errant nucleotide which requires replacement. If a

mismatch or small loop is found, a GATC endonucle-

through the mutation, replaced, and religated.

ase cuts the strand bearing the mutation at a site corre-

sponding to the GATC. An exonuclease then digests

this strand from the GATC through the mutation, thus

E coli MutS protein that is involved in mismatch repair

removing the faulty DNA. This can occur from either

(see above). Mutations of hMSH2 account for 50-60%

end if the defect is bracketed by two GATC sites. This

of HNPCC cases. Another gene, hMLH1, is associated

defect is then filled in by normal cellular enzymes ac-

with most of the other cases. hMLH1 is the human ana-

cording to base pairing rules. In E coli, three proteins

log of the bacterial mismatch repair gene MutL. How

(Mut S, Mut C, and Mut H) are required for recogni-

does faulty mismatch repair result in colon cancer? The

tion of the mutation and nicking of the strand. Other

human genes were localized because microsatellite in-

cellular enzymes, including ligase, polymerase, and

stability was detected. That is, the cancer cells had a mi-

SSBs, remove and replace the strand. The process is

crosatellite of a length different from that found in the

somewhat more complicated in mammalian cells, as

normal cells of the individual. It appears that the af-

about six proteins are involved in the first steps.

fected cells, which harbor a mutated hMSH2

Faulty mismatch repair has been linked to heredi-

hMLH1 mismatch repair enzyme, are unable to remove

tary nonpolyposis colon cancer (HNPCC), one of the

small loops of unpaired DNA, and the microsatellite

most common inherited cancers. Genetic studies linked

thus increases in size. Ultimately, microsatellite DNA

HNPCC in some families to a region of chromosome

expansion must affect either the expression or the func-

2. The gene located, designated hMSH2, was sub-

tion of a protein critical in surveillance of the cell cycle

sequently shown to encode the human analog of the

in these colon cells.

DNA ORGANIZATION, REPLICATION, & REPAIR

337

Base Excision-Repair

3′

5′

C G G C T C A T C C G A T

The depurination of DNA, which happens sponta-

neously owing to the thermal lability of the purine N-

A G C C G A G T A G G C T A

5′

3′

glycosidic bond, occurs at a rate of 5000-10,000/cell/d

Heat energy

at 37 °C. Specific enzymes recognize a depurinated site

and replace the appropriate purine directly, without in-

C G G C T

U A T C C G A T

terruption of the phosphodiester backbone.

Cytosine, adenine, and guanine bases in DNA spon-

A G C C G A

G T A G G C T A

taneously form uracil, hypoxanthine, or xanthine, re-

spectively. Since none of these normally exist in DNA,

URACIL DNA GLYCOSYLASE

it is not surprising that specific N-glycosylases can rec-

ognize these abnormal bases and remove the base itself

C G G C T

T C C G A T

from the DNA. This removal marks the site of the de-

fect and allows an apurinic or apyrimidinic endonu-

A G C C G A G T A G G C T A

clease to excise the abasic sugar. The proper base is

NUCLEASES

then replaced by a repair DNA polymerase, and a ligase

returns the DNA to its original state (Figure 36-23).

C G G C

T C C G A T

This series of events is called base excision-repair. By a

similar series of steps involving initially the recognition

A G C C G A G T A G G C T A

of the defect, alkylated bases and base analogs can be re-

moved from DNA and the DNA returned to its origi-

DNA POLYMERASE + DNA LIGASE

nal informational content. This mechanism is suitable

for replacement of a single base but is not effective at

C G G C T C A T C C G A T

replacing regions of damaged DNA.

A G C C G A G T A G G C T A

Nucleotide Excision-Repair

Figure 36-23. Base excision-repair of DNA. The en-

This mechanism is used to replace regions of damaged

zyme uracil DNA glycosylase removes the uracil created

DNA up to 30 bases in length. Common examples of

by spontaneous deamination of cytosine in the DNA. An

DNA damage include ultraviolet (UV) light, which in-

endonuclease cuts the backbone near the defect; then,

duces the formation of cyclobutane pyrimidine-pyrimi-

after an endonuclease removes a few bases, the defect

dine dimers, and smoking, which causes formation of

is filled in by the action of a repair polymerase and the

benzo[a]pyrene-guanine adducts. Ionizing radiation,

strand is rejoined by a ligase. (Courtesy of B Alberts.)

cancer chemotherapeutic agents, and a variety of chemi-

cals found in the environment cause base modification,

strand breaks, cross-linkage between bases on opposite

strands or between DNA and protein, and numerous

marked sensitivity to sunlight (ultraviolet) with subse-

other defects. These are repaired by a process called nu-

quent formation of multiple skin cancers and prema-

cleotide excision-repair

(Figure 36-24). This complex

ture death. The risk of developing skin cancer is in-

process, which involves more gene products than the two

creased 1000- to 2000-fold. The inherited defect seems

other types of repair, essentially involves the hydrolysis of

to involve the repair of damaged DNA, particularly

two phosphodiester bonds on the strand containing the

thymine dimers. Cells cultured from patients with xero-

defect. A special excision nuclease (exinuclease), consist-

derma pigmentosum exhibit low activity for the nu-

ing of at least three subunits in E coli and 16 polypep-

cleotide excision-repair process. Seven complementa-

tides in humans, accomplishes this task. In eukaryotic

tion groups have been identified using hybrid cell

cells the enzymes cut between the third to fifth phospho-

analyses, so at least seven gene products (XPA-XPG)

diester bond 3′ from the lesion, and on the 5′ side the cut

are involved. Two of these (XPA and XPC) are in-

is somewhere between the twenty-first and twenty-fifth

volved in recognition and excision. XPB and XPD are

bonds. Thus, a fragment of DNA 27-29 nucleotides

helicases and, interestingly, are subunits of the tran-

long is excised. After the strand is removed it is replaced,

scription factor TFIIH (see Chapter 37).

again by exact base pairing, through the action of yet an-

other polymerase (δ/ε in humans), and the ends are

Double-Strand Break Repair

joined to the existing strands by DNA ligase.

Xeroderma pigmentosum (XP) is an autosomal re-

The repair of double-strand breaks is part of the physio-

cessive genetic disease. The clinical syndrome includes

logic process of immunoglobulin gene rearrangement. It

338

CHAPTER 36

3′

5′

ase; and the gaps are filled and closed by DNA ligase.

This repair mechanism is illustrated in Figure 36-25.

5′

3′

RECOGNITION AND UNWINDING

Some Repair Enzymes Are Multifunctional

Somewhat surprising is the recent observation that

3′

5′

DNA repair proteins can serve other purposes. For ex-

ample, some repair enzymes are also found as compo-

5′

3′

nents of the large TFIIH complex that plays a central

role in gene transcription (Chapter 37). Another com-

OLIGONUCLEOTIDE EXCISION

ponent of TFIIH is involved in cell cycle regulation.

BY CUTTING AT TWO SITES

Thus, three critical cellular processes may be linked

3′

5′

through use of common proteins. There is also good

evidence that some repair enzymes are involved in gene

5′

3′

rearrangements that occur normally.

In patients with ataxia-telangiectasia, an autosomal

DEGRADATION OF MUTATED DNA

recessive disease in humans resulting in the development

RESYNTHESIS AND RELIGATION

of cerebellar ataxia and lymphoreticular neoplasms,

3′

5′

there appears to exist an increased sensitivity to damage

by x-ray. Patients with Fanconi’s anemia, an autosomal

5′

3′

recessive anemia characterized also by an increased fre-

Figure 36-24. Nucleotide excision-repair. This

quency of cancer and by chromosomal instability, prob-

mechanism is employed to correct larger defects in

ably have defective repair of cross-linking damage.

DNA and generally involves more proteins than either

mismatch or base excision-repair. After defect recogni-

tion (indicated by XXXX) and unwinding of the DNA en-

compassing the defect, an excision nuclease (exinucle-

Ku and DNA-PK bind

ase) cuts the DNA upstream and downstream of the

defective region. This gap is then filled in by a poly-

merase (δ/ε in humans) and religated.

Approximation

is also an important mechanism for repairing damaged

DNA, such as occurs as a result of ionizing radiation or

oxidative free radical generation. Some chemotherapeu-

Unwinding

tic agents destroy cells by causing ds breaks or prevent-

ing their repair.

Two proteins are initially involved in the nonho-

mologous rejoining of a ds break. Ku, a heterodimer of

70 kDa and 86 kDa subunits, binds to free DNA ends

Alignment and base pairing

and has latent ATP-dependent helicase activity. The

DNA-bound Ku heterodimer recruits a unique protein

kinase, DNA-dependent protein kinase (DNA-PK).

DNA-PK has a binding site for DNA free ends and an-

other for dsDNA just inside these ends. It therefore al-

Ligation

lows for the approximation of the two separated ends.

The free end DNA-Ku-DNA-PK complex activates the

kinase activity in the latter. DNA-PK reciprocally phos-

Figure 36-25. Double-strand break repair of DNA.

phorylates Ku and the other DNA-PK molecule, on the

The proteins Ku and DNA-dependent protein kinase

opposing strand, in trans. DNA-PK then dissociates

combine to approximate the two strands and unwind

from the DNA and Ku, resulting in activation of the

them. The aligned fragments form base pairs; the extra

Ku helicase. This results in unwinding of the two ends.

ends are removed, probably by a DNA-PK-associated

The unwound, approximated DNA forms base pairs;

endo- or exonuclease, and the gaps are filled in; and

the extra nucleotide tails are removed by an exonucle-

continuity is restored by ligation.

DNA ORGANIZATION, REPLICATION, & REPAIR

339

All three of these clinical syndromes are associated

•

Much of the DNA is associated with histone proteins

with an increased frequency of cancer. It is likely that

to form a structure called the nucleosome. Nucleo-

other human diseases resulting from disordered DNA

somes are composed of an octamer of histones and

repair capabilities will be found in the future.

150 bp of DNA.

•

Nucleosomes and higher-order structures formed

DNA & Chromosome Integrity Is

from them serve to compact the DNA.

Monitored Throughout the Cell Cycle

•

As much as 90% of DNA may be transcriptionally

inactive as a result of being nuclease-resistant, highly

Given the importance of normal DNA and chromosome

compacted, and nucleosome-associated.

function to survival, it is not surprising that eukaryotic

•

DNA in transcriptionally active regions is sensitive to

cells have developed elaborate mechanisms to monitor

the integrity of the genetic material. As detailed above, a

nuclease attack; some regions are exceptionally sensi-

tive and are often found to contain transcription

number of complex multi-subunit enzyme systems have

evolved to repair damaged DNA at the nucleotide se-

control sites.

quence level. Similarly, DNA mishaps at the chromo-

•

Transcriptionally active DNA (the genes) is often

some level are also monitored and repaired. As shown in

clustered in regions of each chromosome. Within

Figure

36-20, DNA integrity and chromosomal in-

these regions, genes may be separated by inactive

tegrity are continuously monitored throughout the cell

DNA in nucleosomal structures. The transcription

cycle. The four specific steps at which this monitoring

unit—that portion of a gene that is copied by RNA

occurs have been termed checkpoint controls. If prob-

polymerase—consists of coding regions of DNA

lems are detected at any of these checkpoints, progression

(exons) interrupted by intervening sequences of non-

through the cycle is interrupted and transit through the

coding DNA (introns).

cell cycle is halted until the damage is repaired. The mol-

•

After transcription, during RNA processing, introns

ecular mechanisms underlying detection of DNA dam-

are removed and the exons are ligated together to

age during the G1 and G2 phases of the cycle are under-

form the mature mRNA that appears in the cyto-

stood better than those operative during S and M phases.

plasm.

The tumor suppressor p53, a protein of MW 53

•

DNA in each chromosome is exactly replicated ac-

kDa, plays a key role in both G1 and G2 checkpoint con-

cording to the rules of base pairing during the S

trol. Normally a very unstable protein, p53 is a DNA

phase of the cell cycle.

binding transcription factor, one of a family of related

•

Each strand of the double helix is replicated simulta-

proteins, that is somehow stabilized in response to DNA

neously but by somewhat different mechanisms. A

damage, perhaps by direct p53-DNA interactions. In-

complex of proteins, including DNA polymerase,

creased levels of p53 activate transcription of an ensemble

replicates the leading strand continuously in the 5′ to

of genes that collectively serve to delay transit through the

3′ direction. The lagging strand is replicated discon-

cycle. One of these induced proteins, p21^CIP, is a potent

tinuously, in short pieces of 150-250 nucleotides, in

CDK-cyclin inhibitor (CKI) that is capable of efficiently

the 3′ to 5′ direction.

inhibiting the action of all CDKs. Clearly, inhibition of

•

DNA replication occurs at several sites—called repli-

CDKs will halt progression through the cell cycle (see

cation bubbles—in each chromosome. The entire

Figures 36-19 and 36-20). If DNA damage is too exten-

process takes about 9 hours in a typical cell.

sive to repair, the affected cells undergo apoptosis (pro-

grammed cell death) in a p53-dependent fashion. In this

•

A variety of mechanisms employing different en-

case, p53 induces the activation of a collection of genes

zymes repair damaged DNA, as after exposure to

that induce apoptosis. Cells lacking functional p53 fail to

chemical mutagens or ultraviolet radiation.

undergo apoptosis in response to high levels of radiation

or DNA-active chemotherapeutic agents. It may come as

REFERENCES

no surprise, then, that p53 is one of the most frequently

DePamphilis ML: Origins of DNA replication in metazoan chro-

mutated genes in human cancers. Additional research into

mosomes. J Biol Chem 1993;268:1.

the mechanisms of checkpoint control will prove invalu-

Hartwell LH, Kastan MB: Cell cycle control and cancer. Science

able for the development of effective anticancer therapeu-

1994;266:1821.

tic options.

Jenuwein T, Allis CD: Translating the histone code. Science 2001;

293:1074.

Lander ES et al: Initial sequencing and analysis of the human

SUMMARY

genome. Nature 2001;409:860.

• DNA in eukaryotic cells is associated with a variety

Luger L et al: Crystal structure of the nucleosome core particle at

of proteins, resulting in a structure called chromatin.

2.8 Å resolution. Nature 1997;398:251.

RNA Synthesis, Processing,

& Modification

Daryl K. Granner, MD, & P. Anthony Weil, PhD

BIOMEDICAL IMPORTANCE

following: (1) ribonucleotides are used in RNA synthe-

sis rather than deoxyribonucleotides; (2) U replaces T

The synthesis of an RNA molecule from DNA is a

as the complementary base pair for A in RNA; (3) a

complex process involving one of the group of RNA

primer is not involved in RNA synthesis; (4) only a very

polymerase enzymes and a number of associated pro-

small portion of the genome is transcribed or copied

teins. The general steps required to synthesize the pri-

into RNA, whereas the entire genome must be copied

mary transcript are initiation, elongation, and termina-

during DNA replication; and (5) there is no proofread-

tion. Most is known about initiation. A number of

ing function during RNA transcription.

DNA regions (generally located upstream from the ini-

The process of synthesizing RNA from a DNA tem-

tiation site) and protein factors that bind to these se-

plate has been characterized best in prokaryotes. Al-

quences to regulate the initiation of transcription have

though in mammalian cells the regulation of RNA syn-

been identified. Certain RNAs—mRNAs in particu-

thesis and the processing of the RNA transcripts are

lar—have very different life spans in a cell. It is impor-

different from those in prokaryotes, the process of RNA

tant to understand the basic principles of messenger

synthesis per se is quite similar in these two classes of

RNA synthesis and metabolism, for modulation of this

organisms. Therefore, the description of RNA synthesis

process results in altered rates of protein synthesis and

in prokaryotes, where it is better understood, is applica-

thus a variety of metabolic changes. This is how all or-

ble to eukaryotes even though the enzymes involved

ganisms adapt to changes of environment. It is also how

and the regulatory signals are different.

differentiated cell structures and functions are estab-

lished and maintained. The RNA molecules synthe-

sized in mammalian cells are made as precursor mole-

cules that have to be processed into mature, active

The Template Strand of DNA

RNA. Errors or changes in synthesis, processing, and

Is Transcribed

splicing of mRNA transcripts are a cause of disease.

The sequence of ribonucleotides in an RNA molecule is

complementary to the sequence of deoxyribonu-

RNA EXISTS IN FOUR MAJOR CLASSES

cleotides in one strand of the double-stranded DNA

All eukaryotic cells have four major classes of RNA: ri-

molecule (Figure 35-8). The strand that is transcribed

bosomal RNA (rRNA), messenger RNA (mRNA), trans-

or copied into an RNA molecule is referred to as the

fer RNA (tRNA), and small nuclear RNA (snRNA).

template strand of the DNA. The other DNA strand is

The first three are involved in protein synthesis, and

frequently referred to as the coding strand of that gene.

snRNA is involved in mRNA splicing. As shown in

It is called this because, with the exception of T for U

Table 37-1, these various classes of RNA are different

changes, it corresponds exactly to the sequence of the

in their diversity, stability, and abundance in cells.

primary transcript, which encodes the protein product

of the gene. In the case of a double-stranded DNA mol-

ecule containing many genes, the template strand for

RNA IS SYNTHESIZED FROM A DNA

each gene will not necessarily be the same strand of the

TEMPLATE BY AN RNA POLYMERASE

DNA double helix (Figure 37-1). Thus, a given strand

The processes of DNA and RNA synthesis are similar

of a double-stranded DNA molecule will serve as the

in that they involve (1) the general steps of initiation,

template strand for some genes and the coding strand

elongation, and termination with 5′ to 3′ polarity; (2)

of other genes. Note that the nucleotide sequence of an

large, multicomponent initiation complexes; and (3)

RNA transcript will be the same (except for U replacing

adherence to Watson-Crick base-pairing rules. These

T) as that of the coding strand. The information in the

processes differ in several important ways, including the

template strand is read out in the 3′ to 5′ direction.

341

342

CHAPTER 37

Table 37-1. Classes of eukaryotic RNA.

RNA transcript

5′ P-P-P

β′

RNA

Types

Abundance

Stability

Ribosomal

28S, 18S, 5.8S, 5S

80% of total

Very stable

3′

3′_OH

Transcription

5′

(rRNA)

Messenger

~10⁵ different

2-5% of total

Unstable to

(mRNA)

species

very

5′

stable

3′

Transfer

~60 different

~15% of total

Very stable

(tRNA)

species

RNAP complex

Small nuclear

~30 different

≤ 1% of total

Very stable

Figure 37-2. RNA polymerase (RNAP) catalyzes the

(snRNA)

species

polymerization of ribonucleotides into an RNA se-

quence that is complementary to the template strand

of the gene. The RNA transcript has the same polarity

DNA-Dependent RNA Polymerase

(5′ to 3′) as the coding strand but contains U rather

Initiates Transcription at a Distinct

than T. E coli RNAP consists of a core complex of two

Site, the Promoter

α subunits and two β subunits (β and β′). The holoen-

DNA-dependent RNA polymerase is the enzyme re-

zyme contains the σ subunit bound to the α₂ββ′ core

sponsible for the polymerization of ribonucleotides into

assembly. The ω subunit is not shown. The transcription

a sequence complementary to the template strand of

“bubble” is an approximately 20-bp area of melted

the gene (see Figures 37-2 and 37-3). The enzyme at-

DNA, and the entire complex covers 30-75 bp, depend-

taches at a specific site—the promoter—on the tem-

ing on the conformation of RNAP.

plate strand. This is followed by initiation of RNA syn-

thesis at the starting point, and the process continues

until a termination sequence is reached (Figure 37-3).

(1) Template binding

A transcription unit is defined as that region of DNA

ATP + NTP

that includes the signals for transcription initiation,

elongation, and termination. The RNA product, which

RNAP

is synthesized in the 5′ to 3′ direction, is the primary

(2) Chain initiation

transcript. In prokaryotes, this can represent the prod-

pppApN

uct of several contiguous genes; in mammalian cells, it

pppApN

usually represents the product of a single gene. The 5′

NTPs

terminals of the primary RNA transcript and the ma-

(5) Chain termination

ture cytoplasmic RNA are identical. Thus, the starting

and RNAP release

(3) Promoter

pppApN

clearance

point of transcription corresponds to the 5

nu-

NTPs

cleotide of the mRNA. This is designated position +1,

as is the corresponding nucleotide in the DNA. The

(4) Chain elongation

Figure 37-3. The transcription cycle in bacteria. Bac-

Gene A Gene B Gene C

Gene D

terial RNA transcription is described in four steps:

5′

3′

(1) Template binding: RNA polymerase (RNAP) binds

3′

5′

to DNA and locates a promoter (P) melts the two DNA

strands to form a preinitiation complex (PIC). (2) Chain

Template strands

initiation: RNAP holoenzyme (core + one of multiple

Figure 37-1. This figure illustrates that genes can be

sigma factors) catalyzes the coupling of the first base

transcribed off both strands of DNA. The arrowheads in-

(usually ATP or GTP) to a second ribonucleoside

dicate the direction of transcription (polarity). Note that

triphosphate to form a dinucleotide. (3) Chain elonga-

the template strand is always read in the 3′ to 5′ direc-

tion: Successive residues are added to the 3′-OH termi-

tion. The opposite strand is called the coding strand be-

nus of the nascent RNA molecule. (4) Chain termina-

cause it is identical (except for T for U changes) to the

tion and release: The completed RNA chain and RNAP

mRNA transcript (the primary transcript in eukaryotic

are released from the template. The RNAP holoenzyme

cells) that encodes the protein product of the gene.

re-forms, finds a promoter, and the cycle is repeated.

RNA SYNTHESIS, PROCESSING, & MODIFICATION

343

numbers increase as the sequence proceeds downstream.

Table 37-2. Nomenclature and properties of

This convention makes it easy to locate particular re-

mammalian nuclear DNA-dependent RNA

gions, such as intron and exon boundaries. The nu-

polymerases.

cleotide in the promoter adjacent to the transcription

initiation site is designated −1, and these negative num-

Form of RNA

Sensitivity to

bers increase as the sequence proceeds upstream, away

Polymerase

-Amanitin

Major Products

from the initiation site. This provides a conventional

way of defining the location of regulatory elements in

I (A)

Insensitive

rRNA

the promoter.

II (B)

High sensitivity

mRNA

The primary transcripts generated by RNA polym-

III (C)

Intermediate sensitivity

tRNA/5S rRNA

erase II—one of three distinct nuclear DNA-depen-

dent RNA polymerases in eukaryotes—are promptly

capped by 7-methylguanosine triphosphate caps (Fig-

ferent sets of genes. The sizes of the RNA polymerases

ure 35-10) that persist and eventually appear on the 5′

range from MW 500,000 to MW 600,000. These en-

end of mature cytoplasmic mRNA. These caps are nec-

zymes are much more complex than prokaryotic RNA

essary for the subsequent processing of the primary

polymerases. They all have two large subunits and a

transcript to mRNA, for the translation of the mRNA,

number of smaller subunits—as many as 14 in the case

and for protection of the mRNA against exonucleolytic

of RNA pol III. The eukaryotic RNA polymerases have

attack.

extensive amino acid homologies with prokaryotic

RNA polymerases. This homology has been shown re-

Bacterial DNA-Dependent RNA

cently to extend to the level of three-dimensional struc-

Polymerase Is a Multisubunit Enzyme

tures. The functions of each of the subunits are not yet

fully understood. Many could have regulatory func-

The DNA-dependent RNA polymerase (RNAP) of the

tions, such as serving to assist the polymerase in the

bacterium Escherichia coli exists as an approximately

recognition of specific sequences like promoters and

400 kDa core complex consisting of two identical α

termination signals.

subunits, similar but not identical β and β′ subunits,

One peptide toxin from the mushroom Amanita

and an ω subunit. Beta is thought to be the catalytic

phalloides, α-amanitin, is a specific differential inhibitor

subunit (Figure 37-2). RNAP, a metalloenzyme, also

of the eukaryotic nuclear DNA-dependent RNA polym-

contains two zinc molecules. The core RNA polymerase

erases and as such has proved to be a powerful research

associates with a specific protein factor (the sigma [σ]

tool (Table 37-2). α-Amanitin blocks the translocation

factor) that helps the core enzyme recognize and bind

of RNA polymerase during transcription.

to the specific deoxynucleotide sequence of the pro-

moter region (Figure 37-5) to form the preinitiation

complex (PIC). Sigma factors have a dual role in the

process of promoter recognition; σ association with

RNA SYNTHESIS IS A CYCLICAL PROCESS

core RNA polymerase decreases its affinity for nonpro-

& INVOLVES INITIATION, ELONGATION,

moter DNA while simultaneously increasing holoen-

& TERMINATION

zyme affinity for promoter DNA. Bacteria contain mul-

tiple σ factors, each of which acts as a regulatory

The process of RNA synthesis in bacteria—depicted in

protein that modifies the promoter recognition speci-

Figure 37-3—involves first the binding of the RNA

ficity of the RNA polymerase. The appearance of dif-

holopolymerase molecule to the template at the pro-

ferent σ factors can be correlated temporally with vari-

moter site to form a PIC. Binding is followed by a con-

ous programs of gene expression in prokaryotic systems

formational change of the RNAP, and the first nu-

such as bacteriophage development, sporulation, and

cleotide (almost always a purine) then associates with

the response to heat shock.

the initiation site on the β subunit of the enzyme. In

the presence of the appropriate nucleotide, the RNAP

catalyzes the formation of a phosphodiester bond, and

Mammalian Cells Possess Three

the nascent chain is now attached to the polymerization

Distinct Nuclear DNA-Dependent

site on the β subunit of RNAP. (The analogy to the A

RNA Polymerases

and P sites on the ribosome should be noted; see Figure

The properties of mammalian polymerases are de-

38-9.)

scribed in Table 37-2. Each of these DNA-dependent

Initiation of formation of the RNA molecule at its

RNA polymerases is responsible for transcription of dif-

5′ end then follows, while elongation of the RNA mole-

344

CHAPTER 37

cule from the 5′ to its 3′ end continues cyclically, an-

tiparallel to its template. The enzyme polymerizes the

ribonucleotides in a specific sequence dictated by the

template strand and interpreted by Watson-Crick base-

pairing rules. Pyrophosphate is released in the polymer-

ization reaction. This pyrophosphate (PP_i) is rapidly

degraded to 2 mol of inorganic phosphate (P_i ) by ubiq-

uitous pyrophosphatases, thereby providing irreversibil-

ity on the overall synthetic reaction. In both prokary-

otes and eukaryotes, a purine ribonucleotide is usually

the first to be polymerized into the RNA molecule. As

with eukaryotes, 5′ triphosphate of this first nucleotide

is maintained in prokaryotic mRNA.

As the elongation complex containing the core

RNA polymerase progresses along the DNA molecule,

DNA unwinding must occur in order to provide access

for the appropriate base pairing to the nucleotides of

the coding strand. The extent of this transcription bub-

ble (ie, DNA unwinding) is constant throughout tran-

scription and has been estimated to be about 20 base

pairs per polymerase molecule. Thus, it appears that the

size of the unwound DNA region is dictated by the

polymerase and is independent of the DNA sequence in

the complex. This suggests that RNA polymerase has

associated with it an “unwindase” activity that opens

Figure 37-4.

Electron photomicrograph of multiple

the DNA helix. The fact that the DNA double helix

copies of amphibian ribosomal RNA genes in the

must unwind and the strands part at least transiently

process of being transcribed. The magnification is

for transcription implies some disruption of the nucleo-

about 6000 ×. Note that the length of the transcripts in-

some structure of eukaryotic cells. Topoisomerase both

creases as the RNA polymerase molecules progress

precedes and follows the progressing RNAP to prevent

along the individual ribosomal RNA genes; transcrip-

the formation of superhelical complexes.

Termination of the synthesis of the RNA molecule

tion start sites (filled circles) to transcription termina-

in bacteria is signaled by a sequence in the template

tion sites (open circles). RNA polymerase I (not visual-

strand of the DNA molecule—a signal that is recog-

ized here) is at the base of the nascent rRNA transcripts.

nized by a termination protein, the rho (ρ) factor. Rho

Thus, the proximal end of the transcribed gene has

is an ATP-dependent RNA-stimulated helicase that

short transcripts attached to it, while much longer tran-

disrupts the nascent RNA-DNA complex. After termi-

scripts are attached to the distal end of the gene. The

nation of synthesis of the RNA molecule, the enzyme

arrows indicate the direction (5′ to 3′) of transcription.

separates from the DNA template and probably disso-

(Reproduced with permission, from Miller OL Jr, Beatty BR:

ciates to free core enzyme and free σ factor. With the

Portrait of a gene. J Cell Physiol 1969;74[Suppl 1]:225.)

assistance of another σ factor, the core enzyme then

recognizes a promoter at which the synthesis of a new

RNA molecule commences. In eukaryotic cells, termi-

nation is less well defined. It appears to be somehow

THE FIDELITY & FREQUENCY OF

linked both to initiation and to addition of the 3′

TRANSCRIPTION IS CONTROLLED

polyA tail of mRNA and could involve destabilization

BY PROTEINS BOUND TO CERTAIN

of the RNA-DNA complex at a region of A-U base

DNA SEQUENCES

pairs. More than one RNA polymerase molecule may

transcribe the same template strand of a gene simulta-

The DNA sequence analysis of specific genes has al-

neously, but the process is phased and spaced in such a

lowed the recognition of a number of sequences impor-

way that at any one moment each is transcribing a dif-

tant in gene transcription. From the large number of

ferent portion of the DNA sequence. An electron mi-

bacterial genes studied it is possible to construct con-

crograph of extremely active RNA synthesis is shown

sensus models of transcription initiation and termina-

in Figure 37-4.

tion signals.

RNA SYNTHESIS, PROCESSING, & MODIFICATION

345

The question, “How does RNAP find the correct

tion start site there is a consensus sequence of eight nu-

site to initiate transcription?” is not trivial when the

cleotide pairs (5′-TGTTGACA-3′) to which the RNAP

complexity of the genome is considered. E coli has

binds to form the so-called closed complex. More

4 × 10³ transcription initiation sites in 4 × 10⁶ base

proximal to the transcription start site—about ten nu-

pairs (bp) of DNA. The situation is even more complex

cleotides upstream—is a six-nucleotide-pair A+T-rich

in humans, where perhaps 10⁵ transcription initiation

sequence (5′-TATAAT-3′). These conserved sequence

sites are distributed throughout in 3 × 10⁹ bp of DNA.

elements comprising the promoter are shown schemati-

RNAP can bind to many regions of DNA, but it scans

cally in Figure 37-5. The latter sequence has a low

the DNA sequence—at a rate of ≥ 10³ bp/s—until it

melting temperature because of its deficiency of GC

recognizes certain specific regions of DNA to which it

nucleotide pairs. Thus, the TATA box is thought to

binds with higher affinity. This region is called the pro-

ease the dissociation between the two DNA strands so

moter, and it is the association of RNAP with the pro-

that RNA polymerase bound to the promoter region

moter that ensures accurate initiation of transcription.

can have access to the nucleotide sequence of its imme-

The promoter recognition-utilization process is the tar-

diately downstream template strand. Once this process

get for regulation in both bacteria and humans.

occurs, the combination of RNA polymerase plus pro-

moter is called the open complex. Other bacteria have

slightly different consensus sequences in their promot-

Bacterial Promoters Are Relatively Simple

ers, but all generally have two components to the pro-

Bacterial promoters are approximately 40 nucleotides

moter; these tend to be in the same position relative to

(40 bp or four turns of the DNA double helix) in

the transcription start site, and in all cases the sequences

length, a region small enough to be covered by an

between the boxes have no similarity but still provide

E coli RNA holopolymerase molecule. In this consensus

critical spacing functions facilitating recognition of −35

promoter region are two short, conserved sequence ele-

and −10 sequence by RNA polymerase holoenzyme.

ments. Approximately 35 bp upstream of the transcrip-

Within a bacterial cell, different sets of genes are often

TRANSCRIPTION UNIT

Promoter

Transcribed region

Transcription

start site

Coding strand 5′

Termination

3′

Template strand 3′^{TGTTGACATATAAT}

signals

5′^DNA

−35

−10

region

PPP

RNA

3′

5′

5′ Flanking

3′ Flanking

sequences

Figure 37-5. Bacterial promoters, such as that from E coli shown here,

share two regions of highly conserved nucleotide sequence. These regions

are located 35 and 10 bp upstream (in the 5′ direction of the coding strand)

from the start site of transcription, which is indicated as +1. By convention,

all nucleotides upstream of the transcription initiation site (at +1) are num-

bered in a negative sense and are referred to as 5′-flanking sequences. Also

by convention, the DNA regulatory sequence elements (TATA box, etc) are

described in the 5′ to 3′ direction and as being on the coding strand. These

elements function only in double-stranded DNA, however. Note that the

transcript produced from this transcription unit has the same polarity or

“sense” (ie, 5′ to 3′ orientation) as the coding strand. Termination cis-

elements reside at the end of the transcription unit (see Figure 37-6 for

more detail). By convention the sequences downstream of the site at which

transcription termination occurs are termed 3′-flanking sequences.

346

CHAPTER 37

coordinately regulated. One important way that this is

simplex virus, which utilizes transcription factors of its

accomplished is through the fact that these co-regulated

mammalian host for gene expression, there is a single

genes share unique −35 and −10 promoter sequences.

unique transcription start site, and accurate transcription

These unique promoters are recognized by different σ

from this start site depends upon a nucleotide sequence

factors bound to core RNA polymerase.

located 32 nucleotides upstream from the start site (ie, at

Rho-dependent transcription termination signals

−32) (Figure 37-7). This region has the sequence of

in E coli also appear to have a distinct consensus se-

TATAAAAG and bears remarkable similarity to the

quence, as shown in Figure 37-6. The conserved con-

functionally related TATA box that is located about 10

sensus sequence, which is about 40 nucleotide pairs in

bp upstream from the prokaryotic mRNA start site (Fig-

length, can be seen to contain a hyphenated or inter-

ure 37-5). Mutation or inactivation of the TATA box

rupted inverted repeat followed by a series of AT base

markedly reduces transcription of this and many other

pairs. As transcription proceeds through the hyphen-

genes that contain this consensus cis element (see Figures

ated, inverted repeat, the generated transcript can form

37-7, 37-8). Most mammalian genes have a TATA box

the intramolecular hairpin structure, also depicted in

that is usually located 25-30 bp upstream from the tran-

Figure 37-6.

scription start site. The consensus sequence for a TATA

Transcription continues into the AT region, and

box is TATAAA, though numerous variations have been

with the aid of the ρ termination protein the RNA

characterized. The TATA box is bound by 34 kDa

polymerase stops, dissociates from the DNA template,

TATA binding protein (TBP), which in turn binds sev-

and releases the nascent transcript.

eral other proteins called TBP-associated factors

(TAFs). This complex of TBP and TAFs is referred to as

TFIID. Binding of TFIID to the TATA box sequence is

Eukaryotic Promoters Are More Complex

thought to represent the first step in the formation of the

It is clear that the signals in DNA which control tran-

transcription complex on the promoter.

scription in eukaryotic cells are of several types. Two

A small number of genes lack a TATA box. In such

types of sequence elements are promoter-proximal. One

instances, two additional cis elements, an initiator se-

of these defines where transcription is to commence

quence (Inr) and the so-called downstream promoter

along the DNA, and the other contributes to the mecha-

element (DPE), direct RNA polymerase II to the pro-

nisms that control how frequently this event is to occur.

moter and in so doing provide basal transcription start-

For example, in the thymidine kinase gene of the herpes

ing from the correct site. The Inr element spans the start

Direction of transcription

Coding strand

5′

AGCCCGC

GCGGGCT

TTTTTTTT

3′

DNA

Template strand

3′

TCGGGCG

CGCCCGA

AAAAAAAA

5′

Coding strand

5′

3′

DNA

Template strand

3′

AAAAAAAA

5′

UUUUUUU-3′

RNA transcript

5′

Figure 37-6. The predominant bacterial transcription termination signal contains an inverted, hyphenated re-

peat (the two boxed areas) followed by a stretch of AT base pairs (top figure). The inverted repeat, when tran-

scribed into RNA, can generate the secondary structure in the RNA transcript shown at the bottom of the figure.

Formation of this RNA hairpin causes RNA polymerase to pause and subsequently the ρ termination factor inter-

acts with the paused polymerase and somehow induces chain termination.

RNA SYNTHESIS, PROCESSING, & MODIFICATION

347

Promoter proximal

Promoter

upstream elements

TFIID

Sp1

CAAT

TATA box

tk coding region

CTF

−25

Sp1

Figure 37-7. Transcription elements and binding factors in the herpes simplex virus thymidine ki-

nase (tk) gene. DNA-dependent RNA polymerase II binds to the region of the TATA box (which is bound

by transcription factor TFIID) to form a multicomponent preinitiation complex capable of initiating

transcription at a single nucleotide (+1). The frequency of this event is increased by the presence of up-

stream cis-acting elements (the GC and CAAT boxes). These elements bind trans-acting transcription

factors, in this example Sp1 and CTF (also called C/EBP, NF1, NFY). These cis elements can function inde-

pendently of orientation (arrows).

Regulated expression

“Basal” expression

Distal

Promoter

regulatory

proximal

Promoter

elements

Other

Enhancer (+)

Promoter

regulatory

and

proximal

Inr

DPE

elements

repressor (−)

elements

TATA

Coding region

elements

(GC/CAAT, etc)

Figure 37-8. Schematic diagram showing the transcription control regions in a hypothetical class II

(mRNA-producing) eukaryotic gene. Such a gene can be divided into its coding and regulatory regions,

as defined by the transcription start site (arrow; +1). The coding region contains the DNA sequence that

is transcribed into mRNA, which is ultimately translated into protein. The regulatory region consists of

two classes of elements. One class is responsible for ensuring basal expression. These elements gener-

ally have two components. The proximal component, generally the TATA box, or Inr or DPE elements di-

rect RNA polymerase II to the correct site (fidelity). In TATA-less promoters, an initiator (Inr) element that

spans the initiation site (+1) may direct the polymerase to this site. Another component, the upstream

elements, specifies the frequency of initiation. Among the best studied of these is the CAAT box, but

several other elements (Sp1, NF1, AP1, etc) may be used in various genes. A second class of regulatory

cis-acting elements is responsible for regulated expression. This class consists of elements that enhance

or repress expression and of others that mediate the response to various signals, including hormones,

heat shock, heavy metals, and chemicals. Tissue-specific expression also involves specific sequences of

this sort. The orientation dependence of all the elements is indicated by the arrows within the boxes. For

example, the proximal element (the TATA box) must be in the 5′ to 3′ orientation. The upstream ele-

ments work best in the 5′ to 3′ orientation, but some of them can be reversed. The locations of some el-

ements are not fixed with respect to the transcription start site. Indeed, some elements responsible for

regulated expression can be located either interspersed with the upstream elements, or they can be lo-

cated downstream from the start site.

348

CHAPTER 37

site (from −3 to +5) and consists of the general consen-

below and Figures 37-9 and 37-10). The protein-

sus sequence TCA+1 G/T T T/C which is similar to the

DNA interaction at the TATA box involving RNA

initiation site sequence per se. (A+1 indicates the first

polymerase II and other components of the basal tran-

nucleotide transcribed.) The proteins that bind to Inr in

scription machinery ensures the fidelity of initiation.

order to direct pol II binding include TFIID. Promoters

Together, then, the promoter and promoter-proxi-

that have both a TATA box and an Inr may be stronger

mal cis-active upstream elements confer fidelity and fre-

than those that have just one of these elements. The

quency of initiation upon a gene. The TATA box has a

DPE has the consensus sequence A/GGA/T CGTG and

particularly rigid requirement for both position and ori-

is localized about 25 bp downstream of the +1 start site.

entation. Single-base changes in any of these cis ele-

Like the Inr, DPE sequences are also bound by the TAF

ments have dramatic effects on function by reducing

subunits of TFIID. In a survey of over 200 eukaryotic

the binding affinity of the cognate trans factors (either

genes, roughly 30% contained a TATA box and Inr,

TFIID/TBP or Sp1, CTF, and similar factors). The

25% contained Inr and DPE, 15% contained all three

spacing of these elements with respect to the transcrip-

elements, while ~30% contained just the Inr.

tion start site can also be critical. This is particularly

Sequences farther upstream from the start site deter-

true for the TATA box Inr and DPE.

mine how frequently the transcription event occurs.

A third class of sequence elements can either increase

Mutations in these regions reduce the frequency of

or decrease the rate of transcription initiation of eukary-

transcriptional starts tenfold to twentyfold. Typical of

otic genes. These elements are called either enhancers or

these DNA elements are the GC and CAAT boxes, so

repressors (or silencers), depending on which effect

named because of the DNA sequences involved. As il-

they have. They have been found in a variety of locations

lustrated in Figure 37-7, each of these boxes binds a

both upstream and downstream of the transcription start

protein, Sp1 in the case of the GC box and CTF (or

site and even within the transcribed portions of some

C/EPB,NF1,NFY) by the CAAT box; both bind

genes. In contrast to proximal and upstream promoter el-

through their distinct DNA binding domains (DBDs).

ements, enhancers and silencers can exert their effects

The frequency of transcription initiation is a conse-

when located hundreds or even thousands of bases away

quence of these protein-DNA interactions and complex

from transcription units located on the same chromo-

interactions between particular domains of the tran-

some. Surprisingly, enhancers and silencers can function

scription factors (distinct from the DBD domains—so-

in an orientation-independent fashion. Literally hun-

called activation domains; ADs) of these proteins and

dreds of these elements have been described. In some

the rest of the transcription machinery (RNA polym-

cases, the sequence requirements for binding are rigidly

erase II and the basal factors TFIIA, B, D, E, F). (See

constrained; in others, considerable sequence variation is

TATA

pol II

-50

-30

-10

+10

+30

+50

Figure 37-9. The eukaryotic basal transcription complex. Formation of the basal transcription complex begins

when TFIID binds to the TATA box. It directs the assembly of several other components by protein-DNA and

protein-protein interactions. The entire complex spans DNA from position −30 to +30 relative to the initiation site

(+1, marked by bent arrow). The atomic level, x-ray-derived structures of RNA polymerase II alone and of TBP

bound to TATA promoter DNA in the presence of either TFIIB or TFIIA have all been solved at 3 Å resolution. The

structure of TFIID complexes have been determined by electron microscopy at 30 Å resolution. Thus, the molecu-

lar structures of the transcription machinery are beginning to be elucidated. Much of this structural information is

consistent with the models presented here.

RNA SYNTHESIS, PROCESSING, & MODIFICATION

349

Rate of

transcription

Basal

TBP

complex

TAF

CTF TAF

CCAAT

TBP

Basal complex

CAAT

TATA

nil

CTF

Basal

TBP

complex

TAF

CTF

TAF TAF

CAAT

TATA

nil

TAF

TBP

Basal

Basal complex

TBP

complex

TATA

Figure 37-10. Two models for assembly of the active transcription complex and for how activators and coacti-

vators might enhance transcription. Shown here as a small oval is TBP, which contains TFIID, a large oval that con-

tains all the components of the basal transcription complex illustrated in Figure 37-9 (ie, RNAP II and TFIIA, TFIIB,

TFIIE, TFIIF, and TFIIH). Panel A: The basal transcription complex is assembled on the promoter after the TBP sub-

unit of TFIID is bound to the TATA box. Several TAFs (coactivators) are associated with TBP. In this example, a tran-

scription activator, CTF, is shown bound to the CAAT box, forming a loop complex by interacting with a TAF

bound to TBP. Panel B: The recruitment model. The transcription activator CTF binds to the CAAT box and inter-

acts with a coactivator (TAF in this case). This allows for an interaction with the preformed TBP-basal transcription

complex. TBP can now bind to the TATA box, and the assembled complex is fully active.

allowed. Some sequences bind only a single protein, but

stood. However, it appears that the termination signals

the majority bind several different proteins. Similarly, a

exist far downstream of the coding sequence of eukary-

single protein can bind to more than one element.

otic genes. For example, the transcription termination

Hormone response elements (for steroids, T₃, reti-

signal for mouse β-globin occurs at several positions

noic acid, peptides, etc) act as—or in conjunction with—

1000-2000 bases beyond the site at which the poly(A)

enhancers or silencers (Chapter 43). Other processes

tail will eventually be added. Little is known about the

that enhance or silence gene expression—such as the re-

termination process or whether specific termination

sponse to heat shock, heavy metals (Cd2+ and Zn2+),

factors similar to the bacterial ρ factor are involved.

and some toxic chemicals (eg, dioxin)—are mediated

However, it is known that the mRNA 3′ terminal is

through specific regulatory elements. Tissue-specific ex-

generated posttranscriptionally, is somehow coupled to

pression of genes (eg, the albumin gene in liver, the he-

events or structures formed at the time and site of initi-

moglobin gene in reticulocytes) is also mediated by spe-

ation, depends on a special structure in one of the sub-

cific DNA sequences.

units of RNA polymerase II (the CTD; see below), and

appears to involve at least two steps. After RNA polym-

erase II has traversed the region of the transcription

Specific Signals Regulate

unit encoding the 3′ end of the transcript, an RNA en-

Transcription Termination

donuclease cleaves the primary transcript at a position

The signals for the termination of transcription by

about 15 bases 3′ of the consensus sequence AAUAAA

eukaryotic RNA polymerase II are very poorly under-

that serves in eukaryotic transcripts as a cleavage signal.

350

CHAPTER 37

Finally, this newly formed 3′ terminal is polyadenylated

distinct, polymerase-specific sets of TAFs, is also an im-

in the nucleoplasm, as described below.

portant component of class I and class III initiation

complexes even if they do not contain TATA boxes.

The binding of TBP marks a specific promoter for

THE EUKARYOTIC

transcription and is the only step in the assembly process

TRANSCRIPTION COMPLEX

that is entirely dependent on specific, high-affinity pro-

A complex apparatus consisting of as many as

tein-DNA interaction. Of several subsequent in vitro

unique proteins provides accurate and regulatable tran-

steps, the first is the binding of TFIIB to the TFIID-

scription of eukaryotic genes. The RNA polymerase en-

promoter complex. This results in a stable ternary com-

zymes (pol I, pol II, and pol III for class I, II, and III

plex which is then more precisely located and more

genes, respectively) transcribe information contained in

tightly bound at the transcription initiation site. This

the template strand of DNA into RNA. These polym-

complex then attracts and tethers the pol II-TFIIF com-

erases must recognize a specific site in the promoter in

plex to the promoter. TFIIF is structurally and func-

order to initiate transcription at the proper nucleotide.

tionally similar to the bacterial σ factor and is required

In contrast to the situation in prokaryotes, eukaryotic

for the delivery of pol II to the promoter. TFIIA binds

RNA polymerases alone are not able to discriminate be-

to this assembly and may allow the complex to respond

tween promoter sequences and other regions of DNA;

to activators, perhaps by the displacement of repressors.

thus, other proteins known as general transcription fac-

Addition of TFIIE and TFIIH is the final step in the as-

tors or GTFs facilitate promoter-specific binding of

sembly of the PIC. TFIIE appears to join the complex

these enzymes and formation of the preinitiation com-

with pol II-TFIIF, and TFIIH is then recruited. Each of

plex (PIC). This combination of components can cat-

these binding events extends the size of the complex so

alyze basal or (non)-unregulated transcription in vitro.

that finally about 60 bp (from −30 to +30 relative to +1,

Another set of proteins—coactivators—help regulate

the nucleotide from which transcription commences)

the rate of transcription initiation by interacting with

are covered (Figure 37-9). The PIC is now complete

transcription activators that bind to upstream DNA el-

and capable of basal transcription initiated from the cor-

ements (see below).

rect nucleotide. In genes that lack a TATA box, the

same factors, including TBP, are required. In such cases,

Formation of the Basal

an Inr or the DPEs (see Figure 37-8) position the com-

plex for accurate initiation of transcription.

Transcription Complex

In bacteria, a σ factor-polymerase complex selectively

Phosphorylation Activates Pol II

binds to DNA in the promoter forming the PIC. The

situation is more complex in eukaryotic genes. Class II

Eukaryotic pol II consists of

12 subunits. The two

genes—those transcribed by pol II to make mRNA—

largest subunits, both about 200 kDa, are homologous

are described as an example. In class II genes, the func-

to the bacterial β and β′ subunits. In addition to the in-

tion of σ factors is assumed by a number of proteins.

creased number of subunits, eukaryotic pol II differs

Basal transcription requires, in addition to pol II, a

from its prokaryotic counterpart in that it has a series of

number of GTFs called TFIIA, TFIIB, TFIID,

heptad repeats with consensus sequence Tyr-Ser-Pro-

TFIIE, TFIIF, and TFIIH. These GTFs serve to pro-

Thr-Ser-Pro-Ser at the carboxyl terminal of the largest

mote RNA polymerase II transcription on essentially all

pol II subunit. This carboxyl terminal repeat domain

genes. Some of these GTFs are composed of multiple

(CTD) has 26 repeated units in brewers’ yeast and 52

subunits. TFIID, which binds to the TATA box pro-

units in mammalian cells. The CTD is both a substrate

moter element, is the only one of these factors capa-

for several kinases, including the kinase component of

ble of binding to specific sequences of DNA. As de-

TFIIH, and a binding site for a wide array of proteins.

scribed above, TFIID consists of TATA binding

The CTD has been shown to interact with RNA pro-

protein (TBP) and 14 TBP-associated factors (TAFs).

cessing enzymes; such binding may be involved with

TBP binds to the TATA box in the minor groove of

RNA polyadenylation. The association of the factors

DNA (most transcription factors bind in the major

with the CTD of RNA polymerase II (and other com-

groove) and causes an approximately 100-degree bend

ponents of the basal machinery) somehow serves to

or kink of the DNA helix. This bending is thought to

couple initiation with mRNA 3′ end formation. Pol II

facilitate the interaction of TBP-associated factors with

is activated when phosphorylated on the Ser and Thr

other components of the transcription initiation com-

residues and displays reduced activity when the CTD is

plex and possibly with factors bound to upstream ele-

dephosphorylated. Pol II lacking the CTD tail is inca-

ments. Although defined as a component of class II

pable of activating transcription, which underscores the

gene promoters, TBP, by virtue of its association with

importance of this domain.

RNA SYNTHESIS, PROCESSING, & MODIFICATION

351

Pol II associates with other proteins to form a

TAFs. It is conceivable that different combinations of

holoenzyme complex. In yeast, at least nine gene prod-

TAFs with TBP—or one of several recently discovered

ucts—called Srb

(for suppressor of RNA polymer-

TBP-like factors (TLFs)—may bind to different pro-

ase B)—bind to the CTD. The Srb proteins—or medi-

moters, and recent reports suggest that this may ac-

ators, as they are also called—are essential for pol II

count for selective activation noted in various promot-

transcription, though their exact role in this process has

ers and for the different strengths of certain promoters.

not been defined. Related proteins comprising even

TAFs, since they are required for the action of acti-

more complex forms of RNA polymerase II have been

vators, are often called coactivators. There are thus

described in human cells.

three classes of transcription factors involved in the reg-

ulation of class II genes: basal factors, coactivators, and

The Role of Transcription Activators

activator-repressors (Table 37-4). How these classes of

proteins interact to govern both the site and frequency

& Coactivators

of transcription is a question of central importance.

TFIID was originally considered to be a single protein.

However, several pieces of evidence led to the impor-

Two Models Explain the Assembly

tant discovery that TFIID is actually a complex consist-

of the Preinitiation Complex

ing of TBP and the 14 TAFs. The first evidence that

TFIID was more complex than just the TBP molecules

The formation of the PIC described above is based on

came from the observation that TBP binds to a 10-bp

the sequential addition of purified components in in

segment of DNA, immediately over the TATA box of

vitro experiments. An essential feature of this model is

the gene, whereas native holo-TFIID covers a 35 bp or

that the assembly takes place on the DNA template.

larger region (Figure 37-9). Second, TBP has a molec-

Accordingly, transcription activators, which have au-

ular mass of 20-40 kDa (depending on the species),

tonomous DNA binding and activation domains (see

whereas the TFIID complex has a mass of about 1000

Chapter 39), are thought to function by stimulating ei-

kDa. Finally, and perhaps most importantly, TBP sup-

ther PIC formation or PIC function. The TAF coacti-

ports basal transcription but not the augmented tran-

vators are viewed as bridging factors that communicate

scription provided by certain activators, eg, Sp1 bound

between the upstream activators, the proteins associated

to the GC box. TFIID, on the other hand, supports

with pol II, or the many other components of TFIID.

both basal and enhanced transcription by Sp1, Oct1,

This view, which assumes that there is stepwise assem-

AP1, CTF, ATF, etc. (Table 37-3). The TAFs are es-

bly of the PIC—promoted by various interactions be-

sential for this activator-enhanced transcription. It is

tween activators, coactivators, and PIC components—

not yet clear whether there are one or several forms of

is illustrated in panel A of Figure 37-10. This model

TFIID that might differ slightly in their complement of

was supported by observations that many of these pro-

teins could indeed bind to one another in vitro.

Recent evidence suggests that there is another possi-

ble mechanism of PIC formation and transcription reg-

Table 37-3. Some of the transcription control

ulation. First, large preassembled complexes of GTFs

elements, their consensus sequences, and the

and pol II are found in cell extracts, and this complex

factors that bind to them which are found in

can associate with a promoter in a single step. Second,

mammalian genes transcribed by RNA

the rate of transcription achieved when activators are

polymerase II. A complete list would include

added to limiting concentrations of pol II holoenzyme

dozens of examples. The asterisks mean that

can be matched by increasing the concentration of the

there are several members of this family.

pol II holoenzyme in the absence of activators. Thus,

Element

Consensus Sequence

Factor

TATA box

TATAAA

TBP

Table 37-4. Three classes of transcription factors

CAAT box

CCAATC

C/EBP*, NF-Y*

in class II genes.

GC box

GGGCGG

Sp1*

CAACTGAC

Myo D

General Mechanisms

Specific Components

T/CGGA/CN₅GCCAA

NF1*

lg octamer

ATGCAAAT

Oct1, 2, 4, 6*

Basal components

TBP, TFIIA, B, E, F, and H

AP1

TGAG/CTC/AA

Jun, Fos, ATF*

Coactivators

TAFs (TBP + TAFs) = TFIID; Srbs

Serum response

GATGCCCATA

SRF

Heat shock

(NGAAN)₃

HSF

Activators

SP1, ATF, CTF, AP1, etc

352

CHAPTER 37

activators are not in themselves absolutely essential for

precursor molecules is required for the generation of

PIC formation. These observations led to the “recruit-

the mature functional molecules.

ment” hypothesis, which has now been tested experi-

Nearly all eukaryotic RNA primary transcripts un-

mentally. Simply stated, the role of activators and

dergo extensive processing between the time they are

coactivators may be solely to recruit a preformed

synthesized and the time at which they serve their ulti-

holoenzyme-GTF complex to the promoter. The re-

mate function, whether it be as mRNA or as a com-

quirement for an activation domain is circumvented

ponent of the translation machinery such as rRNA,

when either a component of TFIID or the pol II

5S RNA, or tRNA or RNA processing machinery,

holoenzyme is artificially tethered, using recombinant

snRNAs. Processing occurs primarily within the nu-

DNA techniques, to the DNA binding domain (DBD)

cleus and includes nucleolytic cleavage to smaller mole-

of an activator. This anchoring, through the DBD

cules and coupled nucleolytic and ligation reactions

component of the activator molecule, leads to a tran-

(splicing of exons). In mammalian cells, 50-75% of

scriptionally competent structure, and there is no fur-

the nuclear RNA does not contribute to the cytoplas-

ther requirement for the activation domain of the acti-

mic mRNA. This nuclear RNA loss is significantly

vator. In this view, the role of activation domains and

greater than can be reasonably accounted for by the loss

TAFs is to form an assembly that directs the preformed

of intervening sequences alone (see below). Thus, the

holoenzyme-GTF complex to the promoter; they do

exact function of the seemingly excessive transcripts in

not assist in PIC assembly (see panel B, Figure 37-10).

the nucleus of a mammalian cell is not known.

The efficiency of this recruitment process determines

the rate of transcription at a given promoter.

Hormones—and other effectors that serve to trans-

The Coding Portions (Exons)

mit information related to the extracellular environ-

of Most Eukaryotic Genes

ment—modulate gene expression by influencing the as-

Are Interrupted by Introns

sembly and activity of the activator and coactivator

Interspersed within the amino acid-coding portions

complexes and the subsequent formation of the PIC at

(exons) of many genes are long sequences of DNA that

the promoter of target genes (see Chapter 43). The nu-

do not contribute to the genetic information ultimately

merous components involved provide for an abundance

translated into the amino acid sequence of a protein

of possible combinations and therefore a range of tran-

molecule (see Chapter 36). In fact, these sequences ac-

scriptional activity of a given gene. It is important to

tually interrupt the coding region of structural genes.

note that the two models are not mutually exclusive—

These intervening sequences (introns) exist within

stepwise versus holoenzyme-mediated PIC formation.

most but not all mRNA encoding genes of higher eu-

Indeed, one can envision various more complex models

karyotes. The primary transcripts of the structural genes

invoking elements of both models operating on a gene.

contain RNA complementary to the interspersed se-

quences. However, the intron RNA sequences are

cleaved out of the transcript, and the exons of the tran-

RNA MOLECULES ARE USUALLY

script are appropriately spliced together in the nucleus

PROCESSED BEFORE THEY

before the resulting mRNA molecule appears in the cy-

BECOME FUNCTIONAL

toplasm for translation

(Figures

37-11 and 37-12).

One speculation is that exons, which often encode an

In prokaryotic organisms, the primary transcripts of

activity domain of a protein, represent a convenient

mRNA-encoding genes begin to serve as translation

means of shuffling genetic information, permitting or-

templates even before their transcription has been com-

ganisms to quickly test the results of combining novel

pleted. This is because the site of transcription is not

protein functional domains.

compartmentalized into a nucleus as it is in eukaryotic

organisms. Thus, transcription and translation are cou-

pled in prokaryotic cells. Consequently, prokaryotic

Introns Are Removed & Exons

mRNAs are subjected to little processing prior to carry-

Are Spliced Together

ing out their intended function in protein synthesis. In-

deed, appropriate regulation of some genes (eg, the Trp

The mechanisms whereby introns are removed from

operon) relies upon this coupling of transcription and

the primary transcript in the nucleus, exons are ligated

translation. Prokaryotic rRNA and tRNA molecules are

to form the mRNA molecule, and the mRNA molecule

transcribed in units considerably longer than the ulti-

is transported to the cytoplasm are being elucidated.

mate molecule. In fact, many of the tRNA transcription

Four different splicing reaction mechanisms have been

units contain more than one molecule. Thus, in

described. The one most frequently used in eukaryotic

prokaryotes the processing of these rRNA and tRNA

cells is described below. Although the sequences of nu-

RNA SYNTHESIS, PROCESSING, & MODIFICATION

353

Exon 1

Exon 2

Intron

5′ Cap

A G

A_n

3′ Primary transcript

Cap

G G

A G

A_n

Nucleophilic attack

at 5′ end of intron

Cap

G OH

A G

A Lariat formation

Cap

A_n

Cut at 3′ end of intron

Cap

G—G

A_n

and

Ligation of 3′ end of exon

1 to 5′ end of exon 2

Intron is digested

Figure 37-11. The processing of the primary transcript to mRNA. In this hy-

pothetical transcript, the 5′ (left) end of the intron is cut (↓) and a lariat forms

between the G at the 5′ end of the intron and an A near the 3′ end, in the con-

sensus sequence UACUAAC. This sequence is called the branch site, and it is the

3′ most A that forms the 5′-2’ bond with the G. The 3′ (right) end of the intron is

then cut (⇓). This releases the lariat, which is digested, and exon 1 is joined to

exon 2 at G residues.

cleotides in the introns of the various eukaryotic tran-

mary transcript, five small nuclear RNAs (U1, U2, U5,

scripts—and even those within a single transcript—are

U4, and U6) and more than 60 proteins. Collectively,

quite heterogeneous, there are reasonably conserved se-

these form a small nucleoprotein (snRNP) complex,

quences at each of the two exon-intron (splice) junc-

sometimes called a “snurp.” It is likely that this penta-

tions and at the branch site, which is located 20-40 nu-

snRNP spliceosome forms prior to interaction with

cleotides upstream from the 3′ splice site (see consensus

mRNA precursors. Snurps are thought to position the

sequences in Figure 37-12). A special structure, the

RNA segments for the necessary splicing reactions. The

spliceosome, is involved in converting the primary

splicing reaction starts with a cut at the junction of the

transcript into mRNA. Spliceosomes consist of the pri-

5′ exon (donor or left) and intron (Figure 37-11). This

Consensus sequences

5′

UAAGU

UACUAAC 28-37 nucleotides C

3′

5′ Exon

Intron

Exon 3′

Figure 37-12. Consensus sequences at splice junctions. The 5′ (donor or left) and 3′ (ac-

ceptor or right) sequences are shown. Also shown is the yeast consensus sequence

(UACUAAC) for the branch site. In mammalian cells, this consensus sequence is PyNPyPy-

PuAPy, where Py is a pyrimidine, Pu is a purine, and N is any nucleotide. The branch site is lo-

cated 20-40 nucleotides upstream from the 3′ site.

354

CHAPTER 37

is accomplished by a nucleophilic attack by an adenylyl

above, the sequence of exon-intron splicing events gen-

residue in the branch point sequence located just up-

erally follows a hierarchical order for a given gene. The

stream from the 3′ end of this intron. The free 5′ termi-

fact that very complex RNA structures are formed dur-

nal then forms a loop or lariat structure that is linked

ing splicing—and that a number of snRNAs and pro-

by an unusual 5′-2′ phosphodiester bond to the reac-

teins are involved—affords numerous possibilities for a

tive A in the PyNPyPyPuAPy branch site sequence

change of this order and for the generation of different

(Figure 37-12). This adenylyl residue is typically lo-

mRNAs. Similarly, the use of alternative termination-

cated 28-37 nucleotides upstream from the 3′ end of

cleavage-polyadenylation sites also results in mRNA

the intron being removed. The branch site identifies

heterogeneity. Some schematic examples of these

the 3′ splice site. A second cut is made at the junction

processes, all of which occur in nature, are shown in

of the intron with the 3′ exon (donor on right). In this

Figure 37-13.

second transesterification reaction, the 3′ hydroxyl of

Faulty splicing can cause disease. At least one

the upstream exon attacks the

5′ phosphate at the

form of β-thalassemia, a disease in which the β-globin

downstream exon-intron boundary, and the lariat

gene of hemoglobin is severely underexpressed, appears

structure containing the intron is released and hy-

to result from a nucleotide change at an exon-intron

drolyzed. The 5′ and 3′ exons are ligated to form a con-

junction, precluding removal of the intron and there-

tinuous sequence.

fore leading to diminished or absent synthesis of the

The snRNAs and associated proteins are required

β-chain protein. This is a consequence of the fact that

for formation of the various structures and intermedi-

the normal translation reading frame of the mRNA is

ates. U1 within the snRNP complex binds first by base

disrupted—a defect in this fundamental process (splic-

pairing to the 5′ exon-intron boundary. U2 within the

ing) that underscores the accuracy which the process of

snRNP complex then binds by base pairing to the

RNA-RNA splicing must achieve.

branch site, and this exposes the nucleophilic A residue.

U5/U4/U6 within the snRNP complex mediates an

Alternative Promoter Utilization

ATP-dependent protein-mediated unwinding that re-

Provides a Form of Regulation

sults in disruption of the base-paired U4-U6 complex

with the release of U4. U6 is then able to interact first

Tissue-specific regulation of gene expression can be

with U2, then with U1. These interactions serve to ap-

provided by control elements in the promoter or by the

proximate the 5′ splice site, the branch point with its

reactive A, and the 3′ splice site. This alignment is en-

hanced by U5. This process also results in the forma-

mRNA precursor

tion of the loop or lariat structure. The two ends are

AAUAA AAUAA

(A)_n

cleaved, probably by the U2-U6 within the snRNP

complex. U6 is certainly essential, since yeasts deficient

in this snRNA are not viable. It is important to note

Selective splicing

that RNA serves as the catalytic agent. This sequence is

then repeated in genes containing multiple introns. In

AAUAA AAUAA

(A)_n

such cases, a definite pattern is followed for each gene,

and the introns are not necessarily removed in se-

Alternative 5′ donor site

quence—1, then 2, then 3, etc.

The relationship between hnRNA and the corre-

1′

AAUAA AAUAA

(A)_n

sponding mature mRNA in eukaryotic cells is now ap-

parent. The hnRNA molecules are the primary tran-

Alternative 3′ acceptor site

scripts plus their early processed products, which, after

the addition of caps and poly(A) tails and removal of

2′

AAUAA AAUAA

(A)_n

the portion corresponding to the introns, are trans-

Alternative polyadenylation site

ported to the cytoplasm as mature mRNA molecules.

AAUAA

(A)_n

Alternative Splicing Provides

for Different mRNAs

Figure 37-13. Mechanisms of alternative process-

The processing of hnRNA molecules is a site for reg-

ing of mRNA precursors. This form of RNA processing

ulation of gene expression. Alternative patterns of

involves the selective inclusion or exclusion of exons,

RNA splicing result from tissue-specific adaptive and

the use of alternative 5′ donor or 3′ acceptor sites, and

developmental control mechanisms. As mentioned

the use of different polyadenylation sites.

RNA SYNTHESIS, PROCESSING, & MODIFICATION

355

use of alternative promoters. The glucokinase

(GK)

mature tRNA. A small fraction of tRNAs even contain

gene consists of ten exons interrupted by nine introns.

introns.

The sequence of exons 2-10 is identical in liver and

pancreatic B cells, the primary tissues in which GK pro-

RNAS CAN BE EXTENSIVELY MODIFIED

tein is expressed. Expression of the GK gene is regulated

very differently—by two different promoters—in these

Essentially all RNAs are covalently modified after tran-

two tissues. The liver promoter and exon 1L are located

scription. It is clear that at least some of these modifica-

near exons 2-10; exon 1L is ligated directly to exon 2.

tions are regulatory.

In contrast, the pancreatic B cell promoter is located

about 30 kbp upstream. In this case, the 3′ boundary of

Messenger RNA (mRNA) Is Modified

exon 1B is ligated to the 5′ boundary of exon 2. The

at the 5 & 3 Ends

liver promoter and exon 1L are excluded and removed

As mentioned above, mammalian mRNA molecules

during the splicing reaction (see Figure 37-14). The ex-

contain a 7-methylguanosine cap structure at their 5′

istence of multiple distinct promoters allows for cell-

terminal, and most have a poly(A) tail at the 3′ termi-

and tissue-specific expression patterns of a particular

nal. The cap structure is added to the 5′ end of the

gene (mRNA).

newly transcribed mRNA precursor in the nucleus

prior to transport of the mRNA molecule to the cyto-

plasm. The 5

cap of the RNA transcript is required

Both Ribosomal RNAs & Most

both for efficient translation initiation and protection

Transfer RNAs Are Processed

of the 5′ end of mRNA from attack by 5′ → 3′ exonu-

From Larger Precursors

cleases. The secondary methylations of mRNA mole-

In mammalian cells, the three rRNA molecules are

cules, those on the 2′-hydroxy and the N⁶ of adenylyl

transcribed as part of a single large precursor molecule.

residues, occur after the mRNA molecule has appeared

The precursor is subsequently processed in the nu-

in the cytoplasm.

cleolus to provide the RNA component for the ribo-

Poly(A) tails are added to the 3′ end of mRNA mol-

some subunits found in the cytoplasm. The rRNA

ecules in a posttranscriptional processing step. The

genes are located in the nucleoli of mammalian cells.

mRNA is first cleaved about 20 nucleotides down-

Hundreds of copies of these genes are present in every

stream from an AAUAA recognition sequence. Another

cell. This large number of genes is required to synthe-

enzyme, poly(A) polymerase, adds a poly(A) tail which

size sufficient copies of each type of rRNA to form the

is subsequently extended to as many as 200 A residues.

10⁷

ribosomes required for each cell replication.

The poly(A) tail appears to protect the

3′ end of

Whereas a single mRNA molecule may be copied into

mRNA from 3′ → 5′ exonuclease attack. The presence

10⁵ protein molecules, providing a large amplification,

or absence of the poly(A) tail does not determine

the rRNAs are end products. This lack of amplification

whether a precursor molecule in the nucleus appears in

requires a large number of genes. Similarly, transfer

the cytoplasm, because all poly(A)-tailed hnRNA mole-

RNAs are often synthesized as precursors, with extra se-

cules do not contribute to cytoplasmic mRNA, nor do

quences both 5′ and 3′ of the sequences comprising the

all cytoplasmic mRNA molecules contain poly(A) tails

30 kb)

⁽˜

Liver

2A 2 3

30 kb)

⁽˜

B cell /pituitary

2A 2 3

Figure 37-14. Alternative promoter use in the liver and pancreatic B cell glucokinase

genes. Differential regulation of the glucokinase (GK) gene is accomplished by the use of

tissue-specific promoters. The B cell GK gene promoter and exon 1B are located about

30 kbp upstream from the liver promoter and exon 1L. Each promoter has a unique

structure and is regulated differently. Exons 2-10 are identical in the two genes, and the

GK proteins encoded by the liver and B cell mRNAs have identical kinetic properties.

356

CHAPTER 37

(the histones are most notable in this regard). Cytoplas-

mRNA into protein sequences. The tRNAs contain

mic enzymes in mammalian cells can both add and re-

many modifications of the standard bases A, U, G, and

move adenylyl residues from the poly(A) tails; this

C, including methylation, reduction, deamination, and

process has been associated with an alteration of mRNA

rearranged glycosidic bonds. Further modification of

stability and translatability.

the tRNA molecules includes nucleotide alkylations

The size of some cytoplasmic mRNA molecules,

and the attachment of the characteristic CpCpAOH ter-

even after the poly(A) tail is removed, is still consider-

minal at the 3′ end of the molecule by the enzyme nu-

ably greater than the size required to code for the spe-

cleotidyl transferase. The 3′ OH of the A ribose is the

cific protein for which it is a template, often by a factor

point of attachment for the specific amino acid that is

of 2 or 3. The extra nucleotides occur in untrans-

to enter into the polymerization reaction of protein

lated (non-protein coding) regions both 5′ and 3′ of

synthesis. The methylation of mammalian tRNA pre-

the coding region; the longest untranslated sequences

cursors probably occurs in the nucleus, whereas the

are usually at the 3′ end. The exact function of these se-

cleavage and attachment of CpCpAOH are cytoplasmic

quences is unknown, but they have been implicated in

functions, since the terminals turn over more rapidly

RNA processing, transport, degradation, and transla-

than do the tRNA molecules themselves. Enzymes

tion; each of these reactions potentially contributes ad-

within the cytoplasm of mammalian cells are required

ditional levels of control of gene expression.

for the attachment of amino acids to the CpCpAOH

residues. (See Chapter 38.)

RNA Editing Changes mRNA

After Transcription

RNA CAN ACT AS A CATALYST

The central dogma states that for a given gene and gene

In addition to the catalytic action served by the

product there is a linear relationship between the cod-

snRNAs in the formation of mRNA, several other

ing sequence in DNA, the mRNA sequence, and the

enzymatic functions have been attributed to RNA.

protein sequence (Figure 36-7). Changes in the DNA

Ribozymes are RNA molecules with catalytic activity.

sequence should be reflected in a change in the mRNA

These generally involve transesterification reactions,

sequence and, depending on codon usage, in protein se-

and most are concerned with RNA metabolism (splic-

quence. However, exceptions to this dogma have been

ing and endoribonuclease). Recently, a ribosomal RNA

recently documented. Coding information can be

component was noted to hydrolyze an aminoacyl ester

changed at the mRNA level by RNA editing. In such

and thus to play a central role in peptide bond function

cases, the coding sequence of the mRNA differs from

(peptidyl transferases; see Chapter 38). These observa-

that in the cognate DNA. An example is the apolipo-

tions, made in organelles from plants, yeast, viruses,

protein B (apoB) gene and mRNA. In liver, the single

and higher eukaryotic cells, show that RNA can act as

apoB gene is transcribed into an mRNA that directs the

an enzyme. This has revolutionized thinking about en-

synthesis of a 100-kDa protein, apoB100. In the intes-

zyme action and the origin of life itself.

tine, the same gene directs the synthesis of the primary

transcript; however, a cytidine deaminase converts a

CAA codon in the mRNA to UAA at a single specific

SUMMARY

site. Rather than encoding glutamine, this codon be-

• RNA is synthesized from a DNA template by the en-

comes a termination signal, and a

48-kDa protein

zyme RNA polymerase.

(apoB48) is the result. ApoB100 and apoB48 have dif-

• There are three distinct nuclear DNA-dependent

ferent functions in the two organs. A growing number

RNA polymerases in mammals: RNA polymerases I,

of other examples include a glutamine to arginine

II, and III. These enzymes control the transcriptional

change in the glutamate receptor and several changes

function—the transcription of rRNA, mRNA, and

in trypanosome mitochondrial mRNAs, generally in-

small RNA (tRNA/5S rRNA, snRNA) genes, respec-

volving the addition or deletion of uridine. The exact

tively.

extent of RNA editing is unknown, but current esti-

mates suggest that < 0.01% of mRNAs are edited in

• RNA polymerases interact with unique cis-active re-

this fashion.

gions of genes, termed promoters, in order to form

preinitiation complexes (PICs) capable of initiation.

In eukaryotes the process of PIC formation is facili-

Transfer RNA (tRNA) Is Extensively

tated by multiple general transcription factors

Processed & Modified

(GTFs), TFIIA, B, D, E, F, and H.

As described in Chapters 35 and 38, the tRNA mole-

• Eukaryotic PIC formation can occur either step-

cules serve as adapter molecules for the translation of

wise—by the sequential, ordered interactions of

RNA SYNTHESIS, PROCESSING, & MODIFICATION

357

GTFs and RNA polymerase with promoters—or in

REFERENCES

one step by the recognition of the promoter by a pre-

Busby S, Ebright RH: Promoter structure, promoter recognition,

formed GTF-RNA polymerase holoenzyme complex.

and transcription activation in prokaryotes. Cell

1994;79:

•

Transcription exhibits three phases: initiation, elon-

743.

gation, and termination. All are dependent upon dis-

Cramer P, Bushnell DA, Kornberg R: Structural basis of transcrip-

tinct DNA cis-elements and can be modulated by

tion: RNA polymerase II at 2.8 angstrom resolution. Science

distinct trans-acting protein factors.

2001;292:1863.

•

Most eukaryotic RNAs are synthesized as precursors

Fedor MJ: Ribozymes. Curr Biol 1998;8:R441.

that contain excess sequences which are removed

Gott JM, Emeson RB: Functions and mechanisms of RNA editing.

Ann Rev Genet 2000;34:499.

prior to the generation of mature, functional RNA.

Hirose Y, Manley JL: RNA polymerase II and the integration of

•

Eukaryotic mRNA synthesis results in a pre-mRNA

nuclear events. Genes Dev 2000;14:1415.

precursor that contains extensive amounts of excess

Keaveney M, Struhl K: Activator-mediated recruitment of the

RNA (introns) that must be precisely removed by

RNA polymerase machinery is the predominant mechanism

RNA splicing to generate functional, translatable

for transcriptional activation in yeast. Mol Cell 1998;1:917.

mRNA composed of exonic coding and noncoding

Lemon B, Tjian R: Orchestrated response: a symphony of tran-

sequences.

scription factors for gene control. Genes Dev 2000;14:2551.

•

All steps—from changes in DNA template, sequence,

Maniatis T, Reed R: An extensive network of coupling among gene

and accessibility in chromatin to RNA stability—are

expression machines. Nature 2002;416:499.

subject to modulation and hence are potential con-

Orphanides G, Reinberg D: A unified theory of gene expression.

Cell 2002;108:439.

trol sites for eukaryotic gene regulation.

Shatkin AJ, Manley JL: The ends of the affair: capping and poly-

adenylation. Nat Struct Biol 2000;7:838.

Stevens SW et al: Composition and functional characterization of

the yeast spliceosomal penta-snRNP. Mol Cell 2002;9:31.

Tucker M, Parker R: Mechanisms and control of mRNA decap-

ping in Saccharomyces cerevisiae. Ann Rev Biochem 2000;69:

571.

Woychik NA, Hampsey M: The RNA polymerase II machinery:

structure illuminates function. Cell 2002;108:453.

Protein Synthesis & the

Genetic Code

Daryl K. Granner, MD

BIOMEDICAL IMPORTANCE

The cell must possess the machinery necessary to

translate information accurately and efficiently from

The letters A, G, T, and C correspond to the nu-

the nucleotide sequence of an mRNA into the sequence

cleotides found in DNA. They are organized into three-

of amino acids of the corresponding specific protein.

letter code words called codons, and the collection of

Clarification of our understanding of this process,

these codons makes up the genetic code. It was impos-

which is termed translation, awaited deciphering of the

sible to understand protein synthesis—or to explain

genetic code. It was realized early that mRNA mole-

mutations—before the genetic code was elucidated.

cules themselves have no affinity for amino acids and,

The code provides a foundation for explaining the way

therefore, that the translation of the information in the

in which protein defects may cause genetic disease and

mRNA nucleotide sequence into the amino acid se-

for the diagnosis and perhaps the treatment of these

quence of a protein requires an intermediate adapter

disorders. In addition, the pathophysiology of many

molecule. This adapter molecule must recognize a spe-

viral infections is related to the ability of these agents to

cific nucleotide sequence on the one hand as well as a

disrupt host cell protein synthesis. Many antibacterial

specific amino acid on the other. With such an adapter

agents are effective because they selectively disrupt pro-

molecule, the cell can direct a specific amino acid into

tein synthesis in the invading bacterial cell but do not

the proper sequential position of a protein during its

affect protein synthesis in eukaryotic cells.

synthesis as dictated by the nucleotide sequence of the

specific mRNA. In fact, the functional groups of the

amino acids do not themselves actually come into con-

GENETIC INFORMATION FLOWS

tact with the mRNA template.

FROM DNA TO RNA TO PROTEIN

The genetic information within the nucleotide se-

THE NUCLEOTIDE SEQUENCE

quence of DNA is transcribed in the nucleus into the

specific nucleotide sequence of an RNA molecule. The

OF AN mRNA MOLECULE CONSISTS

sequence of nucleotides in the RNA transcript is com-

OF A SERIES OF CODONS THAT SPECIFY

plementary to the nucleotide sequence of the template

THE AMINO ACID SEQUENCE OF THE

strand of its gene in accordance with the base-pairing

ENCODED PROTEIN

rules. Several different classes of RNA combine to di-

rect the synthesis of proteins.

Twenty different amino acids are required for the syn-

In prokaryotes there is a linear correspondence be-

thesis of the cellular complement of proteins; thus,

tween the gene, the messenger RNA (mRNA) tran-

there must be at least 20 distinct codons that make up

scribed from the gene, and the polypeptide product.

the genetic code. Since there are only four different nu-

The situation is more complicated in higher eukaryotic

cleotides in mRNA, each codon must consist of more

cells, in which the primary transcript is much larger

than a single purine or pyrimidine nucleotide. Codons

than the mature mRNA. The large mRNA precursors

consisting of two nucleotides each could provide for

contain coding regions (exons) that will form the ma-

only 16 (4²) specific codons, whereas codons of three

ture mRNA and long intervening sequences (introns)

nucleotides could provide 64 (4³) specific codons.

that separate the exons. The hnRNA is processed

It is now known that each codon consists of a se-

within the nucleus, and the introns, which often make

quence of three nucleotides; ie, it is a triplet code

up much more of this RNA than the exons, are re-

(see Table 38-1). The deciphering of the genetic code

moved. Exons are spliced together to form mature

depended heavily on the chemical synthesis of nu-

mRNA, which is transported to the cytoplasm, where it

cleotide polymers, particularly triplets in repeated se-

is translated into protein.

quence.

358

PROTEIN SYNTHESIS & THE GENETIC CODE

359

Table 38-1. The genetic code (codon

the code. However, for any specific codon, only a single

assignments in mammalian messenger RNA).¹

amino acid is indicated; with rare exceptions, the ge-

netic code is unambiguous—ie, given a specific codon,

only a single amino acid is indicated. The distinction

First

Second

Third

between ambiguity and degeneracy is an important

Nucleotide

concept.

The unambiguous but degenerate code can be ex-

plained in molecular terms. The recognition of specific

Phe

Ser

Tyr

Cys

Phe

Ser

Tyr

Cys

codons in the mRNA by the tRNA adapter molecules is

Leu

Ser

Term

Term²

dependent upon their anticodon region and specific

Leu

Ser

Term

Trp

base-pairing rules. Each tRNA molecule contains a spe-

cific sequence, complementary to a codon, which is

Leu

Pro

His

Arg

termed its anticodon. For a given codon in the mRNA,

Leu

Pro

His

Arg

only a single species of tRNA molecule possesses the

Leu

Pro

Gln

Arg

proper anticodon. Since each tRNA molecule can be

Leu

Pro

Gln

Arg

charged with only one specific amino acid, each codon

Ile

Thr

Asn

Ser

therefore specifies only one amino acid. However, some

Ile

Thr

Asn

Ser

tRNA molecules can utilize the anticodon to recognize

Ile²

Thr

Lys

Arg²

more than one codon. With few exceptions, given a

Met

Thr

Lys

Arg²

specific codon, only a specific amino acid will be in-

Val

Ala

Asp

Gly

corporated—although, given a specific amino acid,

Val

Ala

Asp

Gly

more than one codon may be used.

Val

Ala

Glu

Gly

As discussed below, the reading of the genetic code

Val

Ala

Glu

Gly

during the process of protein synthesis does not involve

any overlap of codons. Thus, the genetic code is

¹The terms first, second, and third nucleotide refer to the indi-

nonoverlapping. Furthermore, once the reading is

vidual nucleotides of a triplet codon. U, uridine nucleotide;

C, cytosine nucleotide; A, adenine nucleotide; G, guanine nu-

commenced at a specific codon, there is no punctua-

cleotide; Term, chain terminator codon. AUG, which codes for

tion between codons, and the message is read in a con-

Met, serves as the initiator codon in mammalian cells and en-

tinuing sequence of nucleotide triplets until a transla-

codes for internal methionines in a protein. (Abbreviations of

tion stop codon is reached.

amino acids are explained in Chapter 3.)

Until recently, the genetic code was thought to be

²In mammalian mitochondria, AUA codes for Met and UGA for

universal. It has now been shown that the set of tRNA

Trp, and AGA and AGG serve as chain terminators.

molecules in mitochondria (which contain their own

separate and distinct set of translation machinery) from

lower and higher eukaryotes, including humans, reads

THE GENETIC CODE IS DEGENERATE,

four codons differently from the tRNA molecules in

the cytoplasm of even the same cells. As noted in Table

UNAMBIGUOUS, NONOVERLAPPING,

38-1, the codon AUA is read as Met, and UGA codes

WITHOUT PUNCTUATION, & UNIVERSAL

for Trp in mammalian mitochondria. In addition, in

Three of the 64 possible codons do not code for specific

mitochondria, the codons AGA and AGG are read as

amino acids; these have been termed nonsense codons.

stop or chain terminator codons rather than as Arg. As

These nonsense codons are utilized in the cell as termi-

a result, mitochondria require only 22 tRNA molecules

nation signals; they specify where the polymerization

to read their genetic code, whereas the cytoplasmic

of amino acids into a protein molecule is to stop. The

translation system possesses a full complement of 31

remaining 61 codons code for 20 amino acids (Table

tRNA species. These exceptions noted, the genetic

38-1). Thus, there must be “degeneracy” in the ge-

code is universal. The frequency of use of each amino

netic code—ie, multiple codons must decode the same

acid codon varies considerably between species and

amino acid. Some amino acids are encoded by several

among different tissues within a species. The specific

codons; for example, six different codons specify serine.

tRNA levels generally mirror these codon usage biases.

Other amino acids, such as methionine and trypto-

Thus, a particular abundantly used codon is decoded

phan, have a single codon. In general, the third nu-

by a similarly abundant specific tRNA which recognizes

cleotide in a codon is less important than the first two

that particular codon. Tables of codon usage are be-

in determining the specific amino acid to be incorpo-

coming more accurate as more genes are sequenced.

rated, and this accounts for most of the degeneracy of

This is of considerable importance because investigators

360

CHAPTER 38

Table 38-2. Features of the genetic code.

tRNA synthetases. They form an activated intermedi-

ate of aminoacyl-AMP-enzyme complex (Figure 38-1).

The specific aminoacyl-AMP-enzyme complex then

• Degenerate

recognizes a specific tRNA to which it attaches the

• Unambiguous

• Nonoverlapping

aminoacyl moiety at the 3′-hydroxyl adenosyl terminal.

• Not punctuated

The charging reactions have an error rate of less than

• Universal

10−4 and so are extremely accurate. The amino acid re-

mains attached to its specific tRNA in an ester linkage

until it is polymerized at a specific position in the fabri-

cation of a polypeptide precursor of a protein molecule.

often need to deduce mRNA structure from the pri-

The regions of the tRNA molecule referred to in

mary sequence of a portion of protein in order to syn-

Chapter 35 (and illustrated in Figure 35-11) now be-

thesize an oligonucleotide probe and initiate a recombi-

come important. The thymidine-pseudouridine-cyti-

nant DNA cloning project. The main features of the

dine (TΨC) arm is involved in binding of the amino-

genetic code are listed in Table 38-2.

acyl-tRNA to the ribosomal surface at the site of

protein synthesis. The D arm is one of the sites impor-

tant for the proper recognition of a given tRNA species

AT LEAST ONE SPECIES OF TRANSFER

by its proper aminoacyl-tRNA synthetase. The acceptor

arm, located at the 3′-hydroxyl adenosyl terminal, is the

RNA (tRNA) EXISTS FOR EACH OF THE

site of attachment of the specific amino acid.

20 AMINO ACIDS

The anticodon region consists of seven nucleotides,

tRNA molecules have extraordinarily similar functions

and it recognizes the three-letter codon in mRNA (Fig-

and three-dimensional structures. The adapter function

ure 38-2). The sequence read from the 3′ to 5′ direc-

of the tRNA molecules requires the charging of each

tion in that anticodon loop consists of a variable

specific tRNA with its specific amino acid. Since there

base-modified purine-XYZ-pyrimidine-pyrimidine-

is no affinity of nucleic acids for specific functional

5′. Note that this direction of reading the anticodon is

groups of amino acids, this recognition must be carried

3′ to 5′, whereas the genetic code in Table 38-1 is read

out by a protein molecule capable of recognizing both a

5′ to 3′, since the codon and the anticodon loop of the

specific tRNA molecule and a specific amino acid. At

mRNA and tRNA molecules, respectively, are antipar-

least 20 specific enzymes are required for these specific

allel in their complementarity just like all other inter-

recognition functions and for the proper attachment of

molecular interactions between nucleic acid strands.

the 20 amino acids to specific tRNA molecules. The

The degeneracy of the genetic code resides mostly in

process of recognition and attachment

(charging)

the last nucleotide of the codon triplet, suggesting that

proceeds in two steps by one enzyme for each of the 20

the base pairing between this last nucleotide and the

amino acids. These enzymes are termed aminoacyl-

corresponding nucleotide of the anticodon is not strictly

ATP

PP_i

AMP + Enz

HOOC HC R

Enz

•

Adenosine

O P O

H₂N

NH₂

Enzyme (Enz)

Enz•AMP-aa

tRNA

tRNA-aa

(Activated amino acid)

AMINOACYL-

tRNA SYNTHETASE

Amino acid (aa)

Aminoacyl-AMP-enzyme

Aminoacyl-tRNA

complex

Figure 38-1. Formation of aminoacyl-tRNA. A two-step reaction, involving the enzyme

aminoacyl-tRNA synthetase, results in the formation of aminoacyl-tRNA. The first reaction in-

volves the formation of an AMP-amino acid-enzyme complex. This activated amino acid is next

transferred to the corresponding tRNA molecule. The AMP and enzyme are released, and the lat-

ter can be reutilized. The charging reactions have an error rate of less than 10^-4 and so are ex-

tremely accurate.

PROTEIN SYNTHESIS & THE GENETIC CODE

361

mRNA

purine is changed to the other purine. Transversions are

Codon

5′

3′

changes from a purine to either of the two pyrimidines

U • U • U

Anticodon

or the change of a pyrimidine into either of the two

A • A • A

•

purines, as shown in Figure 38-3.

•Pu*

•

Anticodon

If the nucleotide sequence of the gene containing

arm

the mutation is transcribed into an RNA molecule,

then the RNA molecule will possess a complementary

base change at this corresponding locus.

TψC

arm

Single-base changes in the mRNA molecules may

have one of several effects when translated into protein:

Phenylalanyl-

(1) There may be no detectable effect because of the

tRNA

degeneracy of the code. This would be more likely if

5′

the changed base in the mRNA molecule were to be at

•

the third nucleotide of a codon; such mutations are

Acceptor arm

often referred to as silent mutations. Because of wob-

•

ble, the translation of a codon is least sensitive to a

3′

change at the third position.

Phe

(2) A missense effect will occur when a different

amino acid is incorporated at the corresponding site in

Figure 38-2. Recognition of the codon by the anti-

the protein molecule. This mistaken amino acid—or

codon. One of the codons for phenylalanine is UUU.

missense, depending upon its location in the specific

tRNA charged with phenylalanine (Phe) has the com-

protein—might be acceptable, partially acceptable, or

plementary sequence AAA; hence, it forms a base-pair

unacceptable to the function of that protein molecule.

complex with the codon. The anticodon region typi-

From a careful examination of the genetic code, one

cally consists of a sequence of seven nucleotides: vari-

can conclude that most single-base changes would re-

able (N), modified purine ((Pu*), X, Y, Z, and two pyrim-

sult in the replacement of one amino acid by another

idines (Py) in the 3′ to 5′ direction.

with rather similar functional groups. This is an effec-

tive mechanism to avoid drastic change in the physical

properties of a protein molecule. If an acceptable mis-

sense effect occurs, the resulting protein molecule may

by the Watson-Crick rule. This is called wobble; the

pairing of the codon and anticodon can “wobble” at this

not be distinguishable from the normal one. A partially

acceptable missense will result in a protein molecule

specific nucleotide-to-nucleotide pairing site. For exam-

ple, the two codons for arginine, AGA and AGG, can

with partial but abnormal function. If an unacceptable

missense effect occurs, then the protein molecule will

bind to the same anticodon having a uracil at its 5′ end

(UCU). Similarly, three codons for glycine—GGU,

not be capable of functioning in its assigned role.

(3) A nonsense codon may appear that would then

GGC, and GGA—can form a base pair from one anti-

codon, CCI. I is an inosine nucleotide, another of the

result in the premature termination of amino acid in-

corporation into a peptide chain and the production of

peculiar bases appearing in tRNA molecules.

only a fragment of the intended protein molecule. The

probability is high that a prematurely terminated pro-

MUTATIONS RESULT WHEN CHANGES

tein molecule or peptide fragment will not function in

OCCUR IN THE NUCLEOTIDE SEQUENCE

its assigned role.

Although the initial change may not occur in the tem-

plate strand of the double-stranded DNA molecule for

that gene, after replication, daughter DNA molecules

A A

with mutations in the template strand will segregate

and appear in the population of organisms.

Some Mutations Occur

G G

by Base Substitution

Transitions

Transversions

Single-base changes (point mutations) may be transi-

tions or transversions. In the former, a given pyrimi-

Figure 38-3. Diagrammatic representation of transi-

dine is changed to the other pyrimidine or a given

tion mutations and transversion mutations.

362

CHAPTER 38

Hemoglobin Illustrates the Effects of

to possess at position 67 a codon GUA or GUG in

Single-Base Changes in Structural Genes

order that a single nucleotide change could provide for

the appearance of the glutamic acid codons GAA or

Some mutations have no apparent effect. The gene

GAG. Hemoglobin Sydney, which contains an alanine

system that encodes hemoglobin is one of the best-

at position 67, could have arisen by the change of a sin-

studied in humans. The lack of effect of a single-base

gle nucleotide in any of the four codons for valine

change is demonstrable only by sequencing the nu-

(GUU, GUC, GUA, or GUG) to the alanine codons

cleotides in the mRNA molecules or structural genes.

(GCU, GCC, GCA, or GCG, respectively).

The sequencing of a large number of hemoglobin

mRNAs and genes from many individuals has shown

that the codon for valine at position 67 of the β chain

Substitution of Amino Acids Causes

of hemoglobin is not identical in all persons who pos-

Missense Mutations

sess a normally functional β chain of hemoglobin. He-

A. ACCEPTABLE MISSENSE MUTATIONS

moglobin Milwaukee has at position 67 a glutamic

acid; hemoglobin Bristol contains aspartic acid at posi-

An example of an acceptable missense mutation (Figure

tion 67. In order to account for the amino acid change

38-4, top) in the structural gene for the β chain of he-

by the change of a single nucleotide residue in the

moglobin could be detected by the presence of an elec-

codon for amino acid 67, one must infer that the

trophoretically altered hemoglobin in the red cells of an

mRNA encoding hemoglobin Bristol possessed a GUU

apparently healthy individual. Hemoglobin Hikari has

or GUC codon prior to a later change to GAU or

been found in at least two families of Japanese people.

GAC, both codons for aspartic acid. However, the

This hemoglobin has asparagine substituted for lysine

mRNA encoding hemoglobin Milwaukee would have

at the 61 position in the β chain. The corresponding

Protein molecule

Amino acid

Codons

Hb A, β chain

61 Lysine

AAA

AAG

Acceptable

missense

Hb Hikari, β chain

Asparagine

AAU

AAC

Hb A, β chain

6 Glutamate

GAA

GAG

Partially

acceptable

missense

Hb S, β chain

Valine

GUA

GUG

Hb A, α chain

58 Histidine

CAU

CAC

Unacceptable

missense

Hb M (Boston), α chain

Tyrosine

UAU

UAC

Figure 38-4. Examples of three types of missense mutations resulting in abnormal hemoglo-

bin chains. The amino acid alterations and possible alterations in the respective codons are indi-

cated. The hemoglobin Hikari β-chain mutation has apparently normal physiologic properties

but is electrophoretically altered. Hemoglobin S has a β-chain mutation and partial function; he-

moglobin S binds oxygen but precipitates when deoxygenated. Hemoglobin M Boston, an

α-chain mutation, permits the oxidation of the heme ferrous iron to the ferric state and so will

not bind oxygen at all.

PROTEIN SYNTHESIS & THE GENETIC CODE

363

transversion might be either AAA or AAG changed to

mal termination codon (nonsense codon), the reading

either AAU or AAC. The replacement of the specific ly-

of the normal termination signal is disturbed. Such a

sine with asparagine apparently does not alter the nor-

deletion might result in reading through a termination

mal function of the β chain in these individuals.

signal until another nonsense codon is encountered (ex-

ample 1, Figure 38-5). Examples of this phenomenon

B. PARTIALLY ACCEPTABLE MISSENSE MUTATIONS

are described in discussions of hemoglobinopathies.

A partially acceptable missense mutation (Figure 38-4,

Insertions of one or two or nonmultiples of three nu-

center) is best exemplified by hemoglobin S, which is

cleotides into a gene result in an mRNA in which the

found in sickle cell anemia. Here glutamic acid, the

reading frame is distorted upon translation, and the same

normal amino acid in position 6 of the β chain, has

effects that occur with deletions are reflected in the

been replaced by valine. The corresponding single nu-

mRNA translation. This may result in garbled amino

cleotide change within the codon would be GAA or

acid sequences distal to the insertion and the generation

GAG of glutamic acid to GUA or GUG of valine.

of a nonsense codon at or distal to the insertion, or per-

Clearly, this missense mutation hinders normal func-

haps reading through the normal termination codon.

tion and results in sickle cell anemia when the mutant

Following a deletion in a gene, an insertion (or vice

gene is present in the homozygous state. The gluta-

versa) can reestablish the proper reading frame (exam-

mate-to-valine change may be considered to be partially

ple 4, Figure 38-5). The corresponding mRNA, when

acceptable because hemoglobin S does bind and release

translated, would contain a garbled amino acid sequence

oxygen, although abnormally.

between the insertion and deletion. Beyond the reestab-

lishment of the reading frame, the amino acid sequence

C. UNACCEPTABLE MISSENSE MUTATIONS

would be correct. One can imagine that different com-

An unacceptable missense mutation (Figure 38-4, bot-

binations of deletions, of insertions, or of both would

tom) in a hemoglobin gene generates a nonfunctioning

result in formation of a protein wherein a portion is ab-

hemoglobin molecule. For example, the hemoglobin M

normal, but this portion is surrounded by the normal

mutations generate molecules that allow the Fe2+ of the

amino acid sequences. Such phenomena have been

heme moiety to be oxidized to Fe3+, producing methe-

demonstrated convincingly in a number of diseases.

moglobin. Methemoglobin cannot transport oxygen

(see Chapter 6).

Suppressor Mutations Can Counteract

Some of the Effects of Missense,

Frameshift Mutations Result From

Nonsense, & Frameshift Mutations

Deletion or Insertion of Nucleotides in

DNA That Generates Altered mRNAs

The above discussion of the altered protein products of

gene mutations is based on the presence of normally

The deletion of a single nucleotide from the coding

functioning tRNA molecules. However, in prokaryotic

strand of a gene results in an altered reading frame in

and lower eukaryotic organisms, abnormally function-

the mRNA. The machinery translating the mRNA does

ing tRNA molecules have been discovered that are

not recognize that a base was missing, since there is no

themselves the results of mutations. Some of these ab-

punctuation in the reading of codons. Thus, a major al-

normal tRNA molecules are capable of binding to and

teration in the sequence of polymerized amino acids, as

decoding altered codons, thereby suppressing the effects

depicted in example 1, Figure 38-5, results. Altering

of mutations in distant structural genes. These sup-

the reading frame results in a garbled translation of the

pressor tRNA molecules, usually formed as the result

mRNA distal to the single nucleotide deletion. Not

of alterations in their anticodon regions, are capable of

only is the sequence of amino acids distal to this dele-

suppressing missense mutations, nonsense mutations,

tion garbled, but reading of the message can also result

and frameshift mutations. However, since the suppres-

in the appearance of a nonsense codon and thus the

sor tRNA molecules are not capable of distinguishing

production of a polypeptide both garbled and prema-

between a normal codon and one resulting from a gene

turely terminated (example 3, Figure 38-5).

mutation, their presence in a cell usually results in de-

If three nucleotides or a multiple of three are deleted

creased viability. For instance, the nonsense suppressor

from a coding region, the corresponding mRNA when

tRNA molecules can suppress the normal termination

translated will provide a protein from which is missing

signals to allow a read-through when it is not desirable.

the corresponding number of amino acids (example 2,

Frameshift suppressor tRNA molecules may read a nor-

Figure 38-5). Because the reading frame is a triplet, the

mal codon plus a component of a juxtaposed codon to

reading phase will not be disturbed for those codons

provide a frameshift, also when it is not desirable. Sup-

distal to the deletion. If, however, deletion of one or

pressor tRNA molecules may exist in mammalian cells,

two nucleotides occurs just prior to or within the nor-

since read-through transcription occurs.

364

CHAPTER 38

Normal

Wild type

mRNA

5'...

UAG UUUG AUG GCC UCU UGC AAA GGC UAU AGU AGU UAG...

Polypeptide

Met

Ala

Ser

Cys

Lys

Gly

Tyr

Ser

STOP

Example 1

Deletion (-1)

-1 U

mRNA

5'...

UAG UUUG AUG GCC CUU GCA AAG GCU AUA GUA GUU AG...

Polypeptide

Met

Ala

Leu

Ala

Lys

Ala

Thr

Val

Ser

Garbled

Example 2

Deletion (- 3)

- 3 UGC

mRNA

5'...

UAG UUUG AUG GCC UCU AAA GGC UAU AGU AGU UAG...

Polypeptide

Met

Ala

Ser

Lys

Gly

Try

Ser

STOP

Example 3

Insertion (+1)

+1 C

mRNA

5'...

UAG UUUG AUG GCC CUC UUG CAA AGG CUA UAG UAG UUAG...

Polypeptide

Met

Ala

Leu

Gln

Arg

Leu

STOP

Garbled

Example 4

Insertion (+1)

Deletion (-1)

+1 U

-1 C

mRNA

5'...

UAG UUUG AUG GCC UCU UUG CAA AGG UAU AGU AGU UAG...

Polypeptide

Met

Ala

Ser

Leu

Gln

Arg

Tyr

Ser

STOP

Garbled

Figure 38-5. Examples of the effects of deletions and insertions in a gene on the sequence of the mRNA

transcript and of the polypeptide chain translated therefrom. The arrows indicate the sites of deletions or inser-

tions, and the numbers in the ovals indicate the number of nucleotide residues deleted or inserted. Blue type

indicates amino acids in correct order.

LIKE TRANSCRIPTION, PROTEIN

The translation of the mRNA commences near its 5′

terminal with the formation of the corresponding

SYNTHESIS CAN BE DESCRIBED

amino terminal of the protein molecule. The message is

IN THREE PHASES: INITIATION,

read from 5′ to 3′, concluding with the formation of

ELONGATION, & TERMINATION

the carboxyl terminal of the protein. Again, the concept

of polarity is apparent. As described in Chapter 37, the

The general structural characteristics of ribosomes and

transcription of a gene into the corresponding mRNA

their self-assembly process are discussed in Chapter 37.

or its precursor first forms the 5′ terminal of the RNA

These particulate entities serve as the machinery on

molecule. In prokaryotes, this allows for the beginning

which the mRNA nucleotide sequence is translated into

of mRNA translation before the transcription of the

the sequence of amino acids of the specified protein.

gene is completed. In eukaryotic organisms, the process

PROTEIN SYNTHESIS & THE GENETIC CODE

365

of transcription is a nuclear one; mRNA translation oc-

particularly interesting in this regard. This kinase is ac-

curs in the cytoplasm. This precludes simultaneous

tivated by viruses and provides a host defense mecha-

transcription and translation in eukaryotic organisms

nism that decreases protein synthesis, thereby inhibit-

and makes possible the processing necessary to generate

ing viral replication. Phosphorylated eIF-2α binds

mature mRNA from the primary transcript—hnRNA.

tightly to and inactivates the GTP-GDP recycling pro-

tein eIF-2B. This prevents formation of the 43S preini-

tiation complex and blocks protein synthesis.

Initiation Involves Several Protein-RNA

Complexes (Figure 38-6)

OMPLEX

C. FORMATION OF THE 43S INITIATION C

Initiation of protein synthesis requires that an mRNA

The 5′ terminals of most mRNA molecules in eukary-

molecule be selected for translation by a ribosome.

otic cells are “capped,” as described in Chapter 37. This

Once the mRNA binds to the ribosome, the latter finds

methyl-guanosyl triphosphate cap facilitates the bind-

the correct reading frame on the mRNA, and transla-

ing of mRNA to the 43S preinitiation complex. A cap

tion begins. This process involves tRNA, rRNA,

binding protein complex, eIF-4F (4F), which consists

mRNA, and at least ten eukaryotic initiation factors

of eIF-4E and the eIF-4G (4G)-eIF4A (4A) complex,

(eIFs), some of which have multiple (three to eight)

binds to the cap through the 4E protein. Then eIF-4A

subunits. Also involved are GTP, ATP, and amino

(4A) and eIF-4B (4B) bind and reduce the complex sec-

acids. Initiation can be divided into four steps: (1) dis-

ondary structure of the 5′ end of the mRNA through

sociation of the ribosome into its 40S and 60S sub-

ATPase and ATP-dependent helicase activities. The as-

units; (2) binding of a ternary complex consisting of

sociation of mRNA with the 43S preinitiation complex

met-tRNAⁱ, GTP, and eIF-2 to the 40S ribosome to

to form the 48S initiation complex requires ATP hy-

form a preinitiation complex; (3) binding of mRNA to

drolysis. eIF-3 is a key protein because it binds with

the 40S preinitiation complex to form a 43S initiation

high affinity to the 4G component of 4F, and it links

complex; and (4) combination of the 43S initiation

this complex to the 40S ribosomal subunit. Following

complex with the 60S ribosomal subunit to form the

association of the 43S preinitiation complex with the

80S initiation complex.

mRNA cap and reduction (“melting”) of the secondary

structure near the 5′ end of the mRNA, the complex

A. RIBOSOMAL DISSOCIATION

scans the mRNA for a suitable initiation codon. Gener-

Two initiation factors, eIF-3 and eIF-1A, bind to the

ally this is the 5′-most AUG, but the precise initiation

newly dissociated 40S ribosomal subunit. This delays

codon is determined by so-called Kozak consensus se-

its reassociation with the 60S subunit and allows other

quences that surround the AUG:

translation initiation factors to associate with the 40S

subunit.

−3

−1

B. FORMATION OF THE 43S PREINITIATION COMPLEX

GCCA / GCCAUGG

The first step in this process involves the binding of

GTP by eIF-2. This binary complex then binds to met-

Most preferred is the presence of a purine at positions

tRNAⁱ, a tRNA specifically involved in binding to the

−3 and +4 relative to the AUG.

initiation codon AUG. (There are two tRNAs for me-

thionine. One specifies methionine for the initiator

D. ROLE OF THE POLY(A) TAIL IN INITIATION

codon, the other for internal methionines. Each has a

unique nucleotide sequence.) This ternary complex

Biochemical and genetic experiments in yeast have re-

binds to the 40S ribosomal subunit to form the 43S

vealed that the 3′ poly(A) tail and its binding protein,

preinitiation complex, which is stabilized by association

Pab1p, are required for efficient initiation of protein

with eIF-3 and eIF-1A.

synthesis. Further studies showed that the poly(A) tail

eIF-2 is one of two control points for protein syn-

stimulates recruitment of the 40S ribosomal subunit to

thesis initiation in eukaryotic cells. eIF-2 consists of

the mRNA through a complex set of interactions.

α, β, and γ subunits. eIF-2α is phosphorylated (on

Pab1p, bound to the poly(A) tail, interacts with eIF-4G,

serine

51) by at least four different protein kinases

which in turn binds to eIF-4E that is bound to the cap

(HCR, PKR, PERK, and GCN2) that are activated

structure. It is possible that a circular structure is

when a cell is under stress and when the energy expen-

formed and that this helps direct the 40S ribosomal

diture required for protein synthesis would be deleteri-

subunit to the 5′ end of the mRNA. This helps explain

ous. Such conditions include amino acid and glucose

how the cap and poly(A) tail structures have a synergis-

starvation, virus infection, misfolded proteins, serum

tic effect on protein synthesis. It appears that a similar

deprivation, hyperosmolality, and heat shock. PKR is

mechanism is at work in mammalian cells.

Ternary complex

Formation of the 80S

Activation of mRNA

formation

initiation complex

80S

Ribosomal

dissociation

60S

Met

40S

eIF-2C

Cap

AUG

(A)_n

Ternary

ATP

complex

Met

43S Preinitiation

Cap

AUG

(A)

complex

ATP

4A 4B

ADP + P

Met

Cap

AUG

(A)

Cap

AUG

(A)_n

GDP

Met

GTP

ATP

ADP + P_i

Cap

AUG

(A)_n

48S Initiation

complex

Met

eIF-5

+ P

Cap

(A)_n

P site

A site

Met

80S Initiation

complex

Elongation

Figure 38-6.

Diagrammatic representation of the initiation of protein synthesis on the mRNA template contain-

ing a 5′ cap (G^mTP-5′) and 3′ poly(A) terminal [3′(A)_n]. This process proceeds in three steps: (1) activation of mRNA;

(2) formation of the ternary complex consisting of tRNAmetⁱ, initiation factor eIF-2, and GTP; and (3) formation of the

active 80S initiation complex. (See text for details.) GTP, •; GDP, . The various initiation factors appear in abbrevi-

ated form as circles or squares, eg, eIF-3 ( 3 ), eIF-4F ( 4F ). 4•F is a complex consisting of 4E and 4A bound to 4G (see

Figure 38-7). The constellation of protein factors and the 40S ribosomal subunit comprise the 43S preinitiation com-

plex. When bound to mRNA, this forms the 48S preinitiation complex.

PROTEIN SYNTHESIS AND THE GENETIC CODE

367

E. FORMATION OF THE 80S INITIATION COMPLEX

PO₄

The binding of the 60S ribosomal subunit to the 48S

initiation complex involves hydrolysis of the GTP

PO₄

bound to eIF-2 by eIF-5. This reaction results in release

of the initiation factors bound to the 48S initiation

eIF-4E

complex (these factors then are recycled) and the rapid

Insulin

association of the 40S and 60S subunits to form the

(kinase

80S ribosome. At this point, the met-tRNAⁱ is on the P

activation)

eIF-4G

site of the ribosome, ready for the elongation cycle to

commence.

eIF-4A

The Regulation of eIF-4E Controls

the Rate of Initiation

The 4F complex is particularly important in controlling

the rate of protein translation. As described above, 4F is

eIF-4G

eIF-4F

a complex consisting of 4E, which binds to the m⁷G

complex

eIF-4E

eIF-4A

cap structure at the 5′ end of the mRNA, and 4G,

which serves as a scaffolding protein. In addition to

PO₄

binding 4E, 4G binds to eIF-3, which links the com-

plex to the 40S ribosomal subunit. It also binds 4A and

4B, the ATPase-helicase complex that helps unwind the

Cap

AUG

(A)_n

RNA (Figure 38-7).

4E is responsible for recognition of the mRNA cap

Figure 38-7. Activation of eIF-4E by insulin and for-

structure, which is a rate-limiting step in translation.

mation of the cap binding eIF-4F complex. The 4F-cap

This process is regulated at two levels. Insulin and mi-

mRNA complex is depicted as in Figure 38-6. The 4F

togenic growth factors result in the phosphorylation of

complex consists of eIF-4E (4E), eIF-4A, and eIF-4G. 4E is

4E on ser 209 (or thr 210). Phosphorylated 4E binds to

inactive when bound by one of a family of binding pro-

the cap much more avidly than does the nonphospho-

teins (4E-BPs). Insulin and mitogenic factors (eg, IGF-1,

rylated form, thus enhancing the rate of initiation. A

PDGF, interleukin-2, and angiotensin II) activate a serine

component of the MAP kinase pathway (see Figure

protein kinase in the mTOR pathway, and this results in

43-8) appears to be involved in this phosphorylation

the phosphorylation of 4E-BP. Phosphorylated 4E-BP

reaction.

dissociates from 4E, and the latter is then able to form

The activity of 4E is regulated in a second way, and

the 4F complex and bind to the mRNA cap. These

this also involves phosphorylation. A recently discov-

growth peptides also phosphorylate 4E itself by activat-

ered set of proteins bind to and inactivate 4E. These

ing a component of the MAP kinase pathway. Phos-

proteins include 4E-BP1 (BP1, also known as PHAS-1)

phorylated 4E binds much more avidly to the cap than

and the closely related proteins 4E-BP2 and 4E-BP3.

does nonphosphorylated 4E.

BP1 binds with high affinity to 4E. The [4E]•[BP1] as-

sociation prevents 4E from binding to 4G (to form 4F).

Since this interaction is essential for the binding of 4F

to the ribosomal 40S subunit and for correctly posi-

increase of protein synthesis in liver, adipose tissue, and

tioning this on the capped mRNA, BP-1 effectively in-

muscle.

hibits translation initiation.

Insulin and other growth factors result in the phos-

Elongation Also Is a Multistep Process

phorylation of BP-1 at five unique sites. Phosphoryla-

(Figure 38-8)

tion of BP-1 results in its dissociation from 4E, and it

cannot rebind until critical sites are dephosphorylated.

Elongation is a cyclic process on the ribosome in which

The protein kinase responsible has not been identified,

one amino acid at a time is added to the nascent peptide

but it appears to be different from the one that phos-

chain. The peptide sequence is determined by the order

phorylates 4E. A kinase in the mammalian target of

of the codons in the mRNA. Elongation involves several

rapamycin (mTOR) pathway, perhaps mTOR itself, is

steps catalyzed by proteins called elongation factors (EFs).

involved. These effects on the activation of 4E explain

These steps are (1) binding of aminoacyl-tRNA to the A

in part how insulin causes a marked posttranscriptional

site, (2) peptide bond formation, and (3) translocation.

368

CHAPTER 38

n+1

A. BINDING OF AMINOACYL-TRNA TO THE A SITE

Gm TP—5′

3′ (A)_n

In the complete

80S ribosome formed during the

process of initiation, the A site (aminoacyl or acceptor

site) is free. The binding of the proper aminoacyl-

P site

A site

tRNA in the A site requires proper codon recognition.

Elongation factor EF1A forms a ternary complex with

GTP and the entering aminoacyl-tRNA (Figure 38-8).

n-1

Peptidyl-

n-2

This complex then allows the aminoacyl-tRNA to enter

tRNA

the A site with the release of EF1A•GDP and phos-

m et

phate. GTP hydrolysis is catalyzed by an active site on

the ribosome. As shown in Figure 38-8, EF1A-GDP

then recycles to EF1A-GTP with the aid of other solu-

GTP

ble protein factors and GTP.

EFIA

B. PEPTIDE BOND FORMATION

n+1

The α-amino group of the new aminoacyl-tRNA in the

A site carries out a nucleophilic attack on the esterified

GDP

carboxyl group of the peptidyl-tRNA occupying the P

site (peptidyl or polypeptide site). At initiation, this site

GTP

n+1

is occupied by aminoacyl-tRNA metⁱ. This reaction is

5′

3′

catalyzed by a peptidyltransferase, a component of the

28S RNA of the 60S ribosomal subunit. This is another

example of ribozyme activity and indicates an impor-

P_i + GDP

tant—and previously unsuspected—direct role for

EFIA

RNA in protein synthesis (Table 38-3). Because the

n+1

amino acid on the aminoacyl-tRNA is already “acti-

E site

n-1

vated,” no further energy source is required for this re-

n-2

action. The reaction results in attachment of the grow-

ing peptide chain to the tRNA in the A site.

met

C. TRANSLOCATION

n+1

5′

3′

The now deacylated tRNA is attached by its anticodon

to the P site at one end and by the open CCA tail to an

exit

(E) site on the large ribosomal subunit (Figure

38-8). At this point, elongation factor 2 (EF2) binds

to and displaces the peptidyl tRNA from the A site to

n+1

the P site. In turn, the deacylated tRNA is on the E site,

GTP +

from which it leaves the ribosome. The EF2-GTP com-

n-1

plex is hydrolyzed to EF2-GDP, effectively moving the

EF2

n-2

mRNA forward by one codon and leaving the A site

open for occupancy by another ternary complex of

m et

amino acid tRNA-EF1A-GTP and another cycle of

elongation.

n+1

n+2

Codon

Gm TP—5′

3′ (A)_n

P_i + GDP +

Figure 38-8. Diagrammatic representation of the peptide

EF2

elongation process of protein synthesis. The small circles la-

n+1

beled n − 1, n, n + 1, etc, represent the amino acid residues of

n-1

the newly formed protein molecule. EFIA and EF2 represent

n-2

elongation factors 1 and 2, respectively. The peptidyl-tRNA and

aminoacyl-tRNA sites on the ribosome are represented by P site

met

and A site, respectively.

370

CHAPTER 38

Table 38-3. Evidence that rRNA

proteins that hydrolyze the peptidyl-tRNA bond when

is peptidyltransferase.

a stop codon occupies the A site. The mRNA is then re-

leased from the ribosome, which dissociates into its

component 40S and 60S subunits, and another cycle

• Ribosomes can make peptide bonds even when proteins

can be repeated.

are removed or inactivated.

• Certain parts of the rRNA sequence are highly conserved in

all species.

Polysomes Are Assemblies of Ribosomes

• These conserved regions are on the surface of the RNA

Many ribosomes can translate the same mRNA mole-

molecule.

cule simultaneously. Because of their relatively large

• RNA can be catalytic.

size, the ribosome particles cannot attach to an mRNA

• Mutations that result in antibiotic resistance at the level of

protein synthesis are more often found in rRNA than in the

any closer than 35 nucleotides apart. Multiple ribo-

protein components of the ribosome.

somes on the same mRNA molecule form a polyribo-

some, or “polysome.” In an unrestricted system, the

number of ribosomes attached to an mRNA (and thus

the size of polyribosomes) correlates positively with the

The charging of the tRNA molecule with the

length of the mRNA molecule. The mass of the mRNA

aminoacyl moiety requires the hydrolysis of an ATP to

molecule is, of course, quite small compared with the

an AMP, equivalent to the hydrolysis of two ATPs to

mass of even a single ribosome.

two ADPs and phosphates. The entry of the aminoacyl-

A single mammalian ribosome is capable of synthe-

tRNA into the A site results in the hydrolysis of one

sizing about 400 peptide bonds each minute. Polyribo-

GTP to GDP. Translocation of the newly formed pep-

somes actively synthesizing proteins can exist as free

tidyl-tRNA in the A site into the P site by EF2 similarly

particles in the cellular cytoplasm or may be attached to

results in hydrolysis of GTP to GDP and phosphate.

sheets of membranous cytoplasmic material referred to

Thus, the energy requirements for the formation of one

as endoplasmic reticulum. Attachment of the particu-

peptide bond include the equivalent of the hydrolysis of

late polyribosomes to the endoplasmic reticulum is re-

two ATP molecules to ADP and of two GTP molecules

sponsible for its “rough” appearance as seen by electron

to GDP, or the hydrolysis of four high-energy phos-

microscopy. The proteins synthesized by the attached

phate bonds. A eukaryotic ribosome can incorporate as

polyribosomes are extruded into the cisternal space be-

many as six amino acids per second; prokaryotic ribo-

tween the sheets of rough endoplasmic reticulum and

somes incorporate as many as 18 per second. Thus, the

are exported from there. Some of the protein products

process of peptide synthesis occurs with great speed and

of the rough endoplasmic reticulum are packaged by

accuracy until a termination codon is reached.

the Golgi apparatus into zymogen particles for eventual

export (see Chapter 46). The polyribosomal particles

free in the cytosol are responsible for the synthesis of

Termination Occurs When a Stop

proteins required for intracellular functions.

Codon Is Recognized (Figure 38-9)

In comparison to initiation and elongation, termina-

The Machinery of Protein Synthesis Can

tion is a relatively simple process. After multiple cycles

Respond to Environmental Threats

of elongation culminating in polymerization of the spe-

cific amino acids into a protein molecule, the stop or

Ferritin, an iron-binding protein, prevents ionized iron

terminating codon of mRNA (UAA, UAG, UGA) ap-

(Fe2+) from reaching toxic levels within cells. Elemental

pears in the A site. Normally, there is no tRNA with an

iron stimulates ferritin synthesis by causing the release of

anticodon capable of recognizing such a termination

a cytoplasmic protein that binds to a specific region in

signal. Releasing factor RF1 recognizes that a stop

the 5′ nontranslated region of ferritin mRNA. Disrup-

codon resides in the A site (Figure 38-9). RF1 is bound

tion of this protein-mRNA interaction activates ferritin

by a complex consisting of releasing factor RF3 with

mRNA and results in its translation. This mechanism

bound GTP. This complex, with the peptidyl trans-

provides for rapid control of the synthesis of a protein

ferase, promotes hydrolysis of the bond between the

that sequesters Fe2+, a potentially toxic molecule.

peptide and the tRNA occupying the P site. Thus, a

water molecule rather than an amino acid is added.

Many Viruses Co-opt the Host Cell

This hydrolysis releases the protein and the tRNA from

Protein Synthesis Machinery

the P site. Upon hydrolysis and release, the 80S ribo-

some dissociates into its 40S and 60S subunits, which

The protein synthesis machinery can also be modified

are then recycled. Therefore, the releasing factors are

in deleterious ways. Viruses replicate by using host

PROTEIN SYNTHESIS & THE GENETIC CODE

371

cell processes, including those involved in protein syn-

thesis. Some viral mRNAs are translated much more ef-

ficiently than those of the host cell (eg, encephalomyo-

Cap

AUG

carditis virus). Others, such as reovirus and vesicular

stomatitis virus, replicate abundantly, and their mRNAs

have a competitive advantage over host cell mRNAs for

limited translation factors. Other viruses inhibit host

IRES

AUG

cell protein synthesis by preventing the association of

Poliovirus

mRNA with the 40S ribosome.

protease

Poliovirus and other picornaviruses gain a selective

advantage by disrupting the function of the 4F complex

Nil

to their advantage. The mRNAs of these viruses do not

Cap

AUG

have a cap structure to direct the binding of the 40S ri-

bosomal subunit (see above). Instead, the 40S ribosomal

subunit contacts an internal ribosomal entry site

(IRES) in a reaction that requires 4G but not 4E. The

IRES

AUG

virus gains a selective advantage by having a protease that

Figure 38-10. Picornaviruses disrupt the 4F com-

attacks 4G and removes the amino terminal 4E binding

plex. The 4E-4G complex (4F) directs the 40S ribosomal

site. Now the 4E-4G complex (4F) cannot form, so the

subunit to the typical capped mRNA (see text). 4G

40S ribosomal subunit cannot be directed to capped

alone is sufficient for targeting the 40S subunit to the

mRNAs. Host cell translation is thus abolished. The 4G

internal ribosomal entry site (IRES) of viral mRNAs. To

fragment can direct binding of the 40S ribosomal sub-

gain selective advantage, certain viruses (eg, poliovirus)

unit to IRES-containing mRNAs, so viral mRNA trans-

lation is very efficient (Figure 38-10). These viruses also

have a protease that cleaves the 4E binding site from

promote the dephosphorylation of BP1

(PHAS-1),

the amino terminal end of 4G. This truncated 4G can di-

thereby decreasing cap (4E)-dependent translation.

rect the 40S ribosomal subunit to mRNAs that have an

IRES but not to those that have a cap. The widths of the

arrows indicate the rate of translation initiation from

POSTTRANSLATIONAL PROCESSING

the AUG codon in each example.

AFFECTS THE ACTIVITY OF

MANY PROTEINS

Some animal viruses, notably poliovirus and hepatitis A

lagen polypeptide molecules, frequently not identical in

virus, synthesize long polycistronic proteins from one

sequence, align themselves in a particular way that is

long mRNA molecule. These protein molecules are

dependent upon the existence of specific amino termi-

subsequently cleaved at specific sites to provide the sev-

nal peptides. Specific enzymes then carry out hydrox-

eral specific proteins required for viral function. In ani-

ylations and oxidations of specific amino acid residues

mal cells, many proteins are synthesized from the

within the procollagen molecules to provide cross-links

mRNA template as a precursor molecule, which then

for greater stability. Amino terminal peptides are

must be modified to achieve the active protein. The

cleaved off the molecule to form the final product—a

prototype is insulin, which is a low-molecular-weight

strong, insoluble collagen molecule. Many other post-

protein having two polypeptide chains with interchain

translational modifications of proteins occur. Covalent

and intrachain disulfide bridges. The molecule is syn-

modification by acetylation, phosphorylation, methyla-

thesized as a single chain precursor, or prohormone,

tion, ubiquitinylation, and glycosylation is common,

which folds to allow the disulfide bridges to form. A

for example.

specific protease then clips out the segment that con-

nects the two chains which form the functional insulin

MANY ANTIBIOTICS WORK BECAUSE

molecule (see Figure 42-12).

THEY SELECTIVELY INHIBIT PROTEIN

Many other peptides are synthesized as proproteins

SYNTHESIS IN BACTERIA

that require modifications before attaining biologic ac-

tivity. Many of the posttranslational modifications in-

Ribosomes in bacteria and in the mitochondria of

volve the removal of amino terminal amino acid

higher eukaryotic cells differ from the mammalian ribo-

residues by specific aminopeptidases. Collagen, an

some described in Chapter 35. The bacterial ribosome

abundant protein in the extracellular spaces of higher

is smaller (70S rather than 80S) and has a different,

eukaryotes, is synthesized as procollagen. Three procol-

somewhat simpler complement of RNA and protein

372

CHAPTER 38

molecules. This difference is exploited for clinical pur-

Other antibiotics inhibit protein synthesis on all ri-

poses because many effective antibiotics interact specifi-

bosomes (puromycin) or only on those of eukaryotic

cally with the proteins and RNAs of prokaryotic ribo-

cells (cycloheximide). Puromycin (Figure 38-11) is a

somes and thus inhibit protein synthesis. This results in

structural analog of tyrosinyl-tRNA. Puromycin is in-

growth arrest or death of the bacterium. The most use-

corporated via the A site on the ribosome into the car-

ful members of this class of antibiotics (eg, tetracy-

boxyl terminal position of a peptide but causes the pre-

clines, lincomycin, erythromycin, and chlorampheni-

mature release of the polypeptide. Puromycin, as a

col) do not interact with components of eukaryotic

tyrosinyl-tRNA analog, effectively inhibits protein syn-

ribosomal particles and thus are not toxic to eukaryotes.

thesis in both prokaryotes and eukaryotes. Cyclohex-

Tetracycline prevents the binding of aminoacyl-tRNAs

imide inhibits peptidyltransferase in the 60S ribosomal

to the A site. Chloramphenicol and the macrolide class

subunit in eukaryotes, presumably by binding to an

of antibiotics work by binding to 23S rRNA, which is

rRNA component.

interesting in view of the newly appreciated role of

Diphtheria toxin, an exotoxin of Corynebacterium

rRNA in peptide bond formation through its peptidyl-

diphtheriae infected with a specific lysogenic phage, cat-

transferase activity. It should be mentioned that the

alyzes the ADP-ribosylation of EF-2 on the unique

close similarity between prokaryotic and mitochondrial

amino acid diphthamide in mammalian cells. This

ribosomes can lead to complications in the use of some

modification inactivates EF-2 and thereby specifically

antibiotics.

inhibits mammalian protein synthesis. Many animals

(eg, mice) are resistant to diphtheria toxin. This resis-

tance is due to inability of diphtheria toxin to cross the

cell membrane rather than to insensitivity of mouse

N(CH₃)₂

EF-2 to diphtheria toxin-catalyzed ADP-ribosylation

by NAD.

Ricin, an extremely toxic molecule isolated from the

castor bean, inactivates eukaryotic 28S ribosomal RNA

by providing the N-glycolytic cleavage or removal of a

HOCH₂

single adenine.

Many of these compounds—puromycin and cyclo-

H H

heximide in particular—are not clinically useful but

have been important in elucidating the role of protein

NH OH

synthesis in the regulation of metabolic processes, par-

ticularly enzyme induction by hormones.

CH₂

OCH₃

NH₂

SUMMARY

• The flow of genetic information follows the sequence

DNA → RNA → protein.

• The genetic information in the structural region of a

NH₂

gene is transcribed into an RNA molecule such that

the sequence of the latter is complementary to that in

the DNA.

O^-

• Several different types of RNA, including ribosomal

tRNA

O P O

CH₂

RNA (rRNA), transfer RNA (tRNA), and messenger

RNA (mRNA), are involved in protein synthesis.

• The information in mRNA is in a tandem array of

H H

codons, each of which is three nucleotides long.

• The mRNA is read continuously from a start codon

(AUG) to a termination codon (UAA, UAG, UGA).

CH₂

• The open reading frame of the mRNA is the series of

NH₂

codons, each specifying a certain amino acid, that de-

termines the precise amino acid sequence of the pro-

Figure 38-11. The comparative structures of the an-

tein.

tibiotic puromycin (top) and the 3′ terminal portion of

• Protein synthesis, like DNA and RNA synthesis, fol-

tyrosinyl-tRNA (bottom).

lows a 5′ to 3′ polarity and can be divided into three

PROTEIN SYNTHESIS & THE GENETIC CODE

373

processes: initiation, elongation, and termination.

Kozak M: Structural features in eukaryotic mRNAs that modulate

the initiation of translation. J Biol Chem 1991;266:1986.

Mutant proteins arise when single-base substitutions

Lawrence JC, Abraham RT: PHAS/4E-BPs as regulators of mRNA

result in codons that specify a different amino acid at

translation and cell proliferation. Trends Biochem Sci

a given position, when a stop codon results in a trun-

1997;22:345.

cated protein, or when base additions or deletions

Sachs AB, Buratowski S: Common themes in translational and

alter the reading frame, so different codons are read.

transcriptional regulation. Trends Biochem Sci 1997;22:189.

• A variety of compounds, including several antibi-

Sachs AB, Sarnow P, Hentze MW: Starting at the beginning, mid-

otics, inhibit protein synthesis by affecting one or

dle and end: translation initiation in eukaryotes. Cell 1997;

more of the steps involved in protein synthesis.

98:831.

Weatherall DJ et al: The hemoglobinopathies. In: The Metabolic

and Molecular Bases of Inherited Disease, 8th ed. Scriver CR et

REFERENCES

al (editors). McGraw-Hill, 2001.

Crick F et al: The genetic code. Nature 1961;192:1227.

Green R, Noller HF: Ribosomes and translation. Annu Rev

Biochem 1997;66:679.

Regulation of Gene Expression

Daryl K. Granner, MD, & P. Anthony Weil, PhD

BIOMEDICAL IMPORTANCE

suring that the organism can respond to complex envi-

ronmental challenges.

Organisms adapt to environmental changes by altering

In simple terms, there are only two types of gene

gene expression. The process of alteration of gene ex-

regulation: positive regulation and negative regula-

pression has been studied in detail and often involves

tion (Table 39-1). When the expression of genetic in-

modulation of gene transcription. Control of transcrip-

formation is quantitatively increased by the presence of

tion ultimately results from changes in the interaction

a specific regulatory element, regulation is said to be

of specific binding regulatory proteins with various re-

positive; when the expression of genetic information is

gions of DNA in the controlled gene. This can have a

diminished by the presence of a specific regulatory ele-

positive or negative effect on transcription. Transcrip-

ment, regulation is said to be negative. The element or

tion control can result in tissue-specific gene expres-

molecule mediating negative regulation is said to be a

sion, and gene regulation is influenced by hormones,

negative regulator or repressor; that mediating positive

heavy metals, and chemicals. In addition to transcrip-

regulation is a positive regulator or activator. However,

tion level controls, gene expression can also be modu-

a double negative has the effect of acting as a positive.

lated by gene amplification, gene rearrangement, post-

Thus, an effector that inhibits the function of a nega-

transcriptional modifications, and RNA stabilization.

tive regulator will appear to bring about a positive regu-

Many of the mechanisms that control gene expression

lation. Many regulated systems that appear to be in-

are used to respond to hormones and therapeutic

duced are in fact derepressed at the molecular level.

agents. Thus, a molecular understanding of these

(See Chapter 9 for explanation of these terms.)

processes will lead to development of agents that alter

pathophysiologic mechanisms or inhibit the function or

arrest the growth of pathogenic organisms.

BIOLOGIC SYSTEMS EXHIBIT THREE

TYPES OF TEMPORAL RESPONSES

REGULATED EXPRESSION OF GENES

TO A REGULATORY SIGNAL

IS REQUIRED FOR DEVELOPMENT,

Figure 39-1 depicts the extent or amount of gene ex-

DIFFERENTIATION, & ADAPTATION

pression in three types of temporal response to an in-

The genetic information present in each somatic cell of

ducing signal. A type A response is characterized by an

a metazoan organism is practically identical. The excep-

increased extent of gene expression that is dependent

tions are found in those few cells that have amplified or

upon the continued presence of the inducing signal.

rearranged genes in order to perform specialized cellular

When the inducing signal is removed, the amount of

functions. Expression of the genetic information must

gene expression diminishes to its basal level, but the

be regulated during ontogeny and differentiation of the

amount repeatedly increases in response to the reap-

organism and its cellular components. Furthermore, in

pearance of the specific signal. This type of response is

order for the organism to adapt to its environment and

commonly observed in prokaryotes in response to sud-

to conserve energy and nutrients, the expression of

den changes of the intracellular concentration of a nu-

genetic information must be cued to extrinsic signals

trient. It is also observed in many higher organisms

and respond only when necessary. As organisms have

after exposure to inducers such as hormones, nutrients,

evolved, more sophisticated regulatory mechanisms

or growth factors (Chapter 43).

have appeared which provide the organism and its cells

A type B response exhibits an increased amount of

with the responsiveness necessary for survival in a com-

gene expression that is transient even in the continued

plex environment. Mammalian cells possess about 1000

presence of the regulatory signal. After the regulatory

times more genetic information than does the bac-

signal has terminated and the cell has been allowed to

terium Escherichia coli. Much of this additional genetic

recover, a second transient response to a subsequent

information is probably involved in regulation of gene

regulatory signal may be observed. This phenomenon

expression during the differentiation of tissues and bio-

of response-desensitization-recovery characterizes the

logic processes in the multicellular organism and in en-

action of many pharmacologic agents, but it is also a

374

REGULATION OF GENE EXPRESSION

375

Table 39-1. Effects of positive and negative

feature of many naturally occurring processes. This type

regulation on gene expression.

of response commonly occurs during development of

an organism, when only the transient appearance of a

specific gene product is required although the signal

Rate of Gene Expression

persists.

Negative

Positive

The type C response pattern exhibits, in response

Regulation

to the regulatory signal, an increased extent of gene ex-

pression that persists indefinitely even after termination

Regulator present

Decreased

Increased

of the signal. The signal acts as a trigger in this pattern.

Regulator absent

Increased

Decreased

Once expression of the gene is initiated in the cell, it

cannot be terminated even in the daughter cells; it is

therefore an irreversible and inherited alteration. This

type of response typically occurs during the develop-

ment of differentiated function in a tissue or organ.

Type A

Prokaryotes Provide Models for the Study

of Gene Expression in Mammalian Cells

Analysis of the regulation of gene expression in

prokaryotic cells helped establish the principle that in-

formation flows from the gene to a messenger RNA to a

specific protein molecule. These studies were aided by

the advanced genetic analyses that could be performed

in prokaryotic and lower eukaryotic organisms. In re-

Time

cent years, the principles established in these early stud-

Signal

ies, coupled with a variety of molecular biology tech-

Type B

niques, have led to remarkable progress in the analysis

of gene regulation in higher eukaryotic organisms, in-

cluding mammals. In this chapter, the initial discussion

will center on prokaryotic systems. The impressive ge-

netic studies will not be described, but the physiology

of gene expression will be discussed. However, nearly

all of the conclusions about this physiology have been

derived from genetic studies and confirmed by molecu-

lar genetic and biochemical studies.

Recovery

Time

Signal

Some Features of Prokaryotic Gene

Expression Are Unique

Type C

Before the physiology of gene expression can be ex-

plained, a few specialized genetic and regulatory terms

must be defined for prokaryotic systems. In prokary-

otes, the genes involved in a metabolic pathway are

often present in a linear array called an operon, eg, the

lac operon. An operon can be regulated by a single pro-

moter or regulatory region. The cistron is the smallest

unit of genetic expression. As described in Chapter 9,

some enzymes and other protein molecules are com-

posed of two or more nonidentical subunits. Thus, the

Time

Signal

“one gene, one enzyme” concept is not necessarily

valid. The cistron is the genetic unit coding for the

Figure 39-1.

Diagrammatic representations of the

structure of the subunit of a protein molecule, acting as

responses of the extent of expression of a gene to spe-

it does as the smallest unit of genetic expression. Thus,

cific regulatory signals such as a hormone.

the one gene, one enzyme idea might more accurately

376

CHAPTER 39

be regarded as a one cistron, one subunit concept. A

Promoter

Operator

single mRNA that encodes more than one separately

site

translated protein is referred to as a polycistronic

lacI

lacZ

lacY lacA

mRNA. For example, the polycistronic lac operon

mRNA is translated into three separate proteins (see

lac operon

below). Operons and polycistronic mRNAs are com-

mon in bacteria but not in eukaryotes.

Figure 39-2. The positional relationships of the

An inducible gene is one whose expression increases

structural and regulatory genes of the lac operon. lacZ

in response to an inducer or activator, a specific posi-

encodes β-galactosidase, lacY encodes a permease, and

tive regulatory signal. In general, inducible genes have

lacA encodes a thiogalactoside transacetylase. lacI en-

relatively low basal rates of transcription. By contrast,

codes the lac operon repressor protein.

genes with high basal rates of transcription are often

subject to down-regulation by repressors.

The expression of some genes is constitutive, mean-

encodes the primary transcript. Although there are

ing that they are expressed at a reasonably constant rate

many historical exceptions, a gene is generally italicized

and not known to be subject to regulation. These are

in lower case and the encoded protein, when abbrevi-

often referred to as housekeeping genes. As a result of

ated, is expressed in roman type with the first letter cap-

mutation, some inducible gene products become con-

italized. For example, the gene lacI encodes the repres-

stitutively expressed. A mutation resulting in constitu-

sor protein LacI. When E coli is presented with lactose

tive expression of what was formerly a regulated gene is

or some specific lactose analogs under appropriate non-

called a constitutive mutation.

repressing conditions (eg, high concentrations of lac-

tose, no or very low glucose in media; see below), the

expression of the activities of β-galactosidase, galacto-

Analysis of Lactose Metabolism in E coli

side permease, and thiogalactoside transacetylase is in-

Led to the Operon Hypothesis

creased 100-fold to 1000-fold. This is a type A re-

Jacob and Monod in 1961 described their operon

sponse, as depicted in Figure 39-1. The kinetics of

model in a classic paper. Their hypothesis was to a

induction can be quite rapid; lac-specific mRNAs are

large extent based on observations on the regulation of

fully induced within 5-6 minutes after addition of lac-

lactose metabolism by the intestinal bacterium E coli.

tose to a culture; β-galactosidase protein is maximal

The molecular mechanisms responsible for the regula-

within 10 minutes. Under fully induced conditions,

tion of the genes involved in the metabolism of lactose

there can be up to 5000 β-galactosidase molecules per

are now among the best-understood in any organism.

cell, an amount about 1000 times greater than the

β-Galactosidase hydrolyzes the β-galactoside lactose to

basal, uninduced level. Upon removal of the signal, ie,

galactose and glucose. The structural gene for β-galac-

the inducer, the synthesis of these three enzymes de-

tosidase (lacZ) is clustered with the genes responsible

clines.

for the permeation of galactose into the cell (lacY) and

When E coli is exposed to both lactose and glucose

for thiogalactoside transacetylase (lacA). The structural

as sources of carbon, the organisms first metabolize

genes for these three enzymes, along with the lac pro-

the glucose and then temporarily stop growing until the

moter and lac operator (a regulatory region), are physi-

genes of the lac operon become induced to provide the

cally associated to constitute the lac operon as depicted

ability to metabolize lactose as a usable energy source.

in Figure 39-2. This genetic arrangement of the struc-

Although lactose is present from the beginning of the

tural genes and their regulatory genes allows for coordi-

bacterial growth phase, the cell does not induce those

nate expression of the three enzymes concerned with

enzymes necessary for catabolism of lactose until the

lactose metabolism. Each of these linked genes is tran-

glucose has been exhausted. This phenomenon was first

scribed into one large mRNA molecule that contains

thought to be attributable to repression of the lac

multiple independent translation start (AUG) and stop

operon by some catabolite of glucose; hence, it was

(UAA) codons for each cistron. Thus, each protein is

termed catabolite repression. It is now known that

translated separately, and they are not processed from a

catabolite repression is in fact mediated by a catabolite

single large precursor protein. This type of mRNA mol-

gene activator protein

(CAP) in conjunction with

ecule is called a polycistronic mRNA. Polycistronic

cAMP (Figure 18-5). This protein is also referred to as

mRNAs are predominantly found in prokaryotic organ-

the cAMP regulatory protein (CRP). The expression of

isms.

many inducible enzyme systems or operons in E coli

It is now conventional to consider that a gene in-

and other prokaryotes is sensitive to catabolite repres-

cludes regulatory sequences as well as the region that

sion, as discussed below.

REGULATION OF GENE EXPRESSION

377

Operator

Promoter

lacI gene

lacZ gene

lacY gene

lacA gene

RNA polymerase

No inducer

cannot transcribe

↑ RNA

operator or distal

polymerase

genes (Z,Y, A)

Inducer

and

glucose

Repressor

subunits

Repressor

(tetramer)

CAP-cAMP

lacI

lacZ

lacY

lacA

With inducer

and

RNA polymerases transcribing genes

no glucose

Inactive

repressor

mRNA

β-Galacto-

Permease Transacetylase

Inducers

sidase

protein

Figure 39-3. The mechanism of repression and derepression of the lac operon. When either no inducer is

present or inducer is present with glucose (A), the lacI gene products that are synthesized constitutively form

a repressor tetramer molecule which binds at the operator locus to prevent the efficient initiation of transcrip-

tion by RNA polymerase at the promoter locus and thus to prevent the subsequent transcription of the lacZ,

lacY, and lacA structural genes. When inducer is present (B), the constitutively expressed lacI gene forms re-

pressor molecules that are conformationally altered by the inducer and cannot efficiently bind to the operator

locus (affinity of binding reduced > 1000-fold). In the presence of cAMP and its binding protein (CAP), the RNA

polymerase can transcribe the structural genes lacZ, lacY, and lacA, and the polycistronic mRNA molecule

formed can be translated into the corresponding protein molecules β-galactosidase, permease, and

transacetylase, allowing for the catabolism of lactose.

The physiology of induction of the lac operon is

an inverted palindrome (indicated by solid lines about

well understood at the molecular level (Figure 39-3).

the dotted axis) in a region that is 21 base pairs long, as

Expression of the normal lacI gene of the lac operon is

shown below:

constitutive; it is expressed at a constant rate, resulting

in formation of the subunits of the lac repressor. Four

identical subunits with molecular weights of 38,000 as-

5′ − AAT

TGTGAGC G GATAACAATT

semble into a lac repressor molecule. The LacI repressor

3′−

TTA ACACTCG C CTATTGTTAA

protein molecule, the product of lacI, has a high affinity

(K_d about 10−13 mol/L) for the operator locus. The op-

erator locus is a region of double-stranded DNA 27

The minimum effective size of an operator for LacI

base pairs long with a twofold rotational symmetry and

repressor binding is 17 base pairs (boldface letters in the

378

CHAPTER 39

above sequence). At any one time, only two subunits of

priate bioengineered constructs is commonly used to ex-

the repressors appear to bind to the operator, and within

press mammalian recombinant proteins in E coli.

the 17-base-pair region at least one base of each base

In order for the RNA polymerase to efficiently form

pair is involved in LacI recognition and binding. The

a PIC at the promoter site, there must also be present

binding occurs mostly in the major groove without in-

the catabolite gene activator protein (CAP) to which

terrupting the base-paired, double-helical nature of the

cAMP is bound. By an independent mechanism, the

operator DNA. The operator locus is between the pro-

bacterium accumulates cAMP only when it is starved

moter site, at which the DNA-dependent RNA polym-

for a source of carbon. In the presence of glucose—or

erase attaches to commence transcription, and the tran-

of glycerol in concentrations sufficient for growth—the

scription initiation site of the lacZ gene, the structural

bacteria will lack sufficient cAMP to bind to CAP be-

gene for β-galactosidase (Figure 39-2). When attached

cause the glucose inhibits adenylyl cyclase, the enzyme

to the operator locus, the LacI repressor molecule pre-

that converts ATP to cAMP (see Chapter 42). Thus, in

vents transcription of the operator locus as well as of the

the presence of glucose or glycerol, cAMP-saturated

distal structural genes, lacZ, lacY, and lacA. Thus, the

CAP is lacking, so that the DNA-dependent RNA

LacI repressor molecule is a negative regulator; in its

polymerase cannot initiate transcription of the lac

presence (and in the absence of inducer; see below), ex-

operon. In the presence of the CAP-cAMP complex,

pression from the lacZ, lacY, and lacA genes is pre-

which binds to DNA just upstream of the promoter

vented. There are normally 20-40 repressor tetramer

site, transcription then occurs (Figure 39-3). Studies

molecules in the cell, a concentration of tetramer suffi-

indicate that a region of CAP contacts the RNA polym-

cient to effect, at any given time, > 95% occupancy of

erase α subunit and facilitates binding of this enzyme to

the one lac operator element in a bacterium, thus ensur-

the promoter. Thus, the CAP-cAMP regulator is acting

ing low (but not zero) basal lac operon gene transcrip-

as a positive regulator because its presence is required

tion in the absence of inducing signals.

for gene expression. The lac operon is therefore con-

A lactose analog that is capable of inducing the lac

trolled by two distinct, ligand-modulated DNA bind-

operon while not itself serving as a substrate for β-galac-

ing trans factors; one that acts positively (cAMP-CRP

tosidase is an example of a gratuitous inducer. An ex-

complex) and one that acts negatively (LacI repressor).

ample is isopropylthiogalactoside (IPTG). The addition

Maximal activity of the lac operon occurs when glucose

of lactose or of a gratuitous inducer such as IPTG to

levels are low (high cAMP with CAP activation) and

bacteria growing on a poorly utilized carbon source

lactose is present (LacI is prevented from binding to the

(such as succinate) results in prompt induction of the

operator).

lac operon enzymes. Small amounts of the gratuitous in-

When the lacI gene has been mutated so that its

ducer or of lactose are able to enter the cell even in the

product, LacI, is not capable of binding to operator

absence of permease. The LacI repressor molecules—

DNA, the organism will exhibit constitutive expres-

both those attached to the operator loci and those free in

sion of the lac operon. In a contrary manner, an organ-

the cytosol—have a high affinity for the inducer. Bind-

ism with a lacI gene mutation that produces a LacI pro-

ing of the inducer to a repressor molecule attached to

tein which prevents the binding of an inducer to the

the operator locus induces a conformational change in

repressor will remain repressed even in the presence of

the structure of the repressor and causes it to dissociate

the inducer molecule, because the inducer cannot bind

from the DNA because its affinity for the operator is

to the repressor on the operator locus in order to dere-

now 10³ times lower (K_d about 10−9 mol/L) than that of

press the operon. Similarly, bacteria harboring muta-

LacI in the absence of IPTG. If DNA-dependent RNA

tions in their lac operator locus such that the operator

polymerase has already attached to the coding strand at

sequence will not bind a normal repressor molecule

the promoter site, transcription will begin. The polym-

constitutively express the lac operon genes. Mechanisms

erase generates a polycistronic mRNA whose 5′ terminal

of positive and negative regulation comparable to those

is complementary to the template strand of the operator.

described here for the lac system have been observed in

In such a manner, an inducer derepresses the lac

eukaryotic cells (see below).

operon and allows transcription of the structural genes

for β-galactosidase, galactoside permease, and thiogalac-

The Genetic Switch of Bacteriophage

toside transacetylase. Translation of the polycistronic

Lambda ( ) Provides a Paradigm

mRNA can occur even before transcription is com-

for Protein-DNA Interactions

pleted. Derepression of the lac operon allows the cell to

in Eukaryotic Cells

synthesize the enzymes necessary to catabolize lactose as

an energy source. Based on the physiology just de-

Like some eukaryotic viruses (eg, herpes simplex, HIV),

scribed, IPTG-induced expression of transfected plas-

some bacterial viruses can either reside in a dormant

mids bearing the lac operator-promoter ligated to appro-

state within the host chromosomes or can replicate

REGULATION OF GENE EXPRESSION

379

within the bacterium and eventually lead to lysis and

killing of the bacterial host. Some E coli harbor such a

“temperate” virus, bacteriophage lambda

(λ). When

lambda infects an organism of that species it injects its

45,000-bp, double-stranded, linear DNA genome into

the cell (Figure 39-4). Depending upon the nutritional

state of the cell, the lambda DNA will either integrate

into the host genome (lysogenic pathway) and remain

dormant until activated (see below), or it will com-

mence replicating until it has made about 100 copies

of complete, protein-packaged virus, at which point it

causes lysis of its host (lytic pathway). The newly gen-

erated virus particles can then infect other susceptible

hosts.

When integrated into the host genome in its dor-

mant state, lambda will remain in that state until acti-

Lysogenic

Lytic

vated by exposure of its lysogenic bacterial host to

pathway

DNA-damaging agents. In response to such a noxious

stimulus, the dormant bacteriophage becomes

“in-

duced” and begins to transcribe and subsequently trans-

late those genes of its own genome which are necessary

for its excision from the host chromosome, its DNA

replication, and the synthesis of its protein coat and

lysis enzymes. This event acts like a trigger or type C

(Figure 39-1) response; ie, once lambda has committed

itself to induction, there is no turning back until the

cell is lysed and the replicated bacteriophage released.

Ultraviolet

This switch from a dormant or prophage state to a

radiation

Induction

lytic infection is well understood at the genetic and

molecular levels and will be described in detail here.

The switching event in lambda is centered around

an 80-bp region in its double-stranded DNA genome

referred to as the “right operator” (O_R) (Figure 39-5A).

The right operator is flanked on its left side by the

Figure 39-4. Infection of the bacterium E coli by

structural gene for the lambda repressor protein, the cI

phage lambda begins when a virus particle attaches it-

protein, and on its right side by the structural gene en-

self to the bacterial cell (1) and injects its DNA (shaded

coding another regulatory protein called Cro. When

line) into the cell (2, 3). Infection can take either of two

lambda is in its prophage state—ie, integrated into the

courses depending on which of two sets of viral genes

host genome—the cI repressor gene is the only lambda

gene cI protein that is expressed. When the bacterio-

is turned on. In the lysogenic pathway, the viral DNA

phage is undergoing lytic growth, the cI repressor gene

becomes integrated into the bacterial chromosome (4,

is not expressed, but the cro gene—as well as many

5), where it replicates passively as the bacterial cell di-

other genes in lambda—is expressed. That is, when the

vides. The dormant virus is called a prophage, and the

repressor gene is on, the cro gene is off, and when

cell that harbors it is called a lysogen. In the alternative

the cro gene is on, the repressor gene is off. As we

lytic mode of infection, the viral DNA replicates itself (6)

shall see, these two genes regulate each other’s expres-

and directs the synthesis of viral proteins (7). About 100

sion and thus, ultimately, the decision between lytic

new virus particles are formed. The proliferating viruses

and lysogenic growth of lambda. This decision be-

lyse, or burst, the cell (8). A prophage can be “induced”

tween repressor gene transcription and cro gene

by a DNA damaging agent such as ultraviolet radiation

transcription is a paradigmatic example of a molecu-

(9). The inducing agent throws a switch, so that a differ-

lar switch.

ent set of genes is turned on. Viral DNA loops out of the

The 80-bp λ right operator, O_R, can be subdivided

chromosome (10) and replicates; the virus proceeds

into three discrete, evenly spaced,

17-bp cis-active

along the lytic pathway. (Reproduced, with permission,

DNA elements that represent the binding sites for ei-

from Ptashne M, Johnson AD, Pabo CO: A genetic switch

ther of two bacteriophage λ regulatory proteins. Impor-

in a bacterial virus. Sci Am [Nov] 1982;247:128.)

380

CHAPTER 39

Gene for repressor (cl)

Gene for Cro

O_R

Repressor RNA

O_R3

O_R2

O_R1

Repressor promoter

cro Promoter

cro RNA

T A C C T C T G G C G G T G A T A

A T G G A G A C C G C C A C T A T

Figure 39-5. Right operator (O_R) is shown in increasing detail in this series of drawings.

The operator is a region of the viral DNA some 80 base pairs long (A). To its left lies the

gene encoding lambda repressor (cI), to its right the gene (cro) encoding the regulator pro-

tein Cro. When the operator region is enlarged (B), it is seen to include three subregions,

O_R1, O_R2, and O_R3, each 17 base pairs long. They are recognition sites to which both repres-

sor and Cro can bind. The recognition sites overlap two promoters—sequences of bases to

which RNA polymerase binds in order to transcribe these genes into mRNA (wavy lines),

that are translated into protein. Site O_R1 is enlarged (C) to show its base sequence. Note

that in this region of the λ chromosome, both strands of DNA act as a template for tran-

scription (Chapter 39). (Reproduced, with permission, from Ptashne M, Johnson AD, Pabo CO:

A genetic switch in a bacterial virus. Sci Am [Nov] 1982;247:128.)

tantly, the nucleotide sequences of these three tandemly

39-6D). The Cro protein’s single domain

mediates

arranged sites are similar but not identical

(Figure

both operator binding and dimerization.

39-5B). The three related cis elements, termed opera-

In a lysogenic bacterium—ie, a bacterium containing

tors O_R1, O_R2, and O_R3, can be bound by either cI or

a lambda prophage—the lambda repressor dimer binds

Cro proteins. However, the relative affinities of cI and

preferentially to O_R1 but in so doing, by a cooperative

Cro for each of the sites varies, and this differential

interaction, enhances the binding (by a factor of 10) of

binding affinity is central to the appropriate operation

another repressor dimer to O_R2 (Figure 39-7). The

of the λ phage lytic or lysogenic “molecular switch.”

affinity of repressor for O_R3 is the least of the three oper-

The DNA region between the cro and repressor genes

ator subregions. The binding of repressor to O_R1 has two

also contains two promoter sequences that direct the

major effects. The occupation of O_R1 by repressor

binding of RNA polymerase in a specified orientation,

blocks the binding of RNA polymerase to the right-

where it commences transcribing adjacent genes. One

ward promoter and in that way prevents expression of

promoter directs RNA polymerase to transcribe in the

cro. Second, as mentioned above, repressor dimer bound

rightward direction and, thus, to transcribe cro and

to O_R1 enhances the binding of repressor dimer to O_R2.

other distal genes, while the other promoter directs the

The binding of repressor to O_R2 has the important

transcription of the repressor gene in the leftward di-

added effect of enhancing the binding of RNA polym-

rection (Figure 39-5B).

erase to the leftward promoter that overlaps O_R2 and

The product of the repressor gene, the 236-amino-

thereby enhances transcription and subsequent expres-

acid,

27 kDa repressor protein, exists as a two-

sion of the repressor gene. This enhancement of tran-

domain molecule in which the amino terminal domain

scription is apparently mediated through direct protein-

binds to operator DNA and the carboxyl terminal

protein interactions between promoter-bound RNA

domain promotes the association of one repressor

polymerase and O_R2-bound repressor. Thus, the lambda

protein with another to form a dimer. A dimer of re-

repressor is both a negative regulator, by preventing

pressor molecules binds to operator DNA much more

transcription of cro, and a positive regulator, by enhanc-

tightly than does the monomeric form (Figure 39-6A

ing transcription of its own gene, the repressor gene.

to 39-6C).

This dual effect of repressor is responsible for the stable

The product of the cro gene, the 66-amino-acid,

state of the dormant lambda bacteriophage; not only

9 kDa Cro protein, has a single domain but also binds

does the repressor prevent expression of the genes neces-

the operator DNA more tightly as a dimer (Figure

sary for lysis, but it also promotes expression of itself to

REGULATION OF GENE EXPRESSION

381

Amino acids

COOH

COOH COOH

132 - 236

Cro

NH₂

Amino acids

NH₂

1 - 92

O_R1

O_R3

Figure 39-6. Schematic molecular structures of cI (lambda repressor, shown in A, B, and C)

and Cro (D). Lambda repressor protein is a polypeptide chain 236 amino acids long. The chain

folds itself into a dumbbell shape with two substructures: an amino terminal (NH₂) domain and

a carboxyl terminal (COOH) domain. The two domains are linked by a region of the chain that is

susceptible to cleavage by proteases (indicated by the two arrows in A). Single repressor mole-

cules (monomers) tend to associate to form dimers (B); a dimer can dissociate to form

monomers again. A dimer is held together mainly by contact between the carboxyl terminal

domains (hatching). Repressor dimers bind to (and can dissociate from) the recognition sites in

the operator region; they display the greatest affinity for site O_R1 (C). It is the amino terminal

domain of the repressor molecule that makes contact with the DNA (hatching). Cro (D) has a

single domain with sites that promote dimerization and other sites that promote binding of

dimers to operator, preferentially to O_R3. (Reproduced, with permission, from Ptashne M, Johnson

AD, Pabo CO: A genetic switch in a bacterial virus. Sci Am [Nov] 1982;247:128.)

stabilize this state of differentiation. In the event that in-

The resulting newly synthesized Cro protein also

tracellular repressor protein concentration becomes very

binds to the operator region as a dimer, but its order of

high, this excess repressor will bind to O_R3 and by so

preference is opposite to that of repressor

(Figure

doing diminish transcription of the repressor gene from

39-7). That is, Cro binds most tightly to O_R3, but

the leftward promoter until the repressor concentration

there is no cooperative effect of Cro at O_R3 on the

drops and repressor dissociates itself from O_R3.

binding of Cro to O_R2. At increasingly higher concen-

With such a stable, repressive, cI-mediated, lyso-

trations of Cro, the protein will bind to O_R2 and even-

genic state, one might wonder how the lytic cycle could

tually to O_R1.

ever be entered. However, this process does occur quite

Occupancy of O_R3 by Cro immediately turns off

efficiently. When a DNA-damaging signal, such as ul-

transcription from the leftward promoter and in that

traviolet light, strikes the lysogenic host bacterium,

way prevents any further expression of the repressor

fragments of single-stranded DNA are generated that

gene. The molecular switch is thus completely

activate a specific protease coded by a bacterial gene

“thrown” in the lytic direction. The cro gene is now ex-

and referred to as recA (Figure 39-7). The activated

pressed, and the repressor gene is fully turned off. This

recA protease hydrolyzes the portion of the repressor

event is irreversible, and the expression of other lambda

protein that connects the amino terminal and carboxyl

genes begins as part of the lytic cycle. When Cro repres-

terminal domains of that molecule (see Figure 39-6A).

sor concentration becomes quite high, it will eventually

Such cleavage of the repressor domains causes the re-

occupy O_R1 and in so doing reduce the expression of its

pressor dimers to dissociate, which in turn causes dis-

own gene, a process that is necessary in order to effect

sociation of the repressor molecules from O_R2 and

the final stages of the lytic cycle.

eventually from O_R1. The effects of removal of repres-

The three-dimensional structures of Cro and of the

sor from O_R1 and O_R2 are predictable. RNA polym-

lambda repressor protein have been determined by

erase immediately has access to the rightward promoter

x-ray crystallography, and models for their binding and ef-

and commences transcribing the cro gene, and the en-

fecting the above-described molecular and genetic events

hancement effect of the repressor at O_R2 on leftward

have been proposed and tested. Both bind to DNA using

transcription is lost (Figure 39-7).

helix-turn-helix DNA binding domain motifs (see below).

Prophage

O_R3

O_R2

O_R1

RNA polymerase

Repressor promoter

cro promoter

O_R3

O_R2

O_R1

Induction (1)

RNA polymerase

recA

Repressor promoter

cro promoter

Ultraviolet radiation

O_R3

O_R2

O_R1

RNA polymerase

Induction (2)

Repressor promoter

cro promoter

Early lytic growth

O_R3

O_R2

O_R1

RNA polymerase

Repressor promoter

cro promoter

Figure 39-7. Configuration of the switch is shown at four stages of lambda’s life cycle. The lysogenic pathway (in

which the virus remains dormant as a prophage) is selected when a repressor dimer binds to O_R1, thereby making it

likely that O_R2 will be filled immediately by another dimer. In the prophage (top), the repressor dimers bound at O_R1

and O_R2 prevent RNA polymerase from binding to the rightward promoter and so block the synthesis of Cro (nega-

tive control). The repressors also enhance the binding of polymerase to the leftward promoter (positive control),

with the result that the repressor gene is transcribed into RNA (wavy line) and more repressor is synthesized, main-

taining the lysogenic state. The prophage is induced when ultraviolet radiation activates the protease recA, which

cleaves repressor monomers. The equilibrium of free monomers, free dimers, and bound dimers is thereby shifted,

and dimers leave the operator sites. RNA polymerase is no longer encouraged to bind to the leftward promoter, so

that repressor is no longer synthesized. As induction proceeds, all the operator sites become vacant, and so polym-

erase can bind to the rightward promoter and Cro is synthesized. During early lytic growth, a single Cro dimer binds

to O_R3 shaded circles, the site for which it has the highest affinity. Consequently, RNA polymerase cannot bind to the

leftward promoter, but the rightward promoter remains accessible. Polymerase continues to bind there, transcribing

cro and other early lytic genes. Lytic growth ensues. (Reproduced, with permission, from Ptashne M, Johnson AD, Pabo

CO: A genetic switch in a bacterial virus. Sci Am [Nov] 1982;247:128.)

382

REGULATION OF GENE EXPRESSION

383

To date, this system provides the best understanding of

tors with specific DNA regions. The dynamics of the for-

the molecular events involved in gene regulation.

mation and disruption of nucleosome structure are there-

Detailed analysis of the lambda repressor led to the

fore an important part of eukaryotic gene regulation.

important concept that transcription regulatory proteins

Histone acetylation and deacetylation is an im-

have several functional domains. For example, lambda

portant determinant of gene activity. The surprising

repressor binds to DNA with high affinity. Repressor

discovery that histone acetylase activity is associated

monomers form dimers, dimers interact with each

with TAFs and the coactivators involved in hormonal

other, and repressor interacts with RNA polymerase.

regulation of gene transcription (see Chapter 43) has

The protein-DNA interface and the three protein-

provided a new concept of gene regulation. Acetylation

protein interfaces all involve separate and distinct do-

is known to occur on lysine residues in the amino ter-

mains of the repressor molecule. As will be noted below

minal tails of histone molecules. This modification re-

(see Figure 39-17), this is a characteristic shared by

duces the positive charge of these tails and decreases the

most (perhaps all) molecules that regulate transcription.

binding affinity of histone for the negatively charged

DNA. Accordingly, the acetylation of histone could re-

sult in disruption of nucleosomal structure and allow

SPECIAL FEATURES ARE INVOLVED

readier access of transcription factors to cognate regula-

IN REGULATION OF EUKARYOTIC

tory DNA elements. As discussed previously, this

GENE TRANSCRIPTION

would enhance binding of the basal transcription ma-

chinery to the promoter. Histone deacetylation would

Most of the DNA in prokaryotic cells is organized into

have the opposite effect. Different proteins with specific

genes, and the templates can always be transcribed. A

acetylase and deacetylase activities are associated with

very different situation exists in mammalian cells, in

various components of the transcription apparatus. The

which relatively little of the total DNA is organized

specificity of these processes is under investigation, as

into genes and their associated regulatory regions. The

are a variety of mechanisms of action. Some specific ex-

function of the extra DNA is unknown. In addition, as

amples are illustrated in Chapter 43.

described in Chapter 36, the DNA in eukaryotic cells is

There is evidence that the methylation of deoxycy-

extensively folded and packed into the protein-DNA

tidine residues (in the sequence 5′-^mCpG-3′) in DNA

complex called chromatin. Histones are an important

may effect gross changes in chromatin so as to preclude

part of this complex since they both form the structures

its active transcription, as described in Chapter 36. For

known as nucleosomes (see Chapter 36) and also factor

example, in mouse liver, only the unmethylated riboso-

significantly into gene regulatory mechanisms as out-

mal genes can be expressed, and there is evidence that

lined below.

many animal viruses are not transcribed when their

DNA is methylated. Acute demethylation of deoxycyti-

dine residues in a specific region of the tyrosine amino-

Chromatin Remodeling Is an Important

transferase gene—in response to glucocorticoid hor-

Aspect of Eukaryotic Gene Expression

mones—has been associated with an increased rate of

Chromatin structure provides an additional level of

transcription of the gene. However, it is not possible to

control of gene transcription. As discussed in Chapter

generalize that methylated DNA is transcriptionally in-

36, large regions of chromatin are transcriptionally inac-

active, that all inactive chromatin is methylated, or that

tive while others are either active or potentially active.

active DNA is not methylated.

With few exceptions, each cell contains the same com-

Finally, the binding of specific transcription factors

plement of genes (antibody-producing cells are a notable

to cognate DNA elements may result in disruption of

exception). The development of specialized organs, tis-

nucleosomal structure. Many eukaryotic genes have

sues, and cells and their function in the intact organism

multiple protein-binding DNA elements. The serial

depend upon the differential expression of genes.

binding of transcription factors to these elements—in a

Some of this differential expression is achieved by

combinatorial fashion—may either directly disrupt the

having different regions of chromatin available for tran-

structure of the nucleosome or prevent its re-formation

scription in cells from various tissues. For example, the

or recruit, via protein-protein interactions, multipro-

DNA containing the β-globin gene cluster is in “active”

tein coactivator complexes that have the ability to cova-

chromatin in the reticulocyte but in “inactive” chro-

lently modify or remodel nucleosomes. These reactions

matin in muscle cells. All the factors involved in the de-

result in chromatin-level structural changes that in the

termination of active chromatin have not been eluci-

end increase DNA accessibility to other factors and the

dated. The presence of nucleosomes and of complexes of

transcription machinery.

histones and DNA (see Chapter 36) certainly provides a

Eukaryotic DNA that is in an “active” region of

barrier against the ready association of transcription fac-

chromatin can be transcribed. As in prokaryotic cells, a

384

CHAPTER 39

promoter dictates where the RNA polymerase will ini-

(Enhancer

Promoter

Structural gene

response element)

tiate transcription, but this promoter cannot be neatly

defined as containing a −35 and −10 box, particularly

in mammalian cells (Chapter 37). In addition, the

SV40

β globin

trans-acting factors generally come from other chromo-

somes (and so act in trans), whereas this consideration

is moot in the case of the single chromosome-contain-

ing prokaryotic cells. Additional complexity is added by

B SV40

β globin

elements or factors that enhance or repress transcrip-

tion, define tissue-specific expression, and modulate the

actions of many effector molecules.

hGH

Certain DNA Elements Enhance or Repress

Transcription of Eukaryotic Genes

In addition to gross changes in chromatin affecting

GRE

PEPCK

CAT

transcriptional activity, certain DNA elements facilitate

Figure 39-8. A schematic explanation of the action

or enhance initiation at the promoter. For example, in

of enhancers and other cis-acting regulatory elements.

simian virus 40 (SV40) there exists about 200 bp up-

stream from the promoter of the early genes a region of

These model chimeric genes consist of a reporter

two identical, tandem 72-bp lengths that can greatly in-

(structural) gene that encodes a protein which can be

crease the expression of genes in vivo. Each of these

readily assayed, a promoter that ensures accurate initia-

72-bp elements can be subdivided into a series of

tion of transcription, and the putative regulatory ele-

smaller elements; therefore, some enhancers have a very

ments. In all cases, high-level transcription from the in-

complex structure. Enhancer elements differ from the

dicated chimeras depends upon the presence of

promoter in two remarkable ways. They can exert their

enhancers, which stimulate transcription ≥ 100-fold

positive influence on transcription even when separated

over basal transcriptional levels (ie, transcription of the

by thousands of base pairs from a promoter; they work

same chimeric genes containing just promoters fused

when oriented in either direction; and they can work

to the structural genes). Examples A and B illustrate the

upstream (5′) or downstream (3′) from the promoter.

fact that enhancers (eg, SV40) work in either orientation

Enhancers are promiscuous; they can stimulate any

and upon a heterologous promoter. Example C illus-

promoter in the vicinity and may act on more than one

trates that the metallothionein (mt) regulatory element

promoter. The SV40 enhancer element can exert an in-

(which under the influence of cadmium or zinc induces

fluence on, for example, the transcription of β-globin

transcription of the endogenous mt gene and hence

by increasing its transcription 200-fold in cells contain-

the metal-binding mt protein) will work through the

ing both the enhancer and the β-globin gene on the

thymidine kinase (tk) promoter to enhance transcrip-

same plasmid (see below and Figure 39-8). The en-

tion of the human growth hormone (hGH) gene. The

hancer element does not produce a product that in turn

engineered genetic constructions were introduced into

acts on the promoter, since it is active only when it ex-

the male pronuclei of single-cell mouse embryos and

ists within the same DNA molecule as (ie, cis to) the

the embryos placed into the uterus of a surrogate

promoter. Enhancer binding proteins are responsible

for this effect. The exact mechanisms by which these

mother to develop as transgenic animals. Offspring

transcription activators work are subject to much de-

have been generated under these conditions, and in

bate. Certainly, enhancer binding trans factors have

some the addition of zinc ions to their drinking water

been shown to interact with a plethora of other tran-

effects an increase in liver growth hormone. In this

scription proteins. These interactions include chro-

case, these transgenic animals have responded to the

matin-modifying coactivators as well as the individual

high levels of growth hormone by becoming twice as

components of the basal RNA polymerase II transcrip-

large as their normal litter mates. Example D illustrates

tion machinery. Ultimately, trans-factor-enhancer DNA

that a glucocorticoid response element (GRE) will work

binding events result in an increase in the binding of

through homologous (PEPCK gene) or heterologous

the basal transcription machinery to the promoter. En-

promoters (not shown; ie, tk promoter, SV40 promoter,

hancer elements and associated binding proteins often

β-globin promoter, etc).

convey nuclease hypersensitivity to those regions where

they reside (Chapter 36). A summary of the properties

of enhancers is presented in Table 39-2. One of the

REGULATION OF GENE EXPRESSION

385

Table 39-2. Summary of the properties

duces β-interferon gene transcription—rather, it is the

of enhancers.

formation of the enhanceosome proper that provides

appropriate surfaces for the recruitment of coactivators

that results in the enhanced formation of the PIC on

• Work when located long distances from the promoter

the cis-linked promoter and thus transcription activa-

• Work when upstream or downstream from the promoter

tion.

• Work when oriented in either direction

The cis-acting elements that decrease or repress the

• Work through heterologous promoters

expression of specific genes have also been identified.

• Work by binding one or more proteins

• Work by facilitating binding of the basal transcription com-

Because fewer of these elements have been studied, it is

plex to the promoter

not possible to formulate generalizations about their

mechanism of action—though again, as for gene activa-

tion, chromatin level covalent modifications of histones

and other proteins by (repressor)-recruited multisub-

best-understood mammalian enhancer systems is that

unit corepressors have been implicated.

of the β-interferon gene. This gene is induced upon

viral infection of mammalian cells. One goal of the cell,

Tissue-Specific Expression May

once virally infected, is to attempt to mount an antivi-

Result From the Action

ral response—if not to save the infected cell, then to

of Enhancers or Repressors

help to save the entire organism from viral infection.

Interferon production is one mechanism by which this

Many genes are now recognized to harbor enhancer or

is accomplished. This family of proteins is secreted by

activator elements in various locations relative to their

virally infected cells. They interact with neighboring

coding regions. In addition to being able to enhance

cells to cause an inhibition of viral replication by a vari-

gene transcription, some of these enhancer elements

ety of mechanisms, thereby limiting the extent of viral

clearly possess the ability to do so in a tissue-specific

infection. The enhancer element controlling induction

manner. Thus, the enhancer element associated with

of this gene, located between nucleotides −110 and −45

the immunoglobulin genes between the J and C regions

of the β-interferon gene, is well characterized. This en-

enhances the expression of those genes preferentially in

hancer is composed of four distinct clustered cis ele-

lymphoid cells. Similarly to the SV40 enhancer, which

ments, each of which is bound by distinct trans factors.

is capable of promiscuously activating a variety of cis-

One cis element is bound by the trans-acting factor

linked genes, enhancer elements associated with the

NF-κB, one by a member of the IRF (interferon regula-

genes for pancreatic enzymes are capable of enhancing

tory factor) family of trans factors, and a third by the

even unrelated but physically linked genes preferentially

heterodimeric leucine zipper factor ATF-2/c-Jun. The

in the pancreatic cells of mice into which the specifi-

fourth factor is the ubiquitous, architectural transcrip-

cally engineered gene constructions were introduced

tion factor known as HMG I(Y). Upon binding to its

microsurgically at the single-cell embryo stage. This

degenerate, A+T-rich binding sites, HMG I(Y) induces

transgenic animal approach has proved useful in

a significant bend in the DNA. There are four such

studying tissue-specific gene expression. For example,

HMG I(Y) binding sites interspersed throughout the

DNA containing a pancreatic B cell tissue-specific en-

enhancer. These sites play a critical role in forming the

hancer (from the insulin gene), when ligated in a vector

enhanceosome, along with the aforementioned three

to polyoma large-T antigen, an oncogene, produced

trans factors, by inducing a series of critically spaced

B cell tumors in transgenic mice. Tumors did not de-

DNA bends. Consequently, HMG I(Y) induces the co-

velop in any other tissue. Tissue-specific gene expres-

operative formation of a unique, stereospecific, three

sion may therefore be mediated by enhancers or en-

dimensional structure within which all four factors are

hancer-like elements.

active when viral infection signals are sensed by the cell.

The structure formed by the cooperative assembly of

Reporter Genes Are Used to Define

these four factors is termed the β-interferon enhanceo-

Enhancers & Other Regulatory Elements

some (see Figure 39-9), so named because of its obvi-

ous structural similarity to the nucleosome, also a

By ligating regions of DNA suspected of harboring reg-

unique three-dimensional protein DNA structure that

ulatory sequences to various reporter genes

(the re-

wraps DNA about an assembly of proteins (see Figures

porter or chimeric gene approach) (Figures 39-10

36-1 and 36-2). The enhanceosome, once formed, in-

and 39-11), one can determine which regions in the

duces a large increase in β-interferon gene transcription

vicinity of structural genes have an influence on their

upon virus infection. It is not simply the protein occu-

expression. Pieces of DNA thought to harbor regula-

pancy of the linearly apposed cis element sites that in-

tory elements are ligated to a suitable reporter gene and

386

CHAPTER 39

HMG PRDIV HMG PRDI-III PRDII HMG NRDI HMG

HMGI-Y

ATF-2

cJun

NF-κB

IRF(IRF3/7)

HMGI

cJun

ATF-2

HMGI

NF-κB

HMGI

IRF3

IRF7

HMGI

Figure 39-9. Formation and putative structure of the enhanceosome formed on the human β-interferon gene

enhancer. Diagramatically represented at the top is the distribution of the multiple cis-elements (HMG, PRDIV,

PRDI-III, PRDII, NRDI) composing the β-interferon gene enhancer. The intact enhancer mediates transcriptional in-

duction of the β-interferon gene (over 100-fold) upon virus infection of human cells. The cis-elements of this modu-

lar enhancer represent the binding sites for the trans-factors HMG I(Y), cJun-ATF-2, IRF3, IRF7, and NF-κB, respec-

tively. The factors interact with these DNA elements in an obligatory, ordered, and highly cooperative fashion as

indicated by the arrow. Initial binding of four HMG I(Y) proteins induces sharp DNA bends in the enhancer, causing

the entire 70-80 bp region to assume a high level of curvature. This curvature is integral to the subsequent highly

cooperative binding of the other trans-factors since this enables the DNA-bound factors to make important, direct

protein-protein interactions that both contribute to the formation and stability of the enhanceosome and generate

a unique three-dimensional surface that serves to recruit chromatin-modifying activities (eg, Swi/Snf and P/CAF) as

well as the general transcription machinery (RNA polymerase II and GTFs). Although four of the five cis-elements

(PRDIV, PRDI-III, PRDII, NRDI) independently can modestly stimulate (~tenfold) transcription of a reporter gene in

transfected cells (see Figures 39-10 and 39-12), all five cis-elements, in appropriate order, are required to form an

enhancer that can appropriately stimulate mRNA gene transcription (ie, ≥ 100-fold) in response to viral infection of a

human cell. This distinction indicates the strict requirement for appropriate enhanceosome architecture for efficient

trans-activation. Similar enhanceosomes, involving distinct cis- and trans-factors, are proposed to form on many

other mammalian genes.

introduced into a host cell (Figure 39-10). Basal ex-

This strategy, using transfected cells in culture

pression of the reporter gene will be increased if the

and transgenic animals, has led to the identification

DNA contains an enhancer. Addition of a hormone or

of dozens of enhancers, repressors, tissue-specific ele-

heavy metal to the culture medium will increase expres-

ments, and hormone, heavy metal, and drug-response

sion of the reporter gene if the DNA contains a hor-

elements. The activity of a gene at any moment re-

mone or metal response element (Figure 39-11). The

flects the interaction of these numerous cis-acting

location of the element can be pinpointed by using pro-

DNA elements with their respective trans-acting fac-

gressively shorter pieces of DNA, deletions, or point

tors. The challenge now is to figure out how this oc-

mutations (Figure 39-11).

curs.

REGULATION OF GENE EXPRESSION

387

Test promoter

Reporter gene

ner, but the process in most genes, especially in mam-

5′

3′

5′

3′

mals, is much more complicated. Signals representing a

GENE

CAT

number of complex environmental stimuli may con-

ENHANCER-PROMOTER

REPORTER GENE:

verge on a single gene. The response of the gene to

TEST ENHANCER-PROMOTER

DRIVING TRANSCRIPTION

these signals can have several physiologic characteristics.

CAT GENE

First, the response may extend over a considerable

range. This is accomplished by having additive and syn-

CAT

ergistic positive responses counterbalanced by negative

or repressing effects. In some cases, either the positive

or the negative response can be dominant. Also re-

quired is a mechanism whereby an effector such as a

TRANSFECT CELLS USING CaPO₄

PRECIPITATED DNA

hormone can activate some genes in a cell while repress-

ing others and leaving still others unaffected. When all

of these processes are coupled with tissue-specific ele-

Divide and re-plate

ment factors, considerable flexibility is afforded. These

physiologic variables obviously require an arrangement

much more complicated than an on-off switch. The

Cells

array of DNA elements in a promoter specifies—with

associated factors—how a given gene will respond.

Control

Hormones

Some simple examples are illustrated in Figure 39-12.

HARVEST 24 HOURS LATER

ASSAY FOR CAT ACTIVITY

Transcription Domains Can Be Defined by

Identification of

control elements

Locus Control Regions & Insulators

Figure 39-10. The use of reporter genes to define

The large number of genes in eukaryotic cells and the

DNA regulatory elements. A DNA fragment from the

complex arrays of transcription regulatory factors pre-

gene in question—in this example, approximately 2 kb

sents an organizational problem. Why are some genes

of 5′-flanking DNA and cognate promoter—is ligated

available for transcription in a given cell whereas others

are not? If enhancers can regulate several genes and are

into a plasmid vector that contains a suitable reporter

not position- and orientation-dependent, how are they

gene—in this case, the bacterial enzyme chlorampheni-

prevented from triggering transcription randomly? Part

col transferase (CAT). The enzyme luciferase (abbrevi-

of the solution to these problems is arrived at by having

ated LUC) is another popular reporter gene. Neither

the chromatin arranged in functional units that restrict

LUC nor CAT is present in mammalian cells; hence, de-

patterns of gene expression. This may be achieved by

tection of these activities in a cell extract means that

having the chromatin form a structure with the nuclear

the cell was successfully transfected by the plasmid. An

matrix or other physical entity, or compartments

increase of CAT activity over the basal level, eg, after

within the nucleus. Alternatively, some regions are con-

addition of one or more hormones, means that the re-

trolled by complex DNA elements called locus control

gion of DNA inserted into the reporter gene plasmid

regions (LCRs). An LCR—with associated bound pro-

contains functional hormone response elements (HRE).

teins—controls the expression of a cluster of genes. The

Progressively shorter pieces of DNA, regions with inter-

best-defined LCR regulates expression of the globin

nal deletions, or regions with point mutations can be

gene family over a large region of DNA. Another mech-

constructed and inserted to pinpoint the response ele-

anism is provided by insulators. These DNA elements,

ment (see Figure 39-11 for deletion mapping of the rel-

also in association with one or more proteins, prevent

evant HREs).

an enhancer from acting on a promoter on the other

side of an insulator in another transcription domain.

Combinations of DNA Elements

SEVERAL MOTIFS MEDIATE THE BINDING

& Associated Proteins Provide

OF REGULATORY PROTEINS TO DNA

Diversity in Responses

The specificity involved in the control of transcription

Prokaryotic genes are often regulated in an on-off man-

requires that regulatory proteins bind with high affinity

ner in response to simple environmental cues. Some eu-

to the correct region of DNA. Three unique motifs—

karyotic genes are regulated in the simple on-off man-

the helix-turn-helix, the zinc finger, and the leucine

388

CHAPTER 39

REPORTER GENE CONSTRUCTS

HORMONE-DEPENDENT

WITH VARIABLE AMOUNTS

TRANSCRIPTION

OF 5'-FLANKING DNA

INDUCTION

B C

Figure 39-11.

Location of hormone response ele-

CAT

ments (HREs) A, B, and C using the reporter

5′

gene-transfection approach. A family of reporter

2000

1000

genes, constructed as described in Figure 39-10, can

Nucleotide position

be transfected individually into a recipient cell. By an-

alyzing when certain hormone responses are lost in

HRE

comparison to the 5′ deletion, specific hormone-

responsive elements can be located.

zipper—account for many of these specific protein-

DNA interactions. Examples of proteins containing

these motifs are given in Table 39-3.

Gene

Comparison of the binding activities of the proteins

that contain these motifs leads to several important

generalizations.

(1) Binding must be of high affinity to the specific

Gene

site and of low affinity to other DNA.

(2) Small regions of the protein make direct contact

with DNA; the rest of the protein, in addition to pro-

Gene C

Table 39-3. Examples of transcription regulatory

Figure 39-12. Combinations of DNA elements and

proteins that contain the various binding motifs.

proteins provide diversity in the response of a gene.

Gene A is activated (the width of the arrow indicates

Binding Motif

Organism

Regulatory Protein

the extent) by the combination of activators 1, 2, and 3

Helix-turn-helix

E coli

lac repressor

(probably with coactivators, as shown in Figure 37-10).

CAP

Gene B is activated, in this case more effectively, by the

Phage

λcI, cro, and tryptophan and

combination of 1, 3, and 4; note that 4 does not contact

434 repressors

DNA directly in this example. The activators could form

Mammals

homeo box proteins

a linear bridge that links the basal machinery to the

Pit-1, Oct1, Oct2

promoter, or this could be accomplished by looping

Zinc finger

E coli

Gene 32 protein

out of the DNA. In either case, the purpose is to direct

Yeast

GaI4

the basal transcription machinery to the promoter.

Drosophila

Serendipity, Hunchback

Gene C is inactivated by the combination of 1, 5, and 3;

Xenopus

TFIIIA

in this case, factor 5 is shown to preclude the essential

Mammals

steroid receptor family, Sp1

binding of factor 2 to DNA, as occurs in example A. If

Leucine zipper

Yeast

GCN4

activator 1 helps repressor 5 bind and if activator 1

Mammals

C/EBP, fos, Jun, Fra-1,

binding requires a ligand (solid dot), it can be seen how

CRE binding protein,

the ligand could activate one gene in a cell (gene A)

c-myc, n-myc, I-myc

and repress another (gene C).

REGULATION OF GENE EXPRESSION

389

viding the trans-activation domains, may be involved in

The Helix-Turn-Helix Motif

the dimerization of monomers of the binding protein,

may provide a contact surface for the formation of het-

The first motif described—and the one studied most

erodimers, may provide one or more ligand-binding

extensively—is the helix-turn-helix. Analysis of the

sites, or may provide surfaces for interaction with coac-

three-dimensional structure of the λ Cro transcription

tivators or corepressors.

regulator has revealed that each monomer consists of

(3) The protein-DNA interactions are maintained

three antiparallel β sheets and three α helices (Figure

by hydrogen bonds and van der Waals forces.

39-13). The dimer forms by association of the antipar-

(4) The motifs found in these proteins are unique;

allel β₃ sheets. The α₃ helices form the DNA recogni-

their presence in a protein of unknown function sug-

tion surface, and the rest of the molecule appears to be

gests that the protein may bind to DNA.

involved in stabilizing these structures. The average di-

(5) Proteins with the helix-turn-helix or leucine zip-

ameter of an α helix is 1.2 nm, which is the approxi-

per motifs form symmetric dimers, and their respective

mate width of the major groove in the B form of DNA.

DNA binding sites are symmetric palindromes. In pro-

The DNA recognition domain of each Cro monomer

teins with the zinc finger motif, the binding site is re-

interacts with 5 bp and the dimer binding sites span

peated two to nine times. These features allow for co-

3.4 nm, allowing fit into successive half turns of the

operative interactions between binding sites and

major groove on the same surface (Figure 39-13). X-ray

enhance the degree and affinity of binding.

analyses of the λ cI repressor, CAP (the cAMP receptor

α₂

α₃

α₁

N C

α₂

β₁

34 Å

β₂

β₃

α₃

Twofold

axis of

β₃

symmetry

β₂

β₁

α₃

C N

α₃

α₁

34 Å

α₂

Figure 39-13. A schematic representation of the three-dimensional structure of Cro protein and its binding to

DNA by its helix-turn-helix motif. The Cro monomer consists of three antiparallel β sheets (β₁-β₃) and three

α-helices (α₁-α₃). The helix-turn-helix motif is formed because the α₃ and α₂ helices are held at about 90 degrees

to each other by a turn of four amino acids. The α₃ helix of Cro is the DNA recognition surface (shaded). Two

monomers associate through the antiparallel β3 sheets to form a dimer that has a twofold axis of symmetry

(right). A Cro dimer binds to DNA through its α₃ helices, each of which contacts about 5 bp on the same surface of

the major groove. The distance between comparable points on the two DNA α-helices is 34 Å, which is the dis-

tance required for one complete turn of the double helix. (Courtesy of B Mathews.)

390

CHAPTER 39

protein of E coli), tryptophan repressor, and phage 434

The Leucine Zipper Motif

repressor all also display this dimeric helix-turn-helix

Careful analysis of a 30-amino-acid sequence in the car-

structure that is present in eukaryotic DNA proteins as

boxyl terminal region of the enhancer binding protein

well (see Table 39-3).

C/EBP revealed a novel structure. As illustrated in Fig-

ure 39-15, this region of the protein forms an α helix

The Zinc Finger Motif

in which there is a periodic repeat of leucine residues at

every seventh position. This occurs for eight helical

The zinc finger was the second DNA binding motif

turns and four leucine repeats. Similar structures have

whose atomic structure was elucidated. It was known

been found in a number of other proteins associated

that the protein TFIIIA, a positive regulator of 5S RNA

with the regulation of transcription in mammalian and

transcription, required zinc for activity. Structural and

yeast cells. It is thought that this structure allows two

biophysical analyses revealed that each TFIIIA molecule

identical monomers or heterodimers (eg, Fos-Jun or

contains nine zinc ions in a repeating coordination

Jun-Jun) to “zip together” in a coiled coil and form a

complex formed by closely spaced cysteine-cysteine

tight dimeric complex (Figure 39-15). This protein-

residues followed 12-13 amino acids later by a histi-

protein interaction may serve to enhance the associa-

dine-histidine pair (Figure 39-14). In some instances—

tion of the separate DNA binding domains with their

notably the steroid-thyroid receptor family—the His-

target (Figure 39-15).

His doublet is replaced by a second Cys-Cys pair. The

protein containing zinc fingers appears to lie on one

THE DNA BINDING & TRANS-ACTIVATION

face of the DNA helix, with successive fingers alterna-

DOMAINS OF MOST REGULATORY

tively positioned in one turn in the major groove. As is

the case with the recognition domain in the helix-turn-

PROTEINS ARE SEPARATE

helix protein, each TFIIIA zinc finger contacts about

& NONINTERACTIVE

5 bp of DNA. The importance of this motif in the ac-

DNA binding could result in a general conformational

tion of steroid hormones is underscored by an “experi-

change that allows the bound protein to activate tran-

ment of nature.” A single amino acid mutation in either

scription, or these two functions could be served by

of the two zinc fingers of the 1,25(OH)₂-D₃ receptor

separate and independent domains. Domain swap ex-

protein results in resistance to the action of this hor-

periments suggest that the latter is the case.

mone and the clinical syndrome of rickets.

The GAL1 gene product is involved in galactose me-

tabolism in yeast. Transcription of this gene is positively

regulated by the GAL4 protein, which binds to an up-

stream activator sequence (UAS), or enhancer, through

an amino terminal domain. The amino terminal 73-

amino-acid DNA-binding domain (DBD) of GAL4 was

removed and replaced with the DBD of LexA, an E coli

DNA-binding protein. This domain swap resulted in a

molecule that did not bind to the GAL1 UAS and, of

course, did not activate the GAL1 gene (Figure 39-16).

If, however, the lexA operator—the DNA sequence nor-

mally bound by the lexA DBD—was inserted into the

promoter region of the GAL gene, the hybrid protein

bound to this promoter (at the lexA operator) and it ac-

Cys-Cys zinc finger

Cys-His zinc finger

tivated transcription of GAL1. This experiment, which

has been repeated a number of times, affords solid evi-

Figure 39-14. Zinc fingers are a series of repeated

dence that the carboxyl terminal region of GAL4 causes

domains (two to nine) in which each is centered on a

transcriptional activation. These data also demonstrate

tetrahedral coordination with zinc. In the case of TFIIIA,

that the DNA-binding DBD and trans-activation do-

the coordination is provided by a pair of cysteine

mains (ADs) are independent and noninteractive. The

residues (C) separated by 12-13 amino acids from a

hierarchy involved in assembling gene transcription acti-

pair of histidine (H) residues. In other zinc finger pro-

vating complexes includes proteins that bind DNA and

teins, the second pair also consists of C residues. Zinc

trans-activate; others that form protein-protein com-

fingers bind in the major groove, with adjacent fingers

plexes which bridge DNA-binding proteins to trans-

making contact with 5 bp along the same face of the

activating proteins; and others that form protein-protein

helix.

complexes with components of the basal transcription

REGULATION OF GENE EXPRESSION

391

NH₂

COOH

NH₂

Figure 39-15. The leucine zipper motif. A shows a helical wheel analysis of a carboxyl terminal portion of the

DNA binding protein C/EBP. The amino acid sequence is displayed end-to-end down the axis of a schematic

α-helix. The helical wheel consists of seven spokes that correspond to the seven amino acids that comprise every

two turns of the α-helix. Note that leucine residues (L) occur at every seventh position. Other proteins with

“leucine zippers” have a similar helical wheel pattern. B is a schematic model of the DNA binding domain of C/EBP.

Two identical C/EBP polypeptide chains are held in dimer formation by the leucine zipper domain of each

polypeptide (denoted by the rectangles and attached ovals). This association is apparently required to hold the

DNA binding domains of each polypeptide (the shaded rectangles) in the proper conformation for DNA binding.

(Courtesy of S McKnight.)

apparatus. A given protein may thus have several sur-

prokaryotes. These RNA processing steps in eukaryotes,

faces or domains that serve different functions (see Fig-

described in detail in Chapter 37, include capping of

ure 39-17). As described in Chapter 37, the primary

the 5′ ends of the primary transcripts, addition of a

purpose of these complex assemblies is to facilitate the

polyadenylate tail to the 3′ ends of transcripts, and exci-

assembly of the basal transcription apparatus on the cis-

sion of intron regions to generate spliced exons in the

linked promoter.

mature mRNA molecule. To date, analyses of eukary-

otic gene expression provide evidence that regulation

occurs at the level of transcription, nuclear RNA pro-

GENE REGULATION IN PROKARYOTES

cessing, and mRNA stability. In addition, gene ampli-

fication and rearrangement influence gene expression.

& EUKARYOTES DIFFERS IN

Owing to the advent of recombinant DNA technol-

IMPORTANT RESPECTS

ogy, much progress has been made in recent years in

In addition to transcription, eukaryotic cells employ a

the understanding of eukaryotic gene expression. How-

variety of mechanisms to regulate gene expression

ever, because most eukaryotic organisms contain so

(Table 39-4). The nuclear membrane of eukaryotic

much more genetic information than do prokaryotes

cells physically segregates gene transcription from trans-

and because manipulation of their genes is so much

lation, since ribosomes exist only in the cytoplasm.

more limited, molecular aspects of eukaryotic gene reg-

Many more steps, especially in RNA processing, are in-

ulation are less well understood than the examples

volved in the expression of eukaryotic genes than of

discussed earlier in this chapter. This section briefly de-

prokaryotic genes, and these steps provide additional

scribes a few different types of eukaryotic gene regula-

sites for regulatory influences that cannot exist in

tion.

392

CHAPTER 39

GAL4

Active

UAS

GAL1 gene

LexA-GAL4

Inactive

UAS

GAL1 gene

LexA-GAL4

Active

lexA

GAL1 gene

operator

Figure 39-16. Domain-swap experiments demonstrate the independent nature of DNA binding and transcrip-

tion activation domains. The GAL1 gene promoter contains an upstream activating sequence (UAS) or enhancer that

binds the regulatory protein GAL4 (A). This interaction results in a stimulation of GAL1 gene transcription. A chimeric

protein, in which the amino terminal DNA binding domain of GAL4 is removed and replaced with the DNA binding

region of the E coli protein LexA, fails to stimulate GAL1 transcription because the LexA domain cannot bind to the

UAS (B). The LexA-GAL4 fusion protein does increase GAL1 transcription when the lexA operator (its natural target)

is inserted into the GAL1 promoter region (C).

Eukaryotic Genes Can Be Amplified

or Rearranged During Development

or in Response to Drugs

During early development of metazoans, there is an

abrupt increase in the need for specific molecules such

as ribosomal RNA and messenger RNA molecules for

Activation

proteins that make up such organs as the eggshell. One

domains

1- 4

way to increase the rate at which such molecules can be

formed is to increase the number of genes available for

Ligand-binding domain

transcription of these specific molecules. Among the

repetitive DNA sequences are hundreds of copies of ri-

bosomal RNA genes and tRNA genes. These genes pre-

DNA-binding domain

exist repetitively in the genomic material of the gametes

Table 39-4. Gene expression is regulated by

transcription and in numerous other ways in

Figure 39-17. Proteins that regulate transcription

eukaryotic cells.

have several domains. This hypothetical transcription

factor has a DNA-binding domain (DBD) that is distinct

from a ligand-binding domain (LBD) and several activa-

• Gene amplification

tion domains (ADs) (1-4). Other proteins may lack the

• Gene rearrangement

• RNA processing

DBD or LBD and all may have variable numbers of

• Alternate mRNA splicing

domains that contact other proteins, including

• Transport of mRNA from nucleus to cytoplasm

co-regulators and those of the basal transcription

• Regulation of mRNA stability

complex (see also Chapters 42 and 43).

REGULATION OF GENE EXPRESSION

393

and thus are transmitted in high copy numbers from

both immunologic flexibility and specificity. However,

generation to generation. In some specific organisms

a given functional IgG light chain transcription unit—

such as the fruit fly (drosophila), there occurs during

like all other

“normal” mammalian transcription

oogenesis an amplification of a few preexisting genes

units—contains only the coding sequences for a single

such as those for the chorion (eggshell) proteins. Subse-

protein. Thus, before a particular IgG light chain can

quently, these amplified genes, presumably generated

be expressed, single V_L, J_L, and C_L coding sequences

by a process of repeated initiations during DNA syn-

must be recombined to generate a single, contiguous

thesis, provide multiple sites for gene transcription

transcription unit excluding the multiple nonutilized

(Figures 36-16 and 39-18).

segments (ie, the other approximately 300 unused V_L

As noted in Chapter 37, the coding sequences re-

segments, the other four unused J_L segments, and the

sponsible for the generation of specific protein mole-

other nine unused C_L segments). This deletion of un-

cules are frequently not contiguous in the mammalian

used genetic information is accomplished by selective

genome. In the case of antibody encoding genes, this is

DNA recombination that removes the unwanted cod-

particularly true. As described in detail in Chapter 50,

ing DNA while retaining the required coding se-

immunoglobulins are composed of two polypeptides,

quences: one V_L, one J_L, and one C_L sequence. (V_L se-

the so-called heavy (about 50 kDa) and light (about 25

quences are subjected to additional point mutagenesis

kDa) chains. The mRNAs encoding these two protein

to generate even more variability—hence the name.)

subunits are encoded by gene sequences that are sub-

The newly recombined sequences thus form a single

jected to extensive DNA sequence-coding changes.

transcription unit that is competent for RNA polym-

These DNA coding changes are integral to generating

erase II-mediated transcription. Although the IgG

the requisite recognition diversity central to appropriate

genes represent one of the best-studied instances of di-

immune function.

rected DNA rearrangement modulating gene expres-

IgG heavy and light chain mRNAs are encoded by

sion, other cases of gene regulatory DNA rearrange-

several different segments that are tandemly repeated in

ment have been described in the literature. Indeed, as

the germline. Thus, for example, the IgG light chain is

detailed below, drug-induced gene amplification is an

composed of variable (V_L ), joining (J_L ), and constant

important complication of cancer chemotherapy.

(C_L ) domains or segments. For particular subsets of

In recent years, it has been possible to promote the

IgG light chains, there are roughly 300 tandemly re-

amplification of specific genetic regions in cultured

peated V_L gene coding segments, five tandemly

mammalian cells. In some cases, a several thousand-fold

arranged J_L coding sequences, and roughly ten C_L gene

increase in the copy number of specific genes can be

coding segments. All of these multiple, distinct coding

achieved over a period of time involving increasing doses

regions are located in the same region of the same chro-

of selective drugs. In fact, it has been demonstrated in

mosome, and each type of coding segment (V_L, J_L, and

patients receiving methotrexate for cancer that malig-

CL ) is tandemly repeated in head-to-tail fashion within

nant cells can develop drug resistance by increasing the

the segment repeat region. By having multiple V_L, J_L,

number of genes for dihydrofolate reductase, the target

and C_L segments to choose from, an immune cell has a

of methotrexate. Gene amplification events such as these

greater repertoire of sequences to work with to develop

occur spontaneously in vivo—ie, in the absence of ex-

ogenously supplied selective agents—and these unsched-

uled extra rounds of replication can become “frozen” in

the genome under appropriate selective pressures.

Unamplified

s36

s38

Alternative RNA Processing

s36

s38

Is Another Control Mechanism

In addition to affecting the efficiency of promoter uti-

lization, eukaryotic cells employ alternative RNA pro-

Amplified

cessing to control gene expression. This can result when

alternative promoters, intron-exon splice sites, or

polyadenylation sites are used. Occasionally, hetero-

geneity within a cell results, but more commonly the

same primary transcript is processed differently in dif-

Figure 39-18. Schematic representation of the am-

ferent tissues. A few examples of each of these types of

plification of chorion protein genes s36 and s38. (Repro-

regulation are presented below.

duced, with permission, from Chisholm R: Gene amplifica-

The use of alternative transcription start sites re-

tion during development. Trends Biochem Sci 1982;7:161.)

sults in a different 5′ exon on mRNAs corresponding to

394

CHAPTER 39

mouse amylase and myosin light chain, rat glucokinase,

cap structure in eukaryotic mRNA prevents attack by 5′

and drosophila alcohol dehydrogenase and actin. Alter-

exonucleases, and the poly(A) tail prohibits the action

native polyadenylation sites in the µ immunoglobulin

3′ exonucleases. In mRNA molecules with those

heavy chain primary transcript result in mRNAs that

structures, it is presumed that a single endonucleolytic

are either 2700 bases long (µ_m) or 2400 bases long (µ_s).

cut allows exonucleases to attack and digest the entire

This results in a different carboxyl terminal region of

molecule. Other structures (sequences) in the 5′ non-

the encoded proteins such that the µ_m protein remains

coding sequence (5′ NCS), the coding region, and the

attached to the membrane of the B lymphocyte and the

3′ NCS are thought to promote or prevent this initial

µs immunoglobulin is secreted. Alternative splicing

endonucleolytic action (Figure 39-19). A few illustra-

and processing results in the formation of seven

tive examples will be cited.

unique α-tropomyosin mRNAs in seven different tis-

Deletion of the 5′ NCS results in a threefold to five-

sues. It is not clear how these processing-splicing deci-

fold prolongation of the half-life of c-myc mRNA. Short-

sions are made or whether these steps can be regulated.

ening the coding region of histone mRNA results in a

prolonged half-life. A form of autoregulation of mRNA

stability indirectly involves the coding region. Free tubu-

Regulation of Messenger RNA Stability

lin binds to the first four amino acids of a nascent chain

Provides Another Control Mechanism

of tubulin as it emerges from the ribosome. This appears

Although most mRNAs in mammalian cells are very

to activate an RNase associated with the ribosome (RNP)

stable (half-lives measured in hours), some turn over

which then digests the tubulin mRNA.

very rapidly (half-lives of 10-30 minutes). In certain in-

Structures at the 3′ end, including the poly(A) tail,

stances, mRNA stability is subject to regulation. This

enhance or diminish the stability of specific mRNAs.

has important implications since there is usually a di-

The absence of a poly(A) tail is associated with rapid

rect relationship between mRNA amount and the

degradation of mRNA, and the removal of poly(A)

translation of that mRNA into its cognate protein.

from some RNAs results in their destabilization. His-

Changes in the stability of a specific mRNA can there-

tone mRNAs lack a poly(A) tail but have a sequence

fore have major effects on biologic processes.

near the 3′ terminal that can form a stem-loop struc-

Messenger RNAs exist in the cytoplasm as ribonu-

ture, and this appears to provide resistance to exonucle-

cleoprotein particles (RNPs). Some of these proteins

olytic attack. Histone H4 mRNA, for example, is de-

protect the mRNA from digestion by nucleases, while

graded in the 3′ to 5′ direction but only after a single

others may under certain conditions promote nuclease

endonucleolytic cut occurs about nine nucleotides from

attack. It is thought that mRNAs are stabilized or desta-

the 3′ end in the region of the putative stem-loop struc-

bilized by the interaction of proteins with these various

ture. Stem-loop structures in the

3′ noncoding se-

structures or sequences. Certain effectors, such as hor-

quence are also critical for the regulation, by iron, of

mones, may regulate mRNA stability by increasing or

the mRNA encoding the transferrin receptor. Stem-

decreasing the amount of these proteins.

loop structures are also associated with mRNA stability

It appears that the ends of mRNA molecules are

in bacteria, suggesting that this mechanism may be

involved in mRNA stability (Figure 39-19). The 5′

commonly employed.

Cap

5′ NCS

Coding

3′ NCS

A-A-A-A-Aⁿ

AUUUA

Figure 39-19. Structure of a typical eukaryotic mRNA showing

elements that are involved in regulating mRNA stability. The typical

eukaryotic mRNA has a 5′ noncoding sequence (5′ NCS), a coding

region, and a 3′ NCS. All are capped at the 5′ end, and most have a

polyadenylate sequence at the 3′ end. The 5′ cap and 3′ poly(A) tail

protect the mRNA against exonuclease attack. Stem-loop structures

in the 5′ and 3′ NCS, features in the coding sequence, and the AU-

rich region in the 3′ NCS are thought to play roles in mRNA stability.

REGULATION OF GENE EXPRESSION

395

Other sequences in the 3′ ends of certain eukaryotic

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Molecular Genetics, Recombinant

DNA, & Genomic Technology

Daryl K. Granner, MD, & P. Anthony Weil, PhD

BIOMEDICAL IMPORTANCE*

ELUCIDATION OF THE BASIC FEATURES

OF DNA LED TO RECOMBINANT

The development of recombinant DNA, high-density,

high-throughput screening, and other molecular ge-

DNA TECHNOLOGY

netic methodologies has revolutionized biology and is

DNA Is a Complex Biopolymer

having an increasing impact on clinical medicine.

Organized as a Double Helix

Much has been learned about human genetic disease

from pedigree analysis and study of affected proteins,

The fundamental organizational element is the se-

but in many cases where the specific genetic defect is

quence of purine (adenine [A] or guanine [G]) and

unknown, these approaches cannot be used. The new

pyrimidine (cytosine [C] or thymine [T]) bases. These

technologies circumvent these limitations by going di-

bases are attached to the C-1′ position of the sugar de-

rectly to the DNA molecule for information. Manipu-

oxyribose, and the bases are linked together through

lation of a DNA sequence and the construction of

joining of the sugar moieties at their 3′ and 5′ positions

chimeric molecules—so-called genetic engineering—

via a phosphodiester bond (Figure 35-1). The alternat-

provides a means of studying how a specific segment of

ing deoxyribose and phosphate groups form the back-

DNA works. Novel molecular genetic tools allow inves-

bone of the double helix (Figure 35-2). These 3′-5′

tigators to query and manipulate genomic sequences as

linkages also define the orientation of a given strand of

well as to examine both cellular mRNA and protein

the DNA molecule, and, since the two strands run in

profiles at the molecular level.

opposite directions, they are said to be antiparallel.

Understanding this technology is important for sev-

eral reasons: (1) It offers a rational approach to under-

standing the molecular basis of a number of diseases

Base Pairing Is a Fundamental Concept

(eg, familial hypercholesterolemia, sickle cell disease,

of DNA Structure & Function

the thalassemias, cystic fibrosis, muscular dystrophy).

(2) Human proteins can be produced in abundance for

Adenine and thymine always pair, by hydrogen bonding,

therapy (eg, insulin, growth hormone, tissue plasmino-

as do guanine and cytosine (Figure 35-3). These base

gen activator). (3) Proteins for vaccines (eg, hepatitis B)

pairs are said to be complementary, and the guanine

and for diagnostic testing (eg, AIDS tests) can be ob-

content of a fragment of double-stranded DNA will al-

tained. (4) This technology is used to diagnose existing

ways equal its cytosine content; likewise, the thymine

diseases and predict the risk of developing a given dis-

and adenine contents are equal. Base pairing and hy-

ease. (5) Special techniques have led to remarkable ad-

drophobic base-stacking interactions hold the two DNA

vances in forensic medicine. (6) Gene therapy for sickle

strands together. These interactions can be reduced by

cell disease, the thalassemias, adenosine deaminase defi-

heating the DNA to denature it. The laws of base pairing

ciency, and other diseases may be devised.

predict that two complementary DNA strands will rean-

neal exactly in register upon renaturation, as happens

when the temperature of the solution is slowly reduced to

normal. Indeed, the degree of base-pair matching (or

* See glossary of terms at the end of this chapter.

mismatching) can be estimated from the temperature re-

396

MOLECULAR GENETICS, RECOMBINANT DNA, & GENOMIC TECHNOLOGY

397

quired for denaturation-renaturation. Segments of DNA

exons. Regulatory regions for specific eukaryotic genes

with high degrees of base-pair matching require more en-

are usually located in the DNA that flanks the tran-

ergy input (heat) to accomplish denaturation—or, to put

scription initiation site at its

5′ end

(5′ flanking-

it another way, a closely matched segment will withstand

sequence DNA). Occasionally, such sequences are

more heat before the strands separate. This reaction is

found within the gene itself or in the region that flanks

used to determine whether there are significant differ-

the 3′ end of the gene. In mammalian cells, each gene

ences between two DNA sequences, and it underlies the

has its own regulatory region. Many eukaryotic genes

concept of hybridization, which is fundamental to the

(and some viruses that replicate in mammalian cells)

processes described below.

have special regions, called enhancers, that increase the

There are about 3

10⁹ base pairs (bp) in each

rate of transcription. Some genes also have DNA se-

human haploid genome. If an average gene length is

quences, known as silencers, that repress transcription.

3 × 10³ bp (3 kilobases [kb]), the genome could consist

Mammalian genes are obviously complicated, multi-

of 10⁶ genes, assuming that there is no overlap and that

component structures.

transcription proceeds in only one direction. It is

thought that there are < 10⁵ genes in the human and

Genes Are Transcribed Into RNA

that only 1-2% of the DNA codes for proteins. The

Information generally flows from DNA to mRNA to

exact function of the remaining ~98% of the human

protein, as illustrated in Figure 40-1 and discussed in

genome has not yet been defined.

more detail in Chapter 39. This is a rigidly controlled

The double-helical DNA is packaged into a more

process involving a number of complex steps, each of

compact structure by a number of proteins, most

which no doubt is regulated by one or more enzymes or

notably the basic proteins called histones. This con-

factors; faulty function at any of these steps can cause

densation may serve a regulatory role and certainly has

disease.

a practical purpose. The DNA present within the nu-

cleus of a cell, if simply extended, would be about

RECOMBINANT DNA TECHNOLOGY

1 meter long. The chromosomal proteins compact this

long strand of DNA so that it can be packaged into a

INVOLVES ISOLATION & MANIPULATION

nucleus with a volume of a few cubic micrometers.

OF DNA TO MAKE CHIMERIC MOLECULES

Isolation and manipulation of DNA, including end-to-

DNA Is Organized Into Genes

end joining of sequences from very different sources to

make chimeric molecules

(eg, molecules containing

In general, prokaryotic genes consist of a small regula-

both human and bacterial DNA sequences in a se-

tory region (100-500 bp) and a large protein-coding

quence-independent fashion), is the essence of recom-

segment (500-10,000 bp). Several genes are often con-

binant DNA research. This involves several unique

trolled by a single regulatory unit. Most mammalian

techniques and reagents.

genes are more complicated in that the coding regions

are interrupted by noncoding regions that are elimi-

Restriction Enzymes Cut DNA

nated when the primary RNA transcript is processed

Chains at Specific Locations

into mature messenger RNA (mRNA). The coding re-

gions (those regions that appear in the mature RNA

Certain endonucleases—enzymes that cut DNA at spe-

species) are called exons, and the noncoding regions,

cific DNA sequences within the molecule (as opposed

which interpose or intervene between the exons, are

to exonucleases, which digest from the ends of DNA

called introns

(Figure

40-1). Introns are always re-

molecules)—are a key tool in recombinant DNA re-

moved from precursor RNA before transport into the

search. These enzymes were called restriction enzymes

cytoplasm occurs. The process by which introns are re-

because their presence in a given bacterium restricted

moved from precursor RNA and by which exons are

the growth of certain bacterial viruses called bacterio-

ligated together is called RNA splicing. Incorrect pro-

phages. Restriction enzymes cut DNA of any source

cessing of the primary transcript into the mature

into short pieces in a sequence-specific manner—in

mRNA can result in disease in humans (see below); this

contrast to most other enzymatic, chemical, or physical

underscores the importance of these posttranscriptional

methods, which break DNA randomly. These defensive

processing steps. The variation in size and complexity

enzymes (hundreds have been discovered) protect the

of some human genes is illustrated in Table 40-1. Al-

host bacterial DNA from DNA from foreign organisms

though there is a 300-fold difference in the sizes of the

(primarily infective phages). However, they are present

genes illustrated, the mRNA sizes vary only about 20-

only in cells that also have a companion enzyme which

fold. This is because most of the DNA in genes is pres-

methylates the host DNA, rendering it an unsuitable

ent as introns, and introns tend to be much larger than

substrate for digestion by the restriction enzyme. Thus,

398

CHAPTER 40

Regulatory

Basal

Transcription

Poly(A)

region

promoter

start site

addition

region

site

Exon

DNA

5′

CAAT

TATA

AATAAA

3′

5′

Intron

3′

Noncoding

region

Transcription

NUCLEUS

Primary RNA transcript

PPP

Modification of

5′ and 3′ ends

Modified transcript

AAA---A

Poly(A) tail

Cap

Removal of introns

and splicing of exons

Processed nuclear mRNA

AAA---A

Transmembrane

CYTOPLASM

transport

AAA---A

mRNA

Translation

Protein

NH₂

COOH

Figure 40-1. Organization of a eukaryotic transcription unit and the pathway of eukaryotic gene expres-

sion. Eukaryotic genes have structural and regulatory regions. The structural region consists of the coding

DNA and 5′ and 3′ noncoding DNA sequences. The coding regions are divided into two parts: (1) exons, which

eventually are ligated together to become mature RNA, and (2) introns, which are processed out of the pri-

mary transcript. The structural region is bounded at its 5′ end by the transcription initiation site and at its

3′ end by the polyadenylate addition or termination site. The promoter region, which contains specific DNA

sequences that interact with various protein factors to regulate transcription, is discussed in detail in Chap-

ters 37 and 39. The primary transcript has a special structure, a cap, at the 5′ end and a stretch of As at the 3′

end. This transcript is processed to remove the introns; and the mature mRNA is then transported to the cyto-

plasm, where it is translated into protein.

site-specific DNA methylases and restriction enzymes

HpaI) or overlapping (sticky) ends (eg, BamHI) (Figure

always exist in pairs in a bacterium.

40-2), depending on the mechanism used by the en-

Restriction enzymes are named after the bac-

zyme. Sticky ends are particularly useful in constructing

terium from which they are isolated. For example,

hybrid or chimeric DNA molecules (see below). If the

EcoRI is from Escherichia coli, and BamHI is from Bacil-

four nucleotides are distributed randomly in a given

lus amyloliquefaciens (Table 40-2). The first three letters

DNA molecule, one can calculate how frequently a

in the restriction enzyme name consist of the first letter

given enzyme will cut a length of DNA. For each posi-

of the genus (E) and the first two letters of the species

tion in the DNA molecule, there are four possibilities

(co). These may be followed by a strain designation (R)

(A, C, G, and T); therefore, a restriction enzyme that

and a roman numeral (I) to indicate the order of discov-

recognizes a 4-bp sequence cuts, on average, once every

ery (eg, EcoRI, EcoRII). Each enzyme recognizes and

256 bp (4⁴), whereas another enzyme that recognizes a

cleaves a specific double-stranded DNA sequence that is

6-bp sequence cuts once every 4096 bp (4⁶). A given

4-7 bp long. These DNA cuts result in blunt ends (eg,

piece of DNA has a characteristic linear array of sites for

MOLECULAR GENETICS, RECOMBINANT DNA, & GENOMIC TECHNOLOGY

399

Table 40-1. Variations in the size and complexity

Table 40-2. Selected restriction endonucleases

of some human genes and mRNAs.¹

and their sequence specificities.¹

mRNA

Sequence Recognized

Bacterial

Gene Size

Number

Size

Endonuclease

Cleavage Sites Shown

Source

Gene

(kb)

of Introns

(kb)

↓

β-Globin

1.5

0.6

BamHI

GGATCC

Bacillus amylo-

Insulin

1.7

0.4

CCTAGG

liquefaciens H

β-Adrenergic receptor

2.2

↑

Albumin

2.1

↓

LDL receptor

5.5

BgIII

AGATCT

Bacillus glolbigii

Factor VIII

186

9.0

TCTAGA

Thyroglobulin

300

8.7

↑

¹The sizes are given in kilobases (kb). The sizes of the genes in-

↓

clude some proximal promoter and regulatory region sequences;

EcoRI

GAATTC

Escherichia coli

these are generally about the same size for all genes. Genes vary

CTTAAG

RY13

in size from about 1500 base pairs (bp) to over 2 × 10⁶ bp. There is

↑

also great variation in the number of introns and exons. The

↓

β-adrenergic receptor gene is intronless, and the thyroglobulin

EcoRII

CCTGG

Escherichia coli

gene has 36 introns. As noted by the smaller difference in mRNA

GGACC

R245

sizes, introns comprise most of the gene sequence.

↑

↓

HindIII

AAGCTT

Haemophilus

the various enzymes dictated by the linear sequence of

TTCGAA

influenzae R_d

its bases; hence, a restriction map can be constructed.

↑

When DNA is digested with a given enzyme, the ends

↓

of all the fragments have the same DNA sequence. The

Hhal

GCGC

Haemophilus

fragments produced can be isolated by electrophoresis

CGCG

haemolyticus

on agarose or polyacrylamide gels (see the discussion of

↑

blot transfer, below); this is an essential step in cloning

and a major use of these enzymes.

↓

Hpal

GTTAAC

Haemophilus

A number of other enzymes that act on DNA and

CAATTG

parainfluenzae

RNA are an important part of recombinant DNA tech-

↑

nology. Many of these are referred to in this and subse-

quent chapters (Table 40-3).

↓

MstII

CCTNAGG

Microcoleus

GGANTCC

strain

Restriction Enzymes & DNA Ligase Are

↑

Used to Prepare Chimeric DNA Molecules

↓

Sticky-end ligation is technically easy, but some special

PstI

CTGCAG

Providencia

techniques are often required to overcome problems in-

GACGTC

stuartii 164

herent in this approach. Sticky ends of a vector may re-

↑

connect with themselves, with no net gain of DNA.

↓

Sticky ends of fragments can also anneal, so that tandem

Taql

TCGA

Thermus

heterogeneous inserts form. Also, sticky-end sites may

AGCT

aquaticus YTI

not be available or in a convenient position. To circum-

↑

vent these problems, an enzyme that generates blunt

¹A, adenine; C, cytosine; G, guanine, T, thymine. Arrows show the site

ends is used, and new ends are added using the enzyme

of cleavage; depending on the site, sticky ends (BamHI) or blunt ends

terminal transferase. If poly d(G) is added to the 3′ ends

(Hpal) may result. The length of the recognition sequence can be 4 bp

of the vector and poly d(C) is added to the 3′ ends of

(Taql), 5 bp (EcoRII), 6 bp (EcoRI), or 7 bp (MstII) or longer. By conven-

tion, these are written in the 5′ or 3′ direction for the upper strand of

the foreign DNA, the two molecules can only anneal to

each recognition sequence, and the lower strand is shown with the

each other, thus circumventing the problems listed

opposite (ie, 3′ or 5′) polarity. Note that most recognition sequences

above. This procedure is called homopolymer tailing.

are palindromes (ie, the sequence reads the same in opposite direc-

Sometimes, synthetic blunt-ended duplex oligonu-

tions on the two strands). A residue designated N means that any nu-

cleotide linkers with a convenient restriction enzyme se-

cleotide is permitted.

400

CHAPTER 40

A. Sticky or staggered ends

5’

3’

BamHI

Figure 40-2. Results of restriction en-

donuclease digestion. Digestion with a re-

3’

5’

A G

striction endonuclease can result in the for-

B. Blunt ends

mation of DNA fragments with sticky, or

5’

3’

cohesive, ends (A) or blunt ends (B). This is

HpaI

an important consideration in devising

3’

C A A T T G

5’

C A A

T T G

cloning strategies.

quence are ligated to the blunt-ended DNA. Direct

terized or used for other purposes. This technique is

blunt-end ligation is accomplished using the enzyme

based on the fact that chimeric or hybrid DNA molecules

bacteriophage T4 DNA ligase. This technique, though

can be constructed in cloning vectors—typically bacter-

less efficient than sticky-end ligation, has the advantage

ial plasmids, phages, or cosmids—which then continue

of joining together any pairs of ends. The disadvantages

to replicate in a host cell under their own control systems.

are that there is no control over the orientation of inser-

In this way, the chimeric DNA is amplified. The general

tion or the number of molecules annealed together, and

procedure is illustrated in Figure 40-3.

there is no easy way to retrieve the insert.

Bacterial plasmids are small, circular, duplex DNA

molecules whose natural function is to confer antibiotic

resistance to the host cell. Plasmids have several proper-

Cloning Amplifies DNA

ties that make them extremely useful as cloning vectors.

A clone is a large population of identical molecules, bac-

They exist as single or multiple copies within the bac-

teria, or cells that arise from a common ancestor. Molec-

terium and replicate independently from the bacterial

ular cloning allows for the production of a large number

DNA. The complete DNA sequence of many plasmids is

of identical DNA molecules, which can then be charac-

known; hence, the precise location of restriction enzyme

Table 40-3. Some of the enzymes used in recombinant DNA research.¹

Enzyme

Reaction

Primary Use

Alkaline phosphatase

Dephosphorylates 5′ ends of RNA and DNA.

Removal of 5′-PO₄ groups prior to kinase labeling to prevent

self-ligation.

BAL 31 nuclease

Degrades both the 3′ and 5′ ends of DNA.

Progressive shortening of DNA molecules.

DNA ligase

Catalyzes bonds between DNA molecules.

Joining of DNA molecules.

DNA polymerase I

Synthesizes double-stranded DNA from

Synthesis of double-stranded cDNA; nick translation; gener-

single-stranded DNA.

ation of blunt ends from sticky ends.

DNase I

Under appropriate conditions, produces

Nick translation; mapping of hypersensitive sites; mapping

single-stranded nicks in DNA.

protein-DNA interactions.

Exonuclease III

Removes nucleotides from 3′ ends of DNA.

DNA sequencing; mapping of DNA-protein interactions.

λ exonuclease

Removes nucleotides from 5′ ends of DNA.

DNA sequencing.

Polynucleotide kinase

Transfers terminal phosphate (γ position)

³²P labeling of DNA or RNA.

from ATP to 5′-OH groups of DNA or RNA.

Reverse transcriptase

Synthesizes DNA from RNA template.

Synthesis of cDNA from mRNA; RNA (5′ end) mapping

studies.

S1 nuclease

Degrades single-stranded DNA.

Removal of “hairpin” in synthesis of cDNA; RNA mapping

studies (both 5′ and 3′ ends).

Terminal transferase

Adds nucleotides to the 3′ ends of DNA.

Homopolymer tailing.

¹Adapted and reproduced, with permission, from Emery AEH: Page 41 in: An Introduction to Recombinant DNA. Wiley, 1984.

MOLECULAR GENETICS, RECOMBINANT DNA, & GENOMIC TECHNOLOGY

401

EcoRI

restriction

endonuclease

Human DNA

Circular plasmid DNA

Linear plasmid DNA

with sticky ends

EcoRI restriction

endonuclease

AA T T

T T

Piece of human DNA cut with

T AA

DNA

same restriction nuclease and

ligase

containing same sticky ends

Plasmid DNA molecule with human DNA insert

(recombinant DNA molecule)

Figure 40-3. Use of restriction nucleases to make new recombinant or chimeric DNA molecules. When in-

serted back into a bacterial cell (by the process called transformation), typically only a single plasmid is taken up

by a single cell, and the plasmid DNA replicates not only itself but also the physically linked new DNA insert. Since

recombining the sticky ends, as indicated, regenerates the same DNA sequence recognized by the original restric-

tion enzyme, the cloned DNA insert can be cleanly cut back out of the recombinant plasmid circle with this en-

donuclease. If a mixture of all of the DNA pieces created by treatment of total human DNA with a single restriction

nuclease is used as the source of human DNA, a million or so different types of recombinant DNA molecules can

be obtained, each pure in its own bacterial clone. (Modified and reproduced, with permission, from Cohen SN: The

manipulation of genes. Sci Am [July] 1975;233:34.)

cleavage sites for inserting the foreign DNA is available.

Larger fragments of DNA can be cloned in cosmids,

Plasmids are smaller than the host chromosome and are

which combine the best features of plasmids and

therefore easily separated from the latter, and the desired

phages. Cosmids are plasmids that contain the DNA se-

plasmid-inserted DNA is readily removed by cutting the

quences, so-called cos sites, required for packaging

plasmid with the enzyme specific for the restriction site

lambda DNA into the phage particle. These vectors

into which the original piece of DNA was inserted.

grow in the plasmid form in bacteria, but since much of

Phages usually have linear DNA molecules into

the unnecessary lambda DNA has been removed, more

which foreign DNA can be inserted at several restric-

chimeric DNA can be packaged into the particle head.

tion enzyme sites. The chimeric DNA is collected after

It is not unusual for cosmids to carry inserts of chimeric

the phage proceeds through its lytic cycle and produces

DNA that are 35-50 kb long. Even larger pieces of

mature, infective phage particles. A major advantage of

DNA can be incorporated into bacterial artificial chro-

phage vectors is that while plasmids accept DNA pieces

mosome (BAC), yeast artificial chromosome (YAC), or

about 6-10 kb long, phages can accept DNA fragments

E. coli bacteriophage P1-based (PAC) vectors. These

10-20 kb long, a limitation imposed by the amount of

vectors will accept and propagate DNA inserts of sev-

DNA that can be packed into the phage head.

eral hundred kilobases or more and have largely re-

402

CHAPTER 40

Table 40-4. Cloning capacities of common

ferent recombinant clones is called a library. A genomic

cloning vectors.

library is prepared from the total DNA of a cell line or

tissue. A cDNA library comprises complementary

DNA copies of the population of mRNAs in a tissue.

Vector

DNA Insert Size

Genomic DNA libraries are often prepared by perform-

Plasmid pBR322

0.01-10 kb

ing partial digestion of total DNA with a restriction en-

Lambda charon 4A

10-20 kb

zyme that cuts DNA frequently (eg, a four base cutter

Cosmids

35-50 kb

such as TaqI ). The idea is to generate rather large frag-

BAC, P1

50-250 kb

ments so that most genes will be left intact. The BAC,

YAC

500-3000 kb

YAC, and P1 vectors are preferred since they can accept

very large fragments of DNA and thus offer a better

placed the plasmid, phage, and cosmid vectors for some

chance of isolating an intact gene on a single DNA

cloning and gene mapping applications. A comparison

fragment.

of these vectors is shown in Table 40-4.

A vector in which the protein coded by the gene in-

Because insertion of DNA into a functional region

troduced by recombinant DNA technology is actually

of the vector will interfere with the action of this re-

synthesized is known as an expression vector. Such

gion, care must be taken not to interrupt an essential

vectors are now commonly used to detect specific

function of the vector. This concept can be exploited,

cDNA molecules in libraries and to produce proteins

however, to provide a selection technique. For example,

by genetic engineering techniques. These vectors are

the common plasmid vector pBR322 has both tetracy-

specially constructed to contain very active inducible

cline (tet) and ampicillin (amp) resistance genes. A

promoters, proper in-phase translation initiation

single PstI restriction enzyme site within the amp resis-

codons, both transcription and translation termination

tance gene is commonly used as the insertion site for a

signals, and appropriate protein processing signals, if

piece of foreign DNA. In addition to having sticky ends

needed. Some expression vectors even contain genes

(Table 40-2 and Figure 40-2), the DNA inserted at

that code for protease inhibitors, so that the final yield

this site disrupts the amp resistance gene and makes the

of product is enhanced.

bacterium carrying this plasmid amp-sensitive (Figure

40-4). Thus, the parental plasmid, which provides re-

Probes Search Libraries for Specific

sistance to both antibiotics, can be readily separated

Genes or cDNA Molecules

from the chimeric plasmid, which is resistant only to

A variety of molecules can be used to “probe” libraries in

tetracycline. YACs contain replication and segregation

functions that work in both bacteria and yeast cells and

search of a specific gene or cDNA molecule or to define

and quantitate DNA or RNA separated by electrophore-

therefore can be propagated in either organism.

In addition to the vectors described in Table 40-4

sis through various gels. Probes are generally pieces of

DNA or RNA labeled with a ³²P-containing nu-

that are designed primarily for propagation in bacterial

cells, vectors for mammalian cell propagation and insert

cleotide—or fluorescently labeled nucleotides

(more

commonly now). Importantly, neither modification (³²P

gene (cDNA)/protein expression have also been devel-

oped. These vectors are all based upon various eukary-

or fluorescent-label) affects the hybridization properties

of the resulting labeled nucleic acid probes. The probe

otic viruses that are composed of RNA or DNA

genomes. Notable examples of such viral vectors are

must recognize a complementary sequence to be effec-

tive. A cDNA synthesized from a specific mRNA can be

those utilizing adenoviral (DNA-based) and retroviral

(RNA-based) genomes. Though somewhat limited in

used to screen either a cDNA library for a longer cDNA

or a genomic library for a complementary sequence in

the size of DNA sequences that can be inserted, such

mammalian viral cloning vectors make up for this

the coding region of a gene. A popular technique for

finding specific genes entails taking a short amino acid

shortcoming because they will efficiently infect a wide

range of different cell types. For this reason, various

sequence and, employing the codon usage for that

species

(see Chapter 38), making an oligonucleotide

mammalian viral vectors are being investigated for use

in gene therapy experiments.

probe that will detect the corresponding DNA fragment

in a genomic library. If the sequences match exactly,

probes 15-20 nucleotides long will hybridize. cDNA

A Library Is a Collection

probes are used to detect DNA fragments on Southern

of Recombinant Clones

blot transfers and to detect and quantitate RNA on

The combination of restriction enzymes and various

Northern blot transfers. Specific antibodies can also be

cloning vectors allows the entire genome of an organ-

used as probes provided that the vector used synthesizes

ism to be packed into a vector. A collection of these dif-

protein molecules that are recognized by them.

MOLECULAR GENETICS, RECOMBINANT DNA, & GENOMIC TECHNOLOGY

403

Ampicillin

Tetracycline

resistance gene

EcoRI

Tetracycline

HindIII

resistance gene

HindIII

PstI

BamHI

Cut open with

PstI

SalI

PstI

SalI

Then insert

PstI-cut DNA

Amp^r

Amp^s

Tet^r

Host pBR322

Chimeric pBR322

Figure 40-4. A method of screening recombinants for inserted DNA fragments. Using the plasmid pBR322, a

piece of DNA is inserted into the unique PstI site. This insertion disrupts the gene coding for a protein that pro-

vides ampicillin resistance to the host bacterium. Hence, the chimeric plasmid will no longer survive when plated

on a substrate medium that contains this antibiotic. The differential sensitivity to tetracycline and ampicillin can

therefore be used to distinguish clones of plasmid that contain an insert. A similar scheme relying upon produc-

tion of an in-frame fusion of a newly inserted DNA producing a peptide fragment capable of complementing an

inactive, deleted form of the enzyme β-galactosidase allows for blue-white colony formation on agar plates con-

taining a dye hydrolyzable by β-galactoside. β-Galactosidase-positive colonies are blue.

Blotting & Hybridization Techniques Allow

renatured, and analyzed for an interaction by hybridiza-

Visualization of Specific Fragments

tion with a specific labeled DNA probe.

Colony or plaque hybridization is the method by

Visualization of a specific DNA or RNA fragment

which specific clones are identified and purified. Bacte-

among the many thousands of “contaminating” mole-

ria are grown on colonies on an agar plate and overlaid

cules requires the convergence of a number of tech-

with nitrocellulose filter paper. Cells from each colony

niques, collectively termed blot transfer. Figure 40-5

stick to the filter and are permanently fixed thereto by

illustrates the Southern (DNA), Northern (RNA), and

heat, which with NaOH treatment also lyses the cells

Western (protein) blot transfer procedures. (The first is

and denatures the DNA so that it will hybridize with

named for the person who devised the technique, and

the probe. A radioactive probe is added to the filter,

the other names began as laboratory jargon but are now

and (after washing) the hybrid complex is localized by

accepted terms.) These procedures are useful in deter-

exposing the filter to x-ray film. By matching the spot

mining how many copies of a gene are in a given tissue

on the autoradiograph to a colony, the latter can be

or whether there are any gross alterations in a gene

picked from the plate. A similar strategy is used to iden-

(deletions, insertions, or rearrangements). Occasionally,

tify fragments in phage libraries. Successive rounds of

if a specific base is changed and a restriction site is al-

this procedure result in a clonal isolate

(bacterial

tered, these procedures can detect a point mutation.

colony) or individual phage plaque.

The Northern and Western blot transfer techniques are

All of the hybridization procedures discussed in this

used to size and quantitate specific RNA and protein

section depend on the specific base-pairing properties

molecules, respectively. A fourth hybridization tech-

of complementary nucleic acid strands described above.

nique, the Southwestern blot, examines protein•DNA

Perfect matches hybridize readily and withstand high

interactions. Proteins are separated by electrophoresis,

temperatures in the hybridization and washing reac-

404

CHAPTER 40

Southern

Northern

Western

tions. Specific complexes also form in the presence of

low salt concentrations. Less than perfect matches do

DNA

RNA

Protein

not tolerate these stringent conditions (ie, elevated

temperatures and low salt concentrations); thus, hy-

bridization either never occurs or is disrupted during

Gel

the washing step. Gene families, in which there is some

electrophoresis

degree of homology, can be detected by varying the

stringency of the hybridization and washing steps.

Cross-species comparisons of a given gene can also be

made using this approach. Hybridization conditions ca-

pable of detecting just a single base pair mismatch be-

tween probe and target have been devised.

Transfer to paper

Manual & Automatic Techniques

Are Available to Determine

cDNA*

Antibody* Add probe

the Sequence of DNA

The segments of specific DNA molecules obtained by

recombinant DNA technology can be analyzed to de-

Autoradiograph

termine their nucleotide sequence. This method de-

pends upon having a large number of identical DNA

molecules. This requirement can be satisfied by cloning

Figure 40-5. The blot transfer procedure. In a

the fragment of interest, using the techniques described

Southern, or DNA, blot transfer, DNA isolated from a

above. The manual enzymatic method (Sanger) em-

cell line or tissue is digested with one or more restric-

ploys specific dideoxynucleotides that terminate DNA

tion enzymes. This mixture is pipetted into a well in an

strand synthesis at specific nucleotides as the strand is

agarose or polyacrylamide gel and exposed to a direct

synthesized on purified template nucleic acid. The reac-

electrical current. DNA, being negatively charged, mi-

tions are adjusted so that a population of DNA frag-

grates toward the anode; the smaller fragments move

ments representing termination at every nucleotide is

the most rapidly. After a suitable time, the DNA is dena-

obtained. By having a radioactive label incorporated at

tured by exposure to mild alkali and transferred to ni-

the end opposite the termination site, one can separate

trocellulose or nylon paper, in an exact replica of the

the fragments according to size using polyacrylamide

pattern on the gel, by the blotting technique devised

gel electrophoresis. An autoradiograph is made, and

by Southern. The DNA is bound to the paper by expo-

each of the fragments produces an image (band) on an

sure to heat, and the paper is then exposed to the

x-ray film. These are read in order to give the DNA se-

labeled cDNA probe, which hybridizes to complemen-

quence (Figure 40-6). Another manual method, that of

tary fragments on the filter. After thorough washing,

Maxam and Gilbert, employs chemical methods to

the paper is exposed to x-ray film, which is developed

cleave the DNA molecules where they contain the spe-

to reveal several specific bands corresponding to the

cific nucleotides. Techniques that do not require the

DNA fragment that recognized the sequences in the

use of radioisotopes are commonly employed in auto-

mated DNA sequencing. Most commonly employed is

cDNA probe. The RNA, or Northern, blot is conceptually

an automated procedure in which four different fluo-

similar. RNA is subjected to electrophoresis before blot

rescent labels—one representing each nucleotide—are

transfer. This requires some different steps from those

used. Each emits a specific signal upon excitation by a

of DNA transfer, primarily to ensure that the RNA re-

laser beam, and this can be recorded by a computer.

mains intact, and is generally somewhat more difficult.

In the protein, or Western, blot, proteins are elec-

trophoresed and transferred to nitrocellulose and then

Oligonucleotide Synthesis Is Now Routine

probed with a specific antibody or other probe mole-

The automated chemical synthesis of moderately long

cule. (Asterisks signify labeling, either radioactive or

oligonucleotides (about 100 nucleotides) of precise se-

fluorescent.)

quence is now a routine laboratory procedure. Each

synthetic cycle takes but a few minutes, so an entire

molecule can be made by synthesizing relatively short

segments that can then be ligated to one another.

Oligonucleotides are now indispensable for DNA se-

MOLECULAR GENETICS, RECOMBINANT DNA, & GENOMIC TECHNOLOGY

405

Reaction containing radiolabel:

Sequence of original strand:

ddGTP ddATP ddTTP ddCTP

- A - G - T -C - T - T - G - G- A - G - C - T

- 3′

A G T C T T G G A G C T

Bases terminated

Figure 40-6. Sequencing of DNA by the method devised by Sanger. The ladder-like arrays represent from bot-

tom to top all of the successively longer fragments of the original DNA strand. Knowing which specific dideoxynu-

cleotide reaction was conducted to produce each mixture of fragments, one can determine the sequence of nu-

cleotides from the labeled end (asterisk) toward the unlabeled end by reading up the gel. Automated sequencing

involves the reading of chemically modified deoxynucleotides. The base-pairing rules of Watson and Crick (A-T,

G-C) dictate the sequence of the other (complementary) strand. (Asterisks signify radiolabeling.)

quencing, library screening, protein-DNA binding,

quences, and extension of the annealed primers with

DNA mobility shift assays, the polymerase chain reac-

DNA polymerase result in the exponential amplifica-

tion (see below), site-directed mutagenesis, and numer-

tion of DNA segments of defined length. Early PCR re-

ous other applications.

actions used an E coli DNA polymerase that was de-

stroyed by each heat denaturation cycle. Substitution of

a heat-stable DNA polymerase from Thermus aquaticus

The Polymerase Chain Reaction

(or the corresponding DNA polymerase from other

(PCR) Amplifies DNA Sequences

thermophilic bacteria), an organism that lives and repli-

The polymerase chain reaction (PCR) is a method of

cates at 70-80 °C, obviates this problem and has made

amplifying a target sequence of DNA. PCR provides a

possible automation of the reaction, since the polym-

sensitive, selective, and extremely rapid means of ampli-

erase reactions can be run at 70 °C. This has also im-

fying a desired sequence of DNA. Specificity is based

proved the specificity and the yield of DNA.

on the use of two oligonucleotide primers that hy-

DNA sequences as short as 50-100 bp and as long

bridize to complementary sequences on opposite

as 10 kb can be amplified. Twenty cycles provide an

strands of DNA and flank the target sequence (Figure

amplification of 10⁶ and 30 cycles of 10⁹. The PCR al-

40-7). The DNA sample is first heated to separate the

lows the DNA in a single cell, hair follicle, or spermato-

two strands; the primers are allowed to bind to the

zoon to be amplified and analyzed. Thus, the applica-

DNA; and each strand is copied by a DNA polymerase,

tions of PCR to forensic medicine are obvious. The

starting at the primer site. The two DNA strands each

PCR is also used (1) to detect infectious agents, espe-

serve as a template for the synthesis of new DNA from

cially latent viruses; (2) to make prenatal genetic diag-

the two primers. Repeated cycles of heat denaturation,

noses; (3) to detect allelic polymorphisms; (4) to estab-

annealing of the primers to their complementary se-

lish precise tissue types for transplants; and (5) to study

406

CHAPTER 40

evolution, using DNA from archeological samples after

Targeted sequence

RNA copying and mRNA quantitation by the so-called

RT-PCR method (cDNA copies of mRNA generated

START

by a retroviral reverse transcriptase). There are an equal

number of applications of PCR to problems in basic

science, and new uses are developed every year.

CYCLE 1

PRACTICAL APPLICATIONS

OF RECOMBINANT DNA TECHNOLOGY

ARE NUMEROUS

The isolation of a specific gene from an entire genome

CYCLE 2

requires a technique that will discriminate one part in a

million. The identification of a regulatory region that

may be only 10 bp in length requires a sensitivity of

one part in 3 × 10⁸; a disease such as sickle cell anemia

is caused by a single base change, or one part in 3 × 10⁹.

Recombinant DNA technology is powerful enough to

accomplish all these things.

Gene Mapping Localizes Specific

Genes to Distinct Chromosomes

Gene localizing thus can define a map of the human

genome. This is already yielding useful information in

CYCLE 3

the definition of human disease. Somatic cell hybridiza-

tion and in situ hybridization are two techniques used

to accomplish this. In in situ hybridization, the sim-

pler and more direct procedure, a radioactive probe is

added to a metaphase spread of chromosomes on a glass

slide. The exact area of hybridization is localized by lay-

ering photographic emulsion over the slide and, after

exposure, lining up the grains with some histologic

identification of the chromosome. Fluorescence in situ

hybridization (FISH) is a very sensitive technique that

is also used for this purpose. This often places the gene

at a location on a given band or region on the chromo-

some. Some of the human genes localized using these

techniques are listed in Table 40-5. This table repre-

sents only a sampling, since thousands of genes have

been mapped as a result of the recent sequencing of the

Figure 40-7. The polymerase chain reaction is used to

amplify specific gene sequences. Double-stranded DNA is

heated to separate it into individual strands. These bind two

distinct primers that are directed at specific sequences on

opposite strands and that define the segment to be ampli-

fied. DNA polymerase extends the primers in each direction

and synthesizes two strands complementary to the original

two. This cycle is repeated several times, giving an amplified

product of defined length and sequence. Note that the two

CYCLES 4-n

primers are present in excess.

MOLECULAR GENETICS, RECOMBINANT DNA, & GENOMIC TECHNOLOGY

407

Table 40-5. Localization of human genes.¹

Gene

Chromosome

Disease

Insulin

11p15

Prolactin

6p23-q12

Growth hormone

17q21-qter

Growth hormone deficiency

α-Globin

16p12-pter

α-Thalassemia

β-Globin

11p12

β-Thalassemia, sickle cell

Adenosine deaminase

20q13-qter

Adenosine deaminase deficiency

Phenylalanine hydroxylase

12q24

Phenylketonuria

Hypoxanthine-guanine

Xq26-q27

Lesch-Nyhan syndrome

phosphoribosyltransferase

DNA segment G8

Huntington’s chorea

¹This table indicates the chromosomal location of several genes and the diseases asso-

ciated with deficient or abnormal production of the gene products. The chromosome

involved is indicated by the first number or letter. The other numbers and letters refer

to precise localizations, as defined in McKusick VA: Mendelian Inheritance in Man, 6th

ed. John Hopkins Univ Press, 1983.

human genome. Once the defect is localized to a region

Recombinant DNA Technology Is Used

of DNA that has the characteristic structure of a gene

in the Molecular Analysis of Disease

(Figure 40-1), a synthetic gene can be constructed and

A. NORMAL GENE VARIATIONS

expressed in an appropriate vector and its function can

be assessed—or the putative peptide, deduced from the

There is a normal variation of DNA sequence just as is

open reading frame in the coding region, can be synthe-

true of more obvious aspects of human structure. Varia-

sized. Antibodies directed against this peptide can be

tions of DNA sequence, polymorphisms, occur ap-

used to assess whether this peptide is expressed in nor-

proximately once in every 500 nucleotides, or about

mal persons and whether it is absent in those with the

10⁷ times per genome. There are without doubt dele-

genetic syndrome.

tions and insertions of DNA as well as single-base sub-

stitutions. In healthy people, these alterations obviously

occur in noncoding regions of DNA or at sites that

Proteins Can Be Produced

cause no change in function of the encoded protein.

This heritable polymorphism of DNA structure can be

for Research & Diagnosis

associated with certain diseases within a large kindred

A practical goal of recombinant DNA research is the

and can be used to search for the specific gene involved,

production of materials for biomedical applications.

as is illustrated below. It can also be used in a variety of

This technology has two distinct merits: (1) It can sup-

applications in forensic medicine.

ply large amounts of material that could not be ob-

tained by conventional purification methods (eg, inter-

B. GENE VARIATIONS CAUSING DISEASE

feron, tissue plasminogen activating factor). (2) It can

provide human material (eg, insulin, growth hormone).

Classic genetics taught that most genetic diseases were

The advantages in both cases are obvious. Although the

due to point mutations which resulted in an impaired

primary aim is to supply products—generally pro-

protein. This may still be true, but if on reading the

teins—for treatment

(insulin) and diagnosis

(AIDS

initial sections of this chapter one predicted that ge-

testing) of human and other animal diseases and for

netic disease could result from derangement of any of

disease prevention (hepatitis B vaccine), there are other

the steps illustrated in Figure 40-1, one would have

potential commercial applications, especially in agricul-

made a proper assessment. This point is nicely illus-

ture. An example of the latter is the attempt to engineer

trated by examination of the β-globin gene. This gene

plants that are more resistant to drought or temperature

is located in a cluster on chromosome

11 (Figure

extremes, more efficient at fixing nitrogen, or that pro-

40-8), and an expanded version of the gene is illus-

duce seeds containing the complete complement of es-

trated in Figure 40-9. Defective production of β-glo-

sential amino acids (rice, wheat, corn, etc).

bin results in a variety of diseases and is due to many

408

CHAPTER 40

Gγ

Aγ

Ψβ

5′

LCR

3′

10 kb

Hemoglobinopathy

β⁰-Thalassemia

Hemoglobin Lepore

Inverted

(Aγδβ)⁰-Thalassemia

Figure 40-8. Schematic representation of the β-globin gene cluster and of the lesions in some ge-

netic disorders. The β-globin gene is located on chromosome 11 in close association with the two γ-glo-

bin genes and the δ-globin gene. The β-gene family is arranged in the order 5′-ε-Gγ-Aγ-ψβ-δ-β-3′. The

ε locus is expressed in early embryonic life (as a₂ε₂). The γ genes are expressed in fetal life, making fetal

hemoglobin (HbF, α₂γ₂). Adult hemoglobin consists of HbA (α₂β₂) or HbA₂(α₂δ₂). The Ψβ is a pseudo-

gene that has sequence homology with β but contains mutations that prevent its expression. A locus

control region (LCR) located upstream (5′) from the ε gene controls the rate of transcription of the en-

tire β-globin gene cluster. Deletions (solid bar) of the β locus cause β-thalassemia (deficiency or ab-

sence [β⁰] of β-globin). A deletion of δ and β causes hemoglobin Lepore (only hemoglobin α is present).

An inversion (Aγδβ)⁰ in this region (colored bar) disrupts gene function and also results in thalassemia

(type III). Each type of thalassemia tends to be found in a certain group of people, eg, the (Aγδβ)⁰ dele-

tion inversion occurs in persons from India. Many more deletions in this region have been mapped, and

each causes some type of thalassemia.

different lesions in and around the β-globin gene

turn results in an A-to-U change in the mRNA corre-

(Table 40-6).

sponding to the sixth codon of the β-globin gene. The

altered codon specifies a different amino acid (valine

C. POINT MUTATIONS

rather than glutamic acid), and this causes a structural

The classic example is sickle cell disease, which is

abnormality of the β-globin molecule. Other point mu-

caused by mutation of a single base out of the 3 × 10⁹

tations in and around the β-globin gene result in de-

in the genome, a T-to-A DNA substitution, which in

creased production or, in some instances, no produc-

5′

3′

Figure 40-9. Mutations in the β-globin gene causing β-thalassemia. The β-globin gene is shown in the 5′

to 3′ orientation. The cross-hatched areas indicate the 5′ and 3′ nontranslated regions. Reading from the 5′ to

3′ direction, the shaded areas are exons 1-3 and the clear spaces are introns 1 (I₁) and 2 (I₂). Mutations that af-

fect transcription control (•) are located in the 5′ flanking-region DNA. Examples of nonsense mutations (

mutations in RNA processing (

), and RNA cleavage mutations ( ) have been identified and are indicated. In

some regions, many mutations have been found. These are indicated by the brackets.

MOLECULAR GENETICS, RECOMBINANT DNA, & GENOMIC TECHNOLOGY

409

Table 40-6. Structural alterations of the β-globin

E. PEDIGREE ANALYSIS

gene.

Sickle cell disease again provides an excellent example

of how recombinant DNA technology can be applied

Alteration

Function Affected

Disease

to the study of human disease. The substitution of T

for A in the template strand of DNA in the β-globin

Point mutations

Protein folding

Sickle cell disease

gene changes the sequence in the region that corre-

Transcriptional control

β-Thalassemia

sponds to the sixth codon from

Frameshift and non-

β-Thalassemia

sense mutations

↓

RNA processing

β-Thalassemia

CCTGAGG

Coding strand

Deletion

mRNA production

β⁰-Thalassemia

GGAC T CC

Template strand

Hemoglobin

↑

Lepore

Rearrangement

mRNA production

β-Thalassemia

type III

CCTGTGG

Coding strand

GGAC A CC

Template strand

and destroys a recognition site for the restriction en-

tion of β-globin; β-thalassemia is the result of these

zyme MstII (CCTNAGG; denoted by the small vertical

mutations. (The thalassemias are characterized by de-

arrows; Table 40-2). Other MstII sites 5′ and 3′ from

fects in the synthesis of hemoglobin subunits, and so

this site (Figure 40-10) are not affected and so will be

β-thalassemia results when there is insufficient produc-

cut. Therefore, incubation of DNA from normal (AA),

tion of β-globin.) Figure 40-9 illustrates that point

heterozygous (AS), and homozygous (SS) individuals

mutations affecting each of the many processes in-

results in three different patterns on Southern blot

volved in generating a normal mRNA (and therefore a

transfer (Figure 40-10). This illustrates how a DNA

normal protein) have been implicated as a cause of

pedigree can be established using the principles dis-

β-thalassemia.

cussed in this chapter. Pedigree analysis has been ap-

D. DELETIONS, INSERTIONS, &

plied to a number of genetic diseases and is most useful

in those caused by deletions and insertions or the rarer

REARRANGEMENTS OF DNA

instances in which a restriction endonuclease cleavage

Studies of bacteria, viruses, yeasts, and fruit flies show

site is affected, as in the example cited in this para-

that pieces of DNA can move from one place to an-

graph. The analysis is facilitated by the PCR reaction,

other within a genome. The deletion of a critical piece

which can provide sufficient DNA for analysis from

of DNA, the rearrangement of DNA within a gene, or

just a few nucleated red blood cells.

the insertion of a piece of DNA within a coding or reg-

ulatory region can all cause changes in gene expression

F. PRENATAL DIAGNOSIS

resulting in disease. Again, a molecular analysis of

If the genetic lesion is understood and a specific probe

β-thalassemia produces numerous examples of these

is available, prenatal diagnosis is possible. DNA from

processes—particularly deletions—as causes of disease

cells collected from as little as 10 mL of amniotic fluid

(Figure 40-8). The globin gene clusters seem particu-

(or by chorionic villus biopsy) can be analyzed by

larly prone to this lesion. Deletions in the α-globin

Southern blot transfer. A fetus with the restriction pat-

cluster, located on chromosome

16, cause α-thal-

tern AA in Figure 40-10 does not have sickle cell dis-

assemia. There is a strong ethnic association for many

ease, nor is it a carrier. A fetus with the SS pattern will

of these deletions, so that northern Europeans, Fil-

develop the disease. Probes are now available for this

ipinos, blacks, and Mediterranean peoples have differ-

type of analysis of many genetic diseases.

ent lesions all resulting in the absence of hemoglobin A

and α-thalassemia.

G. RESTRICTION FRAGMENT LENGTH

A similar analysis could be made for a number of

POLYMORPHISM (RFLP)

other diseases. Point mutations are usually defined by

sequencing the gene in question, though occasionally, if

The differences in DNA sequence cited above can re-

the mutation destroys or creates a restriction enzyme

sult in variations of restriction sites and thus in the

site, the technique of restriction fragment analysis can

length of restriction fragments. An inherited difference

be used to pinpoint the lesion. Deletions or insertions

in the pattern of restriction (eg, a DNA variation occur-

of DNA larger than 50 bp can often be detected by the

ring in more than 1% of the general population) is

Southern blotting procedure.

known as a restriction fragment length polymorphism,

410

CHAPTER 40

A. MstII restriction sites around and in the β-globin gene

Normal (A)

5′

3′

1.15 kb

0.2

Sickle (S)

5′

3′

1.35 kb

B. Pedigree analysis

Fragment

size

1.35 kb

1.15 kb

Phenotype

Figure 40-10. Pedigree analysis of sickle cell disease. The top part of the fig-

ure (A) shows the first part of the β-globin gene and the MstII restriction en-

zyme sites in the normal (A) and sickle cell (S) β-globin genes. Digestion with

the restriction enzyme MstII results in DNA fragments 1.15 kb and 0.2 kb long in

normal individuals. The T-to-A change in individuals with sickle cell disease

abolishes one of the three MstII sites around the β-globin gene; hence, a single

restriction fragment 1.35 kb in length is generated in response to MstII. This size

difference is easily detected on a Southern blot. (The 0.2-kb fragment would

run off the gel in this illustration.) (B) Pedigree analysis shows three possibili-

ties: AA = normal (open circle); AS = heterozygous (half-solid circles, half-solid

square); SS = homozygous (solid square). This approach allows for prenatal di-

agnosis of sickle cell disease (dash-sided square).

MOLECULAR GENETICS, RECOMBINANT DNA, & GENOMIC TECHNOLOGY

411

or RFLP. An extensive RFLP map of the human

H. MICROSATELLITE DNA POLYMORPHISMS

genome has been constructed. This is proving useful in

Short (2-6 bp), inherited, tandem repeat units of DNA

the human genome sequencing project and is an impor-

occur about

50,000-100,000 times in the human

tant component of the effort to understand various sin-

genome (Chapter 36). Because they occur more fre-

gle-gene and multigenic diseases. RFLPs result from

quently—and in view of the routine application of sen-

single-base changes (eg, sickle cell disease) or from dele-

sitive PCR methods—they are replacing RFLPs as the

tions or insertions of DNA into a restriction fragment

marker loci for various genome searches.

(eg, the thalassemias) and have proved to be useful di-

agnostic tools. They have been found at known gene

I. RFLPS & VNTRS IN FORENSIC MEDICINE

loci and in sequences that have no known function;

Variable numbers of tandemly repeated (VNTR) units

thus, RFLPs may disrupt the function of the gene or

are one common type of “insertion” that results in an

may have no biologic consequences.

RFLP. The VNTRs can be inherited, in which case

RFLPs are inherited, and they segregate in a

they are useful in establishing genetic association with a

mendelian fashion. A major use of RFLPs (thousands

disease in a family or kindred; or they can be unique to

are now known) is in the definition of inherited dis-

an individual and thus serve as a molecular fingerprint

eases in which the functional deficit is unknown.

of that person.

RFLPs can be used to establish linkage groups, which

in turn, by the process of chromosome walking, will

J. GENE THERAPY

eventually define the disease locus. In chromosome

Diseases caused by deficiency of a gene product (Table

walking (Figure 40-11), a fragment representing one

40-5) are amenable to replacement therapy. The strat-

end of a long piece of DNA is used to isolate another

egy is to clone a gene (eg, the gene that codes for

that overlaps but extends the first. The direction of ex-

adenosine deaminase) into a vector that will readily be

tension is determined by restriction mapping, and the

taken up and incorporated into the genome of a host

procedure is repeated sequentially until the desired se-

cell. Bone marrow precursor cells are being investigated

quence is obtained. The X chromosome-linked disor-

for this purpose because they presumably will resettle in

ders are particularly amenable to this approach, since

the marrow and replicate there. The introduced gene

only a single allele is expressed. Hence, 20% of the de-

would begin to direct the expression of its protein prod-

fined RFLPs are on the X chromosome, and a reason-

uct, and this would correct the deficiency in the host

ably complete linkage map of this chromosome exists.

cell.

The gene for the X-linked disorder, Duchenne-type

K. TRANSGENIC ANIMALS

muscular dystrophy, was found using RFLPs. Likewise,

the defect in Huntington’s disease was localized to the

The somatic cell gene replacement described above

terminal region of the short arm of chromosome 4, and

would obviously not be passed on to offspring. Other

the defect that causes polycystic kidney disease is linked

strategies to alter germ cell lines have been devised but

to the α-globin locus on chromosome 16.

have been tested only in experimental animals. A certain

Intact DNA

5′

Gene X

3′

Fragments

Initial

probe

Figure 40-11. The technique of chromosome walking. Gene X is to be isolated from a large piece

of DNA. The exact location of this gene is not known, but a probe (*——) directed against a frag-

ment of DNA (shown at the 5′ end in this representation) is available, as is a library containing a se-

ries of overlapping DNA fragments. For the sake of simplicity, only five of these are shown. The initial

probe will hybridize only with clones containing fragment 1, which can then be isolated and used as

a probe to detect fragment 2. This procedure is repeated until fragment 4 hybridizes with fragment

5, which contains the entire sequence of gene X.

412

CHAPTER 40

percentage of genes injected into a fertilized mouse ovum

square centimeters. By coupling such DNA microarrays

will be incorporated into the genome and found in both

with highly sensitive detection of hybridized fluores-

somatic and germ cells. Hundreds of transgenic animals

cently labeled nucleic acid probes derived from mRNA,

have been established, and these are useful for analysis of

investigators can rapidly and accurately generate profiles

tissue-specific effects on gene expression and effects of

of gene expression (eg, specific cellular mRNA content)

overproduction of gene products (eg, those from the

from cell and tissue samples as small as 1 gram or less.

growth hormone gene or oncogenes) and in discovering

Thus entire transcriptome information (the entire col-

genes involved in development—a process that hereto-

lection of cellular mRNAs) for such cell or tissue sources

fore has been difficult to study. The transgenic approach

can readily be obtained in only a few days. Transcrip-

has recently been used to correct a genetic deficiency in

tome information allows one to predict the collection of

mice. Fertilized ova obtained from mice with genetic hy-

proteins that might be expressed in a particular cell, tis-

pogonadism were injected with DNA containing the

sue, or organ in normal and disease states based upon the

coding sequence for the gonadotropin-releasing hormone

mRNAs present in those cells. Complementing this high-

(GnRH) precursor protein. This gene was expressed and

throughput, transcript-profiling method is the recent de-

regulated normally in the hypothalamus of a certain

velopment of high-sensitivity, high-throughput mass

number of the resultant mice, and these animals were in

spectrometry of complex protein samples. Newer mass

all respects normal. Their offspring also showed no evi-

spectrometry methods allow one to identify hundreds to

dence of GnRH deficiency. This is, therefore, evidence

thousands of proteins in proteins extracted from very

of somatic cell expression of the transgene and of its

small numbers of cells (< 1 g). This critical information

maintenance in germ cells.

tells investigators which of the many mRNAs detected in

transcript microarray mapping studies are actually trans-

lated into protein, generally the ultimate dictator of phe-

Targeted Gene Disruption or Knockout

notype. Microarray techniques and mass spectrometric

In transgenic animals, one is adding one or more copies

protein identification experiments both lead to the gen-

of a gene to the genome, and there is no way to control

eration of huge amounts of data. Appropriate data man-

where that gene eventually resides. A complementary—

agement and interpretation of the deluge of information

and much more difficult—approach involves the selec-

forthcoming from such studies has relied upon statistical

tive removal of a gene from the genome. Gene knock-

methods; and this new technology, coupled with the

out animals

(usually mice) are made by creating a

flood of DNA sequence information, has led to the de-

mutation that totally disrupts the function of a gene.

velopment of the field of bioinformatics, a new disci-

This is then used to replace one of the two genes in an

pline whose goal is to help manage, analyze, and inte-

embryonic stem cell that can be used to create a het-

grate this flood of biologically important information.

erozygous transgenic animal. The mating of two such

Future work at the intersection of bioinformatics and

animals will, by mendelian genetics, result in a ho-

transcript-protein profiling will revolutionize our under-

mozygous mutation in 25% of offspring. Several hun-

standing of biology and medicine.

dred strains of mice with knockouts of specific genes

have been developed.

SUMMARY

RNA Transcript & Protein Profiling

• A variety of very sensitive techniques can now be ap-

plied to the isolation and characterization of genes

The “-omic” revolution of the last several years has cul-

and to the quantitation of gene products.

minated in the determination of the nucleotide se-

• In DNA cloning, a particular segment of DNA is re-

quences of entire genomes, including those of budding

moved from its normal environment using one of

and fission yeasts, various bacteria, the fruit fly, the worm

many restriction endonucleases. This is then ligated

Caenorhabditis elegans, the mouse and, most notably, hu-

into one of several vectors in which the DNA seg-

mans. Additional genomes are being sequenced at an ac-

ment can be amplified and produced in abundance.

celerating pace. The availability of all of this DNA se-

quence information, coupled with engineering advances,

• The cloned DNA can be used as a probe in one of

has lead to the development of several revolutionary

several types of hybridization reactions to detect

methodologies, most of which are based upon high-den-

other related or adjacent pieces of DNA, or it can be

sity microarray technology. We now have the ability to

used to quantitate gene products such as mRNA.

deposit thousands of specific, known, definable DNA se-

• Manipulation of the DNA to change its structure, so-

quences (more typically now synthetic oligonucleotides)

called genetic engineering, is a key element in cloning

on a glass microscope-style slide in the space of a few

(eg, the construction of chimeric molecules) and can

MOLECULAR GENETICS, RECOMBINANT DNA, & GENOMIC TECHNOLOGY

413

also be used to study the function of a certain frag-

quences of a single strand of DNA or RNA.

ment of DNA and to analyze how genes are regulated.

Hybridization: The specific reassociation of com-

• Chimeric DNA molecules are introduced into cells

plementary strands of nucleic acids (DNA with

to make transfected cells or into the fertilized oocyte

DNA, DNA with RNA, or RNA with RNA).

to make transgenic animals.

Insert: An additional length of base pairs in DNA,

generally introduced by the techniques of recom-

• Techniques involving cloned DNA are used to locate

binant DNA technology.

genes to specific regions of chromosomes, to identify

Intron: The sequence of a gene that is transcribed

the genes responsible for diseases, to study how faulty

but excised before translation.

gene regulation causes disease, to diagnose genetic

Library: A collection of cloned fragments that rep-

diseases, and increasingly to treat genetic diseases.

resents the entire genome. Libraries may be either

genomic DNA (in which both introns and exons

GLOSSARY

are represented) or cDNA (in which only exons

are represented).

ARS: Autonomously replicating sequence; the ori-

Ligation: The enzyme-catalyzed joining in phos-

gin of replication in yeast.

phodiester linkage of two stretches of DNA or

Autoradiography: The detection of radioactive

RNA into one; the respective enzymes are DNA

molecules (eg, DNA, RNA, protein) by visualiza-

and RNA ligases.

tion of their effects on photographic film.

Lines: Long interspersed repeat sequences.

Bacteriophage: A virus that infects a bacterium.

Microsatellite polymorphism: Heterozygosity of a

Blunt-ended DNA: Two strands of a DNA duplex

certain microsatellite repeat in an individual.

having ends that are flush with each other.

Microsatellite repeat sequences: Dispersed or

cDNA: A single-stranded DNA molecule that is

group repeat sequences of 2-5 bp repeated up to

complementary to an mRNA molecule and is syn-

50 times. May occur at 50-100 thousand loca-

thesized from it by the action of reverse transcrip-

tions in the genome.

tase.

Nick translation: A technique for labeling DNA

Chimeric molecule: A molecule (eg, DNA, RNA,

based on the ability of the DNA polymerase from

protein) containing sequences derived from two

E coli to degrade a strand of DNA that has been

different species.

nicked and then to resynthesize the strand; if a ra-

Clone: A large number of organisms, cells or mole-

dioactive nucleoside triphosphate is employed, the

cules that are identical with a single parental or-

rebuilt strand becomes labeled and can be used as

ganism cell or molecule.

a radioactive probe.

Cosmid: A plasmid into which the DNA sequences

Northern blot: A method for transferring RNA

from bacteriophage lambda that are necessary for

from an agarose gel to a nitrocellulose filter, on

the packaging of DNA (cos sites) have been in-

which the RNA can be detected by a suitable

serted; this permits the plasmid DNA to be pack-

probe.

aged in vitro.

Oligonucleotide: A short, defined sequence of nu-

Endonuclease: An enzyme that cleaves internal

cleotides joined together in the typical phosphodi-

bonds in DNA or RNA.

ester linkage.

Excinuclease: The excision nuclease involved in nu-

Ori: The origin of DNA replication.

cleotide exchange repair of DNA.

PAC: A high capacity (70-95 kb) cloning vector

Exon: The sequence of a gene that is represented

based upon the lytic E. coli bacteriophage P1 that

(expressed) as mRNA.

replicates in bacteria as an extrachromosomal ele-

Exonuclease: An enzyme that cleaves nucleotides

ment.

from either the 3′ or 5′ ends of DNA or RNA.

Palindrome: A sequence of duplex DNA that is the

Fingerprinting: The use of RFLPs or repeat se-

same when the two strands are read in opposite di-

quence DNA to establish a unique pattern of

rections.

DNA fragments for an individual.

Plasmid: A small, extrachromosomal, circular mole-

Footprinting: DNA with protein bound is resistant

cule of DNA that replicates independently of the

to digestion by DNase enzymes. When a sequenc-

host DNA.

ing reaction is performed using such DNA, a pro-

Polymerase chain reaction (PCR): An enzymatic

tected area, representing the “footprint” of the

method for the repeated copying (and thus ampli-

bound protein, will be detected.

fication) of the two strands of DNA that make up

Hairpin: A double-helical stretch formed by base

a particular gene sequence.

pairing between neighboring complementary se-

414

CHAPTER 40

Primosome: The mobile complex of helicase and

Spliceosome: The macromolecular complex respon-

primase that is involved in DNA replication.

sible for precursor mRNA splicing. The spliceo-

Probe: A molecule used to detect the presence of a

some consists of at least five small nuclear RNAs

specific fragment of DNA or RNA in, for in-

(snRNA; U1, U2, U4, U5, and U6) and many

stance, a bacterial colony that is formed from a ge-

proteins.

netic library or during analysis by blot transfer

Splicing: The removal of introns from RNA ac-

techniques; common probes are cDNA molecules,

companied by the joining of its exons.

synthetic oligodeoxynucleotides of defined se-

Sticky-ended DNA: Complementary single strands

quence, or antibodies to specific proteins.

of DNA that protrude from opposite ends of a

Proteome: The entire collection of expressed pro-

DNA duplex or from the ends of different duplex

teins in an organism.

molecules (see also Blunt-ended DNA, above).

Pseudogene: An inactive segment of DNA arising

Tandem: Used to describe multiple copies of the

by mutation of a parental active gene.

same sequence (eg, DNA) that lie adjacent to one

Recombinant DNA: The altered DNA that results

another.

from the insertion of a sequence of deoxynu-

Terminal transferase: An enzyme that adds nu-

cleotides not previously present into an existing

cleotides of one type (eg, deoxyadenonucleotidyl

molecule of DNA by enzymatic or chemical

residues) to the 3′ end of DNA strands.

means.

Transcription: Template DNA-directed synthesis

Restriction enzyme: An endodeoxynuclease that

of nucleic acids; typically DNA-directed synthesis

causes cleavage of both strands of DNA at highly

of RNA.

specific sites dictated by the base sequence.

Transcriptome: The entire collection of expressed

Reverse transcription: RNA-directed synthesis of

mRNAs in an organism.

DNA, catalyzed by reverse transcriptase.

Transgenic: Describing the introduction of new

RT-PCR: A method used to quantitate mRNA lev-

DNA into germ cells by its injection into the nu-

els that relies upon a first step of cDNA copying of

cleus of the ovum.

mRNAs prior to PCR amplification and quantita-

Translation: Synthesis of protein using mRNA as

tion.

template.

Signal: The end product observed when a specific

Vector: A plasmid or bacteriophage into which for-

sequence of DNA or RNA is detected by autoradi-

eign DNA can be introduced for the purposes of

ography or some other method. Hybridization

cloning.

with a complementary radioactive polynucleotide

Western blot: A method for transferring protein to

(eg, by Southern or Northern blotting) is com-

a nitrocellulose filter, on which the protein can be

monly used to generate the signal.

detected by a suitable probe (eg, an antibody).

Sines: Short interspersed repeat sequences.

SNP: Single nucleotide polymorphism. Refers to

the fact that single nucleotide genetic variation in

REFERENCES

genome sequence exists at discrete loci throughout

Lewin B: Genes VII. Oxford Univ Press, 1999.

the chromosomes. Measurement of allelic SNP

Martin JB, Gusella JF: Huntington’s disease: pathogenesis and

differences is useful for gene mapping studies.

management. N Engl J Med 1986:315:1267.

snRNA: Small nuclear RNA. This family of RNAs

Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Labora-

is best known for its role in mRNA processing.

tory Manual. Cold Spring Harbor Laboratory Press, 1989.

Southern blot: A method for transferring DNA

Spector DL, Goldman RD, Leinwand LA: Cells: A Laboratory

from an agarose gel to nitrocellulose filter, on

Manual. Cold Spring Harbor Laboratory Press, 1998.

which the DNA can be detected by a suitable

Watson JD et al: Recombinant DNA, 2nd ed. Scientific American

probe (eg, complementary DNA or RNA).

Books. Freeman, 1992.

Southwestern blot: A method for detecting pro-

Weatherall DJ: The New Genetics and Clinical Practice, 3rd ed. Ox-

tein-DNA interactions by applying a labeled DNA

ford Univ Press, 1991.

probe to a transfer membrane that contains a rena-

tured protein.

SECTION V

Biochemistry of Extracellular

& Intracellular Communication

Membranes: Structure & Function

Robert K. Murray, MD, PhD, & Daryl K. Granner, MD

BIOMEDICAL IMPORTANCE

MAINTENANCE OF A NORMAL INTRA-

& EXTRACELLULAR ENVIRONMENT

Membranes are highly viscous, plastic structures.

Plasma membranes form closed compartments around

IS FUNDAMENTAL TO LIFE

cellular protoplasm to separate one cell from another

Life originated in an aqueous environment; enzyme re-

and thus permit cellular individuality. The plasma

actions, cellular and subcellular processes, and so forth

membrane has selective permeabilities and acts as a

have therefore evolved to work in this milieu. Since

barrier, thereby maintaining differences in composition

mammals live in a gaseous environment, how is the

between the inside and outside of the cell. The selective

aqueous state maintained? Membranes accomplish this

permeabilities are provided mainly by channels and

by internalizing and compartmentalizing body water.

pumps for ions and substrates. The plasma membrane

also exchanges material with the extracellular environ-

The Body’s Internal Water

ment by exocytosis and endocytosis, and there are spe-

Is Compartmentalized

cial areas of membrane structure—the gap junctions—

through which adjacent cells exchange material. In

Water makes up about 60% of the lean body mass of

addition, the plasma membrane plays key roles in cell-

the human body and is distributed in two large com-

cell interactions and in transmembrane signaling.

partments.

Membranes also form specialized compartments

within the cell. Such intracellular membranes help

A. INTRACELLULAR FLUID (ICF)

shape many of the morphologically distinguishable

This compartment constitutes two-thirds of total body

structures (organelles), eg, mitochondria, ER, sarcoplas-

water and provides the environment for the cell (1) to

mic reticulum, Golgi complexes, secretory granules,

make, store, and utilize energy;

(2) to repair itself;

lysosomes, and the nuclear membrane. Membranes lo-

(3) to replicate; and (4) to perform special functions.

calize enzymes, function as integral elements in excita-

B. EXTRACELLULAR FLUID (ECF)

tion-response coupling, and provide sites of energy

transduction, such as in photosynthesis and oxidative

This compartment contains about one-third of total

phosphorylation.

body water and is distributed between the plasma and

Changes in membrane structure (eg caused by is-

interstitial compartments. The extracellular fluid is a

chemia) can affect water balance and ion flux and there-

delivery system. It brings to the cells nutrients (eg, glu-

fore every process within the cell. Specific deficiencies

cose, fatty acids, amino acids), oxygen, various ions and

or alterations of certain membrane components lead to

trace minerals, and a variety of regulatory molecules

a variety of diseases (see Table 41-5). In short, normal

(hormones) that coordinate the functions of widely sep-

cellular function depends on normal membranes.

arated cells. Extracellular fluid removes CO₂, waste

415

416

CHAPTER 41

products, and toxic or detoxified materials from the im-

mediate cellular environment.

Myelin

0.23

The Ionic Compositions of Intracellular

Mouse

0.85

& Extracellular Fluids Differ Greatly

liver cells

As illustrated in Table 41-1, the internal environment

Retinal rods

is rich in K⁺ and Mg2+, and phosphate is its major

(bovine)

1.0

anion. Extracellular fluid is characterized by high Na⁺

and Ca2+ content, and Cl⁻ is the major anion. Note

Human

1.1

erythrocyte

also that the concentration of glucose is higher in extra-

cellular fluid than in the cell, whereas the opposite is

true for proteins. Why is there such a difference? It is

Ameba

1.3

thought that the primordial sea in which life originated

was rich in K⁺ and Mg2+. It therefore follows that en-

HeLa cells

1.5

zyme reactions and other biologic processes evolved to

function best in that environment—hence the high

concentration of these ions within cells. Cells were

Mitochondrial

1.1

outer membrane

faced with strong selection pressure as the sea gradually

changed to a composition rich in Na⁺ and Ca2+. Vast

Sarcoplasmic

changes would have been required for evolution of a

2.0

reticulum

completely new set of biochemical and physiologic ma-

chinery; instead, as it happened, cells developed barri-

Mitochondrial

ers—membranes with associated “pumps”—to main-

3.2

inner membrane

tain the internal microenvironment.

MEMBRANES ARE COMPLEX

Ratio of protein to lipid

STRUCTURES COMPOSED OF LIPIDS,

Figure 41-1. Ratio of protein to lipid in different

PROTEINS, & CARBOHYDRATES

membranes. Proteins equal or exceed the quantity of

We shall mainly discuss the membranes present in eu-

lipid in nearly all membranes. The outstanding excep-

karyotic cells, although many of the principles de-

tion is myelin, an electrical insulator found on many

scribed also apply to the membranes of prokaryotes.

nerve fibers.

The various cellular membranes have different compo-

sitions, as reflected in the ratio of protein to lipid (Fig-

ure 41-1). This is not surprising, given their divergent

These sheet-like structures are noncovalent assemblies

functions. Membranes are asymmetric sheet-like en-

that are thermodynamically stable and metabolically ac-

closed structures with distinct inner and outer surfaces.

tive. Numerous proteins are located in membranes,

where they carry out specific functions of the organelle,

the cell, or the organism.

Table 41-1. Comparison of the mean

concentrations of various molecules outside and

The Major Lipids in Mammalian

inside a mammalian cell.

Membranes Are Phospholipids,

Glycosphingolipids, & Cholesterol

Substance

Extracellular Fluid

Intracellular Fluid

A. PHOSPHOLIPIDS

Na⁺

140 mmol/L

10 mmol/L

Of the two major phospholipid classes present in mem-

K+

4 mmol/L

140 mmol/L

branes, phosphoglycerides are the more common and

Ca2+ (free)

2.5 mmol/L

0.1 µmol/L

consist of a glycerol backbone to which are attached

Mg2+

1.5 mmol/L

30 mmol/L

two fatty acids in ester linkage and a phosphorylated al-

CI⁻

100 mmol/L

4 mmol/L

cohol (Figure 41-2). The fatty acid constituents are

−

HCO₃

27 mmol/L

10 mmol/L

usually even-numbered carbon molecules, most com-

PO43−

2 mmol/L

60 mmol/L

monly containing 16 or 18 carbons. They are un-

Glucose

5.5 mmol/L

0-1 mmol/L

branched and can be saturated or unsaturated. The sim-

Protein

2 g/dL

16 g/dL

plest phosphoglyceride is phosphatidic acid, which is

MEMBRANES: STRUCTURE & FUNCTION

417

Fatty acids

Each eukaryotic cell membrane has a somewhat dif-

ferent lipid composition, though phospholipids are the

major class in all.

R₁

C O

¹CH₂

Membrane Lipids Are Amphipathic

C O

O^-

All major lipids in membranes contain both hydropho-

³CH₂

R₃

bic and hydrophilic regions and are therefore termed

“amphipathic.” Membranes themselves are thus am-

phipathic. If the hydrophobic regions were separated

Glycerol

Alcohol

from the rest of the molecule, it would be insoluble in

water but soluble in oil. Conversely, if the hydrophilic

Figure 41-2. A phosphoglyceride showing the fatty

region were separated from the rest of the molecule, it

acids (R₁ and R₂), glycerol, and phosphorylated alcohol

would be insoluble in oil but soluble in water. The am-

components. In phosphatidic acid, R₃ is hydrogen.

phipathic nature of a phospholipid is represented in

Figure 41-3. Thus, the polar head groups of the phos-

pholipids and the hydroxyl group of cholesterol inter-

1,2-diacylglycerol 3-phosphate, a key intermediate in

face with the aqueous environment; a similar situation

the formation of all other phosphoglycerides (Chapter

applies to the sugar moieties of the GSLs (see below).

24). In other phosphoglycerides, the 3-phosphate is es-

Saturated fatty acids have straight tails, whereas

terified to an alcohol such as ethanolamine, choline,

unsaturated fatty acids, which generally exist in the cis

serine, glycerol, or inositol (Chapter 14).

form in membranes, make kinked tails (Figure 41-3).

The second major class of phospholipids is com-

As more kinks are inserted in the tails, the membrane

posed of sphingomyelin, which contains a sphingosine

becomes less tightly packed and therefore more fluid.

backbone rather than glycerol. A fatty acid is attached

Detergents are amphipathic molecules that are impor-

by an amide linkage to the amino group of sphingosine,

tant in biochemistry as well as in the household. The

forming ceramide. The primary hydroxyl group of

molecular structure of a detergent is not unlike that of a

sphingosine is esterified to phosphorylcholine. Sphin-

phospholipid. Certain detergents are widely used to sol-

gomyelin, as the name implies, is prominent in myelin

ubilize membrane proteins as a first step in their purifi-

sheaths.

cation. The hydrophobic end of the detergent binds to

The amounts and fatty acid compositions of the var-

ious phospholipids vary among the different cellular

membranes.

B. GLYCOSPHINGOLIPIDS

Polar head group

The glycosphingolipids

(GSLs) are sugar-containing

lipids built on a backbone of ceramide; they include

galactosyl- and glucosylceramide (cerebrosides) and the

gangliosides. Their structures are described in Chapter

14. They are mainly located in the plasma membranes

of cells.

Apolar, hydrocarbon tails

C. STEROLS

The most common sterol in membranes is cholesterol

(Chapter 14), which resides mainly in the plasma mem-

branes of mammalian cells but can also be found in

U S S

lesser quantities in mitochondria, Golgi complexes, and

Figure 41-3. Diagrammatic representation of a

nuclear membranes. Cholesterol intercalates among the

phospholipid or other membrane lipid. The polar head

phospholipids of the membrane, with its hydroxyl

group is hydrophilic, and the hydrocarbon tails are hy-

group at the aqueous interface and the remainder of the

drophobic or lipophilic. The fatty acids in the tails are

molecule within the leaflet. Its effect on the fluidity of

membranes is discussed subsequently.

saturated (S) or unsaturated (U); the former are usually

All of the above lipids can be separated from one an-

attached to carbon 1 of glycerol and the latter to car-

other by techniques such as column, thin layer, and

bon 2. Note the kink in the tail of the unsaturated fatty

gas-liquid chromatography and their structures estab-

acid (U), which is important in conferring increased

lished by mass spectrometry.

membrane fluidity.

418

CHAPTER 41

Aqueous

hydrophobic regions of the proteins, displacing most of

their bound lipids. The polar end of the detergent is

Hydro-

philic

free, bringing the proteins into solution as detergent-

protein complexes, usually also containing some resid-

ual lipids.

Hydro-

phobic

Membrane Lipids Form Bilayers

The amphipathic character of phospholipids suggests

Hydro-

that the two regions of the molecule have incompatible

philic

solubilities; however, in a solvent such as water, phos-

Aqueous

pholipids organize themselves into a form that thermo-

dynamically serves the solubility requirements of both

Figure 41-5. Diagram of a section of a bilayer mem-

regions. A micelle

(Figure 41-4) is such a structure;

brane formed from phospholipid molecules. The unsat-

the hydrophobic regions are shielded from water, while

urated fatty acid tails are kinked and lead to more spac-

the hydrophilic polar groups are immersed in the aque-

ing between the polar head groups, hence to more

ous environment. However, micelles are usually rela-

room for movement. This in turn results in increased

tively small in size

(eg, approximately 200 nm) and

membrane fluidity. (Slightly modified and reproduced,

thus are limited in their potential to form membranes.

with permission, from Stryer L: Biochemistry, 2nd ed. Free-

As was recognized in 1925 by Gorter and Grendel, a

man, 1981.)

bimolecular layer, or lipid bilayer, can also satisfy the

thermodynamic requirements of amphipathic mole-

cules in an aqueous environment. Bilayers, not mi-

favorable environment, but even these exposed edges

celles, are indeed the key structures in biologic mem-

can be eliminated by folding the sheet back upon itself

branes. A bilayer exists as a sheet in which the

to form an enclosed vesicle with no edges. A bilayer can

hydrophobic regions of the phospholipids are protected

extend over relatively large distances (eg, 1 mm). The

from the aqueous environment, while the hydrophilic

closed bilayer provides one of the most essential proper-

regions are immersed in water (Figure 41-5). Only the

ties of membranes. It is impermeable to most water-

ends or edges of the bilayer sheet are exposed to an un-

soluble molecules, since they would be insoluble in the

hydrophobic core of the bilayer.

Lipid bilayers are formed by self-assembly, driven

by the hydrophobic effect. When lipid molecules

come together in a bilayer, the entropy of the surround-

ing solvent molecules increases.

Two questions arise from consideration of the

above. First, how many biologic materials are lipid-

soluble and can therefore readily enter the cell? Gases

such as oxygen, CO₂, and nitrogen—small molecules

with little interaction with solvents—readily diffuse

through the hydrophobic regions of the membrane.

The permeability coefficients of several ions and of a

number of other molecules in a lipid bilayer are shown

in Figure 41-6. The three electrolytes shown (Na⁺, K⁺,

and Cl⁻) cross the bilayer much more slowly than

water. In general, the permeability coefficients of small

molecules in a lipid bilayer correlate with their solubili-

ties in nonpolar solvents. For instance, steroids more

readily traverse the lipid bilayer compared with elec-

trolytes. The high permeability coefficient of water it-

Figure 41-4. Diagrammatic cross-section of a mi-

self is surprising but is partly explained by its small size

celle. The polar head groups are bathed in water,

and relative lack of charge.

whereas the hydrophobic hydrocarbon tails are sur-

The second question concerns molecules that are

rounded by other hydrocarbons and thereby pro-

not lipid-soluble: How are the transmembrane concen-

tected from water. Micelles are relatively small (com-

tration gradients for non-lipid-soluble molecules main-

pared with lipid bilayers) spherical structures.

tained? The answer is that membranes contain proteins,

MEMBRANES: STRUCTURE & FUNCTION

419

ues for their transfer from the interior of a membrane

K⁺

Tryptophan

Indole

to water. Hydrophobic amino acids have positive val-

Na⁺

Cl^-

Glucose

Urea,

H₂O

glycerol

ues; polar amino acids have negative values. The total

free energy values for transferring successive sequences

of 20 amino acids in the protein are plotted, yielding a

so-called hydropathy plot. Values of over 20 kcal⋅mol−1

10^-14

10^-12

10^-10

10^-8

10^-6

10^-4

10^-2

are consistent with—but do not prove—a transmem-

Permeability coefficient (cm/s)

brane location.

Low

High

Another aspect of the interaction of lipids and pro-

Permeability

teins is that some proteins are anchored to one leaflet or

another of the bilayer by covalent linkages to certain

Figure 41-6. Permeability coefficients of water,

lipids. Palmitate and myristate are fatty acids involved

some ions, and other small molecules in lipid bilayer

in such linkages to specific proteins. A number of other

membranes. Molecules that move rapidly through a

proteins

(see Chapter

47) are linked to glycophos-

given membrane are said to have a high permeability

phatidylinositol (GPI) structures.

coefficient. (Slightly modified and reproduced, with per-

mission, from Stryer L: Biochemistry, 2nd ed. Freeman,

Different Membranes Have Different

1981.)

Protein Compositions

The number of different proteins in a membrane

and proteins are also amphipathic molecules that can be

varies from less than a dozen in the sarcoplasmic reticu-

inserted into the correspondingly amphipathic lipid bi-

lum to over 100 in the plasma membrane. Most mem-

layer. Proteins form channels for the movement of ions

brane proteins can be separated from one another using

and small molecules and serve as transporters for larger

sodium dodecyl sulfate polyacrylamide gel electro-

molecules that otherwise could not pass the bilayer.

phoresis (SDS-PAGE), a technique that has revolution-

These processes are described below.

ized their study. In the absence of SDS, few membrane

proteins would remain soluble during electrophoresis.

Proteins are the major functional molecules of mem-

Membrane Proteins Are Associated

branes and consist of enzymes, pumps and channels,

With the Lipid Bilayer

structural components, antigens (eg, for histocompati-

Membrane phospholipids act as a solvent for mem-

bility), and receptors for various molecules. Because

brane proteins, creating an environment in which the

every membrane possesses a different complement of

latter can function. Of the 20 amino acids contributing

proteins, there is no such thing as a typical membrane

to the primary structure of proteins, the functional

structure. The enzymatic properties of several different

groups attached to the α carbon are strongly hydropho-

membranes are shown in Table 41-2.

bic in six, weakly hydrophobic in a few, and hy-

drophilic in the remainder. As described in Chapter 5,

Membranes Are Dynamic Structures

the α-helical structure of proteins minimizes the hy-

drophilic character of the peptide bonds themselves.

Membranes and their components are dynamic struc-

Thus, proteins can be amphipathic and form an inte-

tures. The lipids and proteins in membranes undergo

gral part of the membrane by having hydrophilic re-

turnover there just as they do in other compartments of

gions protruding at the inside and outside faces of the

the cell. Different lipids have different turnover rates,

membrane but connected by a hydrophobic region tra-

and the turnover rates of individual species of mem-

versing the hydrophobic core of the bilayer. In fact,

brane proteins may vary widely. The membrane itself

those portions of membrane proteins that traverse

can turn over even more rapidly than any of its con-

membranes do contain substantial numbers of hy-

stituents. This is discussed in more detail in the section

drophobic amino acids and almost invariably have ei-

on endocytosis.

ther a high α-helical or β-pleated sheet content. For

many membranes, a stretch of approximately 20 amino

Membranes Are Asymmetric Structures

acids in an α helix will span the bilayer.

It is possible to calculate whether a particular se-

This asymmetry can be partially attributed to the irreg-

quence of amino acids present in a protein is consistent

ular distribution of proteins within the membranes. An

with a transmembrane location. This can be done by

inside-outside asymmetry is also provided by the ex-

consulting a table that lists the hydrophobicities of each

ternal location of the carbohydrates attached to mem-

of the 20 common amino acids and the free energy val-

brane proteins. In addition, specific enzymes are lo-

420

CHAPTER 41

present in the two leaflets, contributing to the asym-

Table 41-2. Enzymatic markers of different

metric distribution of these lipid molecules. In addi-

membranes.¹

tion, phospholipid exchange proteins recognize specific

phospholipids and transfer them from one membrane

Membrane

Enzyme

(eg, the endoplasmic reticulum [ER]) to others (eg, mi-

tochondrial and peroxisomal). There is further asym-

Plasma

5’-Nucleotidase

metry with regard to GSLs and also glycoproteins; the

Adenylyl cyclase

sugar moieties of these molecules all protrude outward

Na⁺-K⁺ ATPase

from the plasma membrane and are absent from its

Endoplasmic reticulum

Glucose-6-phosphatase

inner face.

Golgi apparatus

Cis

GlcNAc transferase I

Membranes Contain Integral

Medial

Golgi mannosidase II

& Peripheral Proteins

Trans

Galactosyl transferase

(Figure 41-7)

TGN

Sialyl transferase

It is useful to classify membrane proteins into two

Inner mitochondrial membrane

ATP synthase

types: integral and peripheral. Most membrane pro-

¹Membranes contain many proteins, some of which have enzy-

teins fall into the integral class, meaning that they inter-

matic activity. Some of these enzymes are located only in certain

act extensively with the phospholipids and require the

membranes and can therefore be used as markers to follow the

use of detergents for their solubilization. Also, they gen-

purification of these membranes.

erally span the bilayer. Integral proteins are usually

TGN, trans golgi network.

globular and are themselves amphipathic. They consist

of two hydrophilic ends separated by an intervening hy-

cated exclusively on the outside or inside of mem-

drophobic region that traverses the hydrophobic core of

branes, as in the mitochondrial and plasma membranes.

the bilayer. As the structures of integral membrane pro-

There are regional asymmetries in membranes.

teins were being elucidated, it became apparent that

Some, such as occur at the villous borders of mucosal

certain ones (eg, transporter molecules, various recep-

cells, are almost macroscopically visible. Others, such as

tors, and G proteins) span the bilayer many times (see

those at gap junctions, tight junctions, and synapses,

Figure 46-5). Integral proteins are also asymmetrically

occupy much smaller regions of the membrane and

distributed across the membrane bilayer. This asym-

generate correspondingly smaller local asymmetries.

metric orientation is conferred at the time of their in-

There is also inside-outside (transverse) asymmetry

sertion in the lipid bilayer. The hydrophilic external re-

of the phospholipids. The choline-containing phos-

gion of an amphipathic protein, which is synthesized

pholipids

(phosphatidylcholine and sphingomyelin)

on polyribosomes, must traverse the hydrophobic core

are located mainly in the outer molecular layer; the

of its target membrane and eventually be found on the

aminophospholipids

(phosphatidylserine and phos-

outside of that membrane. The molecular mechanisms

phatidylethanolamine) are preferentially located in the

involved in insertion of proteins into membranes and

inner leaflet. Obviously, if this asymmetry is to exist at

the topic of membrane assembly are discussed in Chap-

all, there must be limited transverse mobility (flip-flop)

ter 46.

of the membrane phospholipids. In fact, phospholipids

Peripheral proteins do not interact directly with

in synthetic bilayers exhibit an extraordinarily slow

the phospholipids in the bilayer and thus do not require

rate of flip-flop; the half-life of the asymmetry can be

use of detergents for their release. They are weakly

measured in several weeks. However, when certain

bound to the hydrophilic regions of specific integral

membrane proteins such as the erythrocyte protein gly-

proteins and can be released from them by treatment

cophorin are inserted artificially into synthetic bilayers,

with salt solutions of high ionic strength. For example,

the frequency of phospholipid flip-flop may increase as

ankyrin, a peripheral protein, is bound to the integral

much as 100-fold.

protein “band 3” of erythrocyte membrane. Spectrin, a

The mechanisms involved in the establishment of

cytoskeletal structure within the erythrocyte, is in turn

lipid asymmetry are not well understood. The enzymes

bound to ankyrin and thereby plays an important role

involved in the synthesis of phospholipids are located

in maintenance of the biconcave shape of the erythro-

on the cytoplasmic side of microsomal membrane vesi-

cyte. Many hormone receptor molecules are integral

cles. Translocases (flippases) exist that transfer certain

proteins, and the specific polypeptide hormones that

phospholipids (eg, phosphatidylcholine) from the inner

bind to these receptor molecules may therefore be con-

to the outer leaflet. Specific proteins that preferen-

sidered peripheral proteins. Peripheral proteins, such as

tially bind individual phospholipids also appear to be

polypeptide hormones, may help organize the distribu-

MEMBRANES: STRUCTURE & FUNCTION

421

Figure 41-7. The fluid mosaic model of membrane structure. The membrane consists of a bimolecu-

lar lipid layer with proteins inserted in it or bound to either surface. Integral membrane proteins are

firmly embedded in the lipid layers. Some of these proteins completely span the bilayer and are called

transmembrane proteins, while others are embedded in either the outer or inner leaflet of the lipid bi-

layer. Loosely bound to the outer or inner surface of the membrane are the peripheral proteins. Many of

the proteins and lipids have externally exposed oligosaccharide chains. (Reproduced, with permission,

from Junqueira LC, Carneiro J: Basic Histology: Text & Atlas, 10th ed. McGraw-Hill, 2003.)

tion of integral proteins, such as their receptors, within

composition to permit systematic examination of the

the plane of the bilayer (see below).

effects of fatty acid composition on certain membrane

functions (eg, transport).

(2) Purified membrane proteins or enzymes can be

ARTIFICIAL MEMBRANES MODEL

incorporated into these vesicles in order to assess what

MEMBRANE FUNCTION

factors (eg, specific lipids or ancillary proteins) the pro-

teins require to reconstitute their function. Investiga-

Artificial membrane systems can be prepared by appro-

tions of purified proteins, eg, the Ca2+ ATPase of the

priate techniques. These systems generally consist of

sarcoplasmic reticulum, have in certain cases suggested

mixtures of one or more phospholipids of natural or

that only a single protein and a single lipid are required

synthetic origin that can be treated (eg, by using mild

to reconstitute an ion pump.

sonication) to form spherical vesicles in which the lipids

(3) The environment of these systems can be rigidly

form a bilayer. Such vesicles, surrounded by a lipid bi-

controlled and systematically varied (eg, ion concentra-

layer, are termed liposomes.

tions). The systems can also be exposed to known lig-

Some of the advantages and uses of artificial mem-

ands if, for example, the liposomes contain specific re-

brane systems in the study of membrane function can

ceptor proteins.

be briefly explained.

(4) When liposomes are formed, they can be made

(1) The lipid content of the membranes can be var-

to entrap certain compounds inside themselves, eg,

ied, allowing systematic examination of the effects of

drugs and isolated genes. There is interest in using lipo-

varying lipid composition on certain functions. For in-

somes to distribute drugs to certain tissues, and if com-

stance, vesicles can be made that are composed solely of

ponents (eg, antibodies to certain cell surface mole-

phosphatidylcholine or, alternatively, of known mix-

cules) could be incorporated into liposomes so that they

tures of different phospholipids, glycolipids, and cho-

would be targeted to specific tissues or tumors, the

lesterol. The fatty acid moieties of the lipids used can

therapeutic impact would be considerable. DNA en-

also be varied by employing synthetic lipids of known

trapped inside liposomes appears to be less sensitive to

422

CHAPTER 41

attack by nucleases; this approach may prove useful in

high cholesterol:phospholipid ratios, transition temper-

attempts at gene therapy.

atures are altogether indistinguishable.

The fluidity of a membrane significantly affects its

functions. As membrane fluidity increases, so does its

THE FLUID MOSAIC MODEL

permeability to water and other small hydrophilic mol-

OF MEMBRANE STRUCTURE

ecules. The lateral mobility of integral proteins in-

IS WIDELY ACCEPTED

creases as the fluidity of the membrane increases. If the

active site of an integral protein involved in a given

The fluid mosaic model of membrane structure pro-

function is exclusively in its hydrophilic regions, chang-

posed in 1972 by Singer and Nicolson (Figure 41-7) is

ing lipid fluidity will probably have little effect on the

now widely accepted. The model is often likened to ice-

activity of the protein; however, if the protein is in-

bergs (membrane proteins) floating in a sea of predomi-

volved in a transport function in which transport com-

nantly phospholipid molecules. Early evidence for the

ponents span the membrane, lipid phase effects may

model was the finding that certain species-specific inte-

significantly alter the transport rate. The insulin recep-

gral proteins

(detected by fluorescent labeling tech-

tor is an excellent example of altered function with

niques) rapidly and randomly redistributed in the

changes in fluidity. As the concentration of unsaturated

plasma membrane of an interspecies hybrid cell formed

fatty acids in the membrane is increased (by growing

by the artificially induced fusion of two different parent

cultured cells in a medium rich in such molecules), flu-

cells. It has subsequently been demonstrated that phos-

idity increases. This alters the receptor so that it binds

pholipids also undergo rapid redistribution in the plane

more insulin.

of the membrane. This diffusion within the plane of

A state of fluidity and thus of translational mobility

the membrane, termed translational diffusion, can be

in a membrane may be confined to certain regions of

quite rapid for a phospholipid; in fact, within the plane

membranes under certain conditions. For example,

of the membrane, one molecule of phospholipid can

protein-protein interactions may take place within the

move several micrometers per second.

plane of the membrane, such that the integral proteins

The phase changes—and thus the fluidity of mem-

form a rigid matrix—in contrast to the more usual situ-

branes—are largely dependent upon the lipid composi-

ation, where the lipid acts as the matrix. Such regions

tion of the membrane. In a lipid bilayer, the hydropho-

of rigid protein matrix can exist side by side in the same

bic chains of the fatty acids can be highly aligned or

membrane with the usual lipid matrix. Gap junctions

ordered to provide a rather stiff structure. As the tem-

and tight junctions are clear examples of such side-by-

perature increases, the hydrophobic side chains undergo

side coexistence of different matrices.

a transition from the ordered state (more gel-like or

crystalline phase) to a disordered one, taking on a more

Lipid Rafts & Caveolae Are Special

liquid-like or fluid arrangement. The temperature at

Features of Some Membranes

which the structure undergoes the transition from or-

dered to disordered (ie, melts) is called the “transition

While the fluid mosaic model of membrane structure

temperature” (T_m). The longer and more saturated

has stood up well to detailed scrutiny, additional fea-

fatty acid chains interact more strongly with each other

tures of membrane structure and function are con-

via their longer hydrocarbon chains and thus cause

stantly emerging. Two structures of particular current

higher values of T_m—ie, higher temperatures are re-

interest, located in surface membranes, are lipid rafts

quired to increase the fluidity of the bilayer. On the

and caveolae. The former are dynamic areas of the exo-

other hand, unsaturated bonds that exist in the cis con-

plasmic leaflet of the lipid bilayer enriched in choles-

figuration tend to increase the fluidity of a bilayer by de-

terol and sphingolipids; they are involved in signal

creasing the compactness of the side chain packing with-

transduction and possibly other processes. Caveolae

out diminishing hydrophobicity

(Figure

41-3). The

may derive from lipid rafts. Many if not all of them

phospholipids of cellular membranes generally contain

contain the protein caveolin-1, which may be involved

at least one unsaturated fatty acid with at least one cis

in their formation from rafts. Caveolae are observable

double bond.

by electron microscopy as flask-shaped indentations of

Cholesterol modifies the fluidity of membranes. At

the cell membrane. Proteins detected in caveolae in-

temperatures below the T_m, it interferes with the inter-

clude various components of the signal-transduction

action of the hydrocarbon tails of fatty acids and thus

system (eg, the insulin receptor and some G proteins),

increases fluidity. At temperatures above the T_m, it lim-

the folate receptor, and endothelial nitric oxide syn-

its disorder because it is more rigid than the hydrocar-

thase (eNOS). Caveolae and lipid rafts are active areas

bon tails of the fatty acids and cannot move in the

of research, and ideas concerning them and their possi-

membrane to the same extent, thus limiting fluidity. At

ble roles in various diseases are rapidly evolving.

MEMBRANES: STRUCTURE & FUNCTION

423

MEMBRANE SELECTIVITY ALLOWS

inversely proportionate to the number of hydrogen

bonds that must be broken in order for a solute in the

SPECIALIZED FUNCTIONS

external aqueous phase to become incorporated in the

If the plasma membrane is relatively impermeable, how

hydrophobic bilayer. Electrolytes, poorly soluble in

do most molecules enter a cell? How is selectivity of

lipid, do not form hydrogen bonds with water, but they

this movement established? Answers to such questions

do acquire a shell of water from hydration by electrosta-

are important in understanding how cells adjust to a

tic interaction. The size of the shell is directly propor-

constantly changing extracellular environment. Meta-

tionate to the charge density of the electrolyte. Elec-

zoan organisms also must have means of communicat-

trolytes with a large charge density have a larger shell of

ing between adjacent and distant cells, so that complex

hydration and thus a slower diffusion rate. Na⁺, for ex-

biologic processes can be coordinated. These signals

ample, has a higher charge density than K⁺. Hydrated

must arrive at and be transmitted by the membrane, or

Na⁺ is therefore larger than hydrated K⁺; hence, the lat-

they must be generated as a consequence of some inter-

ter tends to move more easily through the membrane.

action with the membrane. Some of the major mecha-

The following factors affect net diffusion of a sub-

nisms used to accomplish these different objectives are

stance: (1) Its concentration gradient across the mem-

listed in Table 41-3.

brane. Solutes move from high to low concentration.

(2) The electrical potential across the membrane.

Passive Mechanisms Move Some Small

Solutes move toward the solution that has the opposite

Molecules Across Membranes

charge. The inside of the cell usually has a negative

charge.

(3) The permeability coefficient of the sub-

Molecules can passively traverse the bilayer down elec-

stance for the membrane. (4) The hydrostatic pressure

trochemical gradients by simple diffusion or by facili-

gradient across the membrane. Increased pressure will

tated diffusion. This spontaneous movement toward

increase the rate and force of the collision between the

equilibrium contrasts with active transport, which re-

molecules and the membrane. (5) Temperature. In-

quires energy because it constitutes movement against

creased temperature will increase particle motion and

an electrochemical gradient. Figure 41-8 provides a

thus increase the frequency of collisions between exter-

schematic representation of these mechanisms.

nal particles and the membrane. In addition, a multi-

As described above, some solutes such as gases can

tude of channels exist in membranes that route the

enter the cell by diffusing down an electrochemical gra-

entry of ions into cells.

dient across the membrane and do not require meta-

bolic energy. The simple passive diffusion of a solute

Ion Channels Are Transmembrane

across the membrane is limited by the thermal agitation

of that specific molecule, by the concentration gradient

Proteins That Allow the Selective

across the membrane, and by the solubility of that

Entry of Various Ions

solute (the permeability coefficient, Figure 41-6) in the

In natural membranes, as opposed to synthetic mem-

hydrophobic core of the membrane bilayer. Solubility is

brane bilayers, there are transmembrane channels, pore-

like structures composed of proteins that constitute se-

lective ion channels. Cation-conductive channels have

Table 41-3. Transfer of material and information

an average diameter of about 5-8 nm and are negatively

across membranes.

charged within the channel. The permeability of a chan-

nel depends upon the size, the extent of hydration, and

Cross-membrane movement of small molecules

the extent of charge density on the ion. Specific chan-

Diffusion (passive and facilitated)

nels for Na⁺, K⁺, Ca2+, and Cl⁻ have been identified; two

Active transport

such channels are illustrated in Figure 41-9. Both are

Cross-membrane movement of large molecules

seen to consist of four subunits. Each subunit consists of

Endocytosis

six α-helical transmembrane domains. The amino and

Exocytosis

carboxyl terminals of both ion channels are located in

Signal transmission across membranes

the cytoplasm, with both extracellular and intracellular

Cell surface receptors

loops being present. The actual pores in the channels

1. Signal transduction (eg, glucagon → cAMP)

through which the ions pass are not shown in the figure.

2. Signal internalization (coupled with endocytosis, eg,

They form the center (diameter about 5-8 nm) of a

the LDL receptor)

structure formed by apposition of the subunits. The

Movement to intracellular receptors (steroid hormones; a

channels are very selective, in most cases permitting the

form of diffusion)

passage of only one type of ion (Na⁺, Ca2+, etc). Many

Intercellular contact and communication

variations on the above structural themes are found, but

424

CHAPTER 41

Transported

molecule

Carrier

Channel

protein

Electrochemical

Lipid

gradient

bilayer

Simple

Facilitated

diffusion

Passive transport

Active transport

Figure 41-8. Many small uncharged molecules pass freely through the lipid

bilayer. Charged molecules, larger uncharged molecules, and some small un-

charged molecules are transferred through channels or pores or by specific carrier

proteins. Passive transport is always down an electrochemical gradient, toward

equilibrium. Active transport is against an electrochemical gradient and requires

an input of energy, whereas passive transport does not. (Redrawn and reproduced,

with permission, from Alberts B et al: Molecular Biology of the Cell. Garland, 1983.)

all ion channels are basically made up of transmembrane

Table 41-4; other aspects of ion channels are discussed

subunits that come together to form a central pore

briefly in Chapter 49.

through which ions pass selectively. The combination of

x-ray crystallography (where possible) and site-directed

Ionophores Are Molecules That Act as

mutagenesis affords a powerful approach to delineating

Membrane Shuttles for Various Ions

the structure-function relationships of ion channels.

The membranes of nerve cells contain well-studied

Certain microbes synthesize small organic molecules,

ion channels that are responsible for the action poten-

ionophores, that function as shuttles for the movement

tials generated across the membrane. The activity of

of ions across membranes. These ionophores contain hy-

some of these channels is controlled by neurotransmit-

drophilic centers that bind specific ions and are sur-

ters; hence, channel activity can be regulated. One ion

rounded by peripheral hydrophobic regions; this arrange-

can regulate the activity of the channel of another ion.

ment allows the molecules to dissolve effectively in the

For example, a decrease of Ca2+ concentration in the

membrane and diffuse transversely therein. Others, like

extracellular fluid increases membrane permeability and

the well-studied polypeptide gramicidin, form channels.

increases the diffusion of Na⁺. This depolarizes the

Microbial toxins such as diphtheria toxin and acti-

membrane and triggers nerve discharge, which may ex-

vated serum complement components can produce large

plain the numbness, tingling, and muscle cramps symp-

pores in cellular membranes and thereby provide macro-

tomatic of a low level of plasma Ca2+.

molecules with direct access to the internal milieu.

Channels are open transiently and thus are “gated.”

Gates can be controlled by opening or closing. In lig-

Aquaporins Are Proteins That Form Water

and-gated channels, a specific molecule binds to a re-

Channels in Certain Membranes

ceptor and opens the channel.Voltage-gated channels

open (or close) in response to changes in membrane po-

In certain cells (eg, red cells, cells of the collecting duc-

tential. Some properties of ion channels are listed in

tules of the kidney), the movement of water by simple

426

CHAPTER 41

Table 41-4. Some properties of ion channels.

• They are composed of transmembrane protein subunits.

Lipid

• Most are highly selective for one ion; a few are nonselec-

bilayer

tive.

• They allow impermeable ions to cross membranes at rates

approaching diffusion limits.

• They can permit ion fluxes of 10⁶-10⁷/s.

• Their activities are regulated.

Uniport

Symport

Antiport

• The two main types are voltage-gated and ligand-gated.

• They are usually highly conserved across species.

Cotransport

• Most cells have a variety of Na⁺, K⁺, Ca2+, and CI⁻ channels.

• Mutations in genes encoding them can cause specific

Figure 41-10. Schematic representation of types of

diseases.¹

transport systems. Transporters can be classified with

• Their activities are affected by certain drugs.

regard to the direction of movement and whether one

¹Some diseases caused by mutations of ion channels are briefly

or more unique molecules are moved. (Redrawn and re-

discussed in Chapter 49.

produced, with permission, from Alberts B et al: Molecular

Biology of the Cell. Garland, 1983.)

diffusion is augmented by movement through water

Mutations in bacteria and mammalian cells (including

channels. These channels are composed of tetrameric

some that result in human disease) have supported

transmembrane proteins named aquaporins. At least

these conclusions. Facilitated diffusion and active trans-

five distinct aquaporins (AP-1 to AP-5) have been iden-

port resemble a substrate-enzyme reaction except

tified. Mutations in the gene encoding AP-2 have been

that no covalent interaction occurs. These points of re-

shown to be the cause of one type of nephrogenic dia-

semblance are as follows: (1) There is a specific binding

betes insipidus.

site for the solute. (2) The carrier is saturable, so it has a

maximum rate of transport

(V_max; Figure

41-11).

(3) There is a binding constant (K_m) for the solute, and

PLASMA MEMBRANES ARE INVOLVED

IN FACILITATED DIFFUSION, ACTIVE

TRANSPORT, & OTHER PROCESSES

Passive

Transport systems can be described in a functional

diffusion

V_max

sense according to the number of molecules moved and

the direction of movement (Figure 41-10) or according

to whether movement is toward or away from equilib-

rium. A uniport system moves one type of molecule

Carrier-mediated

diffusion

bidirectionally. In cotransport systems, the transfer of

one solute depends upon the stoichiometric simultane-

ous or sequential transfer of another solute. A symport

moves these solutes in the same direction. Examples are

the proton-sugar transporter in bacteria and the Na+ -

sugar transporters (for glucose and certain other sugars)

and Na⁺-amino acid transporters in mammalian cells.

K_m

Antiport systems move two molecules in opposite di-

Solute concentration

rections (eg, Na⁺ in and Ca2+ out).

Molecules that cannot pass freely through the lipid

Figure 41-11. A comparison of the kinetics of car-

bilayer membrane by themselves do so in association

rier-mediated (facilitated) diffusion with passive diffu-

with carrier proteins. This involves two processes—

sion. The rate of movement in the latter is directly pro-

facilitated diffusion and active transport—and highly

portionate to solute concentration, whereas the

specific transport systems.

process is saturable when carriers are involved. The

Facilitated diffusion and active transport share many

concentration at half-maximal velocity is equal to the

features. Both appear to involve carrier proteins, and

binding constant (K_m) of the carrier for the solute. (V_max,

both show specificity for ions, sugars, and amino acids.

maximal rate.)

MEMBRANES: STRUCTURE & FUNCTION

427

so the whole system has a K_m (Figure 41-11). (4) Struc-

termined by the following factors: (1) The concentra-

turally similar competitive inhibitors block transport.

tion gradient across the membrane. (2) The amount of

Major differences are the following: (1) Facilitated

carrier available (this is a key control step). (3) The ra-

diffusion can operate bidirectionally, whereas active

pidity of the solute-carrier interaction. (4) The rapidity

transport is usually unidirectional. (2) Active transport

of the conformational change for both the loaded and

always occurs against an electrical or chemical gradient,

the unloaded carrier.

and so it requires energy.

Hormones regulate facilitated diffusion by changing

the number of transporters available. Insulin increases

Facilitated Diffusion

glucose transport in fat and muscle by recruiting trans-

porters from an intracellular reservoir. Insulin also en-

Some specific solutes diffuse down electrochemical gra-

hances amino acid transport in liver and other tissues.

dients across membranes more rapidly than might be

One of the coordinated actions of glucocorticoid hor-

expected from their size, charge, or partition coeffi-

mones is to enhance transport of amino acids into liver,

cients. This facilitated diffusion exhibits properties

where the amino acids then serve as a substrate for glu-

distinct from those of simple diffusion. The rate of fa-

coneogenesis. Growth hormone increases amino acid

cilitated diffusion, a uniport system, can be saturated;

transport in all cells, and estrogens do this in the uterus.

ie, the number of sites involved in diffusion of the spe-

There are at least five different carrier systems for

cific solutes appears finite. Many facilitated diffusion

amino acids in animal cells. Each is specific for a group

systems are stereospecific but, like simple diffusion, re-

of closely related amino acids, and most operate as Na⁺-

quire no metabolic energy.

symport systems (Figure 41-10).

As described earlier, the inside-outside asymmetry of

membrane proteins is stable, and mobility of proteins

Active Transport

across (rather than in) the membrane is rare; therefore,

transverse mobility of specific carrier proteins is not

The process of active transport differs from diffusion in

likely to account for facilitated diffusion processes ex-

that molecules are transported away from thermody-

cept in a few unusual cases.

namic equilibrium; hence, energy is required. This en-

A “Ping-Pong” mechanism (Figure 41-12) ex-

ergy can come from the hydrolysis of ATP, from elec-

plains facilitated diffusion. In this model, the carrier

tron movement, or from light. The maintenance of

protein exists in two principal conformations. In the

electrochemical gradients in biologic systems is so im-

“pong” state, it is exposed to high concentrations of

portant that it consumes perhaps 30-40% of the total

solute, and molecules of the solute bind to specific sites

energy expenditure in a cell.

on the carrier protein. Transport occurs when a confor-

In general, cells maintain a low intracellular Na⁺

mational change exposes the carrier to a lower concen-

concentration and a high intracellular K⁺ concentration

tration of solute (“ping” state). This process is com-

(Table 41-1), along with a net negative electrical po-

pletely reversible, and net flux across the membrane

tential inside. The pump that maintains these gradients

depends upon the concentration gradient. The rate at

is an ATPase that is activated by Na⁺ and K⁺ (Na⁺-K⁺

which solutes enter a cell by facilitated diffusion is de-

ATPase; see Figure 41-13). The ATPase is an integral

Pong

Ping

Figure 41-12. The “Ping-Pong” model of facilitated diffusion. A protein carrier (gray structure) in the lipid bi-

layer associates with a solute in high concentration on one side of the membrane. A conformational change en-

sues (“pong” to “ping”), and the solute is discharged on the side favoring the new equilibrium. The empty carrier

then reverts to the original conformation (“ping” to “pong”) to complete the cycle.

428

CHAPTER 41

only where the membrane is free of the insulation. The

INSIDE

OUTSIDE

myelin membrane is composed of phospholipids, cho-

Membrane

lesterol, proteins, and GSLs. Relatively few proteins are

3 Na⁺

found in the myelin membrane; those present appear to

ATP

3 Na⁺

hold together multiple membrane bilayers to form the

hydrophobic insulating structure that is impermeable

to ions and water. Certain diseases, eg, multiple sclero-

Mg2+

sis and the Guillain-Barré syndrome, are characterized

2 K

by demyelination and impaired nerve conduction.

ADP

P_i

2 K⁺

Glucose Transport Involves

Several Mechanisms

Figure 41-13. Stoichiometry of the Na⁺-K⁺ ATPase

A discussion of the transport of glucose summarizes

pump. This pump moves three Na⁺ ions from inside the

many of the points made in this chapter. Glucose must

cell to the outside and brings two K⁺ ions from the out-

enter cells as the first step in energy utilization. In

side to the inside for every molecule of ATP hydrolyzed

adipocytes and muscle, glucose enters by a specific

to ADP by the membrane-associated ATPase. Ouabain

transport system that is enhanced by insulin. Changes

and other cardiac glycosides inhibit this pump by act-

in transport are primarily due to alterations of V_max

ing on the extracellular surface of the membrane.

(presumably from more or fewer active transporters),

(Courtesy of R Post.)

but changes in K_m may also be involved. Glucose trans-

port involves different aspects of the principles of trans-

port discussed above. Glucose and Na⁺ bind to different

membrane protein and requires phospholipids for ac-

sites on the glucose transporter. Na⁺ moves into the cell

tivity. The ATPase has catalytic centers for both ATP

down its electrochemical gradient and “drags” glucose

and Na⁺ on the cytoplasmic side of the membrane, but

with it (Figure 41-14). Therefore, the greater the Na⁺

the K⁺ binding site is located on the extracellular side of

gradient, the more glucose enters; and if Na⁺ in extra-

the membrane. Ouabain or digitalis inhibits this ATP-

cellular fluid is low, glucose transport stops. To main-

ase by binding to the extracellular domain. Inhibition

tain a steep Na⁺ gradient, this Na⁺-glucose symport is

of the ATPase by ouabain can be antagonized by extra-

dependent on gradients generated by an Na⁺-K⁺ pump

cellular K⁺.

that maintains a low intracellular Na⁺ concentration.

Similar mechanisms are used to transport other sugars

as well as amino acids.

Nerve Impulses Are Transmitted

The transcellular movement of sugars involves one

Up & Down Membranes

additional component: a uniport that allows the glucose

The membrane forming the surface of neuronal cells

accumulated within the cell to move across a different

maintains an asymmetry of inside-outside voltage (elec-

surface toward a new equilibrium; this occurs in intesti-

trical potential) and is electrically excitable. When ap-

nal and renal cells, for example.

propriately stimulated by a chemical signal mediated by

a specific synaptic membrane receptor (see discussion of

Cells Transport Certain Macromolecules

the transmission of biochemical signals, below), gates in

Across the Plasma Membrane

the membrane are opened to allow the rapid influx of

Na⁺ or Ca2+ (with or without the efflux of K⁺), so that

The process by which cells take up large molecules is

the voltage difference rapidly collapses and that seg-

called

“endocytosis.” Some of these molecules (eg,

ment of the membrane is depolarized. However, as a re-

polysaccharides, proteins, and polynucleotides), when

sult of the action of the ion pumps in the membrane,

hydrolyzed inside the cell, yield nutrients. Endocytosis

the gradient is quickly restored.

provides a mechanism for regulating the content of cer-

When large areas of the membrane are depolarized

tain membrane components, hormone receptors being

in this manner, the electrochemical disturbance propa-

a case in point. Endocytosis can be used to learn more

gates in wave-like form down the membrane, generat-

about how cells function. DNA from one cell type can

ing a nerve impulse. Myelin sheets, formed by

be used to transfect a different cell and alter the latter’s

Schwann cells, wrap around nerve fibers and provide an

function or phenotype. A specific gene is often em-

electrical insulator that surrounds most of the nerve and

ployed in these experiments, and this provides a unique

greatly speeds up the propagation of the wave (signal)

way to study and analyze the regulation of that gene.

by allowing ions to flow in and out of the membrane

DNA transfection depends upon endocytosis; endocy-

MEMBRANES: STRUCTURE & FUNCTION

429

LUMEN

Glucose

Na⁺

(Symport)

CYTOSOL

Glucose

Na⁺

K⁺

Na⁺

K⁺

Glucose

EXTRACELLULAR FLUID

Figure 41-14. The transcellular movement of glu-

cose in an intestinal cell. Glucose follows Na⁺ across the

luminal epithelial membrane. The Na⁺ gradient that

drives this symport is established by Na⁺ -K⁺ exchange,

which occurs at the basal membrane facing the extra-

Figure 41-15. Two types of endocytosis. An endo-

cellular fluid compartment. Glucose at high concentra-

cytotic vesicle (V) forms as a result of invagination of a

tion within the cell moves “downhill” into the extracel-

portion of the plasma membrane. Fluid-phase endocy-

lular fluid by facilitated diffusion (a uniport mechanism).

tosis (A) is random and nondirected. Receptor-medi-

ated endocytosis (B) is selective and occurs in coated

pits (CP) lined with the protein clathrin (the fuzzy mate-

rial). Targeting is provided by receptors (black symbols)

tosis is responsible for the entry of DNA into the cell.

specific for a variety of molecules. This results in the for-

Such experiments commonly use calcium phosphate,

mation of a coated vesicle (CV).

since Ca²⁺ stimulates endocytosis and precipitates

DNA, which makes the DNA a better object for endo-

cytosis. Cells also release macromolecules by exocyto-

sis. Endocytosis and exocytosis both involve vesicle for-

reused in the cytoplasm. Endocytosis requires (1) en-

mation with or from the plasma membrane.

ergy, usually from the hydrolysis of ATP; (2) Ca2+ in

extracellular fluid; and (3) contractile elements in the

A. ENDOCYTOSIS

cell (probably the microfilament system) (Chapter 49).

All eukaryotic cells are continuously ingesting parts of

There are two general types of endocytosis. Phago-

their plasma membranes. Endocytotic vesicles are gen-

cytosis occurs only in specialized cells such as

erated when segments of the plasma membrane invagi-

macrophages and granulocytes. Phagocytosis involves

nate, enclosing a minute volume of extracellular fluid

the ingestion of large particles such as viruses, bacteria,

and its contents. The vesicle then pinches off as the fu-

cells, or debris. Macrophages are extremely active in

sion of plasma membranes seals the neck of the vesicle

this regard and may ingest 25% of their volume per

at the original site of invagination (Figure 41-15). This

hour. In so doing, a macrophage may internalize 3% of

vesicle fuses with other membrane structures and thus

its plasma membrane each minute or the entire mem-

achieves the transport of its contents to other cellular

brane every 30 minutes.

compartments or even back to the cell exterior. Most

Pinocytosis is a property of all cells and leads to the

endocytotic vesicles fuse with primary lysosomes to

cellular uptake of fluid and fluid contents. There are

form secondary lysosomes, which contain hydrolytic

two types. Fluid-phase pinocytosis is a nonselective

enzymes and are therefore specialized organelles for in-

process in which the uptake of a solute by formation of

tracellular disposal. The macromolecular contents are

small vesicles is simply proportionate to its concentra-

digested to yield amino acids, simple sugars, or nu-

tion in the surrounding extracellular fluid. The forma-

cleotides, and they diffuse out of the vesicles to be

tion of these vesicles is an extremely active process. Fi-

430

CHAPTER 41

broblasts, for example, internalize their plasma mem-

terol—released during the degradation of LDL. Disor-

brane at about one-third the rate of macrophages. This

ders of the LDL receptor and its internalization are

process occurs more rapidly than membranes are made.

medically important and are discussed in Chapter 25.

The surface area and volume of a cell do not change

Absorptive pinocytosis of extracellular glycopro-

much, so membranes must be replaced by exocytosis or

teins requires that the glycoproteins carry specific car-

by being recycled as fast as they are removed by endocy-

bohydrate recognition signals. These recognition signals

tosis.

are bound by membrane receptor molecules, which

The other type of pinocytosis, absorptive pinocyto-

play a role analogous to that of the LDL receptor. A

sis, is a receptor-mediated selective process primarily re-

galactosyl receptor on the surface of hepatocytes is in-

sponsible for the uptake of macromolecules for which

strumental in the absorptive pinocytosis of asialoglyco-

there are a finite number of binding sites on the plasma

proteins from the circulation (Chapter 47). Acid hydro-

membrane. These high-affinity receptors permit the se-

lases taken up by absorptive pinocytosis in fibroblasts

lective concentration of ligands from the medium, min-

are recognized by their mannose 6-phosphate moieties.

imize the uptake of fluid or soluble unbound macro-

Interestingly, the mannose

6-phosphate moiety also

molecules, and markedly increase the rate at which

seems to play an important role in the intracellular tar-

specific molecules enter the cell. The vesicles formed

geting of the acid hydrolases to the lysosomes of the

during absorptive pinocytosis are derived from invagi-

cells in which they are synthesized (Chapter 47).

nations (pits) that are coated on the cytoplasmic side

There is a dark side to receptor-mediated endocyto-

with a filamentous material. In many systems, the pro-

sis in that viruses which cause such diseases as hepatitis

tein clathrin is the filamentous material. It has a three-

(affecting liver cells), poliomyelitis

(affecting motor

limbed structure (called a triskelion), with each limb

neurons), and AIDS (affecting T cells) initiate their

being made up of one light and one heavy chain of

damage by this mechanism. Iron toxicity also begins

clathrin. The polymerization of clathrin into a vesicle is

with excessive uptake due to endocytosis.

directed by assembly particles, composed of four

B. EXOCYTOSIS

adapter proteins. These interact with certain amino

acid sequences in the receptors that become cargo, en-

Most cells release macromolecules to the exterior by ex-

suring selectivity of uptake. The lipid PIP₂ also plays an

ocytosis. This process is also involved in membrane re-

important role in vesicle assembly. In addition, the pro-

modeling, when the components synthesized in the

tein dynamin, which both binds and hydrolyzes GTP,

Golgi apparatus are carried in vesicles to the plasma

is necessary for the pinching off of clathrin-coated vesi-

membrane. The signal for exocytosis is often a hor-

cles from the cell surface. Coated pits may constitute as

mone which, when it binds to a cell-surface receptor,

much as 2% of the surface of some cells.

induces a local and transient change in Ca2+ concentra-

As an example, the low-density lipoprotein (LDL)

tion. Ca2+ triggers exocytosis. Figure 41-16 provides a

molecule and its receptor (Chapter 25) are internalized

comparison of the mechanisms of exocytosis and endo-

by means of coated pits containing the LDL receptor.

cytosis.

These endocytotic vesicles containing LDL and its re-

Molecules released by exocytosis fall into three cate-

ceptor fuse to lysosomes in the cell. The receptor is re-

gories: (1) They can attach to the cell surface and be-

leased and recycled back to the cell surface membrane,

come peripheral proteins, eg, antigens. (2) They can be-

but the apoprotein of LDL is degraded and the choles-

come part of the extracellular matrix, eg, collagen and

teryl esters metabolized. Synthesis of the LDL receptor

glycosaminoglycans. (3) They can enter extracellular

is regulated by secondary or tertiary consequences of

fluid and signal other cells. Insulin, parathyroid hor-

pinocytosis, eg, by metabolic products—such as choles-

mone, and the catecholamines are all packaged in gran-

Exocytosis

Endocytosis

Figure 41-16. A comparison of the mechanisms of endocytosis and exocyto-

sis. Exocytosis involves the contact of two inside surface (cytoplasmic side) mono-

layers, whereas endocytosis results from the contact of two outer surface mono-

layers.

MEMBRANES: STRUCTURE & FUNCTION

431

ules and processed within cells, to be released upon ap-

amino acids, sugars, lipids, urate, anions, cations, water,

propriate stimulation.

and vitamins across the plasma membrane. Mutations

in genes encoding proteins in other membranes can

also have harmful consequences. For example, muta-

Some Signals Are Transmitted

tions in genes encoding mitochondrial membrane

Across Membranes

proteins involved in oxidative phosphorylation can

Specific biochemical signals such as neurotransmitters,

cause neurologic and other problems (eg, Leber’s hered-

hormones, and immunoglobulins bind to specific re-

itary optic neuropathy; LHON). Membrane proteins

ceptors (integral proteins) exposed to the outside of

can also be affected by conditions other than muta-

cellular membranes and transmit information through

tions. Formation of autoantibodies to the acetyl-

these membranes to the cytoplasm. This process, called

choline receptor in skeletal muscle causes myasthenia

transmembrane signaling, involves the generation of a

gravis. Ischemia can quickly affect the integrity of vari-

number of signals, including cyclic nucleotides, cal-

ous ion channels in membranes. Abnormalities of

cium, phosphoinositides, and diacylglycerol. It is dis-

membrane constituents other than proteins can also be

cussed in detail in Chapter 43.

harmful. With regard to lipids, excess of cholesterol

(eg, in familial hypercholesterolemia), of lysophospho-

lipid (eg, after bites by certain snakes, whose venom

Information Can Be Communicated

contains phospholipases), or of glycosphingolipids (eg,

by Intercellular Contact

in a sphingolipidosis) can all affect membrane function.

There are many areas of intercellular contact in a meta-

zoan organism. This necessitates contact between the

Cystic Fibrosis Is Due to Mutations in the

plasma membranes of the individual cells. Cells have

Gene Encoding a Chloride Channel

developed specialized regions on their membranes for

intercellular communication in close proximity. Gap

Cystic fibrosis (CF) is a recessive genetic disorder preva-

junctions mediate and regulate the passage of ions and

lent among whites in North America and certain parts

small molecules (up to 1000-2000 MW) through a

of northern Europe. It is characterized by chronic bac-

narrow hydrophilic core connecting the cytosol of adja-

terial infections of the airways and sinuses, fat maldiges-

cent cells. These structures are primarily composed of

tion due to pancreatic exocrine insufficiency, infertility

the protein connexin, which contains four membrane-

in males due to abnormal development of the vas defer-

spanning α helices. About a dozen genes encoding dif-

ens, and elevated levels of chloride in sweat

ferent connexins have been cloned. An assembly of 12

mmol/L).

connexin molecules forms a structure

(a connexon)

After a Herculean landmark endeavor, the gene for

with a central channel that forms bridges between adja-

CF was identified in 1989 on chromosome 7. It was

cent cells. Ions and small molecules pass from the cy-

found to encode a protein of 1480 amino acids, named

tosol of one cell to that of another through the chan-

cystic fibrosis transmembrane regulator

(CFTR), a

nels, which open and close in a regulated fashion.

cyclic AMP-regulated Cl⁻ channel (see Figure 41-17).

An abnormality of membrane Cl⁻ permeability is be-

lieved to result in the increased viscosity of many bodily

MUTATIONS AFFECTING MEMBRANE

secretions, though the precise mechanisms are still

PROTEINS CAUSE DISEASES

under investigation. The commonest mutation (~70%

In view of the fact that membranes are located in so

in certain Caucasian populations) is deletion of three

many organelles and are involved in so many processes,

bases, resulting in loss of residue 508, a phenylalanine

it is not surprising that mutations affecting their pro-

(∆F₅₀₈). However, more than 900 other mutations have

tein constituents should result in many diseases or dis-

been identified. These mutations affect CFTR in at

orders. Proteins in membranes can be classified as re-

least four ways: (1) its amount is reduced; (2) depend-

ceptors, transporters, ion channels, enzymes, and

ing upon the particular mutation, it may be susceptible

structural components. Members of all of these classes

to misfolding and retention within the ER or Golgi ap-

are often glycosylated, so that mutations affecting this

paratus; (3) mutations in the nucleotide-binding do-

process may alter their function. Examples of diseases

mains may affect the ability of the Cl⁻ channel to open,

or disorders due to abnormalities in membrane proteins

an event affected by ATP; (4) the mutations may also

are listed in Table 41-5; these mainly reflect mutations

reduce the rate of ion flow through a channel, generat-

in proteins of the plasma membrane, with one affect-

ing less of a Cl⁻ current.

ing lysosomal function (I-cell disease). Over 30 genetic

The most serious and life-threatening complication

diseases or disorders have been ascribed to mutations

is recurrent pulmonary infections due to overgrowth of

affecting various proteins involved in the transport of

various pathogens in the viscous secretions of the respi-

432

CHAPTER 41

Table 41-5. Some diseases or pathologic states resulting from or attributed to abnormalities

of membranes.¹

Disease

Abnormality

Achondroplasia

Mutations in the gene encoding the fibroblast growth factor receptor 3

(MIM 100800)

Familial hypercholester-

Mutations in the gene encoding the LDL receptor

olemia (MIM 143890)

Cystic fibrosis

Mutations in the gene encoding the CFTR protein, a Cl⁻ transporter

(MIM 219700)

Congenital long QT syn-

Mutations in genes encoding ion channels in the heart

drome (MIM 192500)

Wilson disease

Mutations in the gene encoding a copper-dependent ATPase

(MIM 277900)

I-cell disease

Mutations in the gene encoding GIcNAc phosphotransferase, resulting in absence of the Man 6-P

(MIM 252500)

signal for lysosomal localization of certain hydrolases

Hereditary spherocytosis

Mutations in the genes encoding spectrin or other structural proteins in the red cell membrane

(MIM 182900)

Metastasis

Abnormalities in the oligosaccharide chains of membrane glycoproteins and glycolipids are thought

to be of importance

Paroxysmal nocturnal

Mutation resulting in deficient attachment of the GPI anchor to certain proteins of the red cell

hemoglobinuria

membrane

(MIM 311770)

¹The disorders listed are discussed further in other chapters. The table lists examples of mutations affecting receptors, a transporter, an

ion channel, an enzyme, and a structural protein. Examples of altered or defective glycosylation of glycoproteins are also presented. Most

of the conditions listed affect the plasma membrane.

ratory tract. Poor nutrition as a result of pancreatic in-

sufficiency worsens the situation. The treatment of CF

thus requires a comprehensive effort to maintain nutri-

tional status, to prevent and combat pulmonary infec-

tions, and to maintain physical and psychologic health.

Advances in molecular genetics mean that mutation

Amino

analysis can be performed for prenatal diagnosis and for

terminal

NBF1

carrier testing in families in which one child already has

R domain

NBF2

the condition. Efforts are in progress to use gene ther-

apy to restore the activity of CFTR. An aerosolized

Carboxyl

preparation of human DNase that digests the DNA of

terminal

microorganisms in the respiratory tract has proved

helpful in therapy.

Figure 41-17. Diagram of the structure of the CFTR

protein (not to scale). The protein contains twelve

SUMMARY

transmembrane segments (probably helical), two nu-

cleotide-binding folds or domains (NBF1 and NBF2),

• Membranes are complex structures composed of

and one regulatory (R) domain. NBF1 and NBF2 proba-

lipids, carbohydrates, and proteins.

bly bind ATP and couple its hydrolysis to transport of

• The basic structure of all membranes is the lipid bi-

Cl⁻. Phe 508, the major locus of mutations in cystic fi-

layer. This bilayer is formed by two sheets of phos-

brosis, is located in NBF1.

pholipids in which the hydrophilic polar head groups

MEMBRANES: STRUCTURE & FUNCTION

433

are directed away from each other and are exposed to

• Receptors may be integral components of mem-

the aqueous environment on the outer and inner sur-

branes (particularly the plasma membrane). The in-

faces of the membrane. The hydrophobic nonpolar

teraction of a ligand with its receptor may not in-

tails of these molecules are oriented toward each

volve the movement of either into the cell, but the

other, in the direction of the center of the mem-

interaction results in the generation of a signal that

brane.

influences intracellular processes

(transmembrane

•

Membrane proteins are classified as integral if they

signaling).

are firmly embedded in the bilayer and as peripheral

• Mutations that affect the structure of membrane pro-

if they are loosely attached to the outer or inner sur-

teins (receptors, transporters, ion channels, enzymes,

face.

and structural proteins) may cause diseases; examples

•

The 20 or so different membranes in a mammalian

include cystic fibrosis and familial hypercholes-

cell have intrinsic functions (eg, enzymatic activity),

terolemia.

and they define compartments, or specialized envi-

ronments, within the cell that have specific functions

(eg, lysosomes).

REFERENCES

•

Certain molecules freely diffuse across membranes,

but the movement of others is restricted because of

Doyle DA et al: The structure of the potassium channel: molecular

basis of K⁺ conductance and selectivity. Science 1998;280:

size, charge, or solubility.

69.

•

Various passive and active mechanisms are employed

Felix R: Channelopathies: ion channel defects linked to heritable

to maintain gradients of such molecules across differ-

clinical disorders. J Med Genet 2000;37:729.

ent membranes.

Garavito RM, Ferguson-Miller S: Detergents as tools in membrane

•

Certain solutes, eg, glucose, enter cells by facilitated

biochemistry. J Biol Chem 2001;276:32403.

diffusion, along a downhill gradient from high to low

Gillooly DJ, Stenmark H: A lipid oils the endocytosis machine. Sci-

concentration. Specific carrier molecules, or trans-

ence 2001;291;993.

porters, are involved in such processes.

Knowles MR, Durie PR: What is cystic fibrosis? N Engl J Med

2002;347:439.

•

Ligand- or voltage-gated ion channels are often em-

Longo N: Inherited defects of membrane transport. In: Harrison’s

ployed to move charged molecules (Na⁺, K⁺, Ca2+,

Principles of Internal Medicine, 15th ed. Braunwald E et al

etc) across membranes.

(editors). McGraw-Hill, 2001.

•

Large molecules can enter or leave cells through

Marx J: Caveolae: a once-elusive structure gets some respect. Sci-

mechanisms such as endocytosis or exocytosis. These

ence 2001;294;1862.

processes often require binding of the molecule to a

White SH et al: How membranes shape protein structure. J Biol

receptor, which affords specificity to the process.

Chem 2001:276:32395.

The Diversity of the

Endocrine System

Daryl K. Granner, MD

ACTH Adrenocorticotropic hormone

GH Growth hormone

ANF Atrial natriuretic factor

IGF-I Insulin-like growth factor-I

cAMP Cyclic adenosine monophosphate

LH Luteotropic hormone

CBG Corticosteroid-binding globulin

LPH Lipotropin

CG Chorionic gonadotropin

MIT Monoiodotyrosine

cGMP Cyclic guanosine monophosphate

MSH Melanocyte-stimulating hormone

CLIP Corticotropin-like intermediate lobe

OHSD Hydroxysteroid dehydrogenase

peptide

PNMT Phenylethanolamine-N-methyltransferase

DBH Dopamine β-hydroxylase

POMC Pro-opiomelanocortin

DHEA Dehydroepiandrosterone

SHBG Sex hormone-binding globulin

DHT Dihydrotestosterone

StAR Steroidogenic acute regulatory (protein)

DIT Diiodotyrosine

TBG Thyroxine-binding globulin

DOC Deoxycorticosterone

TEBG Testosterone-estrogen-binding globulin

EGF Epidermal growth factor

TRH Thyrotropin-releasing hormone

FSH Follicle-stimulating hormone

TSH Thyrotropin-stimulating hormone

BIOMEDICAL IMPORTANCE

tive because hormones can act on adjacent cells

(paracrine action) and on the cell in which they were

The survival of multicellular organisms depends on their

synthesized (autocrine action) without entering the sys-

ability to adapt to a constantly changing environment.

temic circulation. A diverse array of hormones—each

Intercellular communication mechanisms are necessary

with distinctive mechanisms of action and properties of

requirements for this adaptation. The nervous system

biosynthesis, storage, secretion, transport, and metabo-

and the endocrine system provide this intercellular, or-

lism—has evolved to provide homeostatic responses.

ganism-wide communication. The nervous system was

This biochemical diversity is the topic of this chapter.

originally viewed as providing a fixed communication

system, whereas the endocrine system supplied hor-

THE TARGET CELL CONCEPT

mones, which are mobile messages. In fact, there is a re-

markable convergence of these regulatory systems. For

There are about 200 types of differentiated cells in hu-

example, neural regulation of the endocrine system is

mans. Only a few produce hormones, but virtually all of

important in the production and secretion of some hor-

the 75 trillion cells in a human are targets of one or

mones; many neurotransmitters resemble hormones in

more of the over 50 known hormones. The concept of

their synthesis, transport, and mechanism of action; and

the target cell is a useful way of looking at hormone ac-

many hormones are synthesized in the nervous system.

tion. It was thought that hormones affected a single cell

The word “hormone” is derived from a Greek term that

type—or only a few kinds of cells—and that a hormone

means to arouse to activity. As classically defined, a hor-

elicited a unique biochemical or physiologic action. We

mone is a substance that is synthesized in one organ and

now know that a given hormone can affect several dif-

transported by the circulatory system to act on another

ferent cell types; that more than one hormone can affect

tissue. However, this original description is too restric-

a given cell type; and that hormones can exert many dif-

434

THE DIVERSITY OF THE ENDOCRINE SYSTEM

435

ferent effects in one cell or in different cells. With the

Table 42-2. Determinants of the target

discovery of specific cell-surface and intracellular hor-

cell response.

mone receptors, the definition of a target has been ex-

panded to include any cell in which the hormone (lig-

The number, relative activity, and state of occupancy of the

and) binds to its receptor, whether or not a biochemical

specific receptors on the plasma membrane or in the

or physiologic response has yet been determined.

cytoplasm or nucleus.

Several factors determine the response of a target cell

The metabolism (activation or inactivation) of the hormone in

to a hormone. These can be thought of in two general

the target cell.

ways: (1) as factors that affect the concentration of the

The presence of other factors within the cell that are neces-

hormone at the target cell (see Table 42-1) and (2) as

sary for the hormone response.

factors that affect the actual response of the target cell

Up- or down-regulation of the receptor consequent to the

to the hormone (see Table 42-2).

interaction with the ligand.

Postreceptor desensitzation of the cell, including down-

regulation of the receptor.

HORMONE RECEPTORS ARE

OF CENTRAL IMPORTANCE

Receptors Discriminate Precisely

logically relevant: (1) binding should be specific, ie, dis-

placeable by agonist or antagonist; (2) binding should

One of the major challenges faced in making the hor-

be saturable; and (3) binding should occur within the

mone-based communication system work is illustrated

concentration range of the expected biologic response.

in Figure 42-1. Hormones are present at very low con-

centrations in the extracellular fluid, generally in the

range of 10^-15 to 10^-9 mol/L. This concentration is

Both Recognition & Coupling

much lower than that of the many structurally similar

Domains Occur on Receptors

molecules (sterols, amino acids, peptides, proteins) and

All receptors have at least two functional domains. A

other molecules that circulate at concentrations in the

recognition domain binds the hormone ligand and a

10^-5 to 10^-3 mol/L range. Target cells, therefore, must

second region generates a signal that couples hormone

distinguish not only between different hormones pre-

recognition to some intracellular function. Coupling

sent in small amounts but also between a given hor-

(signal transduction) occurs in two general ways.

mone and the 10⁶- to 10⁹-fold excess of other similar

Polypeptide and protein hormones and the cate-

molecules. This high degree of discrimination is pro-

cholamines bind to receptors located in the plasma

vided by cell-associated recognition molecules called re-

membrane and thereby generate a signal that regulates

ceptors. Hormones initiate their biologic effects by

various intracellular functions, often by changing the

binding to specific receptors, and since any effective

activity of an enzyme. In contrast, steroid, retinoid, and

control system also must provide a means of stopping a

thyroid hormones interact with intracellular receptors,

response, hormone-induced actions generally terminate

and it is this ligand-receptor complex that directly pro-

when the effector dissociates from the receptor.

vides the signal, generally to specific genes whose rate of

A target cell is defined by its ability to selectively

transcription is thereby affected.

bind a given hormone to its cognate receptor. Several

The domains responsible for hormone recognition

biochemical features of this interaction are important in

and signal generation have been identified in the pro-

order for hormone-receptor interactions to be physio-

tein polypeptide and catecholamine hormone receptors.

Steroid, thyroid, and retinoid hormone receptors have

several functional domains: one site binds the hormone;

Table 42-1. Determinants of the concentration

another binds to specific DNA regions; a third is in-

of a hormone at the target cell.

volved in the interaction with other coregulator pro-

teins that result in the activation (or repression) of gene

The rate of synthesis and secretion of the hormones.

transcription; and a fourth may specify binding to one

The proximity of the target cell to the hormone source (dilu-

or more other proteins that influence the intracellular

tion effect).

trafficking of the receptor.

The dissociation constants of the hormone with specific

The dual functions of binding and coupling ulti-

plasma transport proteins (if any).

mately define a receptor, and it is the coupling of hor-

The conversion of inactive or suboptimally active forms of the

mone binding to signal transduction—so-called recep-

hormone into the fully active form.

tor-effector coupling—that provides the first step in

The rate of clearance from plasma by other tissues or by

amplification of the hormonal response. This dual pur-

digestion, metabolism, or excretion.

pose also distinguishes the target cell receptor from the

436

CHAPTER 42

◆

❁

✴

❖

✪

❁

◗

❉

Figure 42-1. Specificity and selectivity of

✧

◆

hormone receptors. Many different molecules

❙

✪

✴

❙

ECF

circulate in the extracellular fluid (ECF), but

✧

❃

content

❍

only a few are recognized by hormone recep-

✧

❍

◗

tors. Receptors must select these molecules

❉

❖

✪

❁

from among high concentrations of the other

❉

❁

molecules. This simplified drawing shows that

Hormone

Receptor

a cell may have no hormone receptors (1),

have one receptor (2+5+6), have receptors for

Cell types several hormones (3), or have a receptor but

no hormone in the vicinity (4).

plasma carrier proteins that bind hormone but do not

HORMONES CAN BE CLASSIFIED

generate a signal (see Table 42-6).

IN SEVERAL WAYS

Hormones can be classified according to chemical com-

Receptors Are Proteins

position, solubility properties, location of receptors,

Several classes of peptide hormone receptors have been

and the nature of the signal used to mediate hormonal

defined. For example, the insulin receptor is a het-

action within the cell. A classification based on the last

erotetramer (α₂β₂) linked by multiple disulfide bonds

two properties is illustrated in Table 42-3, and general

in which the extracellular α subunit binds insulin and

features of each group are illustrated in Table 42-4.

the membrane-spanning β subunit transduces the sig-

The hormones in group I are lipophilic. After secre-

nal through the tyrosine protein kinase domain located

tion, these hormones associate with plasma transport or

in the cytoplasmic portion of this polypeptide. The re-

carrier proteins, a process that circumvents the problem

ceptors for insulin-like growth factor I (IGF-I) and

of solubility while prolonging the plasma half-life of the

epidermal growth factor (EGF) are generally similar in

hormone. The relative percentages of bound and free

structure to the insulin receptor. The growth hormone

hormone are determined by the binding affinity and

and prolactin receptors also span the plasma mem-

binding capacity of the transport protein. The free hor-

brane of target cells but do not contain intrinsic pro-

mone, which is the biologically active form, readily tra-

tein kinase activity. Ligand binding to these receptors,

verses the lipophilic plasma membrane of all cells and

however, results in the association and activation of a

encounters receptors in either the cytosol or nucleus of

completely different protein kinase pathway, the Jak-

target cells. The ligand-receptor complex is assumed to

Stat pathway. Polypeptide hormone and catecho-

be the intracellular messenger in this group.

lamine receptors, which transduce signals by altering

The second major group consists of water-soluble

the rate of production of cAMP through G-proteins,

hormones that bind to the plasma membrane of the tar-

are characterized by the presence of seven domains that

get cell. Hormones that bind to the surfaces of cells

span the plasma membrane. Protein kinase activation

communicate with intracellular metabolic processes

and the generation of cyclic AMP,

(cAMP, 3′5′-

through intermediary molecules called second messen-

adenylic acid; see Figure 20-5) is a downstream action

gers (the hormone itself is the first messenger), which

of this class of receptor (see Chapter 43 for further de-

are generated as a consequence of the ligand-receptor

tails).

interaction. The second messenger concept arose from

A comparison of several different steroid receptors

an observation that epinephrine binds to the plasma

with thyroid hormone receptors revealed a remarkable

membrane of certain cells and increases intracellular

conservation of the amino acid sequence in certain re-

cAMP. This was followed by a series of experiments in

gions, particularly in the DNA-binding domains. This

which cAMP was found to mediate the effects of many

led to the realization that receptors of the steroid or

hormones. Hormones that clearly employ this mecha-

thyroid type are members of a large superfamily of nu-

nism are shown in group II.A of Table 42-3. To date,

clear receptors. Many related members of this family

only one hormone, atrial natriuretic factor (ANF), uses

have no known ligand at present and thus are called or-

cGMP as its second messenger, but other hormones

phan receptors. The nuclear receptor superfamily plays

will probably be added to group II.B. Several hor-

a critical role in the regulation of gene transcription by

mones, many of which were previously thought to af-

hormones, as described in Chapter 43.

fect cAMP, appear to use ionic calcium

(Ca2+) or

THE DIVERSITY OF THE ENDOCRINE SYSTEM

437

Table 42-3. Classification of hormones by

Table 42-4. General features of hormone classes.

mechanism of action.

Group I

Group II

I. Hormones that bind to intracellular receptors

Types

Steroids, iodothyro-

Polypeptides, proteins,

Androgens

nines, calcitriol,

glycoproteins, cate-

Calcitriol (1,25[OH]₂-D₃)

retinoids

cholamines

Estrogens

Glucocorticoids

Solubility

Lipophilic

Hydrophilic

Mineralocorticoids

Transport

Yes

Progestins

proteins

Retinoic acid

Thyroid hormones (T₃ and T₄)

Plasma half-

Long (hours to

Short (minutes)

II. Hormones that bind to cell surface receptors

life

days)

A. The second messenger is cAMP:

Receptor

Intracellular

Plasma membrane

α₂-Adrenergic catecholamines

β-Adrenergic catecholamines

Mediator

Receptor-hormone

cAMP, cGMP, Ca2+,

Adrenocorticotropic hormone

complex

metabolites of complex

Antidiuretic hormone

phosphoinositols,

Calcitonin

kinase cascades

Chorionic gonadotropin, human

Corticotropin-releasing hormone

Follicle-stimulating hormone

metabolites of complex phosphoinositides (or both) as

Glucagon

the intracellular signal. These are shown in group II.C

Lipotropin

of the table. The intracellular messenger for group II.D

Luteinizing hormone

is a protein kinase-phosphatase cascade. Several of these

Melanocyte-stimulating hormone

Parathyroid hormone

have been identified, and a given hormone may use

Somatostatin

more than one kinase cascade. A few hormones fit into

Thyroid-stimulating hormone

more than one category, and assignments change as

The second messenger is cGMP:

new information is brought forward.

Atrial natriuretic factor

Nitric oxide

The second messenger is calcium or phosphatidyl-

DIVERSITY OF THE ENDOCRINE SYSTEM

inositols (or both):

Acetylcholine (muscarinic)

Hormones Are Synthesized in a

α₁-Adrenergic catecholamines

Variety of Cellular Arrangements

Angiotensin II

Antidiuretic hormone (vasopressin)

Hormones are synthesized in discrete organs designed

Cholecystokinin

solely for this specific purpose, such as the thyroid (tri-

Gastrin

iodothyronine), adrenal (glucocorticoids and mineralo-

Gonadotropin-releasing hormone

corticoids), and the pituitary (TSH, FSH, LH, growth

Oxytocin

hormone, prolactin, ACTH). Some organs are designed

Platelet-derived growth factor

to perform two distinct but closely related functions.

Substance P

For example, the ovaries produce mature oocytes and

Thyrotropin-releasing hormone

the reproductive hormones estradiol and progesterone.

The second messenger is a kinase or phosphatase

The testes produce mature spermatozoa and testos-

cascade:

terone. Hormones are also produced in specialized cells

Chorionic somatomammotropin

within other organs such as the small intestine

Epidermal growth factor

(glucagon-like peptide), thyroid (calcitonin), and kid-

Erythropoietin

ney (angiotensin II). Finally, the synthesis of some hor-

Fibroblast growth factor

mones requires the parenchymal cells of more than one

Growth hormone

organ—eg, the skin, liver, and kidney are required for

Insulin

the production of 1,25(OH)₂-D₃ (calcitriol). Examples

Insulin-like growth factors I and II

of this diversity in the approach to hormone synthesis,

Nerve growth factor

each of which has evolved to fulfill a specific purpose,

Platelet-derived growth factor

Prolactin

are discussed below.

438

CHAPTER 42

Hormones Are Chemically Diverse

MANY HORMONES ARE MADE

FROM CHOLESTEROL

Hormones are synthesized from a wide variety of chem-

ical building blocks. A large series is derived from cho-

Adrenal Steroidogenesis

lesterol. These include the glucocorticoids, mineralo-

corticoids, estrogens, progestins, and

1,25(OH)₂-D₃

The adrenal steroid hormones are synthesized from

(see Figure 42-2). In some cases, a steroid hormone is

cholesterol. Cholesterol is mostly derived from the

the precursor molecule for another hormone. For ex-

plasma, but a small portion is synthesized in situ from

ample, progesterone is a hormone in its own right but

acetyl-CoA via mevalonate and squalene. Much of the

is also a precursor in the formation of glucocorticoids,

cholesterol in the adrenal is esterified and stored in cy-

mineralocorticoids, testosterone, and estrogens. Testos-

toplasmic lipid droplets. Upon stimulation of the

terone is an obligatory intermediate in the biosynthesis

adrenal by ACTH, an esterase is activated, and the free

of estradiol and in the formation of dihydrotestosterone

cholesterol formed is transported into the mitochon-

(DHT). In these examples, described in detail below,

drion, where a cytochrome P450 side chain cleav-

the final product is determined by the cell type and the

age enzyme (P450scc) converts cholesterol to preg-

associated set of enzymes in which the precursor exists.

nenolone. Cleavage of the side chain involves sequential

The amino acid tyrosine is the starting point in the

hydroxylations, first at C₂₂ and then at C₂₀, followed by

synthesis of the catecholamines and of the thyroid hor-

side chain cleavage (removal of the six-carbon fragment

mones tetraiodothyronine (thyroxine; T₄) and triiodo-

isocaproaldehyde) to give the 21-carbon steroid (Figure

thyronine (T₃) (Figure 42-2). T₃ and T₄ are unique in

42-3, top). An ACTH-dependent steroidogenic acute

that they require the addition of iodine (as I⁻) for bioac-

regulatory (StAR) protein is essential for the transport

tivity. Because dietary iodine is very scarce in many

of cholesterol to P450scc in the inner mitochondrial

parts of the world, an intricate mechanism for accumu-

membrane.

lating and retaining I⁻ has evolved.

All mammalian steroid hormones are formed from

Many hormones are polypeptides or glycoproteins.

cholesterol via pregnenolone through a series of reac-

These range in size from thyrotropin-releasing hor-

tions that occur in either the mitochondria or endoplas-

mone (TRH), a tripeptide, to single-chain polypeptides

mic reticulum of the adrenal cell. Hydroxylases that re-

like adrenocorticotropic hormone (ACTH; 39 amino

quire molecular oxygen and NADPH are essential, and

acids), parathyroid hormone (PTH; 84 amino acids),

dehydrogenases, an isomerase, and a lyase reaction are

and growth hormone (GH; 191 amino acids) (Figure

also necessary for certain steps. There is cellular speci-

42-2). Insulin is an AB chain heterodimer of 21 and 30

ficity in adrenal steroidogenesis. For instance,

18-

amino acids, respectively. Follicle-stimulating hormone

hydroxylase and

19-hydroxysteroid dehydrogenase,

(FSH), luteinizing hormone (LH), thyroid-stimulating

which are required for aldosterone synthesis, are found

hormone (TSH), and chorionic gonadotropin (CG) are

only in the zona glomerulosa cells (the outer region of

glycoprotein hormones of αβ heterodimeric structure.

the adrenal cortex), so that the biosynthesis of this min-

The α chain is identical in all of these hormones, and

eralocorticoid is confined to this region. A schematic

distinct β chains impart hormone uniqueness. These

representation of the pathways involved in the synthesis

hormones have a molecular mass in the range of 25-30

of the three major classes of adrenal steroids is pre-

kDa depending on the degree of glycosylation and the

sented in Figure 42-4. The enzymes are shown in the

length of the β chain.

rectangular boxes, and the modifications at each step

are shaded.

Hormones Are Synthesized & Modified

A. MINERALOCORTICOID SYNTHESIS

For Full Activity in a Variety of Ways

Synthesis of aldosterone follows the mineralocorticoid

pathway and occurs in the zona glomerulosa. Preg-

Some hormones are synthesized in final form and se-

nenolone is converted to progesterone by the action of

creted immediately. Included in this class are the hor-

two smooth endoplasmic reticulum enzymes,

3 -

mones derived from cholesterol. Others such as the cat-

hydroxysteroid dehydrogenase (3

-OHSD) and

^5,4-

echolamines are synthesized in final form and stored in

isomerase. Progesterone is hydroxylated at the C₂₁ posi-

the producing cells. Others are synthesized from pre-

tion to form 11-deoxycorticosterone (DOC), which is an

cursor molecules in the producing cell, then are

active (Na⁺-retaining) mineralocorticoid. The next hy-

processed and secreted upon a physiologic cue (insulin).

droxylation, at C₁₁, produces corticosterone, which has

Finally, still others are converted to active forms from

glucocorticoid activity and is a weak mineralocorticoid (it

precursor molecules in the periphery (T₃ and DHT).

has less than 5% of the potency of aldosterone). In some

All of these examples are discussed in more detail

species (eg, rodents), it is the most potent glucocorticoid.

below.

THE DIVERSITY OF THE ENDOCRINE SYSTEM

439

A. CHOLESTEROL DERIVATIVES

CH₂OH

CH₃

C O

CH₂

17ß-Estradiol

Testosterone

Cortisol

Progesterone

1,25(OH)₂-D₃

B. TYROSINE DERIVATIVES

CH₂CH

COOH

NH₂

H H

T³

Norepinephrine

H CH₃

CH₂CH

COOH

NH₂

H H

T⁴

Epinephrine

C. PEPTIDES OF VARIOUS SIZES

Ser

Tyr

Ser

Mert

Phe

Arg

Lys

Conserved region; required for full biologic activity

Val

Glu Hls Pro

(pyro)

NH₂

Pro

Tyr

Val

Lys Val

Pro

Arg

Lys

Asp

Ala

Gly

Variable region; not required for biologic activity

TRH

Glu

Asp

Ser A

la G

lu A

Phe Pro

Leu Glu

Phe

Structure of human ACTH.

D. GLYCOPROTEINS (TSH, FSH, LH)

ACTH

common

subunits

unique

subunits

Figure 42-2. Chemical diversity of hormones. A. Cholesterol derivatives. B. Tyrosine derivatives.

C. Peptides of various sizes D. Glycoproteins (TSH, FSH, LH) with common α subunits and unique β

subunits.

440

CHAPTER 42

Cholesterol side chain cleavage

CH₃

ACTH

C C C C

(cAMP)

P450scc

Cholesterol

Pregnenolone + isocaproaldehyde

Basic steroid hormone structures

CH₂OH

CH₃

17β—Estradiol

Testosterone

Cortisol

Progesterone

Estrane group (C18)

Androstane group (C19)

Pregnane group (C21)

Figure 42-3. Cholesterol side-chain cleavage and basic steroid hormone structures. The basic sterol rings are iden-

tified by the letters A-D. The carbon atoms are numbered 1-21 starting with the A ring. Note that the estrane group

has 18 carbons (C18), etc.

C₂₁ hydroxylation is necessary for both mineralocorticoid

costerone or aldosterone, depending on the cell type).

and glucocorticoid activity, but most steroids with a C₁₇

17α-Hydroxylase is a smooth endoplasmic reticulum

hydroxyl group have more glucocorticoid and less miner-

enzyme that acts upon either progesterone or, more

alocorticoid action. In the zona glomerulosa, which does

commonly, pregnenolone. 17α-Hydroxyprogesterone is

not have the smooth endoplasmic reticulum enzyme

hydroxylated at C₂₁ to form 11-deoxycortisol, which is

17α-hydroxylase, a mitochondrial 18-hydroxylase is pres-

then hydroxylated at C₁₁ to form cortisol, the most po-

ent. The 18-hydroxylase (aldosterone synthase) acts on

tent natural glucocorticoid hormone in humans. 21-Hy-

corticosterone to form 18-hydroxycorticosterone, which

droxylase is a smooth endoplasmic reticulum enzyme,

is changed to aldosterone by conversion of the 18-alcohol

whereas 11β-hydroxylase is a mitochondrial enzyme.

to an aldehyde. This unique distribution of enzymes and

Steroidogenesis thus involves the repeated shuttling of

the special regulation of the zona glomerulosa by K⁺ and

substrates into and out of the mitochondria.

angiotensin II have led some investigators to suggest that,

in addition to the adrenal being two glands, the adrenal

C. ANDROGEN SYNTHESIS

cortex is actually two separate organs.

The major androgen or androgen precursor produced by

B. GLUCOCORTICOID SYNTHESIS

the adrenal cortex is dehydroepiandrosterone (DHEA).

Most 17-hydroxypregnenolone follows the glucocorticoid

Cortisol synthesis requires three hydroxylases located in

pathway, but a small fraction is subjected to oxidative fis-

the fasciculata and reticularis zones of the adrenal cortex

sion and removal of the two-carbon side chain through

that act sequentially on the C₁₇, C₂₁, and C₁₁ positions.

the action of 17,20-lyase. The lyase activity is actually

The first two reactions are rapid, while C₁₁ hydroxyla-

part of the same enzyme (P450c17) that catalyzes 17α-

tion is relatively slow. If the C₁₁ position is hydroxylated

hydroxylation. This is therefore a dual function protein.

first, the action of 17

-hydroxylase is impeded and the

The lyase activity is important in both the adrenals and

mineralocorticoid pathway is followed (forming corti-

442

CHAPTER 42

the gonads and acts exclusively on 17α-hydroxy-contain-

are generally inactive or less active than the parent com-

ing molecules. Adrenal androgen production increases

pound. Metabolism by the second pathway, which is less

markedly if glucocorticoid biosynthesis is impeded by the

efficient, occurs primarily in target tissues and produces

lack of one of the hydroxylases

(adrenogenital syn-

the potent metabolite dihydrotestosterone (DHT).

drome). DHEA is really a prohormone, since the actions

The most significant metabolic product of testos-

of 3β-OHSD and ∆^5,4-isomerase convert the weak andro-

terone is DHT, since in many tissues, including

gen DHEA into the more potent androstenedione.

prostate, external genitalia, and some areas of the skin,

Small amounts of androstenedione are also formed in the

this is the active form of the hormone. The plasma con-

adrenal by the action of the lyase on 17α-hydroxyproges-

tent of DHT in the adult male is about one-tenth that

terone. Reduction of androstenedione at the C₁₇ position

of testosterone, and approximately 400 µg of DHT is

results in the formation of testosterone, the most potent

produced daily as compared with about 5 mg of testos-

adrenal androgen. Small amounts of testosterone are pro-

terone. About 50-100 µg of DHT are secreted by the

duced in the adrenal by this mechanism, but most of this

testes. The rest is produced peripherally from testos-

conversion occurs in the testes.

terone in a reaction catalyzed by the NADPH-depen-

dent 5

-reductase

(Figure

42-6). Testosterone can

thus be considered a prohormone, since it is converted

Testicular Steroidogenesis

into a much more potent compound (dihydrotestos-

Testicular androgens are synthesized in the interstitial

terone) and since most of this conversion occurs outside

tissue by the Leydig cells. The immediate precursor of

the testes. Some estradiol is formed from the peripheral

the gonadal steroids, as for the adrenal steroids, is cho-

aromatization of testosterone, particularly in males.

lesterol. The rate-limiting step, as in the adrenal, is de-

livery of cholesterol to the inner membrane of the mito-

Ovarian Steroidogenesis

chondria by the transport protein StAR. Once in the

proper location, cholesterol is acted upon by the side

The estrogens are a family of hormones synthesized in a

chain cleavage enzyme P450scc. The conversion of cho-

variety of tissues. 17β-Estradiol is the primary estrogen

lesterol to pregnenolone is identical in adrenal, ovary,

of ovarian origin. In some species, estrone, synthesized

and testis. In the latter two tissues, however, the reac-

in numerous tissues, is more abundant. In pregnancy,

tion is promoted by LH rather than ACTH.

relatively more estriol is produced, and this comes from

The conversion of pregnenolone to testosterone re-

the placenta. The general pathway and the subcellular

quires the action of five enzyme activities contained in

localization of the enzymes involved in the early steps

three proteins: (1) 3β-hydroxysteroid dehydrogenase (3β-

of estradiol synthesis are the same as those involved in

OHSD) and ∆^5,4-isomerase; (2) 17α-hydroxylase and

androgen biosynthesis. Features unique to the ovary are

17,20-lyase; and (3) 17β-hydroxysteroid dehydrogenase

illustrated in Figure 42-7.

(17β-OHSD). This sequence, referred to as the proges-

Estrogens are formed by the aromatization of andro-

terone (or

⁴) pathway, is shown on the right side of Fig-

gens in a complex process that involves three hydroxyla-

ure 42-5. Pregnenolone can also be converted to testos-

tion steps, each of which requires O₂ and NADPH. The

terone by the dehydroepiandrosterone (or

⁵) pathway,

aromatase enzyme complex is thought to include a

which is illustrated on the left side of Figure 42-5. The ∆⁵

P450 monooxygenase. Estradiol is formed if the sub-

route appears to be most used in human testes.

strate of this enzyme complex is testosterone, whereas es-

The five enzyme activities are localized in the micro-

trone results from the aromatization of androstenedione.

somal fraction in rat testes, and there is a close func-

The cellular source of the various ovarian steroids has

tional association between the activities of 3β-OHSD

been difficult to unravel, but a transfer of substrates be-

and ∆^5,4-isomerase and between those of a 17α-hydrox-

tween two cell types is involved. Theca cells are the source

ylase and 17,20-lyase. These enzyme pairs, both con-

of androstenedione and testosterone. These are converted

tained in a single protein, are shown in the general reac-

by the aromatase enzyme in granulosa cells to estrone and

tion sequence in Figure 42-5.

estradiol, respectively. Progesterone, a precursor for all

steroid hormones, is produced and secreted by the corpus

luteum as an end-product hormone because these cells do

Dihydrotestosterone Is Formed From

not contain the enzymes necessary to convert proges-

Testosterone in Peripheral Tissues

terone to other steroid hormones (Figure 42-8).

Testosterone is metabolized by two pathways. One in-

Significant amounts of estrogens are produced by

volves oxidation at the 17 position, and the other in-

the peripheral aromatization of androgens. In human

volves reduction of the A ring double bond and the 3-ke-

males, the peripheral aromatization of testosterone to

tone. Metabolism by the first pathway occurs in many

estradiol (E₂) accounts for 80% of the production of

tissues, including liver, and produces 17-ketosteroids that

the latter. In females, adrenal androgens are important

THE DIVERSITY OF THE ENDOCRINE SYSTEM

445

Acetate

most of the precursor for 1,25(OH)₂-D₃ synthesis is

produced in the malpighian layer of the epidermis from

Cholesterol

7-dehydrocholesterol in an ultraviolet light-mediated,

CH₃

nonenzymatic photolysis reaction. The extent of this

conversion is related directly to the intensity of the ex-

C O

posure and inversely to the extent of pigmentation in

the skin. There is an age-related loss of 7-dehydrocho-

lesterol in the epidermis that may be related to the neg-

ative calcium balance associated with old age.

B. LIVER

A specific transport protein called the vitamin D-bind-

Pregnenolone

ing protein binds vitamin D₃ and its metabolites and

moves vitamin D₃ from the skin or intestine to the

CH₃

liver, where it undergoes

25-hydroxylation, the first

obligatory reaction in the production of 1,25(OH)₂-

C O

D₃. 25-Hydroxylation occurs in the endoplasmic retic-

ulum in a reaction that requires magnesium, NADPH,

molecular oxygen, and an uncharacterized cytoplasmic

factor. Two enzymes are involved: an NADPH-depen-

dent cytochrome P450 reductase and a cytochrome

P450. This reaction is not regulated, and it also occurs

with low efficiency in kidney and intestine. The

25(OH)2-D3 enters the circulation, where it is the

Progesterone

major form of vitamin D found in plasma, and is trans-

ported to the kidney by the vitamin D-binding protein.

Figure 42-8.

Biosynthesis of progesterone in the

corpus luteum.

C. KIDNEY

25(OH)2-D3 is a weak agonist and must be modified

by hydroxylation at position C₁ for full biologic activ-

substrates, since as much as 50% of the E₂ produced

ity. This is accomplished in mitochondria of the renal

during pregnancy comes from the aromatization of an-

proximal convoluted tubule by a three-component

drogens. Finally, conversion of androstenedione to

monooxygenase reaction that requires NADPH, Mg2+,

estrone is the major source of estrogens in post-

molecular oxygen, and at least three enzymes: (1) a

menopausal women. Aromatase activity is present in

flavoprotein, renal ferredoxin reductase; (2) an iron sul-

adipose cells and also in liver, skin, and other tissues.

fur protein, renal ferredoxin; and (3) cytochrome P450.

Increased activity of this enzyme may contribute to the

This system produces 1,25(OH)₂-D₃, which is the most

“estrogenization” that characterizes such diseases as cir-

potent naturally occurring metabolite of vitamin D.

rhosis of the liver, hyperthyroidism, aging, and obesity.

CATECHOLAMINES & THYROID

1,25(OH)₂-D₃ (Calcitriol) Is Synthesized

HORMONES ARE MADE FROM TYROSINE

From a Cholesterol Derivative

Catecholamines Are Synthesized in Final

1,25(OH)₂-D₃ is produced by a complex series of enzy-

Form & Stored in Secretion Granules

matic reactions that involve the plasma transport of pre-

cursor molecules to a number of different tissues (Figure

Three amines—dopamine, norepinephrine, and epi-

42-9). One of these precursors is vitamin D—really not

nephrine—are synthesized from tyrosine in the chro-

a vitamin, but this common name persists. The active

maffin cells of the adrenal medulla. The major product

molecule, 1,25(OH)₂-D₃, is transported to other organs

of the adrenal medulla is epinephrine. This compound

where it activates biologic processes in a manner similar

constitutes about 80% of the catecholamines in the

to that employed by the steroid hormones.

medulla, and it is not made in extramedullary tissue. In

contrast, most of the norepinephrine present in organs

A. SKIN

innervated by sympathetic nerves is made in situ (about

Small amounts of the precursor for 1,25(OH)₂-D₃ syn-

80% of the total), and most of the rest is made in other

thesis are present in food (fish liver oil, egg yolk), but

nerve endings and reaches the target sites via the circu-

446

CHAPTER 42

Sunlight

7-Dehydrocholesterol

Previtamin D₃

Vitamin D₃

25-Hydroxylase

SKIN

LIVER

Other

25-Hydroxycholecalciferol (25[OH]-D₃)

metabolites

24-Hydroxylase

1 α-Hydroxylase

24,25(OH)₂-D₃

KIDNEY

1,25(OH)₂-D₃

1,24,25(OH)₃-D₃

CH₂

7-Dehydrocholesterol

Vitamin D₃

1,25(OH)₂-D₃

Figure 42-9. Formation and hydroxylation of vitamin D₃. 25-Hydroxylation takes place in the liver, and the

other hydroxylations occur in the kidneys. 25,26(OH)₂-D₃ and 1,25,26(OH)₃-D₃ are probably formed as well. The

formulas of 7-dehydrocholesterol, vitamin D₃, and 1,25(OH)₂-D₃ are also shown. (Modified and reproduced, with

permission, from Ganong WF: Review of Medical Physiology, 20th ed. McGraw-Hill, 2001.)

lation. Epinephrine and norepinephrine may be pro-

As the rate-limiting enzyme, tyrosine hydroxylase is regu-

duced and stored in different cells in the adrenal

lated in a variety of ways. The most important mecha-

medulla and other chromaffin tissues.

nism involves feedback inhibition by the catecholamines,

The conversion of tyrosine to epinephrine requires

which compete with the enzyme for the pteridine cofac-

four sequential steps: (1) ring hydroxylation; (2) decar-

tor. Catecholamines cannot cross the blood-brain barrier;

boxylation; (3) side chain hydroxylation to form norepi-

hence, in the brain they must be synthesized locally. In

nephrine; and (4) N-methylation to form epinephrine.

certain central nervous system diseases (eg, Parkinson’s

The biosynthetic pathway and the enzymes involved are

disease), there is a local deficiency of dopamine synthesis.

illustrated in Figure 42-10.

L-Dopa, the precursor of dopamine, readily crosses the

blood-brain barrier and so is an important agent in the

A. TYROSINE HYDROXYLASE IS RATE-LIMITING

treatment of Parkinson’s disease.

FOR CATECHOLAMINE BIOSYNTHESIS

B. DOPA DECARBOXYLASE IS PRESENT IN ALL TISSUES

Tyrosine is the immediate precursor of catecholamines,

and tyrosine hydroxylase is the rate-limiting enzyme in

This soluble enzyme requires pyridoxal phosphate for

catecholamine biosynthesis. Tyrosine hydroxylase is

the conversion of L-dopa to 3,4-dihydroxyphenylethyl-

found in both soluble and particle-bound forms only in

amine (dopamine). Compounds that resemble L-dopa,

tissues that synthesize catecholamines; it functions as an

such as α-methyldopa, are competitive inhibitors of

oxidoreductase, with tetrahydropteridine as a cofactor, to

this reaction. α-Methyldopa is effective in treating

convert L-tyrosine to L-dihydroxyphenylalanine (L-dopa).

some kinds of hypertension.

THE DIVERSITY OF THE ENDOCRINE SYSTEM

447

the conversion of dopamine to norepinephrine occurs

in this organelle.

D. PHENYLETHANOLAMINE-N-METHYLTRANSFERASE

C C

NH₂

(PNMT) CATALYZES THE PRODUCTION

H H

OF EPINEPHRINE

Tyrosine

PNMT catalyzes the N-methylation of norepinephrine

TYROSINE

HYDROXYLASE

to form epinephrine in the epinephrine-forming cells

of the adrenal medulla. Since PNMT is soluble, it is as-

sumed that norepinephrine-to-epinephrine conversion

occurs in the cytoplasm. The synthesis of PNMT is in-

duced by glucocorticoid hormones that reach the

C C

NH₂

medulla via the intra-adrenal portal system. This special

H H

system provides for a 100-fold steroid concentration

Dopa

gradient over systemic arterial blood, and this high

DOPA

intra-adrenal concentration appears to be necessary for

DECARBOXYLASE

the induction of PNMT.

T₃ & T₄ Illustrate the Diversity

in Hormone Synthesis

C C

NH₂

The formation of triiodothyronine (T₃) and tetra-

H H

iodothyronine (thyroxine; T₄) (see Figure 42-2) illus-

DOPAMINE

trates many of the principles of diversity discussed in

DOPAMINE

this chapter. These hormones require a rare element

β-HYDROXYLASE

(iodine) for bioactivity; they are synthesized as part of a

very large precursor molecule (thyroglobulin); they are

stored in an intracellular reservoir (colloid); and there is

peripheral conversion of T₄ to T₃, which is a much

more active hormone.

C C

NH₂

The thyroid hormones T₃ and T₄ are unique in that

iodine (as iodide) is an essential component of both. In

H H

NOREPINEPHRINE

most parts of the world, iodine is a scarce component of

soil, and for that reason there is little in food. A com-

PNMT

plex mechanism has evolved to acquire and retain this

crucial element and to convert it into a form suitable

for incorporation into organic compounds. At the same

time, the thyroid must synthesize thyronine from tyro-

CH₃

sine, and this synthesis takes place in thyroglobulin

(Figure 42-11).

C C

Thyroglobulin is the precursor of T₄ and T₃. It is a

H H

large iodinated, glycosylated protein with a molecular

EPINEPHRINE

mass of 660 kDa. Carbohydrate accounts for 8-10% of

the weight of thyroglobulin and iodide for about

Figure 42-10. Biosynthesis of catecholamines.

0.2-1%, depending upon the iodine content in the

(PNMT, phenylethanolamine-N-methyltransferase.)

diet. Thyroglobulin is composed of two large subunits.

It contains 115 tyrosine residues, each of which is a po-

tential site of iodination. About

70% of the iodide

in thyroglobulin exists in the inactive precursors,

C. DOPAMINE

-HYDROXYLASE (DBH) CATALYZES

monoiodotyrosine (MIT) and diiodotyrosine (DIT),

THE CONVERSION OF DOPAMINE TO NOREPINEPHRINE

while 30% is in the iodothyronyl residues, T₄ and T₃.

DBH is a monooxygenase and uses ascorbate as an elec-

When iodine supplies are sufficient, the T₄:T₃ ratio is

tron donor, copper at the active site, and fumarate as

about 7:1. In iodine deficiency, this ratio decreases, as

modulator. DBH is in the particulate fraction of the

does the DIT:MIT ratio. Thyroglobulin, a large mole-

medullary cells, probably in the secretion granule; thus,

cule of about 5000 amino acids, provides the confor-

448

CHAPTER 42

FOLLICULAR SPACE WITH COLLOID

MIT

T₃

MIT

DIT

Oxidation

Iodination*

Coupling*

DIT

T₄

I ⁺

Tgb

PEROXIDASE

MIT

DIT

H₂O₂

DIT

T₄

Phagocytosis

O₂

and

Tgb

pinocytosis

NADPH

NADP⁺

H⁺

Lysosomes

THYROID CELL

Tgb

Secondary

Tgb

lysosome

Tyrosine

Hydrolysis

Deiodination*

MIT

DEIODINASE

DIT

Concentration

T₃, T₄

Na⁺-K⁺ATPase

Release

Trans-

porter

EXTRACELLULAR SPACE

T₃, T₄

Figure 42-11. Model of iodide metabolism in the thyroid follicle. A follicular cell is shown facing the follicular

lumen (top) and the extracellular space (at bottom). Iodide enters the thyroid primarily through a transporter

(bottom left). Thyroid hormone synthesis occurs in the follicular space through a series of reactions, many of

which are peroxidase-mediated. Thyroid hormones, stored in the colloid in the follicular space, are released from

thyroglobulin by hydrolysis inside the thyroid cell. (Tgb, thyroglobulin; MIT, monoiodotyrosine; DIT, diiodotyro-

sine; T₃, triiodothyronine; T₄, tetraiodothyronine.) Asterisks indicate steps or processes that are inherited enzyme

deficiencies which cause congenital goiter and often result in hypothyroidism.

THE DIVERSITY OF THE ENDOCRINE SYSTEM

449

mation required for tyrosyl coupling and iodide organi-

mones remain as integral parts of thyroglobulin until

fication necessary in the formation of the diaminoacid

the latter is degraded, as described above.

thyroid hormones. It is synthesized in the basal portion

A deiodinase removes I⁻ from the inactive mono-

of the cell and moves to the lumen, where it is a storage

and diiodothyronine molecules in the thyroid. This

form of T₃ and T₄ in the colloid; several weeks’ supply

mechanism provides a substantial amount of the I⁻ used

of these hormones exist in the normal thyroid. Within

in T₃ and T₄ biosynthesis. A peripheral deiodinase in

minutes after stimulation of the thyroid by TSH, col-

target tissues such as pituitary, kidney, and liver selec-

loid reenters the cell and there is a marked increase of

tively removes I⁻ from the 5′ position of T₄ to make T₃

phagolysosome activity. Various acid proteases and

(see Figure 42-2), which is a much more active mole-

peptidases hydrolyze the thyroglobulin into its con-

cule. In this sense, T₄ can be thought of as a prohor-

stituent amino acids, including T₄ and T₃, which are

mone, though it does have some intrinsic activity.

discharged from the basal portion of the cell (see Figure

42-11). Thyroglobulin is thus a very large prohor-

SEVERAL HORMONES ARE MADE FROM

mone.

LARGER PEPTIDE PRECURSORS

Formation of the critical disulfide bridges in insulin re-

Iodide Metabolism Involves

quires that this hormone be first synthesized as part of a

Several Discrete Steps

larger precursor molecule, proinsulin. This is conceptu-

The thyroid is able to concentrate I⁻ against a strong

ally similar to the example of the thyroid hormones,

electrochemical gradient. This is an energy-dependent

which can only be formed in the context of a much

process and is linked to the Na⁺-K⁺ ATPase-dependent

larger molecule. Several other hormones are synthesized

thyroidal I⁻ transporter. The ratio of iodide in thyroid

as parts of large precursor molecules, not because of

to iodide in serum (T:S ratio) is a reflection of the ac-

some special structural requirement but rather as a

tivity of this transporter. This activity is primarily con-

mechanism for controlling the available amount of the

trolled by TSH and ranges from 500:1 in animals

active hormone. PTH and angiotensin II are examples

chronically stimulated with TSH to 5:1 or less in hy-

of this type of regulation. Another interesting example

pophysectomized animals (no TSH). The T:S ratio in

is the POMC protein, which can be processed into

humans on a normal iodine diet is about 25:1.

many different hormones in a tissue-specific manner.

The thyroid is the only tissue that can oxidize I⁻ to a

These examples are discussed in detail below.

higher valence state, an obligatory step in I⁻ organifica-

tion and thyroid hormone biosynthesis. This step in-

Insulin Is Synthesized as a Preprohormone

volves a heme-containing peroxidase and occurs at the

& Modified Within the Cell

luminal surface of the follicular cell. Thyroperoxidase, a

tetrameric protein with a molecular mass of 60 kDa, re-

Insulin has an AB heterodimeric structure with one in-

quires hydrogen peroxide as an oxidizing agent. The

trachain (A6-A11) and two interchain disulfide bridges

H₂O₂ is produced by an NADPH-dependent enzyme

(A7-B7 and A20-B19) (Figure 42-12). The A and B

resembling cytochrome c reductase. A number of com-

chains could be synthesized in the laboratory, but at-

pounds inhibit I⁻ oxidation and therefore its subse-

tempts at a biochemical synthesis of the mature insulin

quent incorporation into MIT and DIT. The most im-

molecule yielded very poor results. The reason for this

portant of these are the thiourea drugs. They are used as

became apparent when it was discovered that insulin is

antithyroid drugs because of their ability to inhibit thy-

synthesized as a preprohormone (molecular weight ap-

roid hormone biosynthesis at this step. Once iodination

proximately 11,500), which is the prototype for peptides

occurs, the iodine does not readily leave the thyroid.

that are processed from larger precursor molecules. The

Free tyrosine can be iodinated, but it is not incorpo-

hydrophobic 23-amino-acid pre-, or leader, sequence di-

rated into proteins since no tRNA recognizes iodinated

rects the molecule into the cisternae of the endoplasmic

tyrosine.

reticulum and then is removed. This results in the 9000-

The coupling of two DIT molecules to form T₄—or

MW proinsulin molecule, which provides the conforma-

of an MIT and DIT to form T₃—occurs within the

tion necessary for the proper and efficient formation of

thyroglobulin molecule. A separate coupling enzyme

the disulfide bridges. As shown in Figure 42-12, the se-

has not been found, and since this is an oxidative

quence of proinsulin, starting from the amino terminal,

process it is assumed that the same thyroperoxidase cat-

is B chain—connecting

alyzes this reaction by stimulating free radical forma-

proinsulin molecule undergoes a series of site-specific

tion of iodotyrosine. This hypothesis is supported by

peptide cleavages that result in the formation of equimo-

the observation that the same drugs which inhibit I⁻ ox-

lar amounts of mature insulin and C peptide. These en-

idation also inhibit coupling. The formed thyroid hor-

zymatic cleavages are summarized in Figure 42-12.

450

CHAPTER 42

Leu Ser

Gly Ala Gly

Pro

Gln

Pro

Leu

Gly

Ala

Gly

Leu

Connecting peptide

Leu

Glu

Gly

Val

Ser

Gln

Leu

Gly

Gln

Val

Lys

Gln

Arg

Leu

Gly

Asp

Ile

Glu

Val

Asn

Glu

Cys

Ala

Phe

Gln

Tyr

Glu

Val

Asn

Cys

A chain

Glu

Arg

Asn

Cys

Leu

Thr

Ser

Gln

Arg

Gln

Ile

Tyr

Cys Ser Leu

Thr

Mis

Leu

Lys

Cys

Pro

Insulin

Thr

Gly

Tyr

Ser

Phe

Mis

B chain

Phe

Leu

Gly

Arg

Val

Glu

Ala

Cys

Gly

Leu Tyr Leu

Val

Figure 42-12. Structure of human proinsulin. Insulin and C-peptide molecules are connected at two sites by

dipeptide links. An initial cleavage by a trypsin-like enzyme (open arrows) followed by several cleavages by a car-

boxypeptidase-like enzyme (solid arrows) results in the production of the heterodimeric (AB) insulin molecule

(light blue) and the C-peptide.

Parathyroid Hormone (PTH) Is Secreted as

mRNA, and this is followed by an increased rate of

an 84-Amino-Acid Peptide

PTH synthesis and secretion. However, about 80-90%

of the proPTH synthesized cannot be accounted for as

The immediate precursor of PTH is proPTH, which

intact PTH in cells or in the incubation medium of ex-

differs from the native 84-amino-acid hormone by hav-

perimental systems. This finding led to the conclusion

ing a highly basic hexapeptide amino terminal exten-

that most of the proPTH synthesized is quickly de-

sion. The primary gene product and the immediate pre-

graded. It was later discovered that this rate of degrada-

cursor for proPTH is the 115-amino-acid preproPTH.

tion decreases when Ca2+ concentrations are low, and it

This differs from proPTH by having an additional 25-

increases when Ca2+ concentrations are high. Very spe-

amino-acid amino terminal extension that, in common

cific fragments of PTH are generated during its prote-

with the other leader or signal sequences characteristic

olytic digestion (Figure 42-13). A number of prote-

of secreted proteins, is hydrophobic. The complete

olytic enzymes, including cathepsins B and D, have

structure of preproPTH and the sequences of proPTH

been identified in parathyroid tissue. Cathepsin B

and PTH are illustrated in Figure 42-13. PTH_1-34 has

cleaves PTH into two fragments: PTH_1-36

and

full biologic activity, and the region 25-34 is primarily

PTH_37-84. PTH_37-84 is not further degraded; however,

responsible for receptor binding.

PTH_1-36 is rapidly and progressively cleaved into di-

The biosynthesis of PTH and its subsequent secre-

and tripeptides. Most of the proteolysis of PTH occurs

tion are regulated by the plasma ionized calcium (Ca2+)

within the gland, but a number of studies confirm that

concentration through a complex process. An acute de-

PTH, once secreted, is proteolytically degraded in other

crease of Ca2+ results in a marked increase of PTH

tissues, especially the liver, by similar mechanisms.

THE DIVERSITY OF THE ENDOCRINE SYSTEM

451

Leader (pre) sequence

–6

-10

-20

-31

Gly Asp Ser Arg Ala Leu Phe Cys Ile Ala Leu Met Val Ile Met Val Lys Val Met Asp Lys Ala Ser Met Met

NH₂

Lys

Pro

Ser

sequence

Val

Lys

(2)

(1)

Lys

-1 Arg

(3)

Ala

Val

Ser

Glu

Gln Phe Met His Asn Leu Gly Lys His Leu Ser Ser Met Glu Arg Val Glu Trp Leu Arg

Ile

Lys

Leu

Full biologic activity sequence

Gln

Asp

Val

His

Asn

C-fragment sequence

Phe

Val

Ala

Asp Glu Lys Lys Arg Pro Arg Gln Ser Ser Gly Asp Arg Tyr Ala

Ile

Ser Ala

Gly Leu

Asn

Val

(4)

Leu

Val

(5)

Glu

Ser

His

Gln

Lys

Ser

Gly Glu Ala Asp Lys Ala Asp Val Asp Val Leu Ile Lys Ala Lys Pro Gln

Leu

Figure 42-13. Structure of bovine preproparathyroid hormone. Arrows indicate sites cleaved by pro-

cessing enzymes in the parathyroid gland (1-5) and in the liver after secretion of the hormone (4-5). The

biologically active region of the molecule is flanked by sequence not required for activity on target re-

ceptors. (Slightly modified and reproduced, with permission, from Habener JF: Recent advances in parathy-

roid hormone research. Clin Biochem 1981;14:223.)

Angiotensin II Is Also Synthesized

tors that decreases fluid volume (dehydration, decreased

From a Large Precursor

blood pressure, fluid or blood loss) or decreases NaCl

concentration stimulates renin release. Renal sympa-

The renin-angiotensin system is involved in the regula-

thetic nerves that terminate in the juxtaglomerular cells

tion of blood pressure and electrolyte metabolism

mediate the central nervous system and postural effects

(through production of aldosterone). The primary hor-

on renin release independently of the baroreceptor and

mone involved in these processes is angiotensin II, an

salt effects, a mechanism that involves the β-adrenergic

octapeptide made from angiotensinogen

(Figure

receptor. Renin acts upon the substrate angiotensino-

42-14). Angiotensinogen, a large α₂-globulin made in

gen to produce the decapeptide angiotensin I.

liver, is the substrate for renin, an enzyme produced in

Angiotensin-converting enzyme, a glycoprotein

the juxtaglomerular cells of the renal afferent arteriole.

found in lung, endothelial cells, and plasma, removes

The position of these cells makes them particularly sen-

two carboxyl terminal amino acids from the decapep-

sitive to blood pressure changes, and many of the physi-

tide angiotensin I to form angiotensin II in a step that

ologic regulators of renin release act through renal

is not thought to be rate-limiting. Various nonapeptide

baroreceptors. The juxtaglomerular cells are also sensi-

analogs of angiotensin I and other compounds act as

tive to changes of Na⁺ and Cl⁻ concentration in the

competitive inhibitors of converting enzyme and are

renal tubular fluid; therefore, any combination of fac-

used to treat renin-dependent hypertension. These are

452

CHAPTER 42

Angiotensinogen

Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu (~ 400 more amino acids)

RENIN

Angiotensin I

Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu

CONVERTING ENZYME

ANGIOTENSIN II

Asp-Arg-Val-Tyr-Ile-His-Pro-Phe

AMINOPEPTIDASE

Angiotensin III

Arg-Val-Tyr-Ile-His-Pro-Phe

ANGIOTENSINASES

Degradation products

Figure 42-14. Formation and metabolism of angiotensins. Small arrows in-

dicate cleavage sites.

referred to as angiotensin-converting enzyme (ACE)

Complex Processing Generates

inhibitors. Angiotensin II increases blood pressure by

the Pro-opiomelanocortin (POMC)

causing vasoconstriction of the arteriole and is a very

Peptide Family

potent vasoactive substance. It inhibits renin release

from the juxtaglomerular cells and is a potent stimula-

The POMC family consists of peptides that act as hor-

tor of aldosterone production. This results in Na⁺ re-

mones (ACTH, LPH, MSH) and others that may serve

tention, volume expansion, and increased blood pres-

as neurotransmitters or neuromodulators (endorphins)

sure.

(see Figure 42-15). POMC is synthesized as a precur-

In some species, angiotensin II is converted to the

sor molecule of 285 amino acids and is processed differ-

heptapeptide angiotensin III (Figure 42-14), an equally

ently in various regions of the pituitary.

potent stimulator of aldosterone production. In hu-

The POMC gene is expressed in the anterior and in-

mans, the plasma level of angiotensin II is four times

termediate lobes of the pituitary. The most conserved

greater than that of angiotensin III, so most effects are

sequences between species are within the amino termi-

exerted by the octapeptide. Angiotensins II and III are

nal fragment, the ACTH region, and the β-endorphin

rapidly inactivated by angiotensinases.

region. POMC or related products are found in several

Angiotensin II binds to specific adrenal cortex

other vertebrate tissues, including the brain, placenta,

glomerulosa cell receptors. The hormone-receptor in-

gastrointestinal tract, reproductive tract, lung, and lym-

teraction does not activate adenylyl cyclase, and cAMP

phocytes.

does not appear to mediate the action of this hormone.

The POMC protein is processed differently in the an-

The actions of angiotensin II, which are to stimulate

terior lobe than in the intermediate lobe. The intermedi-

the conversion of cholesterol to pregnenolone and of

ate lobe of the pituitary is rudimentary in adult humans,

corticosterone to 18-hydroxycorticosterone and aldos-

but it is active in human fetuses and in pregnant women

terone, may involve changes in the concentration of in-

during late gestation and is also active in many animal

tracellular calcium and of phospholipid metabolites by

species. Processing of the POMC protein in the periph-

mechanisms similar to those described in Chapter 43.

eral tissues (gut, placenta, male reproductive tract) resem-

THE DIVERSITY OF THE ENDOCRINE SYSTEM

453

POMC (1-134)

ACTH (1- 39)

β-LPH (42-134)

α-MSH

CLIP

γ-LPH

β-Endorphin

(1-13)

(18-39)

(42-101)

(104 -134)

β-MSH

γ-Endorphin

(84 -101)

(104 -118)

α-Endorphin

(104 -117)

Figure 42-15. Products of pro-opiomelanocortin (POMC) cleavage.

(MSH, melanocyte-stimulating hormone; CLIP, corticotropin-like inter-

mediate lobe peptide; LPH, lipotropin.)

bles that in the intermediate lobe. There are three basic

there is no intracellular reservoir of these hormones.

peptide groups:

(1) ACTH, which can give rise to

The catecholamines, also synthesized in active form, are

α-MSH and corticotropin-like intermediate lobe peptide

stored in granules in the chromaffin cells in the adrenal

(CLIP);

(2) β-lipotropin

(β-LPH), which can yield

medulla. In response to appropriate neural stimulation,

γ-LPH, β-MSH, and β-endorphin (and thus α- and

these granules are released from the cell through exocy-

γ-endorphins); and (3) a large amino terminal peptide,

tosis, and the catecholamines are released into the circu-

which generates γ-MSH. The diversity of these products

lation. A several-hour reserve supply of catecholamines

is due to the many dibasic amino acid clusters that are

exists in the chromaffin cells.

potential cleavage sites for trypsin-like enzymes. Each of

Parathyroid hormone also exists in storage vesicles.

the peptides mentioned is preceded by Lys-Arg, Arg-Lys,

As much as 80-90% of the proPTH synthesized is de-

Arg-Arg, or Lys-Lys residues. After the prehormone seg-

graded before it enters this final storage compartment,

ment is cleaved, the next cleavage, in both anterior and

especially when Ca2+ levels are high in the parathyroid

intermediate lobes, is between ACTH and β-LPH, re-

cell (see above). PTH is secreted when Ca2+ is low in

sulting in an amino terminal peptide with ACTH and a

the parathyroid cells, which contain a several-hour sup-

β-LPH segment (Figure 42-15). ACTH_1-39 is subse-

ply of the hormone.

quently cleaved from the amino terminal peptide, and in

The human pancreas secretes about 40-50 units of in-

the anterior lobe essentially no further cleavages occur. In

sulin daily, which represents about 15-20% of the hor-

the intermediate lobe, ACTH_1-39 is cleaved into α-MSH

mone stored in the B cells. Insulin and the C-peptide (see

(residues 1-13) and CLIP (18-39); β-LPH (42-134) is

Figure

42-12) are normally secreted in equimolar

converted to γ-LPH (42-101) and β-endorphin (104-

amounts. Stimuli such as glucose, which provokes insulin

134). β-MSH (84-101) is derived from γ-LPH.

secretion, therefore trigger the processing of proinsulin to

There are extensive additional tissue-specific modifi-

insulin as an essential part of the secretory response.

cations of these peptides that affect activity. These

A several-week supply of T₃ and T₄ exists in the thy-

modifications include phosphorylation, acetylation,

roglobulin that is stored in colloid in the lumen of the

glycosylation, and amidation.

thyroid follicles. These hormones can be released upon

stimulation by TSH. This is the most exaggerated ex-

ample of a prohormone, as a molecule containing ap-

THERE IS VARIATION IN THE STORAGE

proximately 5000 amino acids must be first synthe-

& SECRETION OF HORMONES

sized, then degraded, to supply a few molecules of the

As mentioned above, the steroid hormones and

active hormones T₄ and T₃.

1,25(OH)₂-D₃

are synthesized in their final active

The diversity in storage and secretion of hormones

form. They are also secreted as they are made, and thus

is illustrated in Table 42-5.

454

CHAPTER 42

Table 42-5. Diversity in the storage of hormones.

lives. A notable exception is IGF-I, which is transported

bound to members of a family of binding proteins.

Hormone

Supply Stored in Cell

Thyroid Hormones Are Transported

Steroids and 1,25(OH)₂-D₃

None

by Thyroid-Binding Globulin

Catecholamines and PTH

Hours

Many of the principles discussed above are illustrated in

Insulin

Days

a discussion of thyroid-binding proteins. One-half to

T₃ and T₄

Weeks

two-thirds of T₄ and T₃ in the body is in an extrathy-

roidal reservoir. Most of this circulates in bound form,

ie, bound to a specific binding protein, thyroxine-

binding globulin (TBG). TBG, a glycoprotein with a

SOME HORMONES HAVE PLASMA

molecular mass of 50 kDa, binds T₄ and T₃ and has the

TRANSPORT PROTEINS

capacity to bind 20 µg/dL of plasma. Under normal

The class I hormones are hydrophobic in chemical na-

circumstances, TBG binds—noncovalently—nearly all

ture and thus are not very soluble in plasma. These hor-

of the T₄ and T₃ in plasma, and it binds T₄ with greater

mones, principally the steroids and thyroid hormones,

affinity than T₃ (Table 42-7). The plasma half-life of

have specialized plasma transport proteins that serve sev-

T₄ is correspondingly four to five times that of T₃. The

eral purposes. First, these proteins circumvent the solu-

small, unbound (free) fraction is responsible for the bi-

bility problem and thereby deliver the hormone to the

ologic activity. Thus, in spite of the great difference in

target cell. They also provide a circulating reservoir of

total amount, the free fraction of T₃ approximates that

the hormone that can be substantial, as in the case of the

of T₄, and given that T₃ is intrinsically more active than

thyroid hormones. Hormones, when bound to the trans-

T₄, most biologic activity is attributed to T₃. TBG does

port proteins, cannot be metabolized, thereby prolonging

not bind any other hormones.

their plasma half-life

(t_1/2). The binding affinity of a

given hormone to its transporter determines the bound

Glucocorticoids Are Transported

versus free ratio of the hormone. This is important be-

by Corticosteroid-Binding Globulin

cause only the free form of a hormone is biologically ac-

Hydrocortisone (cortisol) also circulates in plasma in

tive. In general, the concentration of free hormone in

protein-bound and free forms. The main plasma bind-

plasma is very low, in the range of 10^-15 to 10^-9 mol/L. It

ing protein is an α-globulin called transcortin, or cor-

is important to distinguish between plasma transport

ticosteroid-binding globulin

(CBG). CBG is pro-

proteins and hormone receptors. Both bind hormones

duced in the liver, and its synthesis, like that of TBG, is

but with very different characteristics (Table 42-6).

increased by estrogens. CBG binds most of the hor-

The hydrophilic hormones—generally class II and

mone when plasma cortisol levels are within the normal

of peptide structure—are freely soluble in plasma and

range; much smaller amounts of cortisol are bound to

do not require transport proteins. Hormones such as

albumin. The avidity of binding helps determine the

insulin, growth hormone, ACTH, and TSH circulate

biologic half-lives of various glucocorticoids. Cortisol

in the free, active form and have very short plasma half-

binds tightly to CBG and has a t_1/2 of 1.5-2 hours,

while corticosterone, which binds less tightly, has a t_1/2

Table 42-6. Comparison of receptors with

of less than 1 hour (Table 42-8). The unbound (free)

cortisol constitutes about 8% of the total and represents

transport proteins.

the biologically active fraction. Binding to CBG is not

restricted to glucocorticoids. Deoxycorticosterone and

Feature

Receptors

Transport Proteins

Concentration

Very low

Very high

(thousands/cell)

(billions/µL)

Table 42-7. Comparison of T4 and T3 in plasma.

Binding affinity

High (pmol/L to

Low (µmol/L range)

nmol/L range)

Free Hormone

Binding specificity

Very high

Low

Total

t¹⁄2

Hormone

Percent

in Blood

Saturability

Yes

(µg/dL)

of Total

ng/dL

Molarity

(days)

Reversibility

Yes

T₄

0.03

~2.24

3.0 × 10−11

6.5

Signal transduction

Yes

T₃

0.15

0.3

~0.4

~0.6 × 10−11

1.5

THE DIVERSITY OF THE ENDOCRINE SYSTEM

455

Table 42-8. Approximate affinities of steroids for

binding capacity they probably buffer against sudden

serum-binding proteins.

changes in the plasma level. Because the metabolic

clearance rates of these steroids are inversely related to

the affinity of their binding to SHBG, estrone is cleared

SHBG¹

CBG¹

more rapidly than estradiol, which in turn is cleared

Dihydrotestosterone

> 100

more rapidly than testosterone or DHT.

Testosterone

> 100

Estradiol

> 10

SUMMARY

Estrone

> 10

> 100

Progesterone

> 100

~ 2

• The presence of a specific receptor defines the target

Cortisol

> 100

~ 3

cells for a given hormone.

Corticosterone

> 100

~ 5

• Receptors are proteins that bind specific hormones

¹Affinity expressed as K_d (nmol/L).

and generate an intracellular signal (receptor-effector

coupling).

• Some hormones have intracellular receptors; others

progesterone interact with CBG with sufficient affinity

bind to receptors on the plasma membrane.

to compete for cortisol binding. Aldosterone, the most

• Hormones are synthesized from a number of precur-

potent natural mineralocorticoid, does not have a spe-

sor molecules, including cholesterol, tyrosine per se,

cific plasma transport protein. Gonadal steroids bind

and all the constituent amino acids of peptides and

very weakly to CBG (Table 42-8).

proteins.

• A number of modification processes alter the activity

Gonadal Steroids Are Transported

of hormones. For example, many hormones are syn-

by Sex Hormone-Binding Globulin

thesized from larger precursor molecules.

Most mammals, humans included, have a plasma β-

• The complement of enzymes in a particular cell type

globulin that binds testosterone with specificity, rela-

allows for the production of a specific class of steroid

tively high affinity, and limited capacity (Table 42-8).

hormone.

This protein, usually called sex hormone-binding

• Most of the lipid-soluble hormones are bound to

globulin

(SHBG) or testosterone-estrogen-binding

rather specific plasma transport proteins.

globulin (TEBG), is produced in the liver. Its produc-

tion is increased by estrogens (women have twice the

serum concentration of SHBG as men), certain types of

REFERENCES

liver disease, and hyperthyroidism; it is decreased by

Bartalina L: Thyroid hormone-binding proteins: update 1994. En-

androgens, advancing age, and hypothyroidism. Many

docr Rev 1994;13:140.

of these conditions also affect the production of CBG

Beato M et al: Steroid hormone receptors: many actors in search of

and TBG. Since SHBG and albumin bind 97-99% of

a plot. Cell 1995;83:851.

circulating testosterone, only a small fraction of the

Dai G, Carrasco L, Carrasco N: Cloning and characterization of

hormone in circulation is in the free (biologically ac-

the thyroid iodide transporter. Nature 1996;379:458.

tive) form. The primary function of SHBG may be to

DeLuca HR: The vitamin D story: a collaborative effort of basic

restrict the free concentration of testosterone in the

science and clinical medicine. FASEB J 1988;2:224.

serum. Testosterone binds to SHBG with higher affin-

Douglass J, Civelli O, Herbert E: Polyprotein gene expression:

ity than does estradiol

(Table

42-8). Therefore, a

Generation of diversity of neuroendocrine peptides. Annu

change in the level of SHBG causes a greater change in

Rev Biochem 1984;53:665.

the free testosterone level than in the free estradiol level.

Miller WL: Molecular biology of steroid hormone biosynthesis.

Endocr Rev 1988;9:295.

Estrogens are bound to SHBG and progestins to

CBG. SHBG binds estradiol about five times less avidly

Nagatsu T: Genes for human catecholamine-synthesizing enzymes.

Neurosci Res 1991;12:315.

than it binds testosterone or DHT, while progesterone

Russell DW, Wilson JD: Steroid 5 alpha-reductase: two genes/two

and cortisol have little affinity for this protein (Table

enzymes. Annu Rev Biochem 1994;63:25.

42-8). In contrast, progesterone and cortisol bind with

Russell J et al: Interaction between calcium and 1,25-dihydroxy-

nearly equal affinity to CBG, which in turn has little

vitamin D₃ in the regulation of preproparathyroid hormone

avidity for estradiol and even less for testosterone,

and vitamin D receptor mRNA in avian parathyroids. En-

DHT, or estrone.

docrinology 1993;132:2639.

These binding proteins also provide a circulating

Steiner DF et al: The new enzymology of precursor processing en-

reservoir of hormone, and because of the relatively large

doproteases. J Biol Chem 1992;267:23435.

Hormone Action &

Signal Transduction

Daryl K. Granner, MD

BIOMEDICAL IMPORTANCE

as described in Chapter 42, ie, based on the location of

their specific cellular receptors and the type of signals

The homeostatic adaptations an organism makes to a

generated. Group I hormones interact with an intracel-

constantly changing environment are in large part ac-

lular receptor and group II hormones with receptor

complished through alterations of the activity and

recognition sites located on the extracellular surface of

amount of proteins. Hormones provide a major means

the plasma membrane of target cells. The cytokines, in-

of facilitating these changes. A hormone-receptor inter-

terleukins, and growth factors should also be considered

action results in generation of an intracellular signal

in this latter category. These molecules, of critical im-

that can either regulate the activity of a select set of

portance in homeostatic adaptation, are hormones in

genes, thereby altering the amount of certain proteins

the sense that they are produced in specific cells, have

in the target cell, or affect the activity of specific pro-

the equivalent of autocrine, paracrine, and endocrine

teins, including enzymes and transporter or channel

actions, bind to cell surface receptors, and activate

proteins. The signal can influence the location of pro-

many of the same signal transduction pathways em-

teins in the cell and can affect general processes such as

ployed by the more traditional group II hormones.

protein synthesis, cell growth, and replication, perhaps

through effects on gene expression. Other signaling

molecules—including cytokines, interleukins, growth

SIGNAL GENERATION

factors, and metabolites—use some of the same general

mechanisms and signal transduction pathways. Exces-

The Ligand-Receptor Complex Is the

sive, deficient, or inappropriate production and release

Signal for Group I Hormones

of hormones and of these other regulatory molecules

The lipophilic group I hormones diffuse through the

are major causes of disease. Many pharmacotherapeutic

plasma membrane of all cells but only encounter their

agents are aimed at correcting or otherwise influencing

specific, high-affinity intracellular receptors in target

the pathways discussed in this chapter.

cells. These receptors can be located in the cytoplasm or

in the nucleus of target cells. The hormone-receptor

complex first undergoes an activation reaction. As

HORMONES TRANSDUCE SIGNALS TO

shown in Figure 43-2, receptor activation occurs by at

AFFECT HOMEOSTATIC MECHANISMS

least two mechanisms. For example, glucocorticoids

The general steps involved in producing a coordinated

diffuse across the plasma membrane and encounter

response to a particular stimulus are illustrated in

their cognate receptor in the cytoplasm of target cells.

Figure 43-1. The stimulus can be a challenge or a

Ligand-receptor binding results in the dissociation of

threat to the organism, to an organ, or to the integrity

heat shock protein 90 (hsp90) from the receptor. This

of a single cell within that organism. Recognition of the

step appears to be necessary for subsequent nuclear lo-

stimulus is the first step in the adaptive response. At the

calization of the glucocorticoid receptor. This receptor

organismic level, this generally involves the nervous sys-

also contains nuclear localization sequences that assist

tem and the special senses (sight, hearing, pain, smell,

in the translocation from cytoplasm to nucleus. The

touch). At the organismic or cellular level, recognition

now activated receptor moves into the nucleus (Figure

involves physicochemical factors such as pH, O₂ ten-

43-2) and binds with high affinity to a specific DNA

sion, temperature, nutrient supply, noxious metabo-

sequence called the hormone response element

lites, and osmolarity. Appropriate recognition results in

(HRE). In the case illustrated, this is a glucocorticoid

the release of one or more hormones that will govern

response element, or GRE. Consensus sequences for

generation of the necessary adaptive response. For pur-

HREs are shown in Table 43-1. The DNA-bound, lig-

poses of this discussion, the hormones are categorized

anded receptor serves as a high-affinity binding site for

456

HORMONE ACTION & SIGNAL TRANSDUCTION

457

STIMULUS

Recognition

Group I hormones

Group II hormones

Hormone release

Hormone•receptor complex

Many different signals

Signal generation

Effects

Gene

Transporters

Protein

transcription

Channels

translocation

modification

COORDINATED RESPONSE TO STIMULUS

Figure 43-1. Hormonal involvement in responses to a stimulus. A challenge to the in-

tegrity of the organism elicits a response that includes the release of one or more hormones.

These hormones generate signals at or within target cells, and these signals regulate a vari-

ety of biologic processes which provide for a coordinated response to the stimulus or chal-

lenge. See Figure 43-8 for a specific example.

one or more coactivator proteins, and accelerated gene

gests that these hormones exert their dominant effect

transcription typically ensues when this occurs. By con-

on modulating gene transcription, but they—and many

trast, certain hormones such as the thyroid hormones

of the hormones in the other classes discussed below—

and retinoids diffuse from the extracellular fluid across

can act at any step of the “information pathway” illus-

the plasma membrane and go directly into the nucleus.

trated in Figure 43-3. Direct actions of steroids in the

In this case, the cognate receptor is already bound to

cytoplasm and on various organelles and membranes

the HRE

(the thyroid hormone response element

have also been described.

[TRE], in this example). However, this DNA-bound

receptor fails to activate transcription because it is com-

plexed with a corepressor. Indeed, this receptor-

GROUP II (PEPTIDE &

corepressor complex serves as an active repressor of gene

CATECHOLAMINE) HORMONES

transcription. The association of ligand with these re-

HAVE MEMBRANE RECEPTORS

ceptors results in dissociation of the corepressor. The

& USE INTRACELLULAR MESSENGERS

liganded receptor is now capable of binding one or

more coactivators with high affinity, resulting in the ac-

Many hormones are water-soluble, have no transport

tivation of gene transcription. The relationship of hor-

proteins (and therefore have a short plasma half-life),

mone receptors to other nuclear receptors and to coreg-

and initiate a response by binding to a receptor located

ulators is discussed in more detail below.

in the plasma membrane (see Tables 42-3 and 42-4).

By selectively affecting gene transcription and the

The mechanism of action of this group of hormones

consequent production of appropriate target mRNAs,

can best be discussed in terms of the intracellular sig-

the amounts of specific proteins are changed and meta-

nals they generate. These signals include cAMP (cyclic

bolic processes are influenced. The influence of each of

AMP; 3′,5′-adenylic acid; see Figure

18-5), a nu-

these hormones is quite specific; generally, the hor-

cleotide derived from ATP through the action of

mone affects less than 1% of the genes, mRNA, or pro-

adenylyl cyclase; cGMP, a nucleotide formed by gua-

teins in a target cell; sometimes only a few are affected.

nylyl cyclase; Ca2+; and phosphatidylinositides. Many

The nuclear actions of steroid, thyroid, and retinoid

of these second messengers affect gene transcription, as

hormones are quite well defined. Most evidence sug-

described in the previous paragraph; but they also influ-

458

CHAPTER 43

−

Cytoplasm

−

TRE

GRE

hsp

Nucleus

Figure 43-2. Regulation of gene expression by class I hormones.

Steroid hormones readily gain access to the cytoplasmic compartment

of target cells. Glucocorticoid hormones (solid triangles) encounter

their cognate receptor in the cytoplasm, where it exists in a complex

with heat shock protein 90 (hsp). Ligand binding causes dissociation of

hsp and a conformational change of the receptor. The receptor•ligand

complex then traverses the nuclear membrane and binds to DNA with

specificity and high affinity at a glucocorticoid response element (GRE).

This event triggers the assembly of a number of transcription coregula-

tors ( + ), and enhanced transcription ensues. By contrast, thyroid hor-

mones and retinoic acid (

) directly enter the nucleus, where their

cognate receptors are already bound to the appropriate response ele-

ments with an associated transcription repressor complex ( − ). This

complex, which consists of molecules such as N-CoR or SMRT (see

Table 43-6) in the absence of ligand, actively inhibits transcription. Lig-

and binding results in dissociation of the repressor complex from the

receptor, allowing an activator complex to assemble. The gene is then

actively transcribed.

ence a variety of other biologic processes, as shown in

cloned from various mammalian species. A wide variety

Figure 43-1.

of responses are mediated by the GPCRs.

G Protein-Coupled Receptors (GPCR)

cAMP Is the Intracellular Signal

Many of the group II hormones bind to receptors that

for Many Responses

couple to effectors through a GTP-binding protein in-

Cyclic AMP was the first intracellular signal identified

termediary. These receptors typically have seven hy-

in mammalian cells. Several components comprise a

drophobic plasma membrane-spanning domains. This

system for the generation, degradation, and action of

is illustrated by the seven interconnected cylinders ex-

cAMP.

tending through the lipid bilayer in Figure 43-4. Re-

ceptors of this class, which signal through guanine nu-

A. ADENYLYL CYCLASE

cleotide-bound protein intermediates, are known as

G protein-coupled receptors, or GPCRs. To date,

Different peptide hormones can either stimulate (s) or

over 130 G protein-linked receptor genes have been

inhibit (i) the production of cAMP from adenylyl cy-

HORMONE ACTION & SIGNAL TRANSDUCTION

459

Table 43-1. The DNA sequences of several

Gene

hormone response elements (HREs).¹

TRANSCRIPTION

Hormone or Effector

HRE

DNA Sequence

Primary transcript

Degradation

Glucocorticoids

GRE

Progestins

PRE

GGTACA NNN TGTTCT

←

NUCLEUS

MODIFICATION/PROCESSING

Mineralocorticoids

MRE

Androgens

ARE

mRNA

Degradation

Estrogens

ERE

AGGTCA --- TGA/TCCT

←

Transport

Thyroid hormone

TRE

Retinoic acid

RARE

AGGTCA N3,4,5, AGGTCA

Active

inactive

Vitamin D

VDRE

mRNA

degradation

cAMP

CRE

TGACGTCA

CYTOPLASM

TRANSLATION

¹Letters indicate nucleotide; N means any one of the four can be

used in that position. The arrows pointing in opposite directions

Protein

Modification

degradation

illustrate the slightly imperfect inverted palindromes present in

many HREs; in some cases these are called “half binding sites” be-

Figure 43-3. The “information pathway.” Informa-

cause each binds one monomer of the receptor. The GRE, PRE,

tion flows from the gene to the primary transcript to

MRE, and ARE consist of the same DNA sequence. Specificity may

mRNA to protein. Hormones can affect any of the steps

be conferred by the intracellular concentration of the ligand or

hormone receptor, by flanking DNA sequences not included in

involved and can affect the rates of processing, degra-

the consensus, or by other accessory elements. A second group of

dation, or modification of the various products.

HREs includes those for thyroid hormones, estrogens, retinoic

acid, and vitamin D. These HREs are similar except for the orienta-

tion and spacing between the half palindromes. Spacing deter-

mines the hormone specificity. VDRE (N=3), TRE (N=4), and RARE

tively. In the case of α_s, this modification disrupts the

(N=5) bind to direct repeats rather than to inverted repeats. An-

intrinsic GTP-ase activity; thus, α_s cannot reassociate

other member of the steroid receptor superfamily, the retinoid X

with βγ and is therefore irreversibly activated. ADP-

receptor (RXR), forms heterodimers with VDR, TR, and RARE, and

these constitute the trans-acting factors. cAMP affects gene tran-

ribosylation of α_i-2

prevents the dissociation of α_i-2

scription through the CRE.

from βγ, and free α_i-2 thus cannot be formed. α_s activ-

ity in such cells is therefore unopposed.

There is a large family of G proteins, and these are

clase, which is encoded by at least nine different genes

part of the superfamily of GTPases. The G protein

(Table 43-2). Two parallel systems, a stimulatory (s)

family is classified according to sequence homology

one and an inhibitory (i) one, converge upon a single

into four subfamilies, as illustrated in Table

43-3.

catalytic molecule (C). Each consists of a receptor, R_s or

There are 21 α, 5 β, and 8 γ subunit genes. Various

Ri, and a regulatory complex, Gs and Gi. Gs and Gi are

combinations of these subunits provide a large number

each trimers composed of α, β, and γ subunits. Because

of possible αβγ and cyclase complexes.

the α subunit in G_s differs from that in G_i, the pro-

The α subunits and the βγ complex have actions in-

teins, which are distinct gene products, are designated

dependent of those on adenylyl cyclase

(see Figure

αs and αi. The α subunits bind guanine nucleotides.

43-4 and Table 43-3). Some forms of α_i stimulate K⁺

The β and γ subunits are always associated (βγ) and ap-

channels and inhibit Ca2+ channels, and some α_s mole-

pear to function as a heterodimer. The binding of a hor-

cules have the opposite effects. Members of the G_q fam-

mone to R_s or R_i results in a receptor-mediated activa-

ily activate the phospholipase C group of enzymes. The

tion of G, which entails the exchange of GDP by GTP

βγ complexes have been associated with K⁺ channel

on α and the concomitant dissociation of βγ from α.

stimulation and phospholipase C activation. G proteins

The α_s protein has intrinsic GTPase activity. The

are involved in many important biologic processes in

active form, α_s•GTP, is inactivated upon hydrolysis of

addition to hormone action. Notable examples include

the GTP to GDP; the trimeric G_s complex (αβγ) is

olfaction (α_OLF) and vision (α_t). Some examples are

then re-formed and is ready for another cycle of activa-

listed in Table 43-3. GPCRs are implicated in a num-

tion. Cholera and pertussis toxins catalyze the ADP-

ber of diseases and are major targets for pharmaceutical

ribosylation of α_s and α_i-2 (see Table 43-3), respec-

agents.

460

CHAPTER 43

α_s

No hormone: inactive effector

Bound hormone (H): active effector

Figure 43-4. Components of the hormone receptor-G protein effector system. Receptors that

couple to effectors through G proteins (GPCR) typically have seven membrane-spanning domains. In

the absence of hormone (left), the heterotrimeric G-protein complex (α, β, γ) is in an inactive guano-

sine diphosphate (GDP)-bound form and is probably not associated with the receptor. This complex is

anchored to the plasma membrane through prenylated groups on the βγ subunits (wavy lines) and

perhaps by myristoylated groups on α subunits (not shown). On binding of hormone ( H ) to the re-

ceptor, there is a presumed conformational change of the receptor—as indicated by the tilted mem-

brane spanning domains—and activation of the G-protein complex. This results from the exchange of

GDP with guanosine triphosphate (GTP) on the α subunit, after which α and βγ dissociate. The α sub-

unit binds to and activates the effector (E). E can be adenylyl cyclase, Ca2+, Na⁺, or Cl⁻ channels (α_s), or

it could be a K⁺ channel (α_i), phospholipase Cβ (α_q), or cGMP phosphodiesterase (α_t). The βγ subunit

can also have direct actions on E. (Modified and reproduced, with permission, from Granner DK in: Princi-

ples and Practice of Endocrinology and Metabolism, 3rd ed. Becker KL [editor]. Lippincott, 2000.)

B. PROTEIN KINASE

In prokaryotic cells, cAMP binds to a specific protein

called catabolite regulatory protein (CRP) that binds

Table 43-2. Subclassification of group II.A

directly to DNA and influences gene expression. In eu-

hormones.

karyotic cells, cAMP binds to a protein kinase called

protein kinase A (PKA) that is a heterotetrameric mol-

ecule consisting of two regulatory subunits (R) and two

Hormones That Stimulate

Hormones That Inhibit

catalytic subunits (C). cAMP binding results in the fol-

Adenylyl Cyclase

lowing reaction:

(H_s)

(H_l)

ACTH

Acetylcholine

cAMP + R

⋅(

cAMP) + 2C

ADH

2-Adrenergics

β-Adrenergics

Angiotensin II

Calcitonin

Somatostatin

The R₂C₂ complex has no enzymatic activity, but

CRH

the binding of cAMP by R dissociates R from C,

FSH

thereby activating the latter (Figure 43-5). The active

Glucagon

C subunit catalyzes the transfer of the γ phosphate of

hCG

ATP to a serine or threonine residue in a variety of pro-

teins. The consensus phosphorylation sites are -Arg-

LPH

Arg/Lys-X-Ser/Thr- and -Arg-Lys-X-X-Ser-, where X

MSH

can be any amino acid.

PTH

Protein kinase activities were originally described as

TSH

being “cAMP-dependent” or “cAMP-independent.” This

HORMONE ACTION & SIGNAL TRANSDUCTION

461

Table 43-3. Classes and functions of selected G proteins.^1,2

Class or Type

Stimulus

Effector

Effect

G^s α_s

Glucagon, β-adrenergics

↑ Adenylyl cyclase

Gluconeogenesis, lipolysis,

↑ Cardiac Ca2+, Cl⁻, and Na⁺ channels

glycogenolysis

αolf

Odorant

↑ Adenylyl cyclase

Olfaction

Gⁱ α_i

-1,2,3

Acetylcholine,

↓ Adenylyl cyclase

Slowed heart rate

α₂-adrenergics

↑ Potassium channels

M₂ cholinergics

↓ Calcium channels

α₀

Opioids, endorphins

↑ Potassium channels

Neuronal electrical activity

αt

Light

↑ cGMP phosphodiesterase

Vision

G^qα_q

M₁ cholinergics

α₁-Adrenergics

↑ Phospholipase C-β1

↑ Muscle contraction

and

α₁₁

α₁-Adrenergics

↑ Phospholipase c-β2

↑ Blood pressure

G₁₂

α₁₂

Cl⁻ channel

¹Modified and reproduced, with permission, from Granner DK in: Principles and Practice of Endocrinology and Metabolism, 3rd

ed. Becker KL (editor). Lippincott, 2000.

²The four major classes or families of mammalian G proteins (G_s , G_i , G_q , and G₁₂) are based on protein sequence homology.

Representative members of each are shown, along with known stimuli, effectors, and well-defined biologic effects. Nine iso-

forms of adenylyl cyclase have been identified (isoforms I-IX). All isoforms are stimulated by α_s ; α_i isoforms inhibit types V

and VI, and α₀ inhibits types I and V. At least 16 different α subunits have been identified.

classification has changed, as protein phosphorylation is

ment binding protein (CREB). CREB binds

now recognized as being a major regulatory mecha-

cAMP responsive element (CRE) (see Table 43-1) in

nism. Several hundred protein kinases have now been

its nonphosphorylated state and is a weak activator of

described. The kinases are related in sequence and

transcription. When phosphorylated by PKA, CREB

structure within the catalytic domain, but each is a

binds the coactivator CREB-binding protein CBP/

unique molecule with considerable variability with re-

p300 (see below) and as a result is a much more potent

spect to subunit composition, molecular weight, au-

transcription activator.

tophosphorylation, K_m for ATP, and substrate speci-

D. PHOSPHODIESTERASES

ficity.

Actions caused by hormones that increase cAMP con-

C. PHOSPHOPROTEINS

centration can be terminated in a number of ways, in-

The effects of cAMP in eukaryotic cells are all thought

cluding the hydrolysis of cAMP to 5′-AMP by phos-

to be mediated by protein phosphorylation-dephosphor-

phodiesterases (see Figure 43-5). The presence of these

ylation, principally on serine and threonine residues.

hydrolytic enzymes ensures a rapid turnover of the sig-

The control of any of the effects of cAMP, including

nal (cAMP) and hence a rapid termination of the bio-

such diverse processes as steroidogenesis, secretion, ion

logic process once the hormonal stimulus is removed.

transport, carbohydrate and fat metabolism, enzyme in-

There are at least 11 known members of the phospho-

duction, gene regulation, synaptic transmission, and

diesterase family of enzymes. These are subject to regu-

cell growth and replication, could be conferred by a

lation by their substrates, cAMP and cGMP; by hor-

specific protein kinase, by a specific phosphatase, or by

mones; and by intracellular messengers such as calcium,

specific substrates for phosphorylation. These substrates

probably acting through calmodulin. Inhibitors of

help define a target tissue and are involved in defining

phosphodiesterase, most notably methylated xanthine

the extent of a particular response within a given cell.

derivatives such as caffeine, increase intracellular cAMP

For example, the effects of cAMP on gene transcription

and mimic or prolong the actions of hormones through

are mediated by the protein cyclic AMP response ele-

this signal.

462

CHAPTER 43

ATP • Mg2+

R₂C₂

Inactive PKA

Active

adenylyl

cyclase

cAMP

Phosphodiesterase

C₂

+ R₂

Cell

Active PKA

5′-AMP

membrane

Mg2+ • ATP

Protein

Phosphoprotein

Phosphatase

Physiologic

effects

Figure 43-5. Hormonal regulation of cellular processes through

cAMP-dependent protein kinase (PKA). PKA exists in an inactive form as

an R₂C₂ heterotetramer consisting of two regulatory and two catalytic

subunits. The cAMP generated by the action of adenylyl cyclase (acti-

vated as shown in Figure 43-4) binds to the regulatory (R) subunit of

PKA. This results in dissociation of the regulatory and catalytic subunits

and activation of the latter. The active catalytic subunits phosphorylate

a number of target proteins on serine and threonine residues. Phos-

phatases remove phosphate from these residues and thus terminate

the physiologic response. A phosphodiesterase can also terminate the

response by converting cAMP to 5′-AMP.

E. PHOSPHOPROTEIN PHOSPHATASES

heat-stable protein inhibitors regulate type I phos-

phatase activity. Inhibitor-1 is phosphorylated and acti-

Given the importance of protein phosphorylation, it is

vated by cAMP-dependent protein kinases; and in-

not surprising that regulation of the protein dephos-

hibitor-2, which may be a subunit of the inactive

phorylation reaction is another important control

phosphatase, is also phosphorylated, possibly by glyco-

mechanism (see Figure

43-5). The phosphoprotein

gen synthase kinase-3.

phosphatases are themselves subject to regulation by

phosphorylation-dephosphorylation reactions and by a

variety of other mechanisms, such as protein-protein

cGMP Is Also an Intracellular Signal

interactions. In fact, the substrate specificity of the

phosphoserine-phosphothreonine phosphatases may be

Cyclic GMP is made from GTP by the enzyme gua-

dictated by distinct regulatory subunits whose binding

nylyl cyclase, which exists in soluble and membrane-

is regulated hormonally. The best-studied role of regu-

bound forms. Each of these isozymes has unique physi-

lation by the dephosphorylation of proteins is that of

ologic properties. The atriopeptins, a family of peptides

glycogen metabolism in muscle. Two major types of

produced in cardiac atrial tissues, cause natriuresis, di-

phosphoserine-phosphothreonine phosphatases have

uresis, vasodilation, and inhibition of aldosterone secre-

been described. Type I preferentially dephosphorylates

tion. These peptides (eg, atrial natriuretic factor) bind

the β subunit of phosphorylase kinase, whereas type II

to and activate the membrane-bound form of guanylyl

dephosphorylates the α subunit. Type I phosphatase is

cyclase. This results in an increase of cGMP by as much

implicated in the regulation of glycogen synthase, phos-

as 50-fold in some cases, and this is thought to mediate

phorylase, and phosphorylase kinase. This phosphatase

the effects mentioned above. Other evidence links

is itself regulated by phosphorylation of certain of its

cGMP to vasodilation. A series of compounds, includ-

subunits, and these reactions are reversed by the action

ing nitroprusside, nitroglycerin, nitric oxide, sodium

of one of the type II phosphatases. In addition, two

nitrite, and sodium azide, all cause smooth muscle re-

HORMONE ACTION & SIGNAL TRANSDUCTION

463

laxation and are potent vasodilators. These agents in-

B. CALMODULIN

crease cGMP by activating the soluble form of guanylyl

The calcium-dependent regulatory protein is calmod-

cyclase, and inhibitors of cGMP phosphodiesterase (the

ulin, a 17-kDa protein that is homologous to the mus-

drug sildenafil [Viagra], for example) enhance and pro-

cle protein troponin C in structure and function.

long these responses. The increased cGMP activates

Calmodulin has four Ca2+ binding sites, and full occu-

cGMP-dependent protein kinase (PKG), which in turn

pancy of these sites leads to a marked conformational

phosphorylates a number of smooth muscle proteins.

change, which allows calmodulin to activate enzymes

Presumably, this is involved in relaxation of smooth

and ion channels. The interaction of Ca2+ with calmod-

muscle and vasodilation.

ulin (with the resultant change of activity of the latter)

is conceptually similar to the binding of cAMP to PKA

Several Hormones Act Through

and the subsequent activation of this molecule.

Calcium or Phosphatidylinositols

Calmodulin can be one of numerous subunits of com-

plex proteins and is particularly involved in regulating

Ionized calcium is an important regulator of a variety of

various kinases and enzymes of cyclic nucleotide gener-

cellular processes, including muscle contraction, stimu-

ation and degradation. A partial list of the enzymes reg-

lus-secretion coupling, the blood clotting cascade, en-

ulated directly or indirectly by Ca2+, probably through

zyme activity, and membrane excitability. It is also an

calmodulin, is presented in Table 43-4.

intracellular messenger of hormone action.

In addition to its effects on enzymes and ion trans-

port, Ca2+/calmodulin regulates the activity of many

A. CALCIUM METABOLISM

structural elements in cells. These include the actin-

The extracellular calcium (Ca2+) concentration is about

myosin complex of smooth muscle, which is under β-

5 mmol/L and is very rigidly controlled. Although sub-

adrenergic control, and various microfilament-medi-

stantial amounts of calcium are associated with intracel-

ated processes in noncontractile cells, including cell

lular organelles such as mitochondria and the endoplas-

motility, cell conformation changes, mitosis, granule re-

mic reticulum, the intracellular concentration of free or

lease, and endocytosis.

ionized calcium (Ca2+) is very low: 0.05-10 µmol/L. In

spite of this large concentration gradient and a favor-

C. CALCIUM IS A MEDIATOR OF HORMONE ACTION

able transmembrane electrical gradient, Ca2+ is re-

A role for Ca2+ in hormone action is suggested by the

strained from entering the cell. A considerable amount

observations that the effect of many hormones is (1)

of energy is expended to ensure that the intracellular

blunted by Ca2+-free media or when intracellular cal-

Ca2+ is controlled, as a prolonged elevation of Ca2+ in

cium is depleted; (2) can be mimicked by agents that

the cell is very toxic. A Na⁺/Ca2+ exchange mechanism

increase cytosolic Ca2+, such as the Ca2+ ionophore

that has a high capacity but low affinity pumps Ca2+

A23187; and (3) influences cellular calcium flux. The

out of cells. There also is a Ca2+/proton ATPase-depen-

regulation of glycogen metabolism in liver by vaso-

dent pump that extrudes Ca2+ in exchange for H⁺. This

pressin and α-adrenergic catecholamines provides a

has a high affinity for Ca2+ but a low capacity and is

good example. This is shown schematically in Figures

probably responsible for fine-tuning cytosolic Ca2+.

18-6 and 18-7.

Furthermore, Ca2+ ATPases pump Ca2+ from the cy-

tosol to the lumen of the endoplasmic reticulum. There

are three ways of changing cytosolic Ca2+: (1) Certain

hormones (class II.C, Table 42-3) by binding to recep-

Table 43-4. Enzymes and proteins regulated by

tors that are themselves Ca2+ channels, enhance mem-

brane permeability to Ca2+ and thereby increase Ca2+

calcium or calmodulin.

influx. (2) Hormones also indirectly promote Ca2+ in-

flux by modulating the membrane potential at the

Adenylyl cyclase

plasma membrane. Membrane depolarization opens

Ca2+-dependent protein kinases

voltage-gated Ca2+ channels and allows for Ca2+ influx.

Ca2+-Mg2+ ATPase

(3) Ca2+ can be mobilized from the endoplasmic reticu-

Ca2+-phospholipid-dependent protein kinase

lum, and possibly from mitochondrial pools.

Cyclic nucleotide phosphodiesterase

An important observation linking Ca2+ to hormone

Some cytoskeletal proteins

action involved the definition of the intracellular targets

Some ion channels (eg, L-type calcium channels)

Nitric oxide synthase

of Ca2+ action. The discovery of a Ca2+-dependent reg-

Phosphorylase kinase

ulator of phosphodiesterase activity provided the basis

Phosphoprotein phosphatase 2B

for a broad understanding of how Ca2+ and cAMP in-

Some receptors (eg, NMDA-type glutamate receptor)

teract within cells.

464

CHAPTER 43

A number of critical metabolic enzymes are regu-

face receptors such as those for acetylcholine, antidi-

lated by Ca2+, phosphorylation, or both, including

uretic hormone, and α₁-type catecholamines are, when

glycogen synthase, pyruvate kinase, pyruvate carboxy-

occupied by their respective ligands, potent activators

lase, glycerol-3-phosphate dehydrogenase, and pyruvate

of phospholipase C. Receptor binding and activation of

dehydrogenase.

phospholipase C are coupled by the G_q isoforms (Table

43-3 and Figure 43-6). Phospholipase C catalyzes the

D. PHOSPHATIDYLINOSITIDE METABOLISM AFFECTS

hydrolysis of phosphatidylinositol 4,5-bisphosphate to

CA2+-DEPENDENT HORMONE ACTION

inositol trisphosphate (IP₃) and 1,2-diacylglycerol (Fig-

Some signal must provide communication between the

ure 43-7). Diacylglycerol is itself capable of activating

hormone receptor on the plasma membrane and the in-

protein kinase C (PKC), the activity of which also de-

tracellular Ca2+ reservoirs. This is accomplished by

pends upon Ca2+. IP₃, by interacting with a specific in-

products of phosphatidylinositol metabolism. Cell sur-

tracellular receptor, is an effective releaser of Ca2+ from

Ca2+

Receptor

G protein

Phospholipase C

PIP₂

Diacylglycerol

Endoplasmic reticulum

Protein kinase C

Inositol-P₃

(PKC)

(IP

Mitochondrion

Ca2+

Calmodulin

Ca2+-Calmodulin

Specific

Multifunctional

calmodulin kinase calmodulin kinase

Proteins

Phosphoproteins

Other

proteins

Physiologic responses

Figure 43-6. Certain hormone-receptor interactions result in the activation of phospholipase C. This ap-

pears to involve a specific G protein, which also may activate a calcium channel. Phospholipase C results in

generation of inositol trisphosphate (IP₃), which liberates stored intracellular Ca2+, and diacylglycerol (DAG),

a potent activator of protein kinase C (PKC). In this scheme, the activated PKC phosphorylates specific sub-

strates, which then alter physiologic processes. Likewise, the Ca2+-calmodulin complex can activate specific

kinases, two of which are shown here. These actions result in phosphorylation of substrates, and this leads to

altered physiologic responses. This figure also shows that Ca2+ can enter cells through voltage- or ligand-

gated Ca2+ channels. The intracellular Ca2+ is also regulated through storage and release by the mitochon-

dria and endoplasmic reticulum. (Courtesy of JH Exton.)

HORMONE ACTION & SIGNAL TRANSDUCTION

465

R₁

R₂

R₁

R₂

Phospholipase C

1,2-Diacylglycerol

(DAG)

Phosphatidylinositol 4,5-bisphosphate

(PIP₂)

Figure 43-7. Phospholipase C cleaves PIP₂

into diacylglycerol and inositol trisphosphate. R

generally is stearate, and R₂ is usually arachido-

nate. IP₃

can be dephosphorylated (to the inac-

tive I-1,4-P₂) or phosphorylated (to the potentially

Inositol 1,4,5-trisphosphate

(IP₃)

active I-1,3,4,5-P₄).

intracellular storage sites in the endoplasmic reticulum.

ligand-activated tyrosine kinase activity. Several recep-

Thus, the hydrolysis of phosphatidylinositol 4,5-bis-

tors—generally those involved in binding ligands in-

phosphate leads to activation of PKC and promotes an

volved in growth control, differentiation, and the in-

increase of cytoplasmic Ca2+. As shown in Figure 43-4,

flammatory response—either have intrinsic tyrosine

the activation of G proteins can also have a direct ac-

kinase activity or are associated with proteins that are

tion on Ca2+ channels. The resulting elevations of cy-

tyrosine kinases. Another distinguishing feature of this

tosolic Ca2+ activate Ca2+-calmodulin-dependent kinases

class of hormone action is that these kinases preferen-

and many other Ca2+-calmodulin-dependent enzymes.

tially phosphorylate tyrosine residues, and tyrosine

Steroidogenic agents—including ACTH and cAMP

phosphorylation is infrequent (< 0.03% of total amino

in the adrenal cortex; angiotensin II, K⁺, serotonin,

acid phosphorylation) in mammalian cells. A third dis-

ACTH, and cAMP in the zona glomerulosa of the

tinguishing feature is that the ligand-receptor interac-

adrenal; LH in the ovary; and LH and cAMP in the

tion that results in a tyrosine phosphorylation event ini-

Leydig cells of the testes—have been associated with in-

tiates a cascade that may involve several protein kinases,

creased amounts of phosphatidic acid, phosphatidyl-

phosphatases, and other regulatory proteins.

inositol, and polyphosphoinositides (see Chapter 14) in

A. INSULIN TRANSMITS SIGNALS

the respective target tissues. Several other examples

BY SEVERAL KINASE CASCADES

could be cited.

The roles that Ca2+ and polyphosphoinositide break-

The insulin, epidermal growth factor (EGF), and IGF-I

down products might play in hormone action are pre-

receptors have intrinsic protein tyrosine kinase activities

sented in Figure 43-6. In this scheme the activated pro-

located in their cytoplasmic domains. These activities

tein kinase C can phosphorylate specific substrates,

are stimulated when the receptor binds ligand. The re-

which then alter physiologic processes. Likewise, the

ceptors are then autophosphorylated on tyrosine

Ca2+-calmodulin complex can activate specific kinases.

residues, and this initiates a complex series of events

These then modify substrates and thereby alter physio-

(summarized in simplified fashion in Figure 43-8). The

logic responses.

phosphorylated insulin receptor next phosphorylates

insulin receptor substrates (there are at least four of

these molecules, called IRS 1-4) on tyrosine residues.

Some Hormones Act Through

Phosphorylated IRS binds to the Src homology

a Protein Kinase Cascade

(SH2) domains of a variety of proteins that are directly

Single protein kinases such as PKA, PKC, and Ca2+-

involved in mediating different effects of insulin. One

calmodulin (CaM)-kinases, which result in the phos-

of these proteins, PI-3 kinase, links insulin receptor ac-

phorylation of serine and threonine residues in target

tivation to insulin action through activation of a num-

proteins, play a very important role in hormone action.

ber of molecules, including the kinase PDK1 (phospho-

The discovery that the EGF receptor contains an intrin-

inositide-dependent kinase-1). This enzyme propagates

sic tyrosine kinase activity that is activated by the bind-

the signal through several other kinases, including PKB

ing of the ligand EGF was an important breakthrough.

(akt), SKG, and aPKC (see legend to Figure 43-8 for

The insulin and IGF-I receptors also contain intrinsic

definitions and expanded abbreviations). An alternative

466

CHAPTER 43

RECOGNITION

(HYPERGLYCEMIA)

INSULIN

P-Y

Y-P

IRS 1-4

SIGNAL

P-Y

Y-P

GRB2/mSOS

GENERATION

PI3 -

PTEN

kinase

mTOR

p21Ras

Raf-1

PKB

MEK

SGK

p70S6K

MAP

aPKC

kinase

Protein translocation

Enzyme activity

Gene transcription

Cell growth

DNA synthesis

EFFECTS

Glucose transporter

Insulin receptor

PEPCK

HKII

Early response

Insulin receptor

Protein phosphatases

Glucagon

Glucokinase

genes

IGF-II receptor

Phosphodiesterases*

IGFBP1

> 100 others

Others

Figure 43-8. Insulin signaling pathways. The insulin signaling pathways provide an excellent example of the

“recognition → hormone release → signal generation → effects” paradigm outlined in Figure 43-1. Insulin is re-

leased in response to hyperglycemia. Binding of insulin to a target cell-specific plasma membrane receptor results in

a cascade of intracellular events. Stimulation of the intrinsic tyrosine kinase activity of the insulin receptor marks the

initial event, resulting in increased tyrosine (Y) phosphorylation (Y → Y-P) of the receptor and then one or more of

the insulin receptor substrate molecules (IRS 1-4). This increase in phosphotyrosine stimulates the activity of many

intracellular molecules such as GTPases, protein kinases, and lipid kinases, all of which play a role in certain meta-

bolic actions of insulin. The two best-described pathways are shown. First, phosphorylation of an IRS molecule

(probably IRS-2) results in docking and activation of the lipid kinase, PI-3 kinase, which generates novel inositol lipids

that may act as “second messenger” molecules. These, in turn, activate PDK1 and then a variety of downstream sig-

naling molecules, including protein kinase B (PKB or akt), SGK, and aPKC. An alternative pathway involves the activa-

tion of p70S6K and perhaps other as yet unidentified kinases. Second, phosphorylation of IRS (probably IRS-1) re-

sults in docking of GRB2/mSOS and activation of the small GTPase, p21RAS, which initiates a protein kinase cascade

that activates Raf-1, MEK, and the p42/p44 MAP kinase isoforms. These protein kinases are important in the regula-

tion of proliferation and differentiation of several cell types. The mTOR pathway provides an alternative way of acti-

vating p70S6K and appears to be involved in nutrient signaling as well as insulin action. Each of these cascades may

influence different physiologic processes, as shown. Each of the phosphorylation events is reversible through the

action of specific phosphatases. For example, the lipid phosphatase PTEN dephosphorylates the product of the PI-3

kinase reaction, thereby antagonizing the pathway and terminating the signal. Representative effects of major ac-

tions of insulin are shown in each of the boxes. The asterisk after phosphodiesterase indicates that insulin indirectly

affects the activity of many enzymes by activating phosphodiesterases and reducing intracellular cAMP levels.

(IGFBP, insulin-like growth factor binding protein; IRS 1-4, insulin receptor substrate isoforms 1-4); PI-3 kinase, phos-

phatidylinositol 3-kinase; PTEN, phosphatase and tensin homolog deleted on chromosome 10; PKD1, phosphoinosi-

tide-dependent kinase; PKB, protein kinase B; SGK, serum and glucocorticoid-regulated kinase; aPKC, atypical pro-

tein kinase C; p70S6K, p70 ribosomal protein S6 kinase; mTOR, mammalian target of rapamycin; GRB2, growth factor

receptor binding protein 2; mSOS, mammalian son of sevenless; MEK, MAP kinase kinase and ERK kinase; MAP

kinase, mitogen-activated protein kinase.)

HORMONE ACTION & SIGNAL TRANSDUCTION

467

pathway downstream from PKD1 involves p70S6K and

balancing actions of phosphatases. Two mechanisms

perhaps other as yet unidentified kinases. A second major

are employed to initiate this cascade. Some hormones,

pathway involves mTOR. This enzyme is directly regu-

such as growth hormone, prolactin, erythropoietin, and

lated by amino acids and insulin and is essential for

the cytokines, initiate their action by activating a tyro-

p70S6K activity. This pathway provides a distinction

sine kinase, but this activity is not an integral part of

between the PKB and p70S6K branches downstream

the hormone receptor. The hormone-receptor interac-

from PKD1. These pathways are involved in protein

tion promotes binding and activation of cytoplasmic

translocation, enzyme activity, and the regulation, by

protein tyrosine kinases, such as Tyk-2, Jak1, or

insulin, of genes involved in metabolism (Figure 43-8).

Jak2. These kinases phosphorylate one or more cyto-

Another SH2 domain-containing protein is GRB2,

plasmic proteins, which then associate with other dock-

which binds to IRS-1 and links tyrosine phosphoryla-

ing proteins through binding to SH2 domains. One

tion to several proteins, the result of which is activation

such interaction results in the activation of a family of

of a cascade of threonine and serine kinases. A pathway

cytosolic proteins called signal transducers and activa-

showing how this insulin-receptor interaction activates

tors of transcription (STATs). The phosphorylated

the mitogen-activated protein (MAP) kinase pathway

STAT protein dimerizes and translocates into the nu-

and the anabolic effects of insulin is illustrated in Fig-

cleus, binds to a specific DNA element such as the in-

ure 43-8. The exact roles of many of these docking

terferon response element, and activates transcription.

proteins, kinases, and phosphatases remain to be estab-

This is illustrated in Figure 43-9. Other SH2 docking

lished.

events may result in the activation of PI 3-kinase, the

MAP kinase pathway (through SHC or GRB2), or G

B. THE JAK/STAT PATHWAY IS USED

protein-mediated activation of phospholipase C (PLCγ)

BY HORMONES AND CYTOKINES

with the attendant production of diacylglycerol and ac-

Tyrosine kinase activation can also initiate a phosphor-

tivation of protein kinase C. It is apparent that there is

ylation and dephosphorylation cascade that involves the

a potential for “cross-talk” when different hormones ac-

action of several other protein kinases and the counter-

tivate these various signal transduction pathways.

Ligand

JAK

P JAK

JAK

P P

JAK

STAT

SH2

x =

SHC

Dimerization

GRB2

and

PLCγ

nuclear

PI-3K

translocation

GAP

Figure 43-9. Initiation of signal transduction by receptors linked to Jak ki-

nases. The receptors (R) that bind prolactin, growth hormone, interferons, and cy-

tokines lack endogenous tyrosine kinase. Upon ligand binding, these receptors

dimerize and an associated protein (Jak1, Jak2, or TYK) is phosphorylated. Jak-P,

an active kinase, phosphorylates the receptor on tyrosine residues. The STAT pro-

teins associate with the phosphorylated receptor and then are themselves phos-

phorylated by Jak-P. STAT P dimerizes, translocates to the nucleus, binds to spe-

cific DNA elements, and regulates transcription. The phosphotyrosine residues of

the receptor also bind to several SH2 domain-containing proteins. This results in

activation of the MAP kinase pathway (through SHC or GRB2), PLCγ, or PI-3 kinase.

468

CHAPTER 43

C. THE NF- B PATHWAY IS

Glucocorticoid hormones are therapeutically useful

agents for the treatment of a variety of inflammatory and

REGULATED BY GLUCOCORTICOIDS

immune diseases. Their anti-inflammatory and im-

The transcription factor NF-κB is a heterodimeric

munomodulatory actions are explained in part by the in-

complex typically composed of two subunits termed

hibition of NF-κB and its subsequent actions. Evidence

p50 and p65 (Figure 43-10). Normally, NF-κB is kept

for three mechanisms for the inhibition of NF-κB by

sequestered in the cytoplasm in a transcriptionally inac-

glucocorticoids has been presented: (1) Glucocorticoids

tive form by members of the inhibitor of NF-κB (IκB)

increase IκB mRNA, which leads to an increase of IκB

family. Extracellular stimuli such as proinflammatory

protein and more efficient sequestration of NF-κB in the

cytokines, reactive oxygen species, and mitogens lead to

cytoplasm.

(2) The glucocorticoid receptor competes

activation of the IκB kinase complex, IKK, which is a

with NF-κB for binding to coactivators. (3) The gluco-

heterohexameric structure consisting of α, β, and γ sub-

corticoid receptor directly binds to the p65 subunit of

units. IKK phosphorylates IκB on two serine residues,

NF-κB and inhibits its activation (Figure 43-10).

and this targets IκB for ubiquitination and subsequent

degradation by the proteasome. Following IκB degra-

dation, free NF-κB can now translocate to the nucleus,

HORMONES CAN INFLUENCE

where it binds to a number of gene promoters and acti-

SPECIFIC BIOLOGIC EFFECTS BY

vates transcription, particularly of genes involved in the

MODULATING TRANSCRIPTION

inflammatory response. Transcriptional regulation by

NF-κB is mediated by a variety of coactivators such as

The signals generated as described above have to be

CREB binding protein (CBP), as described below (Fig-

translated into an action that allows the cell to effec-

ure 43-13).

tively adapt to a challenge (Figure 43-1). Much of this

NF-κB Activators

Proinflammatory cytokines

Bacterial and viral infection

Reactive oxygen species

Mitogens

IKK

complex

P P

Proteasome

IκB

β β

Ubiquitin

IκB

p50

p65

Cytoplasm

p50

p65

Nucleus

Coactivators

p50

p65

Target gene

Figure 43-10. Regulation of the NF-κB pathway. NF-κB consists of two sub-

units, p50 and p65, which regulate transcription of many genes when in the

nucleus. NF-κB is restricted from entering the nucleus by IκB, an inhibitor of

NF-κB. IκB binds to—and masks—the nuclear localization signal of NF-κB.

This cytoplasmic protein is phosphorylated by an IKK complex which is acti-

vated by cytokines, reactive oxygen species, and mitogens. Phosphorylated

IκB can be ubiquitinylated and degraded, thus releasing its hold on NF-κB. Glu-

cocorticoids affect many steps in this process, as described in the text.

HORMONE ACTION & SIGNAL TRANSDUCTION

469

Figure 43-11. The hormone response transcrip-

tion unit. The hormone response transcription unit

is an assembly of DNA elements and bound pro-

teins that interact, through protein-protein interac-

tions, with a number of coactivator or corepressor

molecules. An essential component is the hormone

response element which binds the ligand (

bound receptor (R). Also important are the acces-

sory factor elements (AFEs) with bound transcrip-

tion factors. More than two dozen of these

accessory factors (AFs), which are often members of

the nuclear receptor superfamily, have been linked

p160

to hormone effects on transcription. The AFs can in-

teract with each other, with the liganded nuclear

p300

receptors, or with coregulators. These components

communicate with the basal transcription complex

through a coregulator complex that can consist of

BTC

one or more members of the p160, corepressor,

mediator-related, or CBP/p300 families (see

Table 43-6).

adaptation is accomplished through alterations in the

tion initiation site, but they may be located within the

rates of transcription of specific genes. Many different

coding region of the gene, in introns. HREs were de-

observations have led to the current view of how hor-

fined by the strategy illustrated in Figure 39-11. The

mones affect transcription. Some of these are as follows:

consensus sequences illustrated in Table 43-1 were ar-

(1) Actively transcribed genes are in regions of “open”

rived at through analysis of several genes regulated by a

chromatin (defined by a susceptibility to the enzyme

given hormone using simple, heterologous reporter sys-

DNase I), which allows for the access of transcription

tems (see Figure 39-10). Although these simple HREs

factors to DNA. (2) Genes have regulatory regions, and

bind the hormone-receptor complex more avidly than

transcription factors bind to these to modulate the fre-

surrounding DNA—or DNA from an unrelated

quency of transcription initiation. (3) The hormone-

source—and confer hormone responsiveness to a re-

receptor complex can be one of these transcription fac-

porter gene, it soon became apparent that the regula-

tors. The DNA sequence to which this binds is called a

tory circuitry of natural genes must be much more

hormone response element (HRE; see Table 43-1 for

complicated. Glucocorticoids, progestins, mineralocor-

examples). (4) Alternatively, other hormone-generated

ticoids, and androgens have vastly different physiologic

signals can modify the location, amount, or activity of

actions. How could the specificity required for these ef-

transcription factors and thereby influence binding to

fects be achieved through regulation of gene expression

the regulatory or response element. (5) Members of a

by the same HRE (Table 43-1)? Questions like this

large superfamily of nuclear receptors act with—or in a

have led to experiments which have allowed for elabora-

manner analogous to—hormone receptors. (6) These

tion of a very complex model of transcription regula-

nuclear receptors interact with another large group of

tion. For example, the HRE must associate with other

coregulatory molecules to effect changes in the tran-

DNA elements (and associated binding proteins) to

scription of specific genes.

function optimally. The extensive sequence similarity

noted between steroid hormone receptors, particularly

in their DNA-binding domains, led to discovery of the

Several Hormone Response Elements

nuclear receptor superfamily of proteins. These—and

(HREs) Have Been Defined

a large number of coregulator proteins—allow for a

Hormone response elements resemble enhancer ele-

wide variety of DNA-protein and protein-protein inter-

ments in that they are not strictly dependent on posi-

actions and the specificity necessary for highly regulated

tion and location. They generally are found within a

physiologic control. A schematic of such an assembly is

few hundred nucleotides upstream (5′) of the transcrip-

illustrated in Figure 43-11.

470

CHAPTER 43

A/B

AF-1

DBD

Hinge

LBD

AF-2

GR, MR, PR

TR, RAR, VDR

COUP-TF, TR2, GEN8

AR, ER

PPARα, β, γ

HNF-4, TLX

FXR, CAR, LXR,

PXR/SXR

Receptors:

Steroid class

RXR partnered

Orphans

Binding:

Homodimers

Heterodimers

Homodimers

Ligand:

Steroids

9-Cis RA + (x)

DNA element:

Inverted

Direct repeats

repeat

Figure 43-12. The nuclear receptor superfamily. Members of this family are

divided into six structural domains (A-F). Domain A/B is also called AF-1, or the

modulator region, because it is involved in activating transcription. The C do-

main consists of the DNA-binding domain (DBD). The D region contains the

hinge, which provides flexibility between the DBD and the ligand-binding do-

main (LBD, region E). The amino (N) terminal part of region E contains AF-2, an-

other domain important for transactivation. The F region is poorly defined. The

functions of these domains are discussed in more detail in the text. Receptors

with known ligands, such as the steroid hormones, bind as homodimers on in-

verted repeat half-sites. Other receptors form heterodimers with the partner

RXR on direct repeat elements. There can be nucleotide spacers of one to five

bases between these direct repeats (DR1-5). Another class of receptors for

which ligands have not been determined (orphan receptors) bind as homo-

dimers to direct repeats and occasionally as monomers to a single half-site.

receptor [RXR] partner), or as monomers. The target

There Is a Large Family of Nuclear

response element consists of one or two half-site con-

Receptor Proteins

sensus sequences arranged as an inverted or direct re-

The nuclear receptor superfamily consists of a diverse set

peat. The spacing between the latter helps determine

of transcription factors that were discovered because of a

binding specificity. Thus, a direct repeat with three,

sequence similarity in their DNA-binding domains. This

four, or five nucleotide spacer regions specifies the

family, now with more than 50 members, includes the

binding of the vitamin D, thyroid, and retinoic acid re-

nuclear hormone receptors discussed above, a number of

ceptors, respectively, to the same consensus response

other receptors whose ligands were discovered after the

element

(Table

43-1). A multifunctional ligand-

receptors were identified, and many putative or orphan

binding domain (LBD) is located in the carboxyl ter-

receptors for which a ligand has yet to be discovered.

minal half of the receptor. The LBD binds hormones

These nuclear receptors have several common struc-

or metabolites with selectivity and thus specifies a par-

tural features (Figure 43-12). All have a centrally lo-

ticular biologic response. The LBD also contains do-

cated DNA-binding domain (DBD) that allows the

mains that mediate the binding of heat shock proteins,

receptor to bind with high affinity to a response ele-

dimerization, nuclear localization, and transactivation.

ment. The DBD contains two zinc finger binding mo-

The latter function is facilitated by the carboxyl termi-

tifs (see Figure 39-14) that direct binding either as ho-

nal transcription activation function (AF-2 domain),

modimers, as heterodimers (usually with a retinoid X

which forms a surface required for the interaction with

HORMONE ACTION & SIGNAL TRANSDUCTION

471

Group I

GH, Prl,

Insulin,

GPCR

Hormones

Cytokines, etc

EGF, etc

TNF, etc

Jak

cAMP

Retinoic acid,

RAS

Plasma

IRS

estrogen,

membrane

vitamin D,

NFκB•IκB

glucocorticoids,

MEK

etc

STATs

PKA

NFκB

MAPK

Nuclear

CREB

receptors

Nuclear

STATs

membrane

AP-1

NFκB

CBP

p300

Figure 43-13. Several signal transduction pathways converge on

CBP/p300. Ligands that associate with membrane or nuclear receptors even-

tually converge on CBP/p300. Several different signal transduction pathways

are employed. EGF, epidermal growth factor; GH, growth hormone; Prl, pro-

lactin; TNF, tumor necrosis factor; other abbreviations are expanded in the

text.

coactivators. A highly variable hinge region separates

Another group of orphan receptors that as yet have no

the DBD from the LBD. This region provides flexibil-

known ligand bind as homodimers or monomers to di-

ity to the receptor, so it can assume different DNA-

rect repeat sequences.

binding conformations. Finally, there is a highly vari-

As illustrated in Table 43-5, the discovery of the nu-

able amino terminal region that contains another trans-

clear receptor superfamily has led to an important un-

activation domain referred to as AF-1. Less well de-

derstanding of how a variety of metabolites and xenobi-

fined, the AF-1 domain may provide for distinct

otics regulate gene expression and thus the metabolism,

physiologic functions through the binding of different

detoxification, and elimination of normal body prod-

coregulator proteins. This region of the receptor,

ucts and exogenous agents such as pharmaceuticals.

through the use of different promoters, alternative

Not surprisingly, this area is a fertile field for investiga-

splice sites, and multiple translation initiation sites,

tion of new therapeutic interventions.

provides for receptor isoforms that share DBD and

LBD identity but exert different physiologic responses

A Large Number of Nuclear Receptor

because of the association of various coregulators with

Coregulators Also Participate

this variable amino terminal AF-1 domain.

in Regulating Transcription

It is possible to sort this large number of receptors

into groups in a variety of ways. Here they are discussed

Chromatin remodeling, transcription factor modification

according to the way they bind to their respective DNA

by various enzyme activities, and the communication

elements (Figure 43-12). Classic hormone receptors for

between the nuclear receptors and the basal transcrip-

glucocorticoids (GR), mineralocorticoids (MR), estro-

tion apparatus are accomplished by protein-protein in-

gens (ER), androgens (AR), and progestins (PR) bind

teractions with one or more of a class of coregulator

as homodimers to inverted repeat sequences. Other

molecules. The number of these coregulator molecules

hormone receptors such as thyroid (TR), retinoic acid

now exceeds 100, not counting species variations and

(RAR), and vitamin D (VDR) and receptors that bind

splice variants. The first of these to be described was the

various metabolite ligands such as PPAR α β, and γ,

CREB-binding protein, CBP. CBP, through an

FXR, LXR, PXR/SXR, and CAR bind as heterodimers,

amino terminal domain, binds to phosphorylated serine

with retinoid X receptor (RXR) as a partner, to direct

137 of CREB and mediates transactivation in response

repeat sequences (see Figure 43-12 and Table 43-5).

to cAMP. It thus is described as a coactivator. CBP and

472

CHAPTER 43

Table 43-5. Nuclear receptors with special ligands.¹

Receptor

Partner

Ligand

Process Affected

Peroxisome PPARα

RXR (DR1)

Fatty acids

Peroxisome proliferation

Proliferator- PPARβ

Fatty acids

activated PPARγ

Fatty acids

Lipid and carbohydrate metabolism

Eicosanoids,

thiazolidinediones

Farnesoid X FXR

RXR (DR4)

Farnesol, bile acids

Bile acid metabolism

Liver X

LXR

RXR (DR4)

Oxysterols

Cholesterol metabolism

Xenobiotic X CAR

RXR (DR5)

Androstanes

Phenobarbital

Protection against certain drugs, toxic

Xenobiotics

metabolites, and xenobiotics

PXR

RXR (DR3)

Pregnanes

Xenobiotics

¹Many members of the nuclear receptor superfamily were discovered by cloning, and the corresponding

ligands were then identified. These ligands are not hormones in the classic sense, but they do have a

similar function in that they activate specific members of the nuclear receptor superfamily. The recep-

tors described here form heterodimers with RXR and have variable nucleotide sequences separating the

direct repeat binding elements (DR1-5). These receptors regulate a variety of genes encoding cy-

tochrome p450s (CYP), cytosolic binding proteins, and ATP-binding cassette (ABC) transporters to influ-

ence metabolism and protect cells against drugs and noxious agents.

its

relative, p300, interact directly or indirectly

with a number of signaling molecules, including activa-

tor protein-1 (AP-1), signal transducers and activators

of transcription (STATs), nuclear receptors, and CREB

(Figure

39-11). CBP/p300 also binds to the p160

family of coactivators described below and to a number

Table 43-6. Some mammalian coregulator

of other proteins, including viral transcription factor

proteins.

Ela, the p90^rsk protein kinase, and RNA helicase A. It is

important to note that CBP/p300 also has intrinsic

300-kDa family of coactivators

histone acetyltransferase (HAT) activity. The impor-

CBP

CREB-binding protein

tance of this is described below. Some of the many ac-

p300

Protein of 300 kDa

tions of CBP/p300, which appear to depend on intrin-

II.

160-kDa family of coactivators

sic enzyme activities and its ability to serve as a scaffold

A. SRC-1

Steroid receptor coactivator 1

for the binding of other proteins, are illustrated in Fig-

NCoA-1

Nuclear receptor coactivator 1

ure 43-11. Other coregulators may serve similar func-

B. TIF2

Transcriptional intermediary factor 2

tions.

GRIP1

Glucocorticoid receptor-interacting protein

Several other families of coactivator molecules have

NCoA-2

Nuclear receptor coactivator 2

been described. Members of the p160 family of coac-

C. p/CIP

p300/CBP cointegrator-associated protein 1

tivators, all of about 160 kDa, include (1) SRC-1 and

ACTR Activator of the thyroid and retinoic acid

NCoA-1; (2) GRIP 1, TIF2, and NCoA-2; and (3)

receptors

p/CIP, ACTR, AIB1, RAC3, and TRAM-1 (Table

AIB

Amplified in breast cancer

43-6). The different names for members within a sub-

RAC3

Receptor-associated coactivator 3

family often represent species variations or minor splice

TRAM-1

TR activator molecule 1

variants. There is about 35% amino acid identity be-

III. Corepressors

tween members of the different subfamilies. The p160

NCoR

Nuclear receptor corepressor

coactivators share several properties. They

(1) bind

SMRT

Silencing mediator for RXR and TR

IV. Mediator-related proteins

nuclear receptors in an agonist and AF-2 transactiva-

TRAPs

Thyroid hormone receptor-associated

tion domain-dependent manner; (2) have a conserved

proteins

amino terminal basic helix-loop-helix (bHLH) motif

DRIPs

Vitamin D receptor-interacting proteins

(see Chapter 39); (3) have a weak carboxyl terminal

ARC

Activator-recruited cofactor

transactivation domain and a stronger amino terminal

HORMONE ACTION & SIGNAL TRANSDUCTION

473

transactivation domain in a region that is required for

• Many hormone responses are accomplished through

the CBP/p16O interaction; (4) contain at least three of

alterations in the rate of transcription of specific

the LXXLL motifs required for protein-protein inter-

genes.

action with other coactivators; and (5) often have HAT

• The nuclear receptor superfamily of proteins plays a

activity. The role of HAT is particularly interesting, as

central role in the regulation of gene transcription.

mutations of the HAT domain disable many of these

• These receptors, which may have hormones, metabo-

transcription factors. Current thinking holds that these

lites, or drugs as ligands, bind to specific DNA ele-

HAT activities acetylate histones and result in remodel-

ments as homodimers or as heterodimers with RXR.

ing of chromatin into a transcription-efficient environ-

Some—orphan receptors—have no known ligand

ment; however, other protein substrates for HAT-

but bind DNA and influence transcription.

mediated acetylation have been reported. Histone

• Another large family of coregulator proteins remodel

acetylation/deacetylation is proposed to play a critical

chromatin, modify other transcription factors, and

role in gene expression.

bridge the nuclear receptors to the basal transcription

A small number of proteins, including NCoR and

apparatus.

SMRT, comprise the corepressor family. They func-

tion, at least in part, as described in Figure 43-2. An-

other family includes the TRAPs, DRIPs, and ARC

REFERENCES

(Table 43-6). These so-called mediator-related pro-

teins range in size from 80 kDa to 240 kDa and are

Arvanitakis L et al: Constitutively signaling G-protein-coupled re-

ceptors and human disease. Trends Endocrinol Metab

thought to be involved in linking the nuclear receptor-

1998;9:27.

coactivator complex to RNA polymerase II and the

Berridge M: Inositol triphosphate and calcium signalling. Nature

other components of the basal transcription apparatus.

1993;361:315.

The exact role of these coactivators is presently

Chawla A et al: Nuclear receptors and lipid physiology: opening the

under intensive investigation. Many of these proteins

X files. Science 2001;294:1866.

have intrinsic enzymatic activities. This is particularly

Darnell JE Jr, Kerr IM, Stark GR: Jak-STAT pathways and trans-

interesting in view of the fact that acetylation, phos-

criptional activation in response to IFNs and other extracellu-

phorylation, methylation, and ubiquitination—as well

lar signaling proteins. Science 1994;264:1415.

as proteolysis and cellular translocation—have been

Fantl WJ, Johnson DE, Williams LT: Signalling by receptor tyro-

proposed to alter the activity of some of these coregula-

sine kinases. Annu Rev Biochem 1993;62:453.

tors and their targets.

Giguère V: Orphan nuclear receptors: from gene to function. En-

It appears that certain combinations of coregula-

docr Rev 1999;20:689.

tors—and thus different combinations of activators and

Grunstein M: Histone acetylation in chromatin structure and trans-

inhibitors—are responsible for specific ligand-induced

cription. Nature 1997;389:349.

actions through various receptors.

Hanoune J, Defer N: Regulation and role of adenylyl cyclase iso-

forms. Annu Rev Pharmacol Toxicol 2001;41:145.

Hermanson O, Glass CK, Rosenfeld MG: Nuclear receptor coregu-

SUMMARY

lators: multiple modes of receptor modification. Trends En-

• Hormones, cytokines, interleukins, and growth fac-

docrinol Metab 2002;13:55.

tors use a variety of signaling mechanisms to facilitate

Jaken S: Protein kinase C isozymes and substrates. Curr Opin Cell

cellular adaptive responses.

Biol 1996;8:168.

Lucas P, Granner D: Hormone response domains in gene transcrip-

• The ligand-receptor complex serves as the initial sig-

tion. Annu Rev Biochem 1992;61:1131.

nal for members of the nuclear receptor family.

Montminy M: Transcriptional regulation by cyclic AMP. Annu

• Class II hormones, which bind to cell surface recep-

Rev Biochem 1997;66:807

tors, generate a variety of intracellular signals. These

Morris AJ, Malbon CC: Physiological regulation of G protein-

include cAMP, cGMP, Ca2+, phosphatidylinositides,

linked signaling. Physiol Rev 1999;79:1373.

and protein kinase cascades.

Walton KM, Dixon JE: Protein tyrosine phosphatases. Annu Rev

Biochem 1993;62:101.

SECTION VI

Special Topics

Nutrition, Digestion, & Absorption

David A. Bender, PhD, & Peter A. Mayes, PhD, DSc

BIOMEDICAL IMPORTANCE

and steatorrhea. Lactose intolerance is due to lactase

deficiency leading to diarrhea and intestinal discomfort.

Besides water, the diet must provide metabolic fuels

Absorption of intact peptides that stimulate antibody

(mainly carbohydrates and lipids), protein (for growth

responses causes allergic reactions, and celiac disease

and turnover of tissue proteins), fiber (for roughage),

is an allergic reaction to wheat gluten.

minerals (elements with specific metabolic functions),

and vitamins and essential fatty acids (organic com-

pounds needed in small amounts for essential metabolic

DIGESTION & ABSORPTION

and physiologic functions). The polysaccharides, tri-

OF CARBOHYDRATES

acylglycerols, and proteins that make up the bulk of the

diet must be hydrolyzed to their constituent monosac-

The digestion of complex carbohydrates is by hydroly-

charides, fatty acids, and amino acids, respectively, be-

sis to liberate oligosaccharides, then free mono- and di-

fore absorption and utilization. Minerals and vitamins

saccharides. The increase in blood glucose after a test

must be released from the complex matrix of food be-

dose of a carbohydrate compared with that after an

fore they can be absorbed and utilized.

equivalent amount of glucose is known as the glycemic

Globally, undernutrition is widespread, leading to

index. Glucose and galactose have an index of 1, as do

impaired growth, defective immune systems, and re-

lactose, maltose, isomaltose, and trehalose, which give

duced work capacity. By contrast, in developed coun-

rise to these monosaccharides on hydrolysis. Fructose

tries, there is often excessive food consumption (espe-

and the sugar alcohols are absorbed less rapidly and

cially of fat), leading to obesity and to the development

have a lower glycemic index, as does sucrose. The

of cardiovascular disease and some forms of cancer. De-

glycemic index of starch varies between near 1 to near

ficiencies of vitamin A, iron, and iodine pose major

zero due to variable rates of hydrolysis, and that of non-

health concerns in many countries, and deficiencies of

starch polysaccharides is zero. Foods that have a low

other vitamins and minerals are a major cause of ill

glycemic index are considered to be more beneficial

health. In developed countries, nutrient deficiency is

since they cause less fluctuation in insulin secretion.

rare, though there are vulnerable sections of the popula-

tion at risk. Intakes of minerals and vitamins that are

adequate to prevent deficiency may be inadequate to

Amylases Catalyze

promote optimum health and longevity.

the Hydrolysis of Starch

Excessive secretion of gastric acid, associated with

Helicobacter pylori infection, can result in the develop-

The hydrolysis of starch by salivary and pancreatic

ment of gastric and duodenal ulcers; small changes in

amylases catalyze random hydrolysis of α(1→4) glyco-

the composition of bile can result in crystallization of

side bonds, yielding dextrins, then a mixture of glucose,

cholesterol as gallstones; failure of exocrine pancreatic

maltose, and isomaltose (from the branch points in

secretion (as in cystic fibrosis) leads to undernutrition

amylopectin).

474

NUTRITION, DIGESTION, & ABSORPTION

475

Disaccharidases Are Brush

SGLT 1

transporter

Border Enzymes

Glucose

protein

Glucose

Na⁺

The disaccharidases—maltase, sucrase-isomaltase

Galactose

Glucose

bifunctional enzyme catalyzing hydrolysis of sucrose and

Fructose

Galactose

isomaltose), lactase, and trehalase—are located on the

brush border of the intestinal mucosal cells where the re-

GLUT 5

sultant monosaccharides and others arising from the diet

are absorbed. In most people, apart from those of north-

ern European genetic origin, lactase is gradually lost

through adolescence, leading to lactose intolerance.

Brush

border

Lactose remains in the intestinal lumen, where it is a

substrate for bacterial fermentation to lactate, resulting

in discomfort and diarrhea.

Na⁺ -K⁺

Intestinal

pump

There Are Two Separate Mechanisms

epithelium

for the Absorption of Monosaccharides

Na⁺

ATP

in the Small Intestine

3Na⁺

Glucose

Glucose and galactose are absorbed by a sodium-depen-

Fructose

2K⁺

dent process. They are carried by the same transport

Galactose

2K⁺

ADP

protein (SGLT 1) and compete with each other for in-

+ P_i

testinal absorption (Figure 44-1). Other monosaccha-

rides are absorbed by carrier-mediated diffusion. Be-

cause they are not actively transported, fructose and

To capillaries

sugar alcohols are only absorbed down their concentra-

GLUT 2

tion gradient, and after a moderately high intake some

Figure 44-1. Transport of glucose, fructose, and

may remain in the intestinal lumen, acting as a sub-

galactose across the intestinal epithelium. The SGLT 1

strate for bacterial fermentation.

transporter is coupled to the Na⁺-K⁺ pump, allowing

glucose and galactose to be transported against their

DIGESTION & ABSORPTION OF LIPIDS

concentration gradients. The GLUT 5 Na⁺-independent

The major lipids in the diet are triacylglycerols and, to a

facilitative transporter allows fructose as well as glu-

lesser extent, phospholipids. These are hydrophobic

cose and galactose to be transported with their con-

molecules and must be hydrolyzed and emulsified to

centration gradients. Exit from the cell for all the sugars

very small droplets (micelles) before they can be ab-

is via the GLUT 2 facilitative transporter.

sorbed. The fat-soluble vitamins—A, D, E, and K—

and a variety of other lipids (including cholesterol) are

absorbed dissolved in the lipid micelles. Absorption of

the fat-soluble vitamins is impaired on a very low fat

of the products of lipid digestion into micelles and lipo-

diet.

somes together with phospholipids and cholesterol

Hydrolysis of triacylglycerols is initiated by lingual

from the bile. Because the micelles are soluble, they

and gastric lipases that attack the sn-3 ester bond, form-

allow the products of digestion, including the fat-

ing 1,2-diacylglycerols and free fatty acids, aiding emul-

soluble vitamins, to be transported through the aqueous

sification. Pancreatic lipase is secreted into the small

environment of the intestinal lumen and permit close

intestine and requires a further pancreatic protein, coli-

contact with the brush border of the mucosal cells, al-

pase, for activity. It is specific for the primary ester

lowing uptake into the epithelium, mainly of the je-

links—ie, positions 1 and 3 in triacylglycerols—result-

junum. The bile salts pass on to the ileum, where

ing in 2-monoacylglycerols and free fatty acids as the

most are absorbed into the enterohepatic circula-

major end-products of luminal triacylglycerol digestion.

tion (Chapter 26). Within the intestinal epithelium,

Monoacylglycerols are hydrolyzed with difficulty to

1-monoacylglycerols are hydrolyzed to fatty acids and

glycerol and free fatty acids, so that less than 25% of in-

glycerol and 2-monoacylglycerols are re-acylated to tri-

gested triacylglycerol is completely hydrolyzed to glyc-

acylglycerols via the monoacylglycerol pathway. Glyc-

erol and fatty acids (Figure 44-2). Bile salts, formed in

erol released in the intestinal lumen is not reutilized but

the liver and secreted in the bile, enable emulsification

passes into the portal vein; glycerol released within the

NUTRITION, DIGESTION, & ABSORPTION

477

epithelium is reutilized for triacylglycerol synthesis via

several different amino acid transporters, with specificity

the normal phosphatidic acid pathway (Chapter 24).

for the nature of the amino acid side chain (large or

All long-chain fatty acids absorbed are converted to tri-

small; neutral, acidic, or basic). The various amino acids

acylglycerol in the mucosal cells and, together with the

carried by any one transporter compete with each other

other products of lipid digestion, secreted as chylomi-

for absorption and tissue uptake. Dipeptides and tripep-

crons into the lymphatics, entering the blood stream via

tides enter the brush border of the intestinal mucosal

the thoracic duct (Chapter 25).

cells, where they are hydrolyzed to free amino acids,

which are then transported into the hepatic portal vein.

Relatively large peptides may be absorbed intact, either

DIGESTION & ABSORPTION OF PROTEINS

by uptake into mucosal epithelial cells (transcellular) or

Few peptide bonds that are hydrolyzed by proteolytic

by passing between epithelial cells (paracellular). Many

enzymes are accessible without prior denaturation of di-

such peptides are large enough to stimulate antibody for-

etary proteins (by heat in cooking and by the action of

mation—this is the basis of allergic reactions to foods.

gastric acid).

DIGESTION & ABSORPTION

Several Groups of Enzymes Catalyze

OF VITAMINS & MINERALS

the Digestion of Proteins

Vitamins and minerals are released from food during

There are two main classes of proteolytic digestive en-

digestion—though this is not complete—and the avail-

zymes (proteases), with different specificities for the

ability of vitamins and minerals depends on the type of

amino acids forming the peptide bond to be hydrolyzed.

food and, especially for minerals, the presence of chelat-

Endopeptidases hydrolyze peptide bonds between spe-

ing compounds. The fat-soluble vitamins are absorbed

cific amino acids throughout the molecule. They are the

in the lipid micelles that result from fat digestion;

first enzymes to act, yielding a larger number of smaller

water-soluble vitamins and most mineral salts are

fragments, eg, pepsin in the gastric juice and trypsin,

absorbed from the small intestine either by active trans-

chymotrypsin, and elastase secreted into the small in-

port or by carrier-mediated diffusion followed by bind-

testine by the pancreas. Exopeptidases catalyze the hy-

ing to intracellular binding proteins to achieve concen-

drolysis of peptide bonds, one at a time, from the ends

tration upon uptake. Vitamin B₁₂ absorption requires a

of polypeptides. Carboxypeptidases, secreted in the

specific transport protein, intrinsic factor; calcium ab-

pancreatic juice, release amino acids from the free car-

sorption is dependent on vitamin D; zinc absorption

boxyl terminal, and aminopeptidases, secreted by the

probably requires a zinc-binding ligand secreted by the

intestinal mucosal cells, release amino acids from the

exocrine pancreas; and the absorption of iron is limited.

amino terminal. Dipeptides, which are not substrates for

exopeptidases, are hydrolyzed in the brush border of in-

Calcium Absorption Is Dependent

testinal mucosal cells by dipeptidases.

on Vitamin D

The proteases are secreted as inactive zymogens; the

active site of the enzyme is masked by a small region of

In addition to its role in regulating calcium homeosta-

its peptide chain, which is removed by hydrolysis of a

sis, vitamin D is required for the intestinal absorption

specific peptide bond. Pepsinogen is activated to pepsin

of calcium. Synthesis of the intracellular calcium-

by gastric acid and by activated pepsin (autocatalysis). In

binding protein, calbindin, required for calcium ab-

the small intestine, trypsinogen, the precursor of

sorption, is induced by vitamin D, which also affects

trypsin, is activated by enteropeptidase, which is se-

the permeability of the mucosal cells to calcium, an ef-

creted by the duodenal epithelial cells; trypsin can then

fect that is rapid and independent of protein synthesis.

activate chymotrypsinogen to chymotrypsin, proelas-

Phytic acid (inositol hexaphosphate) in cereals binds

tase to elastase, procarboxypeptidase to carboxypepti-

calcium in the intestinal lumen, preventing its absorp-

dase, and proaminopeptidase to aminopeptidase.

tion. Other minerals, including zinc, are also chelated

by phytate. This is mainly a problem among people

who consume large amounts of unleavened whole

Free Amino Acids & Small Peptides Are

wheat products; yeast contains an enzyme, phytase,

Absorbed by Different Mechanisms

which dephosphorylates phytate, so rendering it inac-

The end product of the action of endopeptidases and

tive. High concentrations of fatty acids in the intestinal

exopeptidases is a mixture of free amino acids, di- and

lumen—as a result of impaired fat absorption—can

tripeptides, and oligopeptides, all of which are absorbed.

also reduce calcium absorption by forming insoluble

Free amino acids are absorbed across the intestinal mu-

calcium salts; a high intake of oxalate can sometimes

cosa by sodium-dependent active transport. There are

cause deficiency, since calcium oxalate is insoluble.

478

CHAPTER 44

Iron Absorption Is Limited

lized is carbohydrate, fat, or protein. Measurement of

& Strictly Controlled but Is

the ratio of the volume of carbon dioxide produced to

Enhanced by Vitamin C & Ethanol

volume of oxygen consumed (respiratory quotient; RQ)

is an indication of the mixture of metabolic fuels being

Although iron deficiency is a common problem, about

oxidized (Table 27-1). A more recent technique per-

10% of the population are genetically at risk of iron

mits estimation of total energy expenditure over a pe-

overload (hemochromatosis), and elemental iron can

riod of 1-2 weeks using dual isotopically labeled water,

lead to nonenzymic generation of free radicals. Absorp-

²H₂₁₈O. ²H is lost from the body only in water, while

tion of iron is strictly regulated. Inorganic iron is accu-

¹⁸O is lost in both water and carbon dioxide; the differ-

mulated in intestinal mucosal cells bound to an intra-

ence in the rate of loss of the two labels permits estima-

cellular protein, ferritin. Once the ferritin in the cell is

tion of total carbon dioxide production and thus oxy-

saturated with iron, no more can enter. Iron can only

gen consumption and energy expenditure.

leave the mucosal cell if there is transferrin in plasma

Basal metabolic rate (BMR) is the energy expendi-

to bind to. Once transferrin is saturated with iron, any

ture by the body when at rest—but not asleep—under

that has accumulated in the mucosal cells will be lost

controlled conditions of thermal neutrality, measured

when the cells are shed. As a result of this mucosal bar-

at about 12 hours after the last meal, and depends on

rier, only about 10% of dietary iron is normally ab-

weight, age, and gender. Total energy expenditure de-

sorbed and only 1-5% from many plant foods.

pends on the basal metabolic rate, the energy required

Inorganic iron is absorbed only in the Fe2+ (reduced)

for physical activity, and the energy cost of synthesizing

state, and for that reason the presence of reducing agents

reserves in the fed state. It is therefore possible to calcu-

will enhance absorption. The most effective compound

late an individual’s energy requirement from body

is vitamin C, and while intakes of 40-60 mg of vitamin

weight, age, gender, and level of physical activity. Body

C per day are more than adequate to meet requirements,

weight affects BMR because there is a greater amount

an intake of 25-50 mg per meal will enhance iron ab-

of active tissue in a larger body. The decrease in BMR

sorption, especially when iron salts are used to treat iron

with increasing age, even when body weight remains

deficiency anemia. Ethanol and fructose also enhance

constant, is due to muscle tissue replacement by adi-

iron absorption. Heme iron from meat is absorbed sepa-

pose tissue, which is metabolically much less active.

rately and is considerably more available than inorganic

Similarly, women have a significantly lower BMR than

iron. However, the absorption of both inorganic and

do men of the same body weight because women’s bod-

heme iron is impaired by calcium—a glass of milk with

ies have proportionately more adipose tissue than men.

a meal significantly reduces availability.

Energy Requirements Increase

ENERGY BALANCE:

With Activity

OVER- & UNDERNUTRITION

The most useful way of expressing the energy cost of

After the provision of water, the body’s first requirement

physical activities is as a multiple of BMR. Sedentary

is for metabolic fuels—fats, carbohydrates, and amino

activities use only about 1.1-1.2 × BMR. By contrast,

acids from proteins (and ethanol) (Table 27-1). Food in-

vigorous exertion, such as climbing stairs, cross-country

take in excess of energy expenditure leads to obesity,

skiing, walking uphill, etc, may use 6-8 × BMR.

while intake less than expenditure leads to emaciation

and wasting, as in marasmus and kwashiorkor. Both

Ten Percent of the Energy Yield of a Meal

obesity and severe undernutrition are associated with in-

May Be Expended in Forming Reserves

creased mortality. The body mass index, defined as

weight in kilograms divided by height in meters squared,

There is a considerable increase in metabolic rate after a

is commonly used as a way of expressing relative obesity

meal, a phenomenon known as diet-induced thermo-

to height. A desirable range is between 20 and 25.

genesis. A small part of this is the energy cost of secret-

ing digestive enzymes and of active transport of the

Energy Requirements Are Estimated by

products of digestion; the major part is due to synthe-

sizing reserves of glycogen, triacylglycerol, and protein.

Measurement of Energy Expenditure

Energy expenditure can be determined directly by mea-

There Are Two Extreme Forms

suring heat output from the body but is normally esti-

of Undernutrition

mated indirectly from the consumption of oxygen.

There is an energy expenditure of 20 kJ/L of oxygen

Marasmus can occur in both adults and children and

consumed regardless of whether the fuel being metabo-

occurs in vulnerable groups of all populations. Kwash-

NUTRITION, DIGESTION, & ABSORPTION

479

iorkor only affects children and has only been reported

the liver due to accumulation of fat. It was formerly be-

in developing countries. The distinguishing feature of

lieved that the cause of kwashiorkor was a lack of pro-

kwashiorkor is that there is fluid retention, leading to

tein, with a more or less adequate energy intake; how-

edema. Marasmus is a state of extreme emaciation; it is

ever, analysis of the diets of affected children shows that

the outcome of prolonged negative energy balance. Not

this is not so. Children with kwashiorkor are less

only have the body’s fat reserves been exhausted, but

stunted than those with marasmus, and the edema be-

there is wastage of muscle as well, and as the condition

gins to improve early in treatment, when the child is

progresses there is loss of protein from the heart, liver,

still receiving a low-protein diet. Very commonly, an

and kidneys. The amino acids released by the catabo-

infection precipitates kwashiorkor. Superimposed on

lism of tissue proteins are used as a source of metabolic

general food deficiency, there is probably a deficiency

fuel and as substrates for gluconeogenesis to maintain a

of the antioxidant nutrients such as zinc, copper,

supply of glucose for the brain and red blood cells. As a

carotene, and vitamins C and E. The respiratory burst

result of the reduced synthesis of proteins, there is im-

in response to infection leads to the production of oxy-

paired immune response and more risk from infections.

gen and halogen free radicals as part of the cytotoxic

Impairment of cell proliferation in the intestinal mu-

action of stimulated macrophages. This added oxidant

cosa occurs, resulting in reduction in surface area of the

stress may well trigger the development of kwashiorkor.

intestinal mucosa and reduction in absorption of such

nutrients as are available.

PROTEIN & AMINO ACID REQUIREMENTS

Patients With Advanced Cancer

Protein Requirements Can Be Determined

& AIDS Are Malnourished

by Measuring Nitrogen Balance

Patients with advanced cancer, HIV infection and

The state of protein nutrition can be determined by

AIDS, and a number of other chronic diseases are fre-

measuring the dietary intake and output of nitrogenous

quently undernourished—the condition is called

compounds from the body. Although nucleic acids also

cachexia. Physically, they show all the signs of maras-

contain nitrogen, protein is the major dietary source of

mus, but there is considerably more loss of body pro-

nitrogen and measurement of total nitrogen intake

tein than occurs in starvation. The secretion of cy-

gives a good estimate of protein intake (mg N × 6.25 =

tokines in response to infection and cancer increases the

mg protein, as nitrogen is 16% of most proteins). The

catabolism of tissue protein. This differs from maras-

output of nitrogen from the body is mainly in urea and

mus, in which protein synthesis is reduced but catabo-

smaller quantities of other compounds in urine and

lism in unaffected. Patients are hypermetabolic, ie,

undigested protein in feces, and significant amounts

there is a considerable increase in basal metabolic rate.

may also be lost in sweat and shed skin.

Many tumors metabolize glucose anaerobically to re-

The difference between intake and output of nitroge-

lease lactate. This is used for gluconeogenesis in the

nous compounds is known as nitrogen balance. Three

liver, which is energy-consuming with a net cost of six

states can be defined: In a healthy adult, nitrogen bal-

ATP for each mole of glucose cycled (Chapter 19).

ance is in equilibrium when intake equals output, and

There is increased stimulation of uncoupling proteins

there is no change in the total body content of protein.

by cytokines, leading to thermogenesis and increased

In a growing child, a pregnant woman, or in recovery

oxidation of metabolic fuels. Futile cycling of lipids oc-

from protein loss, the excretion of nitrogenous com-

curs because hormone-sensitive lipase is activated by a

pounds is less than the dietary intake and there is net re-

proteoglycan secreted by tumors, resulting in liberation

tention of nitrogen in the body as protein, ie, positive

of fatty acids from adipose tissue and ATP-expensive

nitrogen balance. In response to trauma or infection—

reesterification in the liver to triacylglycerols, which are

or if the intake of protein is inadequate to meet require-

exported in VLDL.

ments—there is net loss of protein nitrogen from the

body, ie, negative nitrogen balance. The continual ca-

tabolism of tissue proteins creates the requirement for

Kwashiorkor Affects

dietary protein even in an adult who is not growing,

Undernourished Children

though some of the amino acids released can be reuti-

In addition to the wasting of muscle tissue, loss of in-

lized. Nitrogen balance studies show that the average

testinal mucosa, and impaired immune responses seen

daily requirement is 0.6 g of protein per kilogram of

in marasmus, children with kwashiorkor show a num-

body weight (the factor 0.75 should be used to allow for

ber of characteristic features. The defining characteristic

individual variation), or approximately 50 g/d. Average

is edema, associated with a decreased concentration of

intakes of protein in developed countries are about

plasma proteins. In addition, there is enlargement of

80-100 g/d, ie,

14-15% of energy intake. Because

480

CHAPTER 44

growing children are increasing the protein in the body,

aloacetate, and α-ketoglutarate, respectively). The re-

they have a proportionately greater requirement than

maining amino acids are considered as nonessential, but

adults and should be in positive nitrogen balance. Even

under some circumstances the requirement for them

so, the need is relatively small compared with the re-

may outstrip the organism’s capacity for synthesis.

quirement for protein turnover. In some countries, pro-

tein intake may be inadequate to meet these require-

SUMMARY

ments, resulting in stunting of growth.

•

Digestion involves hydrolyzing food molecules into

There Is a Loss of Body Protein in

smaller molecules for absorption through the

Response to Trauma & Infection

gastrointestinal epithelium. Polysaccharides are

absorbed as monosaccharides; triacylglycerols as

One of the metabolic reactions to major trauma, such

2-monoacylglycerols, fatty acids, and glycerol; and

as a burn, a broken limb, or surgery, is an increase in

proteins as amino acids.

the net catabolism of tissue proteins. As much as 6-7%

•

Digestive disorders arise as a result of (1) enzyme de-

of the total body protein may be lost over 10 days. Pro-

ficiency, eg, lactase and sucrase; (2) malabsorption,

longed bed rest results in considerable loss of protein

eg, of glucose and galactose due to defects in the

because of atrophy of muscles. Protein is catabolized as

Na⁺-glucose cotransporter (SGLT 1); (3) absorption

normal, but without the stimulus of exercise it is not

of unhydrolyzed polypeptides, leading to immuno-

completely replaced. Lost protein is replaced during

logic responses, eg, as in celiac disease; and (4) pre-

convalescence, when there is positive nitrogen balance.

cipitation of cholesterol from bile as gallstones.

A normal diet is adequate to permit this replacement.

•

Besides water, the diet must provide metabolic fuels

(carbohydrate and fat) for bodily growth and activity;

The Requirement Is Not for Protein Itself

protein for synthesis of tissue proteins; fiber for

but for Specific Amino Acids

roughage; minerals for specific metabolic functions;

Not all proteins are nutritionally equivalent. More of

certain polyunsaturated fatty acids of the n-3 and n-6

some than of others is needed to maintain nitrogen

families for eicosanoid synthesis and other functions;

balance because different proteins contain different

and vitamins, organic compounds needed in small

amounts of the various amino acids. The body’s re-

amounts for many varied essential functions.

quirement is for specific amino acids in the correct

•

Twenty different amino acids are required for pro-

proportions to replace the body proteins. The amino

tein synthesis, of which nine are essential in the

acids can be divided into two groups: essential and

human diet. The quantity of protein required is af-

nonessential. There are nine essential or indispensable

fected by protein quality, energy intake, and physical

amino acids, which cannot be synthesized in the body:

activity.

histidine, isoleucine, leucine, lysine, methionine, phen-

•

Undernutrition occurs in two extreme forms: maras-

ylalanine, threonine, tryptophan, and valine. If one of

mus in adults and children and kwashiorkor in chil-

these is lacking or inadequate, then—regardless of the

dren. Overnutrition from excess energy intake is as-

total intake of protein—it will not be possible to main-

sociated with diseases such as obesity, type 2 diabetes

tain nitrogen balance since there will not be enough of

mellitus, atherosclerosis, cancer, and hypertension.

that amino acid for protein synthesis.

Two amino acids—cysteine and tyrosine—can be

synthesized in the body, but only from essential amino

REFERENCES

acid precursors (cysteine from methionine and tyrosine

Bender DA, Bender AE: Nutrition: A Reference Handbook. Oxford

from phenylalanine). The dietary intakes of cysteine

Univ Press, 1997.

and tyrosine thus affect the requirements for methio-

Büller HA, Grand RJ: Lactose intolerance. Annu Rev Med

nine and phenylalanine. The remaining 11 amino acids

1990;41:141.

in proteins are considered to be nonessential or dispens-

Fuller MF, Garlick PJ: Human amino acid requirements. Annu

able, since they can be synthesized as long as there is

Rev Nutr 1994;14:217.

enough total protein in the diet—ie, if one of these

Garrow JS, James WPT, Ralph A: Human Nutrition and Dietetics,

amino acids is omitted from the diet, nitrogen balance

10th ed. Churchill-Livingstone, 2000.

can still be maintained. However, only three amino

National Academy of Sciences report on diet and health. Nutr Rev

acids—alanine, aspartate, and glutamate—can be con-

1989;47:142.

sidered to be truly dispensable; they are synthesized

Nielsen FH: Nutritional significance of the ultratrace elements.

from common metabolic intermediates (pyruvate, ox-

Nutr Rev 1988;46:337.

Vitamins & Minerals

David A. Bender, PhD, & Peter A. Mayes, PhD, DSc

BIOMEDICAL IMPORTANCE

mia (iron), cretinism and goiter (iodine). If present in

excess as with selenium, toxicity symptoms may occur.

Vitamins are a group of organic nutrients required in

small quantities for a variety of biochemical functions

THE DETERMINATION OF NUTRIENT

and which, generally, cannot be synthesized by the

body and must therefore be supplied in the diet.

REQUIREMENTS DEPENDS ON THE

The lipid-soluble vitamins are apolar hydrophobic

CRITERIA OF ADEQUACY CHOSEN

compounds that can only be absorbed efficiently when

For any nutrient, particularly minerals and vitamins,

there is normal fat absorption. They are transported in

there is a range of intakes between that which is clearly

the blood, like any other apolar lipid, in lipoproteins or

inadequate, leading to clinical deficiency disease, and

attached to specific binding proteins. They have diverse

that which is so much in excess of the body’s metabolic

functions, eg, vitamin A, vision; vitamin D, calcium

capacity that there may be signs of toxicity. Between

and phosphate metabolism; vitamin E, antioxidant; vi-

these two extremes is a level of intake that is adequate

tamin K, blood clotting. As well as dietary inadequacy,

for normal health and the maintenance of metabolic in-

conditions affecting the digestion and absorption of the

tegrity. Individuals do not all have the same require-

lipid-soluble vitamins—such as steatorrhea and disor-

ment for nutrients even when calculated on the basis of

ders of the biliary system—can all lead to deficiency

body size or energy expenditure. There is a range of in-

syndromes, including: night blindness and xeroph-

dividual requirements of up to 25% around the mean.

thalmia (vitamin A); rickets in young children and os-

Therefore, in order to assess the adequacy of diets, it is

teomalacia in adults (vitamin D); neurologic disorders

necessary to set a reference level of intake high enough

and anemia of the newborn (vitamin E); and hemor-

to ensure that no one will either suffer from deficiency

rhage of the newborn (vitamin K). Toxicity can result

or be at risk of toxicity. If it is assumed that individual

from excessive intake of vitamins A and D. Vitamin A

requirements are distributed in a statistically normal

and β-carotene (provitamin A), as well as vitamin E, are

fashion around the observed mean requirement, then a

antioxidants and have possible roles in atherosclerosis

range of +/− 2 × the standard deviation (SD) around

and cancer prevention.

the mean will include the requirements of 95% of the

The water-soluble vitamins comprise the B complex

population.

and vitamin C and function as enzyme cofactors. Folic

acid acts as a carrier of one-carbon units. Deficiency of

a single vitamin of the B complex is rare, since poor

THE VITAMINS ARE A DISPARATE GROUP

diets are most often associated with multiple deficiency

OF COMPOUNDS WITH A VARIETY

states. Nevertheless, specific syndromes are characteris-

OF METABOLIC FUNCTIONS

tic of deficiencies of individual vitamins, eg, beriberi

(thiamin); cheilosis, glossitis, seborrhea

(riboflavin);

A vitamin is defined as an organic compound that is re-

pellagra

(niacin); peripheral neuritis

(pyridoxine);

quired in the diet in small amounts for the maintenance

megaloblastic anemia, methylmalonic aciduria, and

of normal metabolic integrity. Deficiency causes a spe-

pernicious anemia

(vitamin B₁₂); and megaloblastic

cific disease, which is cured or prevented only by restor-

anemia (folic acid). Vitamin C deficiency leads to

ing the vitamin to the diet (Table 45-1). However, vit-

scurvy.

amin D, which can be made in the skin after exposure

Inorganic mineral elements that have a function in

to sunlight, and niacin, which can be formed from the

the body must be provided in the diet. When the intake

essential amino acid tryptophan, do not strictly con-

is insufficient, deficiency symptoms may arise, eg, ane-

form to this definition.

481

482

CHAPTER 45

Table 45-1. The vitamins.

Vitamin

Functions

Deficiency Disease

Retinol, β-carotene

Visual pigments in the retina; regulation of

Night blindness, xerophthalmia;

gene expression and cell differentiation;

keratinization of skin

β-carotene is an antioxidant

Calciferol

Maintenance of calcium balance; enhances

Rickets = poor mineralization of bone;

intestinal absorption of Ca2+ and mobilizes

osteomalacia = bone demineralization

bone mineral

Tocopherols, tocotrienols

Antioxidant, especially in cell membranes

Extremely rare—serious neurologic

dysfunction

Phylloquinone,

Coenzyme in formation of γ -carboxyglutamate

Impaired blood clotting, hemor-

menaquinones

in enzymes of blood clotting and bone matrix

rhagic disease

B₁

Thiamin

Coenzyme in pyruvate and α−ketoglutarate,

Peripheral nerve damage (beriberi) or

dehydrogenases, and transketolase; poorly

central nervous system lesions

defined function in nerve conduction

(Wernicke-Korsakoff syndrome)

B₂

Riboflavin

Coenzyme in oxidation and reduction reactions;

Lesions of corner of mouth, lips, and

prosthetic group of flavoproteins

tongue; seborrheic dermatitis

Niacin

Nicotinic acid,

Coenzyme in oxidation and reduction reactions,

Pellagra—photosensitive dermatitis,

nicotinamide

functional part of NAD and NADP

depressive psychosis

B₆

Pyridoxine, pyridoxal,

Coenzyme in transamination and decarboxy-

Disorders of amino acid metabolism,

pyridoxamine

lation of amino acids and glycogen

convulsions

phosphorylase; role in steroid hormone action

Folic acid

Coenzyme in transfer of one-carbon fragments

Megaloblastic anemia

B₁₂

Cobalamin

Coenzyme in transfer of one-carbon fragments

Pernicious anemia = megaloblastic

and metabolism of folic acid

anemia with degeneration of the

spinal cord

Pantothenic acid

Functional part of CoA and acyl carrier protein:

fatty acid synthesis and metabolism

Biotin

Coenzyme in carboxylation reactions in gluco-

Impaired fat and carbohydrate metab-

neogenesis and fatty acid synthesis

olism, dermatitis

Ascorbic acid

Coenzyme in hydroxylation of proline and

Scurvy—impaired wound healing,

lysine in collagen synthesis; antioxidant;

loss of dental cement, subcutaneous

enhances absorption of iron

hemorrhage

provitamin A, as they can be cleaved to yield retinalde-

hyde and thence retinol and retinoic acid. The α-, β-,

LIPID-SOLUBLE VITAMINS

and γ-carotenes and cryptoxanthin are quantitatively

the most important provitamin A carotenoids. Al-

RETINOIDS & CAROTENOIDS

though it would appear that one molecule of β-

carotene should yield two of retinol, this is not so in

HAVE VITAMIN A ACTIVITY

practice; 6 µg of β-carotene is equivalent to 1 µg of

(Figure 45-1)

preformed retinol. The total amount of vitamin A in

foods is therefore expressed as micrograms of retinol

Retinoids comprise retinol, retinaldehyde, and

equivalents. Beta-carotene and other provitamin A

retinoic acid (preformed vitamin A, found only in

carotenoids are cleaved in the intestinal mucosa by

foods of animal origin); carotenoids, found in plants,

carotene dioxygenase, yielding retinaldehyde, which is

comprise carotenes and related compounds, known as

reduced to retinol, esterified, and secreted in chylomi-

VITAMINS & MINERALS

483

H₃C

CH₃

H₃C

CH₃

H₃C

CH₃

-Carotene

CH₃

H₃C

CH₃

H₃C

CH₃

CH₂OH

CHO

CH₃

Retinol

CH₃

Retinaldehyde

CH₃

H₃C

CH₃

H₃C

CH₃

COOH

CH₃

Figure 45-1. β-Carotene and the major vita-

min A vitamers. * Shows the site of cleavage of

H₃C

All-trans-retinoic acid

β-carotene into two molecules of retinaldehyde

COOH

by carotene dioxygenase.

9-cis-retinoic acid

crons together with esters formed from dietary retinol.

ciency, both the time taken to adapt to darkness and the

The intestinal activity of carotene dioxygenase is low,

ability to see in poor light are impaired.

so that a relatively large proportion of ingested β-

carotene may appear in the circulation unchanged.

Retinoic Acid Has a Role

While the principal site of carotene dioxygenase attack

in the Regulation of Gene

is the central bond of β-carotene, asymmetric cleavage

Expression & Tissue Differentiation

may also occur, leading to the formation of 8′-, 10′-,

and 12′-apo-carotenals, which are oxidized to retinoic

A most important function of vitamin A is in the con-

acid but cannot be used as sources of retinol or retin-

trol of cell differentiation and turnover. All-trans-

aldehyde.

retinoic acid and 9-cis-retinoic acid (Figure 45-1) regu-

late growth, development, and tissue differentiation;

they have different actions in different tissues. Like the

Vitamin A Has a Function in Vision

steroid hormones and vitamin D, retinoic acid binds to

In the retina, retinaldehyde functions as the prosthetic

nuclear receptors that bind to response elements of

group of the light-sensitive opsin proteins, forming

DNA and regulate the transcription of specific genes.

rhodopsin (in rods) and iodopsin (in cones). Any one

There are two families of nuclear retinoid receptors: the

cone cell contains only one type of opsin and is sensitive

retinoic acid receptors

(RARs) bind all-trans-retinoic

to only one color. In the pigment epithelium of the

acid or 9-cis-retinoic acid, and the retinoid X receptors

retina, all-trans-retinol is isomerized to

11-cis-retinol

(RXRs) bind 9-cis-retinoic acid.

and oxidized to 11-cis-retinaldehyde. This reacts with a

lysine residue in opsin, forming the holoprotein

Vitamin A Deficiency Is a Major Public

rhodopsin. As shown in Figure 45-2, the absorption of

Health Problem Worldwide

light by rhodopsin causes isomerization of the retinalde-

hyde from 11-cis to all-trans, and a conformational

Vitamin A deficiency is the most important preventable

change in opsin. This results in the release of retinalde-

cause of blindness. The earliest sign of deficiency is a

hyde from the protein and the initiation of a nerve im-

loss of sensitivity to green light, followed by impair-

pulse. The formation of the initial excited form of

ment of adaptation to dim light, followed by night

rhodopsin, bathorhodopsin, occurs within picoseconds

blindness. More prolonged deficiency leads to xeroph-

of illumination. There is then a series of conformational

thalmia: keratinization of the cornea and skin and

changes leading to the formation of metarhodopsin II,

blindness. Vitamin A also has an important role in dif-

which initiates a guanine nucleotide amplification cas-

ferentiation of immune system cells, and mild defi-

cade and then a nerve impulse. The final step is hydroly-

ciency leads to increased susceptibility to infectious dis-

sis to release all-trans-retinaldehyde and opsin. The key

eases. Furthermore, the synthesis of retinol-binding

to initiation of the visual cycle is the availability of

protein in response to infection is reduced (it is a nega-

11-cis-retinaldehyde, and hence vitamin A. In defi-

tive acute phase protein), decreasing the circulating vi-

484

CHAPTER 45

CH₃

ataxia, and anorexia, all associated with increased cere-

H₃C

CH₃

CH₂OH

brospinal fluid pressure), the liver (hepatomegaly with

histologic changes and hyperlipidemia), calcium ho-

CH₃

All-trans-retinol

meostasis (thickening of the long bones, hypercalcemia

CH₃

and calcification of soft tissues), and the skin (excessive

H₃C

CH₃

dryness, desquamation, and alopecia).

11-cis-Retinol

CH₃ H₃C

CH₂OH

VITAMIN D IS REALLY A HORMONE

CH₃

H₃C

CH₃

Vitamin D is not strictly a vitamin since it can be syn-

11-cis-Retinaldehyde

thesized in the skin, and under most conditions that is

CH₃ H₃C

its major source. Only when sunlight is inadequate is a

HC=O

C=O

dietary source required. The main function of vitamin

H₂N

D is in the regulation of calcium absorption and ho-

CH₃

Lysine residue

H₃C

CH₃

meostasis; most of its actions are mediated by way

in opsin

of nuclear receptors that regulate gene expression.

CH₃ H₃C

Deficiency—leading to rickets in children and osteo-

C=O

HC=N

malacia in adults—continues to be a problem in north-

Rhodopsin (visual purple)

ern latitudes, where sunlight exposure is poor.

LIGHT

10^-15sec

CH₃

C=O

Vitamin D Is Synthesized in the Skin

H₃C

CH₃

C=N

7-Dehydrocholesterol (an intermediate in the synthesis

CH₃

of cholesterol that accumulates in the skin), undergoes

Photorhodopsin

a nonenzymic reaction on exposure to ultraviolet light,

45 psec

yielding previtamin D (Figure 45-3). This undergoes a

5'GMP

Bathorhodopsin

further reaction over a period of hours to form the vita-

cGMP

Na channel closed

30 nsec

min itself, cholecalciferol, which is absorbed into the

Na channel open

Lumirhodopsin

bloodstream. In temperate climates, the plasma concen-

75 µsec

Inactive

Active

tration of vitamin D is highest at the end of summer

phosphodiesterase

Metarhodopsin I

and lowest at the end of winter. Beyond about 40 de-

GDP

Transducin-GTP

grees north or south in winter, there is very little ultra-

10 msec

Metarhodopsin II

violet radiation of appropriate wavelength.

minutes

GTP

Transducin-GDP

Metarhodopsin III

Vitamin D Is Metabolized to the Active

Metabolite, Calcitriol, in Liver & Kidney

CH₃

H₃C

CH₃

C=O

In the liver, cholecalciferol, which has been synthesized

in the skin or derived from food, is hydroxylated to

All-trans-retinaldehyde + opsin

CH₃

form the 25-hydroxy derivative calcidiol (Figure 45-4).

This is released into the circulation bound to a vitamin

The role of retinaldehyde in vision.

Figure 45-2.

D-binding globulin which is the main storage form of

the vitamin. In the kidney, calcidiol undergoes either

1-hydroxylation to yield the active metabolite 1,25-dihy-

tamin, and therefore there is further impairment of im-

droxyvitamin D (calcitriol) or 24-hydroxylation to yield

an inactive metabolite, 24,25-dihydroxyvitamin D (24-

mune responses.

hydroxycalcidiol). Ergocalciferol from fortified foods

undergoes similar hydroxylations to yield ercalcitriol.

Vitamin A Is Toxic in Excess

There is only a limited capacity to metabolize vitamin

Vitamin D Metabolism Both Regulates

A, and excessive intakes lead to accumulation beyond

& Is Regulated by Calcium Homeostasis

the capacity of binding proteins, so that unbound vita-

min A causes tissue damage. Symptoms of toxicity af-

The main function of vitamin D is in the control of cal-

fect the central nervous system

(headache, nausea,

cium homeostasis, and in turn vitamin D metabolism is

VITAMINS & MINERALS

485

Thermal isomerization

LIGHT

Cholecalciferol

(calciol;vitamin D₃)

CH₃

CH₂

7-Dehydrocholesterol

Previtamin D

Figure 45-3. Synthesis of vitamin D in the skin.

regulated by factors that respond to plasma concentra-

Vitamin D Deficiency Affects

tions of calcium and phosphate. Calcitriol acts to reduce

Children & Adults

its own synthesis by inducing the 24-hydroxylase and

In the vitamin D deficiency disease rickets, the bones

repressing the 1-hydroxylase in the kidney. Its principal

of children are undermineralized as a result of poor ab-

function is to maintain the plasma calcium concentra-

sorption of calcium. Similar problems occur in adoles-

tion. Calcitriol achieves this in three ways: it increases

cents who are deficient during their growth spurt. Os-

intestinal absorption of calcium, reduces excretion of

teomalacia in adults results from demineralization of

calcium (by stimulating resorption in the distal renal

bone in women who have little exposure to sunlight,

tubules), and mobilizes bone mineral. In addition, cal-

often after several pregnancies. Although vitamin D is

citriol is involved in insulin secretion, synthesis and se-

essential for prevention and treatment of osteomalacia

cretion of parathyroid and thyroid hormones, inhibition

in the elderly, there is little evidence that it is beneficial

of production of interleukin by activated T lymphocytes

in treating osteoporosis.

and of immunoglobulin by activated B lymphocytes,

differentiation of monocyte precursor cells, and mod-

Vitamin D Is Toxic in Excess

ulation of cell proliferation. In its actions, it behaves like

a steroid hormone, binding to a nuclear receptor

Some infants are sensitive to intakes of vitamin D as

protein.

low as 50 µg/d, resulting in an elevated plasma concen-

Calciol-25-hydroxylase

Calcidiol-1-hydroxylase

CH₂

Calcitriol

Calcidiol

(1,25-hydroxycholecalciferol)

Cholecalciferol

(25-hydroxycholecalciferol)

(calciol;vitamin D₃)

Calcidiol-24-hydroxylase

Calcidiol-1-hydroxylase

CH₂

24-hydroxycalcidiol

Calcitetrol

Figure 45-4. Metabolism of vitamin D.

486

CHAPTER 45

tration of calcium. This can lead to contraction of

reduced back to tocopherol by reaction with vitamin

blood vessels, high blood pressure, and calcinosis—the

C from plasma (Figure 45-6). The resultant monode-

calcification of soft tissues. Although excess dietary vita-

hydroascorbate free radical then undergoes enzymic or

min D is toxic, excessive exposure to sunlight does not

nonenzymic reaction to yield ascorbate and dehy-

lead to vitamin D poisoning because there is a limited

droascorbate, neither of which is a free radical. The

capacity to form the precursor

7-dehydrocholesterol

stability of the tocopheroxyl free radical means that it

and to take up cholecalciferol from the skin.

can penetrate farther into cells and, potentially, propa-

gate a chain reaction. Therefore, vitamin E may, like

other antioxidants, also have pro-oxidant actions, es-

VITAMIN E DOES NOT HAVE A PRECISELY

pecially at high concentrations. This may explain why,

DEFINED METABOLIC FUNCTION

although studies have shown an association between

No unequivocal unique function for vitamin E has

high blood concentrations of vitamin E and a lower

been defined. However, it does act as a lipid-soluble an-

incidence of atherosclerosis, the effect of high doses of

tioxidant in cell membranes, where many of its func-

vitamin E have been disappointing.

tions can be provided by synthetic antioxidants. Vita-

min E is the generic descriptor for two families of

Dietary Vitamin E Deficiency

compounds, the tocopherols and the tocotrienols (Fig-

in Humans Is Unknown

ure 45-5). The different vitamers (compounds having

similar vitamin activity) have different biologic poten-

In experimental animals, vitamin E deficiency results in

cies; the most active is D-α-tocopherol, and it is usual

resorption of fetuses and testicular atrophy. Dietary de-

to express vitamin E intake in milligrams of D-α-tocoph-

ficiency of vitamin E in humans is unknown, though

erol equivalents. Synthetic DL-α-tocopherol does not

patients with severe fat malabsorption, cystic fibrosis,

have the same biologic potency as the naturally occur-

and some forms of chronic liver disease suffer defi-

ring compound.

ciency because they are unable to absorb the vitamin or

transport it, exhibiting nerve and muscle membrane

Vitamin E Is the Major Lipid-Soluble

damage. Premature infants are born with inadequate re-

Antioxidant in Cell Membranes

serves of the vitamin. Their erythrocyte membranes are

& Plasma Lipoproteins

abnormally fragile as a result of peroxidation, which

leads to hemolytic anemia.

The main function of vitamin E is as a chain-break-

ing, free radical trapping antioxidant in cell mem-

branes and plasma lipoproteins. It reacts with the lipid

VITAMIN K IS REQUIRED FOR SYNTHESIS

peroxide radicals formed by peroxidation of polyun-

OF BLOOD-CLOTTING PROTEINS

saturated fatty acids before they can establish a chain

Vitamin K was discovered as a result of investigations

reaction. The tocopheroxyl free radical product is rela-

into the cause of a bleeding disorder—hemorrhagic

tively unreactive and ultimately forms nonradical

(sweet clover) disease—of cattle, and of chickens fed on

compounds. Commonly, the tocopheroxyl radical is

a fat-free diet. The missing factor in the diet of the

chickens was vitamin K, while the cattle feed contained

dicumarol, an antagonist of the vitamin. Antagonists

of vitamin K are used to reduce blood coagulation in

R₁

patients at risk of thrombosis—the most widely used

agent is warfarin.

R₂

Three compounds have the biologic activity of vita-

CH₃

R₃

Tocopherol

min K (Figure 45-7): phylloquinone, the normal di-

etary source, found in green vegetables; menaqui-

R₁

nones, synthesized by intestinal bacteria, with differing

lengths of side-chain; menadione, menadiol, and

R₂

menadiol diacetate, synthetic compounds that can be

CH₃

R₃

Tocotrienol

metabolized to phylloquinone. Menaquinones are ab-

sorbed to some extent but it is not clear to what extent

Figure 45-5. The vitamin E vitamers. In α-tocoph-

they are biologically active as it is possible to induce

erol and tocotrienol R₁, R₂, and R₃ are all CH₃ groups.

signs of vitamin K deficiency simply by feeding a phyl-

In the β-vitamers R₂ is H; in the γ-vitamers R₁ is H, and in

loquinone deficient diet, without inhibiting intestinal

the δ-vitamers R₁ and R₂ are both H.

bacterial action.

VITAMINS & MINERALS

487

Free radical

chain reaction

PUFA

PUFA OOH

TocOH

TocO

PHOSPHOLIPASE

O₂

A₂

PUFA H

(in phospholipid)

MEMBRANES

CYTOSOL

Vitamin C_ox

Vitamin C_red,

PUFA OOH,

GS SG

GSH

H₂O₂

GSH

GLUTATHIONE

SUPEROXIDE

CATALASE

PEROXIDASE

DISMUTASE

–

O₂

H2O,

GS SG

Superoxide

PUFA OH

Figure 45-6. Interaction and synergism between antioxidant systems operating in the lipid

phase (membranes) of the cell and the aqueous phase (cytosol). (R•, free radical; PUFA-OO•, peroxyl

free radical of polyunsaturated fatty acid in membrane phospholipid; PUFA-OOH, hydroperoxy

polyunsaturated fatty acid in membrane phospholipid released as hydroperoxy free fatty acid into

cytosol by the action of phospholipase A₂; PUFA-OH, hydroxy polyunsaturated fatty acid; TocOH,

vitamin E (α-tocopherol); TocO•, free radical of α-tocopherol; Se, selenium; GSH, reduced glu-

tathione; GS-SG, oxidized glutathione, which is returned to the reduced state after reaction with

NADPH catalyzed by glutathione reductase; PUFA-H, polyunsaturated fatty acid.)

Vitamin K Is the Coenzyme

quinone reductase. In the presence of warfarin, vitamin

for Carboxylation of Glutamate

K epoxide cannot be reduced but accumulates, and is

in the Postsynthetic Modification

excreted. If enough vitamin K (a quinone) is provided

in the diet, it can be reduced to the active hydro-

of Calcium-Binding Proteins

quinone by the warfarin-insensitive enzyme, and car-

Vitamin K is the cofactor for the carboxylation of gluta-

boxylation can continue, with stoichiometric utilization

mate residues in the post-synthetic modification of pro-

of vitamin K and excretion of the epoxide. A high dose

teins to form the unusual amino acid γ-carboxygluta-

of vitamin K is the antidote to an overdose of warfarin.

mate (Gla), which chelates the calcium ion. Initially,

Prothrombin and several other proteins of the blood

vitamin K hydroquinone is oxidized to the epoxide

clotting system (Factors VII, IX and X, and proteins C

(Figure 45-8), which activates a glutamate residue in

and S) each contain between four and six γ-carboxygluta-

the protein substrate to a carbanion, that reacts non-

mate residues which chelate calcium ions and so permit

enzymically with carbon dioxide to form γ-carboxyglut-

the binding of the blood clotting proteins to membranes.

amate. Vitamin K epoxide is reduced to the quinone by

In vitamin K deficiency or in the presence of warfarin, an

a warfarin-sensitive reductase, and the quinone is re-

abnormal precursor of prothrombin (preprothrombin)

duced to the active hydroquinone by either the same

containing little or no γ-carboxyglutamate, and incapable

warfarin-sensitive reductase or a warfarin-insensitive

of chelating calcium, is released into the circulation.

488

CHAPTER 45

Vitamin K Is Also Important

CH₃

in the Synthesis of Bone

Calcium-Binding Proteins

Treatment of pregnant women with warfarin can lead to

Phylloquinone

fetal bone abnormalities (fetal warfarin syndrome). Two

proteins are present in bone that contain γ-carboxygluta-

mate, osteocalcin and bone matrix Gla protein. Osteocal-

CH₃

cin also contains hydroxyproline, so its synthesis is depen-

dent on both vitamins K and C; in addition, its synthesis

is induced by vitamin D. The release into the circulation

Menaquinone

CH₃

of osteocalcin provides an index of vitamin D status.

CH₃

WATER-SOLUBLE VITAMINS

Menadiol

VITAMIN B₁ (THIAMIN) HAS A KEY ROLE

CH₃

IN CARBOHYDRATE METABOLISM

Menadiolo diacetate

Thiamin has a central role in energy-yielding metabo-

(acetomenaphthone)

lism, and especially the metabolism of carbohydrate

Figure 45-7. The vitamin K vitamers. Menadiol (or

(Figure 45-9). Thiamin diphosphate is the coenzyme

menadione) and menadiol diacetate are synthetic com-

for three multi-enzyme complexes that catalyze oxida-

pounds that are converted to menaquinone in the liver

tive decarboxylation reactions: pyruvate dehydrogenase

and have vitamin K activity.

in carbohydrate metabolism; α-ketoglutarate dehydro-

OOC

COO

CH₂

CH C

Carboxyglutamate residue

CO ₂

non-

enzymic

CH COO

O₂

CH COO

CH₂

HN CH C O

VITAMIN K

EPOXIDASE

Glutamate residue

Glutamate carbanion

CH₃

Vitamin K hydroquinone

Vitamin K epoxide

Disulfide

Sulfhydryl

NADP+

VITAMIN K QUINONE

VITAMIN K EPOXIDE

QUINONE

REDUCTASE

Sulfhydryl

NADPH

CH₃

Disulfide

Figure 45-8.

The role of vitamin K in the

Vitamin K quinone

biosynthesis of γ-carboxyglutamate.

VITAMINS & MINERALS

489

H₃C

NH₂

H₃C

CH₂

CH₂OH

CH₂

P O P O

CH₂

H+

Thiamin

Thiamin diphosphate

Carbanion

Figure 45-9. Thiamin, thiamin diphosphate, and the carbanion form.

genase in the citric acid cycle; and the branched-chain

pyruvate dehydrogenase means that in deficiency there is

keto-acid dehydrogenase involved in the metabolism of

impaired conversion of pyruvate to acetyl CoA. In sub-

leucine, isoleucine, and valine. It is also the coenzyme

jects on a relatively high carbohydrate diet, this results in

for transketolase, in the pentose phosphate pathway. In

increased plasma concentrations of lactate and pyruvate,

each case, the thiamin diphosphate provides a reactive

which may cause life-threatening lactic acidosis.

carbon on the thiazole moiety that forms a carbanion,

which then adds to the carbonyl group of, for instance,

Thiamin Nutritional Status Can

pyruvate. The addition compound then decarboxylates,

Be Assessed by Erythrocyte

eliminating CO₂. Electrical stimulation of nerve leads

to a fall in membrane thiamin triphosphate and release

Transketolase Activation

of free thiamin. It is likely that thiamin triphosphate

The activation of apo-transketolase(the enzyme pro-

acts as a phosphate donor for phosphorylation of the

tein) in erythrocyte lysate by thiamin diphosphate

nerve membrane sodium transport channel.

added in vitro has become the accepted index of thi-

amin nutritional status.

Thiamin Deficiency Affects

the Nervous System & Heart

VITAMIN B₂ (RIBOFLAVIN) HAS

Thiamin deficiency can result in three distinct syn-

A CENTRAL ROLE IN ENERGY-

dromes: a chronic peripheral neuritis, beriberi, which

YIELDING METABOLISM

may or may not be associated with heart failure and

edema; acute pernicious (fulminating) beriberi (shoshin

Riboflavin fulfills its role in metabolism as the coenzymes

beriberi), in which heart failure and metabolic abnor-

flavin mononucleotide

(FMN) and flavin adenine

malities predominate, without peripheral neuritis; and

dinucleotide (FAD) (Figure 45-10). FMN is formed by

Wernicke’s encephalopathy with Korsakoff’s psy-

ATP-dependent phosphorylation of riboflavin, whereas

chosis, which is associated especially with alcohol and

FAD is synthesized by further reaction of FMN with

drug abuse. The central role of thiamin diphosphate in

ATP in which its AMP moiety is transferred to the

OH OH OH

CH₂

CH CH CH

CH₂OH

CH₂

CH CH CH

CH₂

O P O

H₃C

N N

H₃C

N N

H₃C

Riboflavin

FMN

NH₂

OH OH OH

CH₂

CH CH CH

CH₂

O P O

CH₂

N N

H₃C

N N

H₃C

FAD

Figure 45-10. Riboflavin and the coenzymes flavin mononucleotide (FMN) and flavin

adenine dinucleotide (FAD).

490

CHAPTER 45

FMN. The main dietary sources of riboflavin are milk

COO

CONH₂

and dairy products. In addition, because of its intense

yellow color, riboflavin is widely used as a food additive.

Nicotinic acid

Nicotinamide

Flavin Coenzymes Are Electron Carriers

CONH₂

NH₂

in Oxidoreduction Reactions

These include the mitochondrial respiratory chain, key

CH₂

O P O

CH₂

N N

enzymes in fatty acid and amino acid oxidation, and

the citric acid cycle. Reoxidation of the reduced flavin

in oxygenases and mixed-function oxidases proceeds by

way of formation of the flavin radical and flavin hy-

NAD

droperoxide, with the intermediate generation of super-

oxide and perhydroxyl radicals and hydrogen peroxide.

Figure 45-11. Niacin (nicotinic acid and nicotin-

Because of this, flavin oxidases make a significant con-

amide) and nicotinamide adenine dinucleotide (NAD).

tribution to the total oxidant stress of the body.

* Shows the site of phosphorylation in NADP.

Riboflavin Deficiency Is

Widespread But Not Fatal

diarrhea, and, if untreated, death. Although the nutri-

Although riboflavin is fundamentally involved in me-

tional etiology of pellagra is well established and trypto-

tabolism, and deficiencies are found in most countries,

phan or niacin will prevent or cure the disease, addi-

it is not fatal as there is very efficient conservation of

tional factors, including deficiency of riboflavin or

tissue riboflavin. Riboflavin deficiency is characterized

vitamin B₆, both of which are required for synthesis of

by cheilosis, lingual desquamation and a seborrheic der-

nicotinamide from tryptophan, may be important. In

matitis. Riboflavin nutritional status is assessed by mea-

most outbreaks of pellagra twice as many women as

surement of the activation of erythrocyte glutathione

men are affected, probably the result of inhibition of

reductase by FAD added in vitro.

tryptophan metabolism by estrogen metabolites.

NIACIN IS NOT STRICTLY A VITAMIN

Pellagra Can Occur as a Result

Niacin was discovered as a nutrient during studies of pel-

of Disease Despite an Adequate

lagra. It is not strictly a vitamin since it can be syn-

Intake of Tryptophan & Niacin

thesized in the body from the essential amino acid

A number of genetic diseases that result in defects of

tryptophan. Two compounds, nicotinic acid and nico-

tinamide, have the biologic activity of niacin; its meta-

tryptophan metabolism are associated with the develop-

ment of pellagra despite an apparently adequate intake

bolic function is as the nicotinamide ring of the coen-

zymes NAD and NADP in oxidation-reduction reactions

of both tryptophan and niacin. Hartnup disease is a

rare genetic condition in which there is a defect of the

(Figure 45-11). About 60 mg of tryptophan is equivalent

to 1 mg of dietary niacin. The niacin content of foods is

membrane transport mechanism for tryptophan, result-

ing in large losses due to intestinal malabsorption and

expressed as mg niacin equivalents = mg preformed niacin

+ 1/60 × mg tryptophan. Because most of the niacin in

failure of the renal resorption mechanism. In carcinoid

syndrome there is metastasis of a primary liver tumor

cereals is biologically unavailable, this is discounted.

of enterochromaffin cells which synthesize 5-hydroxy-

tryptamine. Overproduction of

5-hydroxytryptamine

NAD Is the Source of ADP-Ribose

may account for as much as 60% of the body’s trypto-

In addition to its coenzyme role, NAD is the source of

phan metabolism, causing pellagra because of the diver-

ADP-ribose for the ADP-ribosylation of proteins and

sion away from NAD synthesis.

polyADP-ribosylation of nucleoproteins involved in the

DNA repair mechanism.

Niacin Is Toxic in Excess

Nicotinic acid has been used to treat hyperlipidemia

Pellagra Is Caused by Deficiency

when of the order of 1-6 g/d are required, causing dila-

of Tryptophan & Niacin

tion of blood vessels and flushing, with skin irritation.

Pellagra is characterized by a photosensitive dermatitis.

Intakes of both nicotinic acid and nicotinamide in ex-

As the condition progresses, there is dementia, possibly

cess of 500 mg/d can cause liver damage.

VITAMINS & MINERALS

491

VITAMIN B₆ IS IMPORTANT IN AMINO

Vitamin B₆ Deficiency Is Rare

ACID & GLYCOGEN METABOLISM

Although clinical deficiency disease is rare, there is evi-

& IN STEROID HORMONE ACTION

dence that a significant proportion of the population

have marginal vitamin B₆ status. Moderate deficiency

Six compounds have vitamin B₆ activity (Figure 45-12):

results in abnormalities of tryptophan and methionine

pyridoxine, pyridoxal, pyridoxamine, and their 5′-

metabolism. Increased sensitivity to steroid hormone ac-

phosphates. The active coenzyme is pyridoxal 5′-phos-

tion may be important in the development of hormone-

phate. Approximately 80% of the body’s total vitamin B₆

dependent cancer of the breast, uterus, and prostate,

is present as pyridoxal phosphate in muscle, mostly asso-

and vitamin B₆ status may affect the prognosis.

ciated with glycogen phosphorylase. This is not available

in B₆ deficiency but is released in starvation, when glyco-

gen reserves become depleted, and is then available, espe-

Vitamin B₆ Status Is Assessed by Assaying

cially in liver and kidney, to meet increased requirement

Erythrocyte Aminotransferases

for gluconeogenesis from amino acids.

The most widely used method of assessing vitamin B₆

status is by the activation of erythrocyte aminotrans-

Vitamin B₆ Has Several Roles

ferases by pyridoxal phosphate added in vitro, expressed

in Metabolism

as the activation coefficient.

Pyridoxal phosphate is a coenzyme for many enzymes

involved in amino acid metabolism, especially in

In Excess, Vitamin B₆ Causes

transamination and decarboxylation. It is also the co-

Sensory Neuropathy

factor of glycogen phosphorylase, where the phosphate

group is catalytically important. In addition, vitamin B₆

The development of sensory neuropathy has been re-

is important in steroid hormone action where it re-

ported in patients taking 2-7 g of pyridoxine per day for

moves the hormone-receptor complex from DNA

a variety of reasons (there is some slight evidence that it

binding, terminating the action of the hormones. In vi-

is effective in treating premenstrual syndrome). There

tamin B₆ deficiency, this results in increased sensitivity

was some residual damage after withdrawal of these high

to the actions of low concentrations of estrogens, an-

doses; other reports suggest that intakes in excess of 200

drogens, cortisol, and vitamin D.

mg/d are associated with neurologic damage.

VITAMIN B₁₂ IS FOUND ONLY

IN FOODS OF ANIMAL ORIGIN

CH₂OH

KINASE

The term “vitamin B₁₂” is used as a generic descriptor

HOCH₂

POCH₂

for the cobalamins—those corrinoids (cobalt con-

PHOSPHATASE

N CH₃

taining compounds possessing the corrin ring) having

Pyridoxine

Pyridoxine phosphate

the biologic activity of the vitamin (Figure 45-13).

Some corrinoids that are growth factors for microor-

OXIDASE

ganisms not only have no vitamin B₁₂ activity but may

also be antimetabolites of the vitamin. Although it is

HC=O

synthesized exclusively by microorganisms, for practi-

KINASE

HOCH₂

POCH₂

cal purposes vitamin B₁₂ is found only in foods of ani-

PHOSPHATASE

mal origin, there being no plant sources of this vita-

N CH₃

min. This means that strict vegetarians (vegans) are at

Pyridoxal

Pyridoxal phosphate

risk of developing B₁₂ deficiency. The small amounts

of the vitamin formed by bacteria on the surface of

AMINOTRANSFERASES

OXIDASE

fruits may be adequate to meet requirements, but

preparations of vitamin B₁₂ made by bacterial fermen-

CH₂NH₂

KINASE

tation are available.

HOCH₂

POCH₂

PHOSPHATASE

N CH₃

Vitamin B₁₂ Absorption Requires Two

Pyridoxamine

Pyridoxamine phosphate

Binding Proteins

Figure 45-12. Interconversion of the vitamin B₆

Vitamin B₁₂ is absorbed bound to intrinsic factor, a

vitamers.

small glycoprotein secreted by the parietal cells of the

492

CHAPTER 45

CH₂CONH₂

mentation in ruminants. It undergoes vitamin B₁₂-

H₃C

CH₂CH₂CONH₂

dependent rearrangement to succinyl-CoA, catalyzed

H₃C

by methylmalonyl-CoA isomerase (Figure 19-2). The

H₂NCOCH₂CH₂

activity of this enzyme is greatly reduced in vitamin B₁₂

CH₃

H₂NCOCH₂

deficiency, leading to an accumulation of methyl-

H₃C

malonyl-CoA and urinary excretion of methylmalonic

H₃C

acid, which provides a means of assessing vitamin B₁₂

CH₂CH₂CONH₂

CH₃

nutritional status.

H₂NCOCH₂

CH₂

Vitamin B₁₂ Deficiency Causes

CH₂

Pernicious Anemia

Pernicious anemia arises when vitamin B₁₂ deficiency

blocks the metabolism of folic acid, leading to func-

tional folate deficiency. This impairs erythropoiesis,

causing immature precursors of erythrocytes to be re-

CH₃

H₃C C O

leased into the circulation (megaloblastic anemia). The

CH₃

commonest cause of pernicious anemia is failure of the

absorption of vitamin B₁₂

rather than dietary defi-

ciency. This can be due to failure of intrinsic factor se-

HOCH₂ O

cretion caused by autoimmune disease of parietal cells

or to generation of anti-intrinsic factor antibodies.

Figure 45-13. Vitamin B₁₂(cobalamin). R may be

varied to give the various forms of the vitamin, eg,

THERE ARE MULTIPLE FORMS

in hydroxocobal-

R = CN^- in cyanocobalamin; R = OH^-

OF FOLATE IN THE DIET

amin; R = 5′-deoxyadenosyl in 5′-deoxyadenosylcobal-

amin; R = H₂O in aquocobalamin; and R = CH₃ in

The active form of folic acid (pteroyl glutamate) is

methylcobalamin.

tetrahydrofolate (Figure 45-15). The folates in foods

may have up to seven additional glutamate residues

linked by γ-peptide bonds. In addition, all of the one-

carbon substituted folates in Figure 45-15 may also be

gastric mucosa. Gastric acid and pepsin release the vita-

present in foods.

min from protein binding in food and make it available

The extent to which the different forms of folate can

to bind to cobalophilin, a binding protein secreted in

be absorbed varies, and this must be allowed for in cal-

the saliva. In the duodenum, cobalophilin is hy-

culating folate intakes.

drolyzed, releasing the vitamin for binding to intrinsic

factor. Pancreatic insufficiency can therefore be a fac-

tor in the development of vitamin B₁₂ deficiency, re-

sulting in the excretion of cobalophilin-bound vitamin

B₁₂. Intrinsic factor binds the various vitamin B₁₂

vita-

H₃C S

mers, but not other corrinoids. Vitamin B₁₂ is absorbed

(CH₂ )₂

from the distal third of the ileum via receptors that

bind the intrinsic factor-vitamin B₁₂ complex but not

H C NH

free intrinsic factor or free vitamin.

COO

COO-

Homocysteine

Methionine

There Are Three Vitamin

METHIONINE

B₁₂-Dependent Enzymes

SYNTHASE

Methylmalonyl CoA mutase, leucine aminomutase,

and methionine synthase (Figure 45-14) are vitamin

Methylcobalamin

Methyl

B₁₂

H₄

folate

B₁₂-dependent enzymes. Methylmalonyl CoA is formed

4folate

as an intermediate in the catabolism of valine and by

the carboxylation of propionyl CoA arising in the ca-

Figure 45-14. Homocysteinuria and the folate trap.

tabolism of isoleucine, cholesterol, and, rarely, fatty

Vitamin B₁₂ deficiency leads to inhibition of methionine

acids with an odd number of carbon atoms—or directly

synthase activity causing homocysteinuria and the

from propionate, a major product of microbial fer-

trapping of folate as methyltetrahydrofolate.

VITAMINS & MINERALS

493

COO

CH₂

H₂N

N N

Tetrahydrofolate (THF)

(Glu)

CH₂

5-Formyl THF

10-Formyl THF

H₂N

N N

H₂N N N

HC NH

CH₂

5-Formimino THF

5,10-Methylene THF

H₂N

N N

H₂N N N

CH₃

CH₂

Figure 45-15.

Tetrahydrofolic acid and the

5-Methyl THF

5,10-Methenyl THF

H₂N

N N

H₂N N N

one-carbon substituted folates.

Tetrahydrofolate Is a Carrier

ceutically in the agent known as folinic acid and in the

of One-Carbon Units

synthetic (racemic) compound leucovorin.

The major point of entry for one-carbon fragments

Tetrahydrofolate can carry one-carbon fragments at-

into substituted folates is methylene tetrahydrofolate

tached to N-5 (formyl, formimino, or methyl groups),

(Figure 45-16), which is formed by the reaction of

N-10 (formyl group), or bridging N-5 to N-10 (meth-

glycine, serine, and choline with tetrahydrofolate. Serine

ylene or methenyl groups). 5-Formyl-tetrahydrofolate is

is the most important source of substituted folates

more stable than folate and is therefore used pharma-

for biosynthetic reactions, and the activity of serine hy-

Sources of one-carbon units

Synthesis using one-carbon units

Serine

Glycine

Methylene-THF

Methyl-THF

Methionine

Choline

TMP + dihydrofolate

Histidine

Formimino-THF

Methenyl-THF

DNA

Formyl-methionine

Formate

Formyl-THF

Purines

CO₂

Figure 45-16. Sources and utilization of one-carbon substituted folates.

494

CHAPTER 45

droxymethyltransferase is regulated by the state of folate

Folic Acid Supplements Reduce

substitution and the availability of folate. The reaction is

the Risk of Neural Tube Defects

reversible, and in liver it can form serine from glycine as

& Hyperhomocysteinemia

a substrate for gluconeogenesis. Methylene, methenyl,

Supplements of 400 µg/d of folate begun before con-

and 10-formyl tetrahydrofolates are interconvertible.

When one-carbon folates are not required, the oxidation

ception result in a significant reduction in the incidence

of neural tube defects as found in spina bifida. Ele-

of formyl tetrahydrofolate to yield carbon dioxide pro-

vides a means of maintaining a pool of free folate.

vated blood homocysteine is an associated risk factor

for atherosclerosis, thrombosis, and hypertension.

The condition is due to impaired ability to form

Inhibitors of Folate Metabolism

methyl-tetrahydrofolate by methylene-tetrahydrofolate

Provide Cancer Chemotherapy &

reductase, causing functional folate deficiency and re-

Antibacterial & Antimalarial Drugs

sulting in failure to remethylate homocysteine to me-

The methylation of deoxyuridine monophosphate

thionine. People with the causative abnormal variant of

(dUMP) to thymidine monophosphate (TMP), cat-

methylene-tetrahydrofolate reductase do not develop

alyzed by thymidylate synthase, is essential for the syn-

hyperhomocysteinemia if they have a relatively high in-

thesis of DNA. The one-carbon fragment of methy-

take of folate, but it is not yet known whether this af-

lene-tetrahydrofolate is reduced to a methyl group with

fects the incidence of cardiovascular disease.

release of dihydrofolate, which is then reduced back to

tetrahydrofolate by dihydrofolate reductase. Thymi-

Folate Enrichment of Foods

dylate synthase and dihydrofolate reductase are espe-

May Put Some People at Risk

cially active in tissues with a high rate of cell division.

Folate supplements will rectify the megaloblastic anemia

Methotrexate, an analog of 10-methyl-tetrahydrofo-

of vitamin B₁₂ deficiency but may hasten the develop-

late, inhibits dihydrofolate reductase and has been ex-

ment of the (irreversible) nerve damage found in B₁₂ de-

ploited as an anticancer drug. The dihydrofolate reduc-

ficiency. There is also antagonism between folic acid and

tases of some bacteria and parasites differ from the

the anticonvulsants used in the treatment of epilepsy.

human enzyme; inhibitors of these enzymes can be used

as antibacterial drugs, eg, trimethoprim, and anti-

malarial drugs, eg, pyrimethamine.

DIETARY BIOTIN DEFICIENCY

IS UNKNOWN

Vitamin B₁₂ Deficiency Causes Functional

The structures of biotin, biocytin, and carboxy-biotin

Folate Deficiency—the Folate Trap

(the active metabolic intermediate) are shown in Figure

When acting as a methyl donor, S-adenosylmethionine

45-17. Biotin is widely distributed in many foods as

forms homocysteine, which may be remethylated by

biocytin (ε-amino-biotinyl lysine), which is released on

methyltetrahydrofolate catalyzed by methionine syn-

proteolysis. It is synthesized by intestinal flora in excess

thase, a vitamin B₁₂-dependent enzyme (Figure 45-14).

of requirements. Deficiency is unknown except among

The reduction of methylene-tetrahydrofolate to methyl-

people maintained for many months on parenteral nu-

tetrahydrofolate is irreversible, and since the major source

trition and a very small number who eat abnormally

of tetrahydrofolate for tissues is methyl-tetrahydrofolate,

large amounts of uncooked egg white, which contains

the role of methionine synthase is vital and provides a link

avidin, a protein that binds biotin and renders it un-

between the functions of folate and vitamin B₁₂. Impair-

available for absorption.

ment of methionine synthase in B₁₂ deficiency results in

the accumulation of methyl-tetrahydrofolate—the “fo-

Biotin Is a Coenzyme

late trap.” There is therefore functional deficiency of fo-

of Carboxylase Enzymes

late secondary to the deficiency of vitamin B₁₂.

Biotin functions to transfer carbon dioxide in a small

number of carboxylation reactions. A holocarboxylase

Folate Deficiency Causes

synthetase acts on a lysine residue of the apoenzymes of

Megaloblastic Anemia

acetyl-CoA carboxylase, pyruvate carboxylase, propi-

Deficiency of folic acid itself—or deficiency of vitamin

onyl-CoA carboxylase, or methylcrotonyl-CoA carboxy-

B₁₂, which leads to functional folic acid deficiency—af-

lase to react with free biotin to form the biocytin residue

fects cells that are dividing rapidly because they have a

of the holoenzyme. The reactive intermediate is 1-N-

large requirement for thymidine for DNA synthesis.

carboxybiocytin, formed from bicarbonate in an ATP-

Clinically, this affects the bone marrow, leading to

dependent reaction. The carboxyl group is then trans-

megaloblastic anemia.

ferred to the substrate for carboxylation (Figure 21-1).

VITAMINS & MINERALS

495

the SH prosthetic group of CoA and ACP. CoA

takes part in reactions of the citric acid cycle, fatty acid

HN NH

Biotin

synthesis and oxidation, acetylations, and cholesterol

synthesis. ACP participates in fatty acid synthesis. The

COO

vitamin is widely distributed in all foodstuffs, and defi-

ciency has not been unequivocally reported in human

beings except in specific depletion studies.

HN NH

Biotinyl-lysine (biocytin)

C O

ASCORBIC ACID IS A VITAMIN

FOR ONLY SOME SPECIES

Vitamin C (Figure 45-19) is a vitamin for human beings

and other primates, the guinea pig, bats, passerine birds,

OOC

N NH

Carboxy-biocytin

and most fishes and invertebrates; other animals synthe-

C O

size it as an intermediate in the uronic acid pathway of

glucose metabolism (Chapter 20). In those species for

which it is a vitamin, there is a block in that pathway due

to absence of gulonolactone oxidase. Both ascorbic acid

and dehydroascorbic acid have vitamin activity.

Figure 45-17. Biotin, biocytin, and carboxy-biocytin.

Vitamin C Is the Coenzyme

for Two Groups of Hydroxylases

Biotin also has a role in regulation of the cell cycle,

Ascorbic acid has specific roles in the copper-containing

acting to biotinylate key nuclear proteins.

hydroxylases and the α-ketoglutarate-linked iron-con-

taining hydroxylases. It also increases the activity of a

AS PART OF CoA AND ACP,

number of other enzymes in vitro, though this is a non-

specific reducing action. In addition, it has a number of

PANTOTHENIC ACID ACTS AS

nonenzymic effects due to its action as a reducing agent

A CARRIER OF ACYL RADICALS

and oxygen radical quencher.

Pantothenic acid has a central role in acyl group metab-

Dopamine

-hydroxylase is a copper-containing

olism when acting as the pantetheine functional moiety

enzyme involved in the synthesis of the catecholamines

of coenzyme A or acyl carrier protein (ACP) (Figure

norepinephrine and epinephrine from tyrosine in the

45-18). The pantetheine moiety is formed after combi-

adrenal medulla and central nervous system. During hy-

nation of pantothenate with cysteine, which provides

droxylation, the Cu⁺ is oxidized to Cu2+; reduction back

O C

CH₂

NH₂

H₃C

CH₃

H₃C

CH₃

CH₂OH

CH₂

N N

Pantothenic acid

Coenzyme A (CoASH)

Figure 45-18. Pantothenic acid and

coenzyme A. * Shows the site of acylation

O P O

by fatty acids.

496

CHAPTER 45

CH₂OH

CH₂

Ascorbate

Monodehydroascorbate

Dehydroascorbate

(semidehydroascorbate)

Figure 45-19. Vitamin C.

vitamin C prevent the common cold or reduce the du-

to Cu⁺ specifically requires ascorbate, which is oxidized

ration of its symptoms.

to monodehydroascorbate. Similar actions of ascorbate

occur in tyrosine degradation at the p-hydroxy-

phenylpyruvate hydroxylase step and at the homogenti-

sate dioxygenase step, which needs Fe2+ (Figure 30-12).

A number of peptide hormones have a carboxyl ter-

MINERALS ARE REQUIRED

minal amide which is derived from a glycine terminal

FOR BOTH PHYSIOLOGIC &

residue. This glycine is hydroxylated on the α-carbon

by a copper-containing enzyme, peptidylglycine hy-

BIOCHEMICAL FUNCTIONS

droxylase, which, again, requires ascorbate for reduc-

tion of Cu2+.

Many of the essential minerals (Table 45-2) are widely

A number of iron-containing, ascorbate-requiring

distributed in foods, and most people eating a normal

hydroxylases share a common reaction mechanism in

mixed diet are likely to receive adequate intakes. The

which hydroxylation of the substrate is linked to decar-

boxylation of α-ketoglutarate (Figure 28-11). Many of

these enzymes are involved in the modification of pre-

cursor proteins. Proline and lysine hydroxylases are

Table 45-2. Classification of essential minerals

required for the postsynthetic modification of procol-

according to their function.

lagen to collagen, and proline hydroxylase is also re-

quired in formation of osteocalcin and the C1q com-

Function

Mineral

ponent of complement. Aspartate β-hydroxylase is

required for the postsynthetic modification of the pre-

Structural function

Calcium, magnesium, phosphate

cursor of protein C, the vitamin K-dependent protease

Involved in membrane

Sodium, potassium

which hydrolyzes activated factor V in the blood clot-

function: principal

ting cascade. Trimethyllysine and γ-butyrobetaine hy-

cations of extracellular-

droxylases are required for the synthesis of carnitine.

and intracellular fluids,

respectively

Vitamin C Deficiency Causes Scurvy

Function as prosthetic

Cobalt, copper, iron, molybde-

Signs of vitamin C deficiency in scurvy include skin

groups in enzymes

num, selenium, zinc

changes, fragility of blood capillaries, gum decay, tooth

Regulatory role or role

Calcium, chromium, iodine,

loss, and bone fracture, many of which can be attrib-

in hormone action

magnesium, manganese, sodium,

uted to deficient collagen synthesis.

potassium

Known to be essential,

Silicon, vanadium, nickel, tin

There May Be Benefits From Higher

but function unknown

Intakes of Vitamin C

Have effects in the

Fluoride, lithium

At intakes above approximately 100 mg/d, the body’s

body, but essentiality is

capacity to metabolize vitamin C is saturated, and any

not established

further intake is excreted in the urine. However, in ad-

Without known nutritional

Aluminum, arsenic, antimony,

dition to its other roles, vitamin C enhances the absorp-

function but toxic in

boron, bromine, cadmium, ce-

tion of iron, and this depends on the presence of the vi-

excess

sium, germanium, lead, mercury,

tamin in the gut. Therefore, increased intakes may be

silver, strontium

beneficial. Evidence is unconvincing that high doses of

VITAMINS & MINERALS

497

amounts required vary from the order of grams per day

decarboxylation of α-keto acids and of transketolase

for sodium and calcium, through milligrams per day

in the pentose phosphate pathway. Riboflavin and

(eg, iron) to micrograms per day for the trace elements.

niacin are important cofactors in oxidoreduction re-

In general, mineral deficiencies are encountered when

actions, respectively present in flavoprotein enzymes

foods come from one region, where the soil may be de-

and in NAD and NADP.

ficient in some minerals, eg, iodine deficiency. Where

•

Pantothenic acid is present in coenzyme A and acyl

the diet comes from a variety of different regions, min-

carrier protein, which act as carriers for acyl groups

eral deficiencies are unlikely. However, iron deficiency

in metabolic reactions. Pyridoxine, as pyridoxal

is a general problem because if iron losses from the

phosphate, is the coenzyme for several enzymes of

body are relatively high

(eg, from heavy menstrual

amino acid metabolism, including the aminotrans-

blood loss), it is difficult to achieve an adequate intake

ferases, and of glycogen phosphorylase. Biotin is the

to replace the losses. Foods from soils containing high

coenzyme for several carboxylase enzymes.

levels of selenium cause toxicity, and increased intakes

•

Besides other functions, vitamin B₁₂ and folic acid

of common salt (sodium chloride) cause hypertension

take part in providing one-carbon residues for DNA

in susceptible individuals.

synthesis, deficiency resulting in megaloblastic ane-

mia. Vitamin C is a water-soluble antioxidant that

SUMMARY

maintains vitamin E and many metal cofactors in the

reduced state.

• Vitamins are organic nutrients with essential meta-

•

Inorganic mineral elements that have a function in

bolic functions, generally required in small amounts

the body must be provided in the diet. When insuffi-

in the diet because they cannot be synthesized by the

cient, deficiency symptoms may arise, and if present

body. The lipid-soluble vitamins (A, D, E, and K)

in excess they may be toxic.

are hydrophobic molecules requiring normal fat ab-

sorption for their efficient absorption and the avoid-

ance of deficiency symptoms.

REFERENCES

• Vitamin A (retinol), present in carnivorous diets, and

Bender DA, Bender AE: Nutrition: A Reference Handbook. Oxford

the provitamin (β-carotene), found in plants, form

Univ Press, 1997.

retinaldehyde, utilized in vision, and retinoic acid,

Bender DA: Nutritional Biochemistry of the Vitamins. 2nd ed. Cam-

which acts in the control of gene expression. Vitamin

bridge Univ Press, 2003.

D is a steroid prohormone yielding the active hor-

Garrow JS, James WPT, Ralph A: Human Nutrition and Dietetics,

mone derivative calcitriol, which regulates calcium

10th ed. Churchill-Livingstone, 2000.

and phosphate metabolism. Vitamin D deficiency

Halliwell B, Chirico S: Lipid peroxidation: its mechanism, mea-

leads to rickets and osteomalacia.

surement, and significance. Am J Clin Nutr

1993;57(5

Suppl):715S.

• Vitamin E (tocopherol) is the most important an-

tioxidant in the body, acting in the lipid phase of

Krinsky NI: Actions of carotenoids in biological systems. Annu Rev

Nutr 1993;13:561.

membranes and protecting against the effects of free

Padh H: Vitamin C: newer insights into its biochemical functions.

radicals. Vitamin K functions as cofactor to a car-

Nutr Rev 1991;49:65.

boxylase that acts on glutamate residues of clotting

Shane B: Folylpolyglutamate synthesis and role in the regulation of

factor precursor proteins to enable them to chelate

one-carbon metabolism. Vitam Horm 1989;45:263.

calcium.

Wiseman H, Halliwell B: Damage to DNA by reactive oxygen and

• The water-soluble vitamins of the B complex act as

nitrogen species: role in inflammatory disease and progression

enzyme cofactors. Thiamin is a cofactor in oxidative

to cancer. Biochem J 1996;313:17.

Intracellular Traffic & Sorting

of Proteins

Robert K. Murray, MD, PhD

BIOMEDICAL IMPORTANCE

the signal peptide are given below. Proteins synthesized

on free polyribosomes lack this particular signal pep-

Proteins must travel from polyribosomes to many dif-

tide and are delivered into the cytosol. There they are

ferent sites in the cell to perform their particular func-

directed to mitochondria, nuclei, and peroxisomes by

tions. Some are destined to be components of specific

specific signals—or remain in the cytosol if they lack a

organelles, others for the cytosol or for export, and yet

signal. Any protein that contains a targeting sequence

others will be located in the various cellular mem-

that is subsequently removed is designated as a prepro-

branes. Thus, there is considerable intracellular traffic

tein. In some cases a second peptide is also removed,

of proteins. Many studies have shown that the Golgi

and in that event the original protein is known as a pre-

apparatus plays a major role in the sorting of proteins

proprotein (eg, preproalbumin; Chapter 50).

for their correct destinations. A major insight was the

Proteins synthesized and sorted in the rough ER

recognition that for proteins to attain their proper loca-

branch (Figure 46-2) include many destined for vari-

tions, they generally contain information (a signal or

ous membranes (eg, of the ER, Golgi apparatus, lyso-

coding sequence) that targets them appropriately. Once

somes, and plasma membrane) and for secretion. Lyso-

a number of the signals were defined, it became appar-

somal enzymes are also included. Thus, such proteins

ent that certain diseases result from mutations that af-

may reside in the membranes or lumens of the ER or

fect these signals. In this chapter we discuss the intracel-

follow the major transport route of intracellular pro-

lular traffic of proteins and their sorting and briefly

teins to the Golgi apparatus. Further signal-mediated

consider some of the disorders that result when abnor-

sorting of certain proteins occurs in the Golgi appara-

malities occur.

tus, resulting in delivery to lysosomes, membranes of

the Golgi apparatus, and other sites. Proteins destined

MANY PROTEINS ARE TARGETED

for the plasma membrane or for secretion pass through

the Golgi apparatus but generally are not thought to

BY SIGNAL SEQUENCES TO THEIR

carry specific sorting signals; they are believed to reach

CORRECT DESTINATIONS

their destinations by default.

The protein biosynthetic pathways in cells can be con-

The entire pathway of ER → Golgi apparatus →

sidered to be one large sorting system. Many proteins

plasma membrane is often called the secretory or exo-

carry signals (usually but not always specific sequences

cytotic pathway. Events along this route will be given

of amino acids) that direct them to their destination,

special attention. Most of the proteins reaching the

thus ensuring that they will end up in the appropriate

Golgi apparatus or the plasma membrane are carried in

membrane or cell compartment; these signals are a fun-

transport vesicles; a brief description of the formation

damental component of the sorting system. Usually the

of these important particles will be given subsequently.

signal sequences are recognized and interact with com-

Other proteins destined for secretion are carried in se-

plementary areas of proteins that serve as receptors for

cretory vesicles (Figure 46-2). These are prominent in

the proteins that contain them.

the pancreas and certain other glands. Their mobiliza-

A major sorting decision is made early in protein

tion and discharge are regulated and often referred to as

biosynthesis, when specific proteins are synthesized ei-

“regulated secretion,” whereas the secretory pathway

ther on free or on membrane-bound polyribosomes.

involving transport vesicles is called

“constitutive.”

This results in two sorting branches called the cytosolic

Experimental approaches that have afforded major

branch and the rough endoplasmic reticulum (RER)

insights to the processes described in this chapter in-

branch (Figure 46-1). This sorting occurs because pro-

clude (1) use of yeast mutants; (2) application of re-

teins synthesized on membrane-bound polyribosomes

combinant DNA techniques (eg, mutating or eliminat-

contain a signal peptide that mediates their attach-

ing particular sequences in proteins, or fusing new

ment to the membrane of the ER. Further details on

sequences onto them; and (3) development of in vitro

498

INTRACELLULAR TRAFFIC & SORTING OF PROTEINS

499

Proteins

about 20-80 amino acids in length, which is not highly

Mitochondrial

conserved but contains many positively charged amino

acids (eg, Lys or Arg). The presequence is equivalent to

Nuclear

(1) Cytosolic

a signal peptide mediating attachment of polyribosomes

Peroxisomal

to membranes of the ER (see below), but in this in-

Cytosolic

stance targeting proteins to the matrix; if the leader se-

Polyribosomes

quence is cleaved off, potential matrix proteins will not

ER membrane

reach their destination.

GA membrane

Translocation is believed to occur posttranslation-

(2) Rough ER

Plasma membrane

ally, after the matrix proteins are released from the cy-

tosolic polyribosomes. Interactions with a number of

Secretory

cytosolic proteins that act as chaperones (see below)

Lysosomal enzymes

and as targeting factors occur prior to translocation.

Figure 46-1. Diagrammatic representation of the

Two distinct translocation complexes are situated

two branches of protein sorting occurring by synthesis

in the outer and inner mitochondrial membranes, re-

ferred to

(respectively) as TOM

(translocase-of-the-

on (1) cytosolic and (2) membrane-bound polyribo-

outer membrane) and TIM (translocase-of-the-inner

somes. The mitochondrial proteins listed are encoded

membrane). Each complex has been analyzed and

by nuclear genes. Some of the signals used in further

found to be composed of a number of proteins, some of

sorting of these proteins are listed in Table 46-4. (ER,

which act as receptors for the incoming proteins and

endoplasmic reticulum; GA, Golgi apparatus.)

others as components of the transmembrane pores

through which these proteins must pass. Proteins must

be in the unfolded state to pass through the com-

systems (eg, to study translocation in the ER and mech-

plexes, and this is made possible by ATP-dependent

anisms of vesicle formation).

binding to several chaperone proteins. The roles of

The sorting of proteins belonging to the cytosolic

chaperone proteins in protein folding are discussed later

branch referred to above is described next, starting with

in this chapter. In mitochondria, they are involved in

mitochondrial proteins.

translocation, sorting, folding, assembly, and degrada-

tion of imported proteins. A proton-motive force

across the inner membrane is required for import; it is

made up of the electric potential across the membrane

THE MITOCHONDRION BOTH IMPORTS

(inside negative) and the pH gradient (see Chapter

& SYNTHESIZES PROTEINS

12). The positively charged leader sequence may be

Mitochondria contain many proteins. Thirteen pro-

helped through the membrane by the negative charge

teins

(mostly membrane components of the electron

in the matrix. The presequence is split off in the matrix

transport chain) are encoded by the mitochondrial

by a matrix-processing peptidase

(MPP). Contact

genome and synthesized in that organelle using its own

with other chaperones present in the matrix is essential

protein-synthesizing system. However, the majority (at

to complete the overall process of import. Interaction

least several hundred) are encoded by nuclear genes,

with mt-Hsp70 (Hsp = heat shock protein) ensures

are synthesized outside the mitochondria on cytosolic

proper import into the matrix and prevents misfolding

polyribosomes, and must be imported. Yeast cells have

or aggregation, while interaction with the mt-Hsp60-

proved to be a particularly useful system for analyzing

Hsp10 system ensures proper folding. The latter pro-

the mechanisms of import of mitochondrial proteins,

teins resemble the bacterial GroEL chaperonins, a sub-

partly because it has proved possible to generate a vari-

class of chaperones that form complex cage-like

ety of mutants that have illuminated the fundamental

assemblies made up of heptameric ring structures. The

processes involved. Most progress has been made in the

interactions of imported proteins with the above chap-

study of proteins present in the mitochondrial matrix,

erones require hydrolysis of ATP to drive them.

such as the F₁ ATPase subunits. Only the pathway of

The details of how preproteins are translocated have

import of matrix proteins will be discussed in any detail

not been fully elucidated. It is possible that the electric

here.

potential associated with the inner mitochondrial mem-

Matrix proteins must pass from cytosolic polyribo-

brane causes a conformational change in the unfolded

somes through the outer and inner mitochondrial

preprotein being translocated and that this helps to pull

membranes to reach their destination. Passage through

it across. Furthermore, the fact that the matrix is more

the two membranes is called translocation. They have

negative than the intermembrane space may “attract”

an amino terminal leader sequence

(presequence),

the positively charged amino terminal of the preprotein

Cytosol

Early

Secretory

endosome

Constitutive

storage

(excretory)

granule

transport

Prelysosome

vesicle

(or late endosome)

Lysosome

TGN

Golgi

trans

apparatus

medial

cis

Endoplasmic

reticulum

Nuclear

envelope

Figure 46-2. Diagrammatic representation of the rough endoplasmic reticu-

lum branch of protein sorting. Newly synthesized proteins are inserted into the

ER membrane or lumen from membrane-bound polyribosomes (small black cir-

cles studding the cytosolic face of the ER). Those proteins that are transported

out of the ER (indicated by solid black arrows) do so from ribosome-free transi-

tional elements. Such proteins may then pass through the various subcompart-

ments of the Golgi until they reach the TGN, the exit side of the Golgi. In the TGN,

proteins are segregated and sorted. Secretory proteins accumulate in secretory

storage granules from which they may be expelled as shown in the upper right-

hand side of the figure. Proteins destined for the plasma membrane or those that

are secreted in a constitutive manner are carried out to the cell surface in trans-

port vesicles, as indicated in the upper middle area of the figure. Some proteins

may reach the cell surface via late and early endosomes. Other proteins enter

prelysosomes (late endosomes) and are selectively transferred to lysosomes. The

endocytic pathway illustrated in the upper left-hand area of the figure is consid-

ered elsewhere in this chapter. Retrieval from the Golgi apparatus to the ER is not

considered in this scheme. (CGN, cis-Golgi network; TGN, trans-Golgi network.)

(Courtesy of E Degen.)

500

INTRACELLULAR TRAFFIC & SORTING OF PROTEINS

501

to enter the matrix. Close contact between the mem-

These macromolecules include histones, ribosomal pro-

brane sites in the outer and inner membranes involved

teins and ribosomal subunits, transcription factors, and

in translocation is necessary.

mRNA molecules. The transport is bidirectional and

The above describes the major pathway of proteins

occurs through the nuclear pore complexes (NPCs).

destined for the mitochondrial matrix. However, cer-

These are complex structures with a mass approxi-

tain proteins insert into the outer mitochondrial

mately 30 times that of a ribosome and are composed

membrane facilitated by the TOM complex. Others

of about 100 different proteins. The diameter of an

stop in the intermembrane space, and some insert into

NPC is approximately 9 nm but can increase up to ap-

the inner membrane. Yet others proceed into the ma-

proximately 28 nm. Molecules smaller than about 40

trix and then return to the inner membrane or inter-

kDa can pass through the channel of the NPC by diffu-

membrane space. A number of proteins contain two

sion, but special translocation mechanisms exist for

signaling sequences—one to enter the mitochondrial

larger molecules. These mechanisms are under intensive

matrix and the other to mediate subsequent relocation

investigation, but some important features have already

(eg, into the inner membrane). Certain mitochondrial

emerged.

proteins do not contain presequences (eg, cytochrome

Here we shall mainly describe nuclear import of

c, which locates in the inter membrane space), and oth-

certain macromolecules. The general picture that has

ers contain internal presequences. Overall, proteins

emerged is that proteins to be imported (cargo mole-

employ a variety of mechanisms and routes to attain

cules) carry a nuclear localization signal (NLS). One

their final destinations in mitochondria.

example of an NLS is the amino acid sequence (Pro)₂-

General features that apply to the import of proteins

(Lys)₄-Ala-Lys-Val, which is markedly rich in basic ly-

into organelles, including mitochondria and some of

sine residues. Depending on which NLS it contains, a

the other organelles to be discussed below, are summa-

cargo molecule interacts with one of a family of soluble

rized in Table 46-1.

proteins called importins, and the complex docks at

the NPC. Another family of proteins called Ran plays a

IMPORTINS & EXPORTINS ARE

critical regulatory role in the interaction of the complex

with the NPC and in its translocation through the

INVOLVED IN TRANSPORT

NPC. Ran proteins are small monomeric nuclear GTP-

OF MACROMOLECULES IN

ases and, like other GTPases, exist in either GTP-

& OUT OF THE NUCLEUS

bound or GDP-bound states. They are themselves reg-

ulated by guanine nucleotide exchange factors

It has been estimated that more than a million macro-

(GEFs; eg, the protein RCC1 in eukaryotes), which are

molecules per minute are transported between the nu-

located in the nucleus, and Ran guanine-activating

cleus and the cytoplasm in an active eukaryotic cell.

proteins (GAPs), which are predominantly cytoplas-

mic. The GTP-bound state of Ran is favored in the nu-

cleus and the GDP-bound state in the cytoplasm. The

Table 46-1. Some general features of protein

conformations and activities of Ran molecules vary de-

import to organelles.¹

pending on whether GTP or GDP is bound to them

(the GTP-bound state is active; see discussion of G pro-

• Import of a protein into an organelle usually occurs in three

teins in Chapter 43). The asymmetry between nucleus

stages: recognition, translocation, and maturation.

and cytoplasm—with respect to which of these two nu-

• Targeting sequences on the protein are recognized in the

cleotides is bound to Ran molecules—is thought to be

cytoplasm or on the surface of the organelle.

crucial in understanding the roles of Ran in transferring

• The protein is unfolded for translocation, a state main-

complexes unidirectionally across the NPC. When

tained in the cytoplasm by chaperones.

cargo molecules are released inside the nucleus, the im-

• Threading of the protein through a membrane requires en-

ergy and organellar chaperones on the trans side of the

portins recirculate to the cytoplasm to be used again.

membrane.

Figure 46-3 summarizes some of the principal features

• Cycles of binding and release of the protein to the chaper-

in the above process.

one result in pulling of its polypeptide chain through the

Other small monomeric GTPases (eg, ARF, Rab,

membrane.

Ras, and Rho) are important in various cellular pro-

• Other proteins within the organelle catalyze folding of the

cesses such as vesicle formation and transport (ARF and

protein, often attaching cofactors or oligosaccharides and

Rab; see below), certain growth and differentiation

assembling them into active monomers or oligomers.

processes (Ras), and formation of the actin cytoskele-

ton. A process involving GTP and GDP is also crucial

¹Data from McNew JA, Goodman JM: The targeting and assembly

of peroxisomal proteins: some old rules do not apply. Trends

in the transport of proteins across the membrane of the

Biochem Sci 1998;21:54.

ER (see below).

502

CHAPTER 46

Targeting

RanGTP

GDP

OFF

GTP

Ran

GAP

Ran

Docking

Ran exchange

GEF

RanGDP

Ran

GTP

GDP

Ran

GEF ?

RanBP1

GDP

Ran

GTP

Termination

Translocation

Ran

GTP

Ran

GAP

GTP

Ran

GTP

GDP

Recycle factors

Figure 46-3. Schematic representation of the proposed role of Ran in the import of cargo

carrying an NLS signal. (1) The targeting complex forms when the NLS receptor (α, an importin)

binds NLS cargo and the docking factor (β). (2) Docking occurs at filamentous sites that pro-

trude from the NPC. Ran-GDP docks independently. (3) Transfer to the translocation channel is

triggered when a RanGEF converts Ran-GDP to Ran-GTP. (4) The NPC catalyzes translocation of

the targeting complex. (5) Ran-GTP is recycled to Ran-GDP by docked RanGAP. (6) Ran-GTP dis-

rupts the targeting complex by binding to a site on β that overlaps with a binding site. (7) NLS

cargo dissociates from α, and Ran-GTP may dissociate from β. (8) α and β factors are recycled to

the cytoplasm. Inset: The Ran translocation switch is off in the cytoplasm and on in the nucleus.

Ran-GTP promotes NLS- and NES-directed translocation. However, cytoplasmic Ran is enriched

in Ran-GDP (OFF) by an active RanGAP, and nuclear pools are enriched in Ran-GTP (ON) by an

active GEF. RanBP1 promotes the contrary activities of these two factors. Direct linkage of nu-

clear and cytoplasmic pools of Ran occurs through the NPC by an unknown shuttling mecha-

nism. P_i, inorganic phosphate; NLS, nuclear localization signal; NPC, nuclear pore complex; GEF,

guanine nucleotide exchange factor; GAP, guanine-activating protein; NES, nuclear export sig-

nal; BP, binding protein. (Reprinted, with permission, from Goldfarb DS: Whose finger is on the

switch? Science 1997;276:1814.)

INTRACELLULAR TRAFFIC & SORTING OF PROTEINS

503

Proteins similar to importins, referred to as ex-

the synthesis of bile acids, and a marked reduction of

portins, are involved in export of many macromole-

plasmalogens. The condition is believed to be due to

cules from the nucleus. Cargo molecules for export

mutations in genes encoding certain proteins—so

carry nuclear export signals (NESs). Ran proteins are

called peroxins—involved in various steps of peroxi-

involved in this process also, and it is now established

some biogenesis (such as the import of proteins de-

that the processes of import and export share a number

scribed above), or in genes encoding certain peroxiso-

of common features.

mal enzymes themselves. Two closely related conditions

are neonatal adrenoleukodystrophy and infantile

Refsum disease. Zellweger syndrome and these two

MOST CASES OF ZELLWEGER SYNDROME

conditions represent a spectrum of overlapping fea-

ARE DUE TO MUTATIONS IN GENES

tures, with Zellweger syndrome being the most severe

(many proteins affected) and infantile Refsum disease

INVOLVED IN THE BIOGENESIS

the least severe (only one or a few proteins affected).

OF PEROXISOMES

Table 46-2 lists some features of these and related con-

The peroxisome is an important organelle involved in

ditions.

aspects of the metabolism of many molecules, including

fatty acids and other lipids (eg, plasmalogens, choles-

THE SIGNAL HYPOTHESIS EXPLAINS

terol, bile acids), purines, amino acids, and hydrogen

HOW POLYRIBOSOMES BIND TO THE

peroxide. The peroxisome is bounded by a single mem-

ENDOPLASMIC RETICULUM

brane and contains more than 50 enzymes; catalase and

urate oxidase are marker enzymes for this organelle. Its

As indicated above, the rough ER branch is the second

proteins are synthesized on cytosolic polyribosomes and

of the two branches involved in the synthesis and sort-

fold prior to import. The pathways of import of a num-

ing of proteins. In this branch, proteins are synthesized

ber of its proteins and enzymes have been studied, some

on membrane-bound polyribosomes and translocated

being matrix components and others membrane com-

into the lumen of the rough ER prior to further sorting

ponents. At least two peroxisomal-matrix targeting

(Figure 46-2).

sequences (PTSs) have been discovered. One, PTS1, is

The signal hypothesis was proposed by Blobel and

a tripeptide (ie, Ser-Lys-Leu [SKL], but variations of

Sabatini partly to explain the distinction between free

this sequence have been detected) located at the car-

and membrane-bound polyribosomes. They found that

boxyl terminal of a number of matrix proteins, includ-

proteins synthesized on membrane-bound polyribo-

ing catalase. Another, PTS2, consisting of about 26-36

somes contained a peptide extension (signal peptide)

amino acids, has been found in at least four matrix pro-

teins (eg, thiolase) and, unlike PTS1, is cleaved after

Table 46-2. Disorders due to peroxisomal

entry into the matrix. Proteins containing PTS1 se-

quences form complexes with a soluble receptor protein

abnormalities.¹

(PTS1R) and proteins containing PTS2 sequences

complex with another, PTS2R. The resulting com-

MIM Number²

plexes then interact with a membrane receptor, Pex14p.

Zellweger syndrome

214100

Proteins involved in further transport of proteins into

Neonatal adrenoleukodystrophy

202370

the matrix are also present. Most peroxisomal mem-

Infantile Refsum disease

266510

brane proteins have been found to contain neither of

Hyperpipecolic acidemia

239400

the above two targeting sequences, but apparently con-

Rhizomelic chondrodysplasia punctata

215100

tain others. The import system can handle intact

Adrenoleukodystrophy

300100

oligomers (eg, tetrameric catalase). Import of matrix

Pseudo-neonatal adrenoleukodystrophy

264470

proteins requires ATP, whereas import of membrane

Pseudo-Zellweger syndrome

261510

proteins does not.

Hyperoxaluria type 1

259900

Interest in import of proteins into peroxisomes has

Acatalasemia

115500

been stimulated by studies on Zellweger syndrome.

Glutaryl-CoA oxidase deficiency

231690

This condition is apparent at birth and is characterized

¹Reproduced, with permission, from Seashore MR, Wappner RS:

by profound neurologic impairment, victims often

Genetics in Primary Care & Clinical Medicine. Appleton & Lange,

dying within a year. The number of peroxisomes can

1996.

vary from being almost normal to being virtually absent

²MIM = Mendelian Inheritance in Man. Each number specifies a ref-

in some patients. Biochemical findings include an accu-

erence in which information regarding each of the above condi-

mulation of very long chain fatty acids, abnormalities of

tions can be found.

504

CHAPTER 46

at their amino terminals which mediated their attach-

and the β subunit spans the membrane. When the SRP-

ment to the membranes of the ER. As noted above,

signal peptide complex interacts with the receptor, the

proteins whose entire synthesis occurs on free polyribo-

exchange of GDP for GTP is stimulated. This form of

somes lack this signal peptide. An important aspect of

the receptor (with GTP bound) has a high affinity for

the signal hypothesis was that it suggested—as turns

the SRP and thus releases the signal peptide, which binds

out to be the case—that all ribosomes have the same

to the translocation machinery (translocon) also present

structure and that the distinction between membrane-

in the ER membrane. The α subunit then hydrolyzes its

bound and free ribosomes depends solely on the for-

bound GTP, restoring GDP and completing a GTP-

mer’s carrying proteins that have signal peptides. Much

GDP cycle. The unidirectionality of this cycle helps drive

evidence has confirmed the original hypothesis. Because

the interaction of the polyribosome and its signal peptide

many membrane proteins are synthesized on mem-

with the ER membrane in the forward direction.

brane-bound polyribosomes, the signal hypothesis plays

The translocon consists of a number of membrane

an important role in concepts of membrane assembly.

proteins that form a protein-conducting channel in the

Some characteristics of signal peptides are summarized

ER membrane through which the newly synthesized

in Table 46-3.

protein may pass. The channel appears to be open only

Figure 46-4 illustrates the principal features in rela-

when a signal peptide is present, preserving conductance

tion to the passage of a secreted protein through the

across the ER membrane when it closes. The conduc-

membrane of the ER. It incorporates features from the

tance of the channel has been measured experimentally.

original signal hypothesis and from subsequent work.

Specific functions of a number of components of the

The mRNA for such a protein encodes an amino termi-

translocon have been identified or suggested. TRAM

nal signal peptide (also variously called a leader se-

(translocating chain-associated membrane) protein may

quence, a transient insertion signal, a signal sequence,

bind the signal sequence as it initially interacts with the

or a presequence). The signal hypothesis proposed that

translocon and the Sec61p complex (consisting of three

the protein is inserted into the ER membrane at the

proteins) binds the heavy subunit of the ribosome.

same time as its mRNA is being translated on polyribo-

The insertion of the signal peptide into the conduct-

somes, so-called cotranslational insertion. As the sig-

ing channel, while the other end of the parent protein is

nal peptide emerges from the large subunit of the ribo-

still attached to ribosomes, is termed

“cotranslational

some, it is recognized by a signal recognition particle

insertion.” The process of elongation of the remaining

(SRP) that blocks further translation after about 70

portion of the protein probably facilitates passage of the

amino acids have been polymerized (40 buried in the

nascent protein across the lipid bilayer as the ribosomes

large ribosomal subunit and 30 exposed). The block is

remain attached to the membrane of the ER. Thus, the

referred to as elongation arrest. The SRP contains six

rough (or ribosome-studded) ER is formed. It is impor-

proteins and has a 7S RNA associated with it that is

tant that the protein be kept in an unfolded state prior

closely related to the Alu family of highly repeated

to entering the conducting channel—otherwise, it may

DNA sequences (Chapter 36). The SRP-imposed block

not be able to gain access to the channel.

is not released until the SRP-signal peptide-polyribo-

Ribosomes remain attached to the ER during syn-

some complex has bound to the so-called docking pro-

thesis of signal peptide-containing proteins but are re-

tein (SRP-R, a receptor for the SRP) on the ER mem-

leased and dissociated into their two types of subunits

brane; the SRP thus guides the signal peptide to the

when the process is completed. The signal peptide is

SRP-R and prevents premature folding and expulsion

hydrolyzed by signal peptidase, located on the luminal

of the protein being synthesized into the cytosol.

side of the ER membrane (Figure 46-4), and then is

The SRP-R is an integral membrane protein com-

apparently rapidly degraded by proteases.

posed of α and β subunits. The α subunit binds GDP

Cytochrome P450 (Chapter 53), an integral ER

membrane protein, does not completely cross the mem-

brane. Instead, it resides in the membrane with its sig-

Table 46-3. Some properties of signal peptides.

nal peptide intact. Its passage through the membrane is

prevented by a sequence of amino acids called a halt- or

• Usually, but not always, located at the amino terminal

stop-transfer signal.

• Contain approximately 12-35 amino acids

Secretory proteins and proteins destined for mem-

• Methionine is usually the amino terminal amino acid

branes distal to the ER completely traverse the mem-

• Contain a central cluster of hydrophobic amino acids

brane bilayer and are discharged into the lumen of the

• Contain at least one positively charged amino acid near

ER. N-Glycan chains, if present, are added (Chapter

their amino terminal

47) as these proteins traverse the inner part of the ER

• Usually cleaved off at the carboxyl terminal end of an Ala

membrane—a process called “cotranslational glycosyla-

residue by signal peptidase

tion.” Subsequently, the proteins are found in the

INTRACELLULAR TRAFFIC & SORTING OF PROTEINS

505

3′

Signal codons

Signal peptide

SRP

Signal peptidase

Ribosome receptor

Signal receptor

Figure 46-4. Diagram of the signal hypothesis for the transport of secreted proteins across the

ER membrane. The ribosomes synthesizing a protein move along the messenger RNA specifying the

amino acid sequence of the protein. (The messenger is represented by the line between 5′ and 3′.)

The codon AUG marks the start of the message for the protein; the hatched lines that follow AUG

represent the codons for the signal sequence. As the protein grows out from the larger ribosomal

subunit, the signal sequence is exposed and bound by the signal recognition particle (SRP). Transla-

tion is blocked until the complex binds to the “docking protein,” also designated SRP-R (repre-

sented by the solid bar) on the ER membrane. There is also a receptor (open bar) for the ribosome

itself. The interaction of the ribosome and growing peptide chain with the ER membrane results in

the opening of a channel through which the protein is transported to the interior space of the ER.

During translocation, the signal sequence of most proteins is removed by an enzyme called the

“signal peptidase,” located at the luminal surface of the ER membrane. The completed protein is

eventually released by the ribosome, which then separates into its two components, the large and

small ribosomal subunits. The protein ends up inside the ER. See text for further details. (Slightly

modified and reproduced, with permission, from Marx JL: Newly made proteins zip through the cell. Sci-

lumen of the Golgi apparatus, where further changes in

PROTEINS FOLLOW SEVERAL ROUTES

glycan chains occur (Figure 47-9) prior to intracellular

TO BE INSERTED INTO OR ATTACHED

distribution or secretion. There is strong evidence that

TO THE MEMBRANES OF THE

the signal peptide is involved in the process of protein

ENDOPLASMIC RETICULUM

insertion into ER membranes. Mutant proteins, con-

taining altered signal peptides in which a hydrophobic

The routes that proteins follow to be inserted into the

amino acid is replaced by a hydrophilic one, are not in-

membranes of the ER include the following.

serted into ER membranes. Nonmembrane proteins

A. COTRANSLATIONAL INSERTION

(eg, α-globin) to which signal peptides have been at-

tached by genetic engineering can be inserted into the

Figure 46-5 shows a variety of ways in which proteins

lumen of the ER or even secreted.

are distributed in the plasma membrane. In particular,

There is considerable evidence that a second trans-

the amino terminals of certain proteins (eg, the LDL re-

poson in the ER membrane is involved in retrograde

ceptor) can be seen to be on the extracytoplasmic face,

transport of various molecules from the ER lumen to

whereas for other proteins (eg, the asialoglycoprotein re-

the cytosol. These molecules include unfolded or mis-

ceptor) the carboxyl terminals are on this face. To ex-

folded glycoproteins, glycopeptides, and oligosaccha-

plain these dispositions, one must consider the initial

rides. Some at least of these molecules are degraded in

biosynthetic events at the ER membrane. The LDL re-

proteasomes. Thus, there is two-way traffic across the

ceptor enters the ER membrane in a manner analogous

ER membrane.

to a secretory protein (Figure 46-4); it partly traverses

506

CHAPTER 46

EXTRACYTOPLASMIC

FACE

C C

Various transporters (eg, glucose)

Insulin and

Influenza neuraminidase

IGF-I receptors

G protein-coupled receptors

Asialoglycoprotein receptor

Transferrin receptor

HLA-DR invariant chain

LDL receptor

HLA-A heavy chain

Influenza hemagglutinin

Figure 46-5. Variations in the way in which proteins are inserted into membranes. This

schematic representation, which illustrates a number of possible orientations, shows the seg-

ments of the proteins within the membrane as α-helices and the other segments as lines. The

LDL receptor, which crosses the membrane once and has its amino terminal on the exterior, is

called a type I transmembrane protein. The asialoglycoprotein receptor, which also crosses the

membrane once but has its carboxyl terminal on the exterior, is called a type II transmembrane

protein. The various transporters indicated (eg, glucose) cross the membrane a number of times

and are called type III transmembrane proteins; they are also referred to as polytopic membrane

proteins. (N, amino terminal; C, carboxyl terminal.) (Adapted, with permission, from Wickner WT,

Lodish HF: Multiple mechanisms of protein insertion into and across membranes. Science

the ER membrane, its signal peptide is cleaved, and its

cleaved insertion sequences and as halt-transfer signals,

amino terminal protrudes into the lumen. However, it is

respectively. Each pair of helical segments is inserted as a

retained in the membrane because it contains a highly

hairpin. Sequences that determine the structure of a

hydrophobic segment, the halt- or stop-transfer signal.

protein in a membrane are called topogenic sequences.

This sequence forms the single transmembrane segment

As explained in the legend to Figure 46-5, the above

of the protein and is its membrane-anchoring domain.

three proteins are examples of type I, type II, and type

The small patch of ER membrane in which the newly

III transmembrane proteins.

synthesized LDL receptor is located subsequently buds

off as a component of a transport vesicle, probably from

B. SYNTHESIS ON FREE POLYRIBOSOMES

the transitional elements of the ER (Figure 46-2). As

& SUBSEQUENT ATTACHMENT TO THE

described below in the discussion of asymmetry of pro-

ENDOPLASMIC RETICULUM MEMBRANE

teins and lipids in membrane assembly, the disposition

of the receptor in the ER membrane is preserved in the

An example is cytochrome b₅, which enters the ER

vesicle, which eventually fuses with the plasma mem-

membrane spontaneously.

brane. In contrast, the asialoglycoprotein receptor pos-

sesses an internal insertion sequence, which inserts into

C. RETENTION AT THE LUMINAL ASPECT

the membrane but is not cleaved. This acts as an anchor,

OF THE ENDOPLASMIC RETICULUM

and its carboxyl terminal is extruded through the mem-

BY SPECIFIC AMINO ACID SEQUENCES

brane. The more complex disposition of the trans-

porters (eg, for glucose) can be explained by the fact

A number of proteins possess the amino acid sequence

that alternating transmembrane α-helices act as un-

KDEL (Lys-Asp-Glu-Leu) at their carboxyl terminal.

INTRACELLULAR TRAFFIC & SORTING OF PROTEINS

507

This sequence specifies that such proteins will be at-

denote transport steps that may be independent of tar-

tached to the inner aspect of the ER in a relatively loose

geting signals, whereas the vertical open arrows repre-

manner. The chaperone BiP (see below) is one such

sent steps that depend on specific signals. Thus, flow of

protein. Actually, KDEL-containing proteins first travel

certain proteins

(including membrane proteins) from

to the Golgi, interact there with a specific KDEL recep-

the ER to the plasma membrane (designated “bulk

tor protein, and then return in transport vesicles to the

flow,” as it is nonselective) probably occurs without any

ER, where they dissociate from the receptor.

targeting sequences being involved, ie, by default. On

the other hand, insertion of resident proteins into the

D. RETROGRADE TRANSPORT FROM

ER and Golgi membranes is dependent upon specific

THE GOLGI APPARATUS

signals

(eg, KDEL or halt-transfer sequences for the

Certain other non-KDEL-containing proteins destined

ER). Similarly, transport of many enzymes to lysosomes

for the membranes of the ER also pass to the Golgi and

is dependent upon the Man 6-P signal (Chapter 47),

then return, by retrograde vesicular transport, to the ER

and a signal may be involved for entry of proteins into

to be inserted therein (see below).

secretory granules. Table 46-4 summarizes informa-

The foregoing paragraphs demonstrate that a vari-

tion on sequences that are known to be involved in tar-

ety of routes are involved in assembly of the proteins of

geting various proteins to their correct intracellular sites.

the ER membranes; a similar situation probably holds

for other membranes

(eg, the mitochondrial mem-

CHAPERONES ARE PROTEINS

branes and the plasma membrane). Precise targeting se-

THAT PREVENT FAULTY FOLDING

quences have been identified in some instances (eg,

& UNPRODUCTIVE INTERACTIONS

KDEL sequences).

The topic of membrane biogenesis is discussed fur-

OF OTHER PROTEINS

ther later in this chapter.

Exit from the ER may be the rate-limiting step in the

secretory pathway. In this context, it has been found

PROTEINS MOVE THROUGH CELLULAR

that certain proteins play a role in the assembly or

proper folding of other proteins without themselves

COMPARTMENTS TO SPECIFIC

being components of the latter. Such proteins are called

DESTINATIONS

molecular chaperones; a number of important proper-

A scheme representing the possible flow of proteins

ties of these proteins are listed in Table 46-5, and the

along the ER → Golgi apparatus → plasma membrane

names of some of particular importance in the ER are

route is shown in Figure 46-6. The horizontal arrows

listed in Table 46-6. Basically, they stabilize unfolded

Lysosomes

cis

medial

trans

Cell

Golgi

surface

Secretory

storage vesicles

Figure 46-6. Flow of membrane proteins from the endoplas-

mic reticulum (ER) to the cell surface. Horizontal arrows denote

steps that have been proposed to be signal independent and

thus represent bulk flow. The open vertical arrows in the boxes

denote retention of proteins that are resident in the membranes

of the organelle indicated. The open vertical arrows outside the

boxes indicate signal-mediated transport to lysosomes and secre-

tory storage granules. (Reproduced, with permission, from Pfeffer

SR, Rothman JE: Biosynthetic protein transport and sorting by the en-

doplasmic reticulum and Golgi. Annu Rev Biochem 1987;56:829.)

508

CHAPTER 46

Table 46-4. Some sequences or compounds that

Table 46-6. Some chaperones and enzymes

direct proteins to specific organelles.

involved in folding that are located in the rough

endoplasmic reticulum.

Targeting Sequence

or Compound

Organelle Targeted

• BiP (immunoglobulin heavy chain binding protein)

• GRP94 (glucose-regulated protein)

Signal peptide sequence

Membrane of ER

• Calnexin

Amino terminal

Luminal surface of ER

• Calreticulin

KDEL sequence

• PDI (protein disulfide isomerase)

(Lys-Asp-Glu-Leu)

• PPI (peptidyl prolyl cis-trans isomerase)

Amino terminal sequence

Mitochondrial matrix

(20-80 residues)

NLS¹ (eg, Pro₂-Lys₂-Ala-

Nucleus

Several examples of chaperones were introduced

Lys-Val)

above when the sorting of mitochondrial proteins was

discussed. The immunoglobulin heavy chain binding

PTS¹ (eg, Ser-Lys-Leu)

Peroxisome

protein (BiP) is located in the lumen of the ER. This

Mannose 6-phosphate

Lysosome

protein will bind abnormally folded immunoglobulin

¹NLS, nuclear localization signal; PTS, peroxisomal-matrix target-

heavy chains and certain other proteins and prevent

ing sequence.

them from leaving the ER, in which they are degraded.

Another important chaperone is calnexin, a calcium-

binding protein located in the ER membrane. This pro-

tein binds a wide variety of proteins, including mixed

histocompatibility

(MHC) antigens and a variety of

or partially folded intermediates, allowing them time to

serum proteins. As mentioned in Chapter 47, calnexin

fold properly, and prevent inappropriate interactions,

binds the monoglycosylated species of glycoproteins

thus combating the formation of nonfunctional struc-

that occur during processing of glycoproteins, retaining

tures. Most chaperones exhibit ATPase activity and

them in the ER until the glycoprotein has folded prop-

bind ADP and ATP. This activity is important for their

erly. Calreticulin, which is also a calcium-binding pro-

effect on folding. The ADP-chaperone complex often

tein, has properties similar to those of calnexin; it is not

has a high affinity for the unfolded protein, which,

membrane-bound. Chaperones are not the only pro-

when bound, stimulates release of ADP with replace-

teins in the ER lumen that are concerned with proper

ment by ATP. The ATP-chaperone complex, in turn,

folding of proteins. Two enzymes are present that play

releases segments of the protein that have folded prop-

an active role in folding. Protein disulfide isomerase

erly, and the cycle involving ADP and ATP binding is

(PDI) promotes rapid reshuffling of disulfide bonds

repeated until the folded protein is released.

until the correct set is achieved. Peptidyl prolyl isom-

erase (PPI) accelerates folding of proline-containing

proteins by catalyzing the cis-trans isomerization of

X-Pro bonds, where X is any amino acid residue.

Table 46-5. Some properties of chaperone

proteins.

TRANSPORT VESICLES ARE KEY PLAYERS

IN INTRACELLULAR PROTEIN TRAFFIC

• Present in a wide range of species from bacteria to humans

• Many are so-called heat shock proteins (Hsp)

Most proteins that are synthesized on membrane-

• Some are inducible by conditions that cause unfolding of

bound polyribosomes and are destined for the Golgi

newly synthesized proteins (eg, elevated temperature and

apparatus or plasma membrane reach these sites inside

various chemicals)

transport vesicles. The precise mechanisms by which

• They bind to predominantly hydrophobic regions of un-

proteins synthesized in the rough ER are inserted into

folded and aggregated proteins

these vesicles are not known. Those involved in trans-

• They act in part as a quality control or editing mechanism

port from the ER to the Golgi apparatus and vice

for detecting misfolded or otherwise defective proteins

versa—and from the Golgi to the plasma membrane—

• Most chaperones show associated ATPase activity, with ATP

are mainly clathrin-free, unlike the coated vesicles in-

or ADP being involved in the protein-chaperone interaction

volved in endocytosis (see discussions of the LDL re-

• Found in various cellular compartments such as cytosol,

ceptor in Chapters 25 and 26). For the sake of clarity,

mitochondria, and the lumen of the endoplasmic reticulum

the non-clathrin-coated vesicles will be referred to in

INTRACELLULAR TRAFFIC & SORTING OF PROTEINS

509

this text as transport vesicles. There is evidence that

Table 46-7. Factors involved in the formation of

proteins destined for the membranes of the Golgi appa-

non-clathrin-coated vesicles and their transport.

ratus contain specific signal sequences. On the other

hand, most proteins destined for the plasma membrane

•

ARF: ADP-ribosylation factor, a GTPase

or for secretion do not appear to contain specific sig-

•

Coatomer: A family of at least seven coat proteins (α, β, γ, δ,

nals, reaching these destinations by default.

ε, β′, and ζ). Different transport vesicles have different com-

plements of coat proteins.

The Golgi Apparatus Is Involved in

•

SNAP: Soluble NSF attachment factor

•

SNARE: SNAP receptor

Glycosylation & Sorting of Proteins

•

v-SNARE: Vesicle SNARE

The Golgi apparatus plays two important roles in mem-

•

t-SNARE: Target SNARE

brane synthesis. First, it is involved in the processing

•

GTP-γ-S: A nonhydrolyzable analog of GTP, used to test the

of the oligosaccharide chains of membrane and other

involvement of GTP

N-linked glycoproteins and also contains enzymes in-

•

NEM: N-Ethylmaleimide, a chemical that alkylates sulfhy-

volved in O-glycosylation (see Chapter 47). Second, it

dryl groups

is involved in the sorting of various proteins prior to

•

NSF: NEM-sensitive factor, an ATPase

their delivery to their appropriate intracellular destina-

•

Rab proteins: A family of ras-related proteins first observed

in rat brain; they are GTPases and are active when GTP is

tions. All parts of the Golgi apparatus participate in the

found

first role, whereas the trans-Golgi is particularly in-

•

Sec1: A member of a family of proteins that attach to

volved in the second and is very rich in vesicles. Because

t-SNAREs and are displaced from them by Rab proteins,

of their central role in protein transport, considerable

thereby allowing v-SNARE-t-SNARE interactions to occur.

research has been conducted in recent years concerning

the formation and fate of transport vesicles.

A Model of Non-Clathrin-Coated Vesicles

Step 2: Membrane-associated ARF recruits the coat

proteins that comprise the coatomer shell from the

Involves SNAREs & Other Factors

cytosol, forming a coated bud.

Vesicles lie at the heart of intracellular transport of

Step 3: The bud pinches off in a process involving

many proteins. Recently, significant progress has been

acyl-CoA—and probably ATP—to complete the

made in understanding the events involved in vesicle

formation of the coated vesicle.

formation and transport. This has transpired because of

Step 4: Coat disassembly (involving dissociation of

the use of a number of approaches. These include es-

ARF and coatomer shell) follows hydrolysis of

tablishment of cell-free systems with which to study

bound GTP; uncoating is necessary for fusion to

vesicle formation. For instance, it is possible to observe,

occur.

by electron microscopy, budding of vesicles from Golgi

Step 5: Vesicle targeting is achieved via members of

preparations incubated with cytosol and ATP. The de-

a family of integral proteins, termed v-SNAREs,

velopment of genetic approaches for studying vesicles

that tag the vesicle during its budding. v-SNAREs

in yeast has also been crucial. The picture is complex,

pair with cognate t-SNAREs in the target membrane

with its own nomenclature (Table 46-7), and involves

to dock the vesicle.

a variety of cytosolic and membrane proteins, GTP,

ATP, and accessory factors.

It is presumed that steps 4 and 5 are closely coupled

Based largely on a proposal by Rothman and col-

and that step 4 may follow step 5, with ARF and the

leagues, anterograde vesicular transport can be consid-

coatomer shell rapidly dissociating after docking.

ered to occur in eight steps (Figure 46-7). The basic

concept is that each transport vesicle bears a unique ad-

Step 6: The general fusion machinery then assem-

dress marker consisting of one or more v-SNARE pro-

bles on the paired SNARE complex; it includes an

teins, while each target membrane bears one or more

ATPase (NSF; NEM-sensitive factor) and the SNAP

complementary t-SNARE proteins with which the

(soluble NSF attachment factor) proteins. SNAPs

former interact specifically.

bind to the SNARE (SNAP receptor) complex, en-

abling NSF to bind.

Step 1: Coat assembly is initiated when ARF is ac-

tivated by binding GTP, which is exchanged for

Step 7: Hydrolysis of ATP by NSF is essential for

GDP. This leads to the association of GTP-bound

fusion, a process that can be inhibited by NEM (N-

ARF with its putative receptor (hatched in Figure

ethylmaleimide). Certain other proteins and calcium

46-7) in the donor membrane.

are also required.

510

CHAPTER 46

Coated

vesicle

t-SNARE

Coated

GTP-γ-S

bud

Acyl-CoA

SNAPs

NSF

ATP

GDP

Budding

SNAPs

NSF

Ca2+

20S fusion

Coatomer

particle

v-SNARE

ATP

GTP

NEM

Fusion

GTP

BFA

GDP

ARF

Nocodazole

Donor

Target

membrane

(eg, ER)

(eg, CGN)

Figure 46-7. Model of the steps in a round of anterograde vesicular transport. The

cycle starts in the bottom left-hand side of the figure, where two molecules of ARF

are represented as small ovals containing GDP. The steps in the cycle are described in

the text. Most of the abbreviations used are explained in Table 46-7. The roles of Rab

and Sec1 proteins (see text) in the overall process are not dealt with in this figure.

(CGN, cis-Golgi network; BFA, Brefeldin A.) (Adapted from Rothman JE: Mechanisms of

intracellular protein transport. Nature 1994;372:55.) (Courtesy of E Degen.)

Step 8: Retrograde transport occurs to restart the

doubt remain to be discovered. COPI vesicles are in-

cycle. This last step may retrieve certain proteins

volved in bidirectional transport from the ER to the

or recycle v-SNAREs. Nocodazole, a microtubule-

Golgi and in the reverse direction, whereas COPII vesi-

disrupting agent, inhibits this step.

cles are involved mainly in transport in the former di-

rection. Clathrin-containing vesicles are involved in

transport from the trans-Golgi network to prelysosomes

Brefeldin A Inhibits the Coating Process

and from the plasma membrane to endosomes, respec-

tively. Regarding selection of cargo molecules by vesi-

The following points expand and clarify the above.

cles, this appears to be primarily a function of the coat

(a) To participate in step 1, ARF must first be modi-

proteins of vesicles. Cargo molecules may interact with

fied by addition of myristic acid (C14:0), employing

coat proteins either directly or via intermediary proteins

myristoyl-CoA as the acyl donor. Myristoylation is one

that attach to coat proteins, and they then become en-

of a number of enzyme-catalyzed posttranslational mod-

closed in their appropriate vesicles.

ifications, involving addition of certain lipids to specific

residues of proteins, that facilitate the binding of pro-

GTP from binding to ARF in step 1 and thus inhibits

teins to the cytosolic surfaces of membranes or vesicles.

the entire coating process. In its presence, the Golgi ap-

Others are addition of palmitate, farnesyl, and geranyl-

paratus appears to disintegrate, and fragments are lost.

geranyl; the two latter molecules are polyisoprenoids

It may do this by inhibiting the guanine nucleotide ex-

containing 15 and 20 carbon atoms, respectively.

changer involved in step 1.

(b) At least three different types of coated vesicles

(d) GTP- -S (a nonhydrolyzable analog of GTP

have been distinguished: COPI, COPII, and clathrin-

often used in investigations of the role of GTP in bio-

coated vesicles; the first two are referred to here as

chemical processes) blocks disassembly of the coat from

transport vesicles. Many other types of vesicles no

coated vesicles, leading to a build-up of coated vesicles.

INTRACELLULAR TRAFFIC & SORTING OF PROTEINS

511

(e) A family of Ras-like proteins, called the Rab pro-

Asymmetry of Both Proteins & Lipids Is

tein family, are required in several steps of intracellular

Maintained During Membrane Assembly

protein transport, regulated secretion, and endocytosis.

Vesicles formed from membranes of the ER and Golgi

They are small monomeric GTPases that attach to the

apparatus, either naturally or pinched off by homoge-

cytosolic faces of membranes via geranylgeranyl chains.

nization, exhibit transverse asymmetries of both lipid

They attach in the GTP-bound state (not shown in

and protein. These asymmetries are maintained during

Figure 46-7) to the budding vesicle. Another family of

fusion of transport vesicles with the plasma membrane.

proteins (Sec1) binds to t-SNAREs and prevents inter-

The inside of the vesicles after fusion becomes the out-

action with them and their complementary v-SNAREs.

side of the plasma membrane, and the cytoplasmic side

When a vesicle interacts with its target membrane, Rab

of the vesicles remains the cytoplasmic side of the mem-

proteins displace Sec1 proteins and the v-SNARE-

brane (Figure 46-8). Since the transverse asymmetry of

t-SNARE interaction is free to occur. It appears that

the membranes already exists in the vesicles of the ER

the Rab and Sec1 families of proteins regulate the speed

well before they are fused to the plasma membrane, a

of vesicle formation, opposing each other. Rab proteins

major problem of membrane assembly becomes under-

have been likened to throttles and Sec1 proteins to

standing how the integral proteins are inserted into the

dampers on the overall process of vesicle formation.

lipid bilayer of the ER. This problem was addressed

(f) Studies using v- and t-SNARE proteins reconsti-

earlier in this chapter.

tuted into separate lipid bilayer vesicles have indicated

Phospholipids are the major class of lipid in mem-

that they form SNAREpins, ie, SNARE complexes that

branes. The enzymes responsible for the synthesis of

link two membranes (vesicles). SNAPs and NSF are re-

phospholipids reside in the cytoplasmic surface of the

quired for formation of SNAREpins, but once they

cisternae of the ER. As phospholipids are synthesized at

have formed they can apparently lead to spontaneous

that site, they probably self-assemble into thermody-

fusion of membranes at physiologic temperature, sug-

namically stable bimolecular layers, thereby expanding

gesting that they are the minimal machinery required

the membrane and perhaps promoting the detachment

for membrane fusion.

of so-called lipid vesicles from it. It has been proposed

(g) The fusion of synaptic vesicles with the plasma

that these vesicles travel to other sites, donating their

membrane of neurons involves a series of events similar

lipids to other membranes; however, little is known

to that described above. For example, one v-SNARE is

about this matter. As indicated above, cytosolic pro-

designated synaptobrevin and two t-SNAREs are des-

teins that take up phospholipids from one membrane

ignated syntaxin and SNAP 25 (synaptosome-associ-

and release them to another (ie, phospholipid exchange

ated protein of 25 kDa). Botulinum B toxin is one of

proteins) have been demonstrated; they probably play a

the most lethal toxins known and the most serious

role in contributing to the specific lipid composition of

cause of food poisoning. One component of this toxin

various membranes.

is a protease that appears to cleave only synaptobrevin,

thus inhibiting release of acetylcholine at the neuro-

muscular junction and possibly proving fatal, depend-

Lipids & Proteins Undergo Turnover at

ing on the dose taken.

Different Rates in Different Membranes

(h) Although the above model describes non-

clathrin-coated vesicles, it appears likely that many of

It has been shown that the half-lives of the lipids of the

the events described above apply, at least in principle,

ER membranes of rat liver are generally shorter than

to clathrin-coated vesicles.

those of its proteins, so that the turnover rates of

lipids and proteins are independent. Indeed, differ-

ent lipids have been found to have different half-lives.

Furthermore, the half-lives of the proteins of these

THE ASSEMBLY OF MEMBRANES

membranes vary quite widely, some exhibiting short

IS COMPLEX

(hours) and others long (days) half-lives. Thus, individ-

There are many cellular membranes, each with its own

ual lipids and proteins of the ER membranes appear to

specific features. No satisfactory scheme describing the

be inserted into it relatively independently; this is the

assembly of any one of these membranes is available.

case for many other membranes.

How various proteins are initially inserted into the

The biogenesis of membranes is thus a complex

membrane of the ER has been discussed above. The

process about which much remains to be learned. One

transport of proteins, including membrane proteins, to

indication of the complexity involved is to consider the

various parts of the cell inside vesicles has also been de-

number of posttranslational modifications that mem-

scribed. Some general points about membrane assembly

brane proteins may be subjected to prior to attaining

remain to be addressed.

their mature state. These include proteolysis, assembly

512

CHAPTER 46

Membrane protein

Exterior surface

Table 46-8. Major features of membrane

assembly.

• Lipids and proteins are inserted independently into mem-

branes.

• Individual membrane lipids and proteins turn over indepen-

dently and at different rates.

Plasma

membrane

• Topogenic sequences (eg, signal [amino terminal or inter-

Lumen

nal] and stop-transfer) are important in determining the in-

sertion and disposition of proteins in membranes.

Cytoplasm

Integral

• Membrane proteins inside transport vesicles bud off the en-

protein

doplasmic reticulum on their way to the Golgi; final sorting

of many membrane proteins occurs in the trans-Golgi net-

Vesicle

work.

membrane

• Specific sorting sequences guide proteins to particular

organelles such as lysosomes, peroxisomes, and mitochon-

dria.

into multimers, glycosylation, addition of a glycophos-

phatidylinositol (GPI) anchor, sulfation on tyrosine or

carbohydrate moieties, phosphorylation, acylation, and

prenylation—a list that is undoubtedly not complete.

Nevertheless, significant progress has been made; Table

46-8 summarizes some of the major features of mem-

brane assembly that have emerged to date.

Table 46-9. Some disorders due to mutations in

genes encoding proteins involved in intracellular

membrane transport.¹

Disorder²

Protein Involved

Chédiak-Higashi syndrome,

Lysosomal trafficking regula-

214500

tor

Figure 46-8. Fusion of a vesicle with the plasma

Combined deficiency of factors

ERGIC-53, a mannose-

membrane preserves the orientation of any integral

V and VIII, 227300

binding lectin

proteins embedded in the vesicle bilayer. Initially, the

Hermansky-Pudlak syndrome,

AP-3 adaptor complex β3A

amino terminal of the protein faces the lumen, or inner

203300

subunit

cavity, of such a vesicle. After fusion, the amino termi-

I-cell disease, 252500

N-Acetylglucosamine

nal is on the exterior surface of the plasma membrane.

1-phosphotransferase

That the orientation of the protein has not been re-

versed can be perceived by noting that the other end

Oculocerebrorenal syndrome,

OCRL-1, an inositol poly-

of the molecule, the carboxyl terminal, is always im-

30900

phosphate 5-phosphatase

mersed in the cytoplasm. The lumen of a vesicle and

¹Modified from Olkonnen VM, Ikonen E: Genetic defects of intra-

the outside of the cell are topologically equivalent. (Re-

cellular-membrane transport. N Eng J Med 2000;343:1095. Certain

drawn and modified, with permission, from Lodish HF,

related conditions not listed here are also described in this publi-

Rothman JE: The assembly of cell membranes. Sci Am

cation. I-cell disease is described in Chapter 47. The majority of

the disorders listed above affect lysosomal function; readers

[Jan] 1979;240:43.)

should consult a textbook of medicine for information on the

clinical manifestations of these conditions.

²The numbers after each disorder are the OMIM numbers.

INTRACELLULAR TRAFFIC & SORTING OF PROTEINS

513

Various Disorders Result From Mutations

and attachment of transport vesicles to a target mem-

in Genes Encoding Proteins Involved

brane is summarized.

in Intracellular Transport

• Membrane assembly is discussed and shown to be

complex. Asymmetry of both lipids and proteins is

Some of these are listed in Table 46-9; the majority af-

maintained during membrane assembly.

fect lysosomal function. A number of other mutations

• A number of disorders have been shown to be due to

affecting intracellular protein transport have been re-

mutations in genes encoding proteins involved in

ported but are not included here.

various aspects of protein traffic and sorting.

SUMMARY

REFERENCES

• Many proteins are targeted to their destinations by

Fuller GM, Shields DL: Molecular Basis of Medical Cell Biology.

signal sequences. A major sorting decision is made

McGraw-Hill, 1998.

when proteins are partitioned between cytosolic and

Gould SJ et al: The peroxisome biogenesis disorders. In: The Meta-

membrane-bound polyribosomes by virtue of the ab-

bolic and Molecular Bases of Inherited Disease, 8th ed. Scriver

sence or presence of a signal peptide.

CR et al (editors). McGraw-Hill, 2001.

• The pathways of protein import into mitochondria,

Graham JM, Higgins JA: Membrane Analysis. BIOS Scientific,

1997.

nuclei, peroxisomes, and the endoplasmic reticulum

are described.

Griffith J, Sansom C: The Transporter Facts Book. Academic Press,

1998.

• Many proteins synthesized on membrane-bound

Lodish H et al: Molecular Cell Biology, 4th ed. Freeman, 2000.

polyribosomes proceed to the Golgi apparatus and

(Chapter 17 contains comprehensive coverage of protein sort-

the plasma membrane in transport vesicles.

ing and organelle biogenesis.)

• A number of glycosylation reactions occur in com-

Olkkonen VM, Ikonen E: Genetic defects of intracellular-mem-

partments of the Golgi, and proteins are further

brane transport. N Engl J Med 2000;343:1095.

sorted in the trans-Golgi network.

Reithmeier RAF: Assembly of proteins into membranes. In: Bio-

chemistry of Lipids, Lipoproteins and Membranes. Vance DE,

• Most proteins destined for the plasma membrane

Vance JE (editors). Elsevier, 1996.

and for secretion appear to lack specific signals—a

Sabatini DD, Adesnik MB: The biogenesis of membranes and or-

default mechanism.

ganelles. In: The Metabolic and Molecular Bases of Inherited

• The role of chaperone proteins in the folding of pro-

Disease,

8th ed. Scriver CR et al (editors). McGraw-Hill,

teins is presented, and a model describing budding

2001.

Glycoproteins

Robert K. Murray, MD, PhD

BIOMEDICAL IMPORTANCE

are firmly established; others are still under investi-

gation.

Glycoproteins are proteins that contain oligosaccha-

ride

(glycan) chains covalently attached to their

OLIGOSACCHARIDE CHAINS ENCODE

polypeptide backbones. They are one class of glycocon-

jugate or complex carbohydrates—equivalent terms

BIOLOGIC INFORMATION

used to denote molecules containing one or more car-

An enormous number of glycosidic linkages can be gen-

bohydrate chains covalently linked to protein (to form

erated between sugars. For example, three different hex-

glycoproteins or proteoglycans) or lipid (to form glyco-

oses may be linked to each other to form over 1000 dif-

lipids). (Proteoglycans are discussed in Chapter 48 and

ferent trisaccharides. The conformations of the sugars in

glycolipids in Chapter 14). Almost all the plasma pro-

oligosaccharide chains vary depending on their linkages

teins of humans—except albumin—are glycoproteins.

and proximity to other molecules with which the oligo-

Many proteins of cellular membranes (Chapter 41)

saccharides may interact. It is now established that certain

contain substantial amounts of carbohydrate. A num-

oligosaccharide chains encode considerable biologic in-

ber of the blood group substances are glycoproteins,

formation and that this depends upon their constituent

whereas others are glycosphingolipids. Certain hor-

sugars, their sequences, and their linkages. For instance,

mones (eg, chorionic gonadotropin) are glycoproteins.

mannose 6-phosphate residues target newly synthesized

A major problem in cancer is metastasis, the phenome-

lysosomal enzymes to that organelle (see below).

non whereby cancer cells leave their tissue of origin (eg,

the breast), migrate through the bloodstream to some

distant site in the body (eg, the brain), and grow there

TECHNIQUES ARE AVAILABLE

in an unregulated manner, with catastrophic results for

FOR DETECTION, PURIFICATION,

the affected individual. Many cancer researchers think

& STRUCTURAL ANALYSIS

that alterations in the structures of glycoproteins and

OF GLYCOPROTEINS

other glycoconjugates on the surfaces of cancer cells are

important in the phenomenon of metastasis.

A variety of methods used in the detection, purifica-

tion, and structural analysis of glycoproteins are listed

in Table 47-3. The conventional methods used to pu-

GLYCOPROTEINS OCCUR WIDELY

rify proteins and enzymes are also applicable to the pu-

& PERFORM NUMEROUS FUNCTIONS

rification of glycoproteins. Once a glycoprotein has

Glycoproteins occur in most organisms, from bacteria

been purified, the use of mass spectrometry and high-

to humans. Many viruses also contain glycoproteins,

resolution NMR spectroscopy can often identify the

some of which have been much investigated, in part be-

structures of its glycan chains. Analysis of glycoproteins

cause they are very suitable for biosynthetic studies.

can be complicated by the fact that they often exist as

Numerous proteins with diverse functions are glycopro-

glycoforms; these are proteins with identical amino

teins (Table 47-1); their carbohydrate content ranges

acid sequences but somewhat different oligosaccharide

from 1% to over 85% by weight.

compositions. Although linkage details are not stressed

Many studies have been conducted in an attempt to

in this chapter, it is critical to appreciate that the precise

define the precise roles oligosaccharide chains play in

natures of the linkages between the sugars of glycopro-

the functions of glycoproteins. Table 47-2 summarizes

teins are of fundamental importance in determining the

results from such studies. Some of the functions listed

structures and functions of these molecules.

514

GLYCOPROTEINS

515

Table 47-1. Some functions served

Table 47-3. Some important methods used to

by glycoproteins.

study glycoproteins.

Function

Glycoproteins

Method

Use

Structural molecule

Collagens

Periodic acid-Schiff reagent

Detects glycoproteins as pink

bands after electrophoretic sep-

Lubricant and

Mucins

aration.

protective agent

Incubation of cultured cells

Leads to detection of a radio-

Transport molecule

Transferrin, ceruloplasmin

with glycoproteins as

active sugar after electropho-

Immunologic molecule

Immunoglobulins, histocompatibil-

radioactive bands

retic separation.

ity antigens

Treatment with appropriate

Resultant shifts in electropho-

Hormone

Chorionic gonadotropin, thyroid-

endo- or exoglycosidase

retic migration help distinguish

stimulating hormone (TSH)

or phospholipases

among proteins with N-glycan,

O-glycan, or GPI linkages and

Enzyme

Various, eg, alkaline phosphatase

also between high mannose

Cell attachment-

Various proteins involved in cell-cell

and complex N-glycans.

recognition site

(eg, sperm-oocyte), virus-cell,

Sepharose-lectin column

To purify glycoproteins or gly-

bacterium-cell, and hormone-cell

chromatography

copeptides that bind the par-

interactions

ticular lectin used.

Antifreeze

Certain plasma proteins of cold

Compositional analysis

Identifies sugars that the gly-

water fish

following acid hydrolysis

coprotein contains and their

Interact with specific

Lectins, selectins (cell adhesion

stoichiometry.

carbohydrates

lectins), antibodies

Mass spectrometry

Provides information on molec-

Receptor

Various proteins involved in hor-

ular mass, composition, se-

mone and drug action

quence, and sometimes branch-

ing of a glycan chain.

Affect folding of

Calnexin, calreticulin

certain proteins

NMR spectroscopy

To identify specific sugars, their

sequence, linkages, and the

anomeric nature of glycosidic

linkages.

Methylation (linkage)

To determine linkages between

analysis

sugars.

Amino acid or cDNA

Determination of amino acid

Table 47-2. Some functions of the

sequencing

sequence.

oligosaccharide chains of glycoproteins.¹

• Modulate physicochemical properties, eg, solubility, vis-

cosity, charge, conformation, denaturation, and binding

sites for bacteria and viruses

EIGHT SUGARS PREDOMINATE

• Protect against proteolysis, from inside and outside of cell

IN HUMAN GLYCOPROTEINS

• Affect proteolytic processing of precursor proteins to

smaller products

About 200 monosaccharides are found in nature; how-

• Are involved in biologic activity, eg, of human chorionic

ever, only eight are commonly found in the oligosac-

gonadotropin (hCG)

charide chains of glycoproteins (Table 47-4). Most of

• Affect insertion into membranes, intracellular migration,

these sugars were described in Chapter 13. N-Acetyl-

sorting and secretion

neuraminic acid (NeuAc) is usually found at the ter-

• Affect embryonic development and differentiation

mini of oligosaccharide chains, attached to subterminal

• May affect sites of metastases selected by cancer cells

galactose

(Gal) or N-acetylgalactosamine

(GalNAc)

residues. The other sugars listed are generally found in

¹Adapted from Schachter H: Biosynthetic controls that determine

the branching and heterogeneity of protein-bound oligosaccha-

more internal positions. Sulfate is often found in glyco-

rides. Biochem Cell Biol 1986;64:163.

proteins, usually attached to Gal, GalNAc, or GlcNAc.

516

CHAPTER 47

Table 47-4. The principal sugars found in human glycoproteins. Their structures are illustrated in

Chapter 13.

Nucleotide

Sugar

Type

Abbreviation

Sugar

Comments

Galactose

Hexose

Gal

UDP-Gal

Often found subterminal to NeuAc in N-linked gly-

coproteins. Also found in core trisaccharide of pro-

teoglycans.

Glucose

Hexose

Glc

UDP-Glc

Present during the biosynthesis of N-linked glyco-

proteins but not usually present in mature glyco-

proteins. Present in some clotting factors.

Mannose

Hexose

Man

GDP-Man

Common sugar in N-linked glycoproteins.

N-Acetylneur-

Sialic acid (nine

NeuAc

CMP-NeuAc

Often the terminal sugar in both N- and O-linked

aminic acid

C atoms)

glycoproteins. Other types of sialic acid are also

found, but NeuAc is the major species found in hu-

mans. Acetyl groups may also occur as O-acetyl

species as well as N-acetyl.

Fucose

Deoxyhexose

Fuc

GDP-Fuc

May be external in both N- and O-linked glycopro-

teins or internal, linked to the GlcNAc residue at-

tached to Asn in N-linked species. Can also occur

internally attached to the OH of Ser (eg, in t-PA and

certain clotting factors).

N-Acetylgalactosamine

Aminohexose

GalNAc

UDP-GalNAc

Present in both N- and O-linked glycoproteins.

N-Acetylglucosamine

Aminohexose

GlcNAc

UDP-GlcNAc

The sugar attached to the polypeptide chain via

Asn in N-linked glycoproteins; also found at other

sites in the oligosaccharides of these proteins.

Many nuclear proteins have GlcNAc attached to the

OH of Ser or Thr as a single sugar.

Xylose

Pentose

Xyl

UDP-Xyl

Xyl is attached to the OH of Ser in many proteogly-

cans. Xyl in turn is attached to two Gal residues,

forming a link trisaccharide. Xyl is also found in t-PA

and certain clotting factors.

NUCLEOTIDE SUGARS ACT

suitable acceptors provided appropriate transferases are

AS SUGAR DONORS IN MANY

available.

Most nucleotide sugars are formed in the cytosol,

BIOSYNTHETIC REACTIONS

generally from reactions involving the corresponding

The first nucleotide sugar to be reported was uridine

nucleoside triphosphate. CMP-sialic acids are formed

diphosphate glucose (UDP-Glc); its structure is shown

in the nucleus. Formation of uridine diphosphate galac-

in Figure 18-2. The common nucleotide sugars in-

tose (UDP-Gal) requires the following two reactions in

volved in the biosynthesis of glycoproteins are listed in

mammalian tissues:

Table 47-4; the reasons some contain UDP and others

UDP-Glc

guanosine diphosphate (GDP) or cytidine monophos-

PYROPHOS-

phate (CMP) are obscure. Many of the glycosylation re-

PHORYLASE

actions involved in the biosynthesis of glycoproteins

UTP + Glucose 1-phosphate

utilize these compounds (see below). The anhydro na-

UDP-Glc + Pyrophosphate

ture of the linkage between the phosphate group and

the sugars is of the high-energy, high-group-transfer-

UDP-Glc

potential type (Chapter 10). The sugars of these com-

EPIMERASE

pounds are thus “activated” and can be transferred to

UDP-Glc

UDP-Gal

GLYCOPROTEINS

517

Because many glycosylation reactions occur within

tide backbone (ie, at internal sites; Figure 47-5) and are

the lumen of the Golgi apparatus, carrier systems (per-

thus useful in releasing large oligosaccharide chains for

meases, transporters) are necessary to transport nu-

structural analyses. A glycoprotein can be treated with

cleotide sugars across the Golgi membrane. Systems

one or more of the above glycosidases to analyze the

transporting UDP-Gal, GDP-Man, and CMP-NeuAc

effects on its biologic behavior of removal of specific

into the cisternae of the Golgi apparatus have been de-

sugars.

scribed. They are antiport systems; ie, the influx of one

molecule of nucleotide sugar is balanced by the efflux

THE MAMMALIAN

of one molecule of the corresponding nucleotide (eg,

ASIALOGLYCOPROTEIN RECEPTOR

UMP, GMP, or CMP) formed from the nucleotide

IS INVOLVED IN CLEARANCE

sugars. This mechanism ensures an adequate concentra-

tion of each nucleotide sugar inside the Golgi appara-

OF CERTAIN GLYCOPROTEINS

tus. UMP is formed from UDP-Gal in the above

FROM PLASMA BY HEPATOCYTES

process as follows:

Experiments performed by Ashwell and his colleagues

in the early 1970s played an important role in focusing

GALACTOSYL-

TRANSFERASE

attention on the functional significance of the oligosac-

UDP-Gal + Protein

Protein Gal + UDP

charide chains of glycoproteins. They treated rabbit

ceruloplasmin (a plasma protein; see Chapter 50) with

neuraminidase in vitro. This procedure exposed subter-

NUCLEOSIDE

DIPHOSPHATE

minal Gal residues that were normally masked by ter-

PHOSPHATASE

minal NeuAc residues. Neuraminidase-treated radioac-

UDP

UMP + P_i

tive ceruloplasmin was found to disappear rapidly from

the circulation, in contrast to the slow clearance of the

untreated protein. Very significantly, when the Gal

EXO- & ENDOGLYCOSIDASES

residues exposed to treatment with neuraminidase were

FACILITATE STUDY

removed by treatment with a galactosidase, the clear-

OF GLYCOPROTEINS

ance rate of the protein returned to normal. Further

studies demonstrated that liver cells contain a mam-

A number of glycosidases of defined specificity have

malian asialoglycoprotein receptor that recognizes

proved useful in examining structural and functional

the Gal moiety of many desialylated plasma proteins

aspects of glycoproteins (Table 47-5). These enzymes

and leads to their endocytosis. This work indicated that

act at either external (exoglycosidases) or internal (en-

an individual sugar, such as Gal, could play an impor-

doglycosidases) positions of oligosaccharide chains. Ex-

tant role in governing at least one of the biologic prop-

amples of exoglycosidases are neuraminidases and

erties (ie, time of residence in the circulation) of certain

galactosidases; their sequential use removes terminal

glycoproteins. This greatly strengthened the concept

NeuAc and subterminal Gal residues from most glyco-

that oligosaccharide chains could contain biologic in-

proteins. Endoglycosidases F and H are examples of

formation.

the latter class; these enzymes cleave the oligosaccharide

chains at specific GlcNAc residues close to the polypep-

LECTINS CAN BE USED TO

PURIFY GLYCOPROTEINS

Table 47-5. Some glycosidases used to study the

& TO PROBE THEIR FUNCTIONS

structure and function of glycoproteins.¹

Lectins are carbohydrate-binding proteins that aggluti-

nate cells or precipitate glycoconjugates; a number of

Enzymes

Type

lectins are themselves glycoproteins. Immunoglobulins

Neuraminidases

Exoglycosidase

that react with sugars are not considered lectins. Lectins

Galactosidases

Exo- or endoglycosidase

contain at least two sugar-binding sites; proteins with a

Endoglycosidase F

Endoglycosidase

single sugar-binding site will not agglutinate cells or

Endoglycosidase H

Endoglycosidase

precipitate glycoconjugates. The specificity of a lectin is

usually defined by the sugars that are best at inhibiting

¹The enzymes are available from a variety of sources and are often

its ability to cause agglutination or precipitation. En-

specific for certain types of glycosidic linkages and also for their

zymes, toxins, and transport proteins can be classified

anomeric natures. The sites of action of endoglycosidases F and H

are shown in Figure 47-5. F acts on both high-mannose and com-

as lectins if they bind carbohydrate. Lectins were first

plex oligosaccharides, whereas H acts on the former.

discovered in plants and microbes, but many lectins of

518

CHAPTER 47

animal origin are now known. The mammalian asialo-

O-linked), involving the hydroxyl side chain of serine

glycoprotein receptor described above is an important

or threonine and a sugar such as N-acetylgalactosamine

example of an animal lectin. Some important lectins are

(GalNAc-Ser[Thr]); (2) those containing an N-glyco-

listed in Table 47-6. Much current research is centered

sidic linkage (ie, N-linked), involving the amide nitro-

on the roles of various animal lectins (eg, the selectins)

gen of asparagine and N-acetylglucosamine (GlcNAc-

in cell-cell interactions that occur in pathologic condi-

Asn); and (3) those linked to the carboxyl terminal

tions such as inflammation and cancer metastasis (see

amino acid of a protein via a phosphoryl-ethanolamine

below).

moiety joined to an oligosaccharide (glycan), which in

Numerous lectins have been purified and are com-

turn is linked via glucosamine to phosphatidylinositol

mercially available; three plant lectins that have been

(PI). This latter class is referred to as glycosylphos-

widely used experimentally are listed in Table 47-7.

phatidylinositol-anchored (GPI-anchored, or GPI-

Among many uses, lectins have been employed to pu-

linked) glycoproteins. Other minor classes of glycopro-

rify specific glycoproteins, as tools for probing the gly-

teins also exist.

coprotein profiles of cell surfaces, and as reagents for

The number of oligosaccharide chains attached to

generating mutant cells deficient in certain enzymes in-

one protein can vary from one to 30 or more, with the

volved in the biosynthesis of oligosaccharide chains.

sugar chains ranging from one or two residues in

length to much larger structures. Many proteins con-

tain more than one type of linkage; for instance, gly-

THERE ARE THREE MAJOR CLASSES

cophorin, an important red cell membrane glycopro-

OF GLYCOPROTEINS

tein

(Chapter 52), contains both O- and N-linked

Based on the nature of the linkage between their poly-

oligosaccharides.

peptide chains and their oligosaccharide chains, glyco-

proteins can be divided into three major classes (Figure

GLYCOPROTEINS CONTAIN SEVERAL

47-1): (1) those containing an O-glycosidic linkage (ie,

TYPES OF O-GLYCOSIDIC LINKAGES

At least four subclasses of O-glycosidic linkages are

found in human glycoproteins:

(1) The GalNAc-

Table 47-6. Some important lectins.

Ser(Thr) linkage shown in Figure 47-1 is the predomi-

nant linkage. Two typical oligosaccharide chains found

in members of this subclass are shown in Figure 47-2.

Lectins

Examples or Comments

Usually a Gal or a NeuAc residue is attached to the

Legume lectins

Concanavalin A, pea lectin

GalNAc, but many variations in the sugar compositions

Wheat germ

Widely used in studies of surfaces of nor-

and lengths of such oligosaccharide chains are found.

agglutinin

mal cells and cancer cells

This type of linkage is found in mucins (see below).

(2) Proteoglycans contain a Gal-Gal-Xyl-Ser trisac-

Ricin

Cytotoxic glycoprotein derived from seeds

charide (the so-called link trisaccharide). (3) Collagens

of the castor plant

contain a Gal-hydroxylysine (Hyl) linkage. (Subclasses

Bacterial toxins

Heat-labile enterotoxin of E coli and

[2] and

[3] are discussed further in Chapter

48.)

cholera toxin

(4) Many nuclear proteins (eg, certain transcription

factors) and cytosolic proteins contain side chains con-

Influenza virus

Responsible for host-cell attachment and

sisting of a single GlcNAc attached to a serine or threo-

hemagglutinin

membrane fusion

nine residue (GlcNAc-Ser[Thr]).

C-type lectins

Characterized by a Ca2+-dependent carbo-

hydrate recognition domain (CRD); in-

cludes the mammalian asialoglycoprotein

receptor, the selectins, and the mannose-

Table 47-7. Three plant lectins and the sugars

binding protein

with which they interact.¹

S-type lectins

β-Galactoside-binding animal lectins with

roles in cell-cell and cell-matrix interac-

Lectin

Abbreviation

Sugars

tions

Concanavalin A

ConA

Man and Glc

P-type lectins

Mannose 6-P receptor

Soybean lectin

Gal and GalNAc

l-type lectins

Members of the immunoglobulin super-

Wheat germ agglutinin

WGA

Glc and NeuAc

family, eg, sialoadhesin mediating adhe-

¹In most cases, lectins show specificity for the anomeric nature of

sion of macrophages to various cells

the glycosidic linkage (α or β); this is not indicated in the table.

GLYCOPROTEINS

519

CH₂OH

NH₂

Protein

CH₂

Glycine

Ser

Ethanolamine

H H

Mannose

Ethanolamine

Mannose

CH₃

Mannose

CH₂OH

Glucosamine

Inositol

PI-PLC

Additional fatty acid

Plasma

Asn

membrane

H H

CH₃

Figure 47-1.

Depictions of (A) an O-linkage (N-acetylgalactosamine to serine); (B) an N-linkage (N-acetylglu-

cosamine to asparagine) and (C) a glycosylphosphatidylinositol (GPI) linkage. The GPI structure shown is that

linking acetylcholinesterase to the plasma membrane of the human red blood cell. The carboxyl terminal amino

acid is glycine joined in amide linkage via its COOH group to the NH₂ group of phosphorylethanolamine, which

in turn is joined to a mannose residue. The core glycan contains three mannose and one glucosamine residues.

The glucosamine is linked to inositol, which is attached to phosphatidic acid. The site of action of PI-phospholi-

pase C (PI-PLC) is indicated. The structure of the core glycan is shown in the text. This particular GPI contains an

extra fatty acid attached to inositol and also an extra phosphorylethanolamine moiety attached to the middle of

the three mannose residues. Variations found among different GPI structures include the identity of the carboxyl

terminal amino acid, the molecules attached to the mannose residues, and the precise nature of the lipid moiety.

Mucins Have a High Content of O-Linked

Oligosaccharides & Exhibit Repeating

Amino Acid Sequences

α 2,6

NeuAc

GalNAc

Ser(Thr)

Mucins are glycoproteins with two major characteris-

tics: (1) a high content of O-linked oligosaccharides

(the carbohydrate content of mucins is generally more

than 50%); and (2) the presence of repeating amino

β 1,3

Gal

GalNAc

Ser(Thr)

acid sequences (tandem repeats) in the center of their

α 2,3

α 2,6

polypeptide backbones, to which the O-glycan chains

NeuAc

are attached in clusters (Figure 47-3). These sequences

are rich in serine, threonine, and proline. Although O-

Figure 47-2. Structures of two O-linked oligosac-

glycans predominate, mucins often contain a number

charides found in (A) submaxillary mucins and (B) fe-

of N-glycan chains. Both secretory and membrane-

tuin and in the sialoglycoprotein of the membrane of

bound mucins occur. The former are found in the

human red blood cells. (Modified and reproduced, with

mucus present in the secretions of the gastrointestinal,

permission, from Lennarz WJ: The Biochemistry of Glyco-

respiratory, and reproductive tracts. Mucus consists of

proteins and Proteoglycans. Plenum Press, 1980.)

about 94% water and 5% mucins, with the remainder

520

CHAPTER 47

O-glycan chain

epitopes have been used to stimulate an immune re-

N-glycan chain

sponse against cancer cells.

The genes encoding the polypeptide backbones of a

number of mucins derived from various tissues (eg,

pancreas, small intestine, trachea and bronchi, stomach,

and salivary glands) have been cloned and sequenced.

These studies have revealed new information about the

polypeptide backbones of mucins (size of tandem re-

peats, potential sites of N-glycosylation, etc) and ulti-

Tandem repeat sequence

mately should reveal aspects of their genetic control.

Some important properties of mucins are summarized

Figure 47-3. Schematic diagram of a mucin. O-gly-

in Table 47-8.

can chains are shown attached to the central region of

the extended polypeptide chain and N-glycan chains to

The Biosynthesis of O-Linked

the carboxyl terminal region. The narrow rectangles

Glycoproteins Uses Nucleotide Sugars

represent a series of tandem repeat amino acid se-

The polypeptide chains of O-linked and other glyco-

quences. Many mucins contain cysteine residues whose

proteins are encoded by mRNA species; because most

SH groups form interchain linkages; these are not

glycoproteins are membrane-bound or secreted, they

shown in the figure. (Adapted from Strous GJ, Dekker J:

are generally translated on membrane-bound polyribo-

Mucin-type glycoproteins. Crit Rev Biochem Mol Biol

somes (Chapter 38). Hundreds of different oligosaccha-

1992;27:57.)

ride chains of the O-glycosidic type exist. These glyco-

proteins are built up by the stepwise donation of

sugars from nucleotide sugars, such as UDP-GalNAc,

being a mixture of various cell molecules, electrolytes,

UDP-Gal, and CMP-NeuAc. The enzymes catalyzing

and remnants of cells. Secretory mucins generally have

this type of reaction are membrane-bound glycopro-

an oligomeric structure and thus often have a very high

tein glycosyltransferases. Generally, synthesis of one

molecular mass. The oligomers are composed of

specific type of linkage requires the activity of a corre-

monomers linked by disulfide bonds. Mucus exhibits a

spondingly specific transferase. The factors that deter-

high viscosity and often forms a gel. These qualities are

mine which specific serine and threonine residues are

functions of its content of mucins. The high content of

glycosylated have not been identified but are probably

O-glycans confers an extended structure on mucins.

found in the peptide structure surrounding the glycosy-

This is in part explained by steric interactions between

lation site. The enzymes assembling O-linked chains are

their GalNAc moieties and adjacent amino acids, re-

located in the Golgi apparatus, sequentially arranged in

sulting in a chain-stiffening effect so that the conforma-

an assembly line with terminal reactions occurring in

tions of mucins often become those of rigid rods. Inter-

the trans-Golgi compartments.

molecular noncovalent interactions between various

The major features of the biosynthesis of O-linked

sugars on neighboring glycan chains contribute to gel

glycoproteins are summarized in Table 47-9.

formation. The high content of NeuAc and sulfate

residues found in many mucins confers a negative

charge on them. With regard to function, mucins help

lubricate and form a protective physical barrier on

Table 47-8. Some properties of mucins.

epithelial surfaces. Membrane-bound mucins partici-

pate in various cell-cell interactions (eg, involving se-

• Found in secretions of the gastrointestinal, respiratory,

lectins; see below). The density of oligosaccharide

and reproductive tracts and also in membranes of various

chains makes it difficult for proteases to approach their

cells.

polypeptide backbones, so that mucins are often resis-

• Exhibit high content of O-glycan chains, usually containing

tant to their action. Mucins also tend to “mask” certain

NeuAc.

surface antigens. Many cancer cells form excessive

• Contain repeating amino acid sequences rich in serine, thre-

amounts of mucins; perhaps the mucins may mask cer-

onine, and proline.

tain surface antigens on such cells and thus protect the

• Extended structure contributes to their high visco-

elasticity.

cells from immune surveillance. Mucins also carry can-

• Form protective physical barrier on epithelial surfaces, are

cer-specific peptide and carbohydrate epitopes (an epi-

involved in cell-cell interactions, and may contain or mask

tope is a site on an antigen recognized by an antibody,

certain surface antigens.

also called an antigenic determinant). Some of these

GLYCOPROTEINS

521

Table 47-9. Summary of main features

penta-antennary structures may all be found. A bewil-

of O-glycosylation.

dering number of chains of the complex type exist, and

that indicated in Figure 47-4 is only one of many.

Other complex chains may terminate in Gal or Fuc.

• Involves a battery of membrane-bound glycoprotein glyco-

High-mannose oligosaccharides typically have two to

syltransferases acting in a stepwise manner; each trans-

six additional Man residues linked to the pentasaccha-

ferase is generally specific for a particular type of linkage.

ride core. Hybrid molecules contain features of both of

• The enzymes involved are located in various subcompart-

the two other classes.

ments of the Golgi apparatus.

• Each glycosylation reaction involves the appropriate

nucleotide-sugar.

The Biosynthesis of N-Linked

• Dolichol-P-P-oligosaccharide is not involved, nor are gly-

Glycoproteins Involves

cosidases; and the reactions are not inhibited by tuni-

Dolichol-P-P-Oligosaccharide

camycin.

• O-Glycosylation occurs posttranslationally at certain Ser and

Leloir and his colleagues described the occurrence of

Thr residues.

a dolichol-pyrophosphate-oligosaccharide (Dol-P-P-

oligosaccharide), which subsequent research showed

to play a key role in the biosynthesis of N-linked glyco-

proteins. The oligosaccharide chain of this compound

N-LINKED GLYCOPROTEINS CONTAIN

generally has the structure R-GlcNAc₂Man₉Glc₃ (R =

AN Asn-GlcNAc LINKAGE

Dol-P-P). The sugars of this compound are first assem-

N-Linked glycoproteins are distinguished by the pres-

bled on the Dol-P-P backbone, and the oligosaccharide

ence of the Asn-GlcNAc linkage (Figure 47-1). It is the

chain is then transferred en bloc to suitable Asn resi-

major class of glycoproteins and has been much stud-

dues of acceptor apoglycoproteins during their synthe-

ied, since the most readily accessible glycoproteins (eg,

sis on membrane-bound polyribosomes. All N-glycans

plasma proteins) mainly belong to this group. It in-

have a common pentasaccharide core structure (Fig-

cludes both membrane-bound and circulating glyco-

ure 47-5).

proteins. The principal difference between this and the

To form high-mannose chains, only the Glc

previous class, apart from the nature of the amino acid

residues plus certain of the peripheral Man residues are

to which the oligosaccharide chain is attached (Asn ver-

removed. To form an oligosaccharide chain of the com-

sus Ser or Thr), concerns their biosynthesis.

plex type, the Glc residues and four of the Man

residues are removed by glycosidases in the endoplas-

mic reticulum and Golgi. The sugars characteristic of

Complex, Hybrid, & High-Mannose

complex chains (GlcNAc, Gal, NeuAc) are added by

Are the Three Major Classes

the action of individual glycosyltransferases located in

of N-Linked Oligosaccharides

the Golgi apparatus. The phenomenon whereby the

There are three major classes of N-linked oligosaccha-

glycan chains of N-linked glycoproteins are first par-

rides: complex, hybrid, and high-mannose (Figure

tially degraded and then in some cases rebuilt is referred

47-4). Each type shares a common pentasaccharide,

to as oligosaccharide processing. Hybrid chains are

Man₃GlcNAc₂—shown within the boxed area in Figure

formed by partial processing, forming complex chains

47-4 and depicted also in Figure 47-5—but they differ

on one arm and Man structures on the other arm.

in their outer branches. The presence of the common

Thus, the initial steps involved in the biosynthesis of

pentasaccharide is explained by the fact that all three

the N-linked glycoproteins differ markedly from those

classes share an initial common mechanism of biosyn-

involved in the biosynthesis of the O-linked glycopro-

thesis. Glycoproteins of the complex type generally

teins. The former involves Dol-P-P-oligosaccharide; the

contain terminal NeuAc residues and underlying Gal

latter, as described earlier, does not.

and GlcNAc residues, the latter often constituting the

The process of N-glycosylation can be broken down

disaccharide N-acetyllactosamine. Repeating N-acetyl-

into two stages: (1) assembly of Dol-P-P-oligosaccha-

lactosamine units—[Galβ1-3/4GlcNAcβ1-3]_n (poly-

ride and transfer of the oligosaccharide; and (2) pro-

N-acetyllactosaminoglycans)—are often found on N-

cessing of the oligosaccharide chain.

linked glycan chains. I/i blood group substances belong

A. ASSEMBLY & TRANSFER OF

to this class. The majority of complex-type oligosaccha-

DOLICHOL-P-P-OLIGOSACCHARIDE

rides contain two, three, or four outer branches (Figure

47-4), but structures containing five branches have also

Polyisoprenol compounds exist in both bacteria and

been described. The oligosaccharide branches are often

eukaryotic cells. They participate in the synthesis of

referred to as antennae, so that bi-, tri-, tetra-, and

bacterial polysaccharides and in the biosynthesis of N-

522

CHAPTER 47

Sialic acid

α2,3 or 2,6

Gal

Man

β1,4

α1,2

GlcNAc

Man

β1,2

α1,3

α1,6

α1,2

α1,3

α1,6

Man

α1,6

α1,3

α1,6

α1,3

α1,6

β1,4

±GlcNAc

Man

GlcNAc

Man

β1,4

GlcNAc

β1,4

α1,6

±Fuc

GlcNAc

Asn

Complex

Hybrid

High-mannose

Figure 47-4. Structures of the major types of asparagine-linked oligosaccharides. The boxed area en-

closes the pentasaccharide core common to all N-linked glycoproteins. (Reproduced, with permission,

from Kornfeld R, Kornfeld S: Assembly of asparagine-linked oligosaccharides. Annu Rev Biochem

1985;54:631.)

linked glycoproteins and GPI anchors. The polyiso-

in the membranes of the endoplasmic reticulum from

prenol used in eukaryotic tissues is dolichol, which is,

Dol-P and UDP-GlcNAc in the following reaction,

next to rubber, the longest naturally occurring hydro-

catalyzed by GlcNAc-P transferase:

carbon made up of a single repeating unit. Dolichol is

composed of 17-20 repeating isoprenoid units (Figure

Dol - P + UDP - GlcNAc → Dol - P - P - GlcNAc + UMP

47-6).

Before it participates in the biosynthesis of Dol-P-P-

The above reaction—which is the first step in the as-

oligosaccharide, dolichol must first be phosphorylated

sembly of Dol-P-P-oligosaccharide—and the other later

to form dolichol phosphate (Dol-P) in a reaction cat-

reactions are summarized in Figure 47-7. The essential

alyzed by dolichol kinase and using ATP as the phos-

features of the subsequent steps in the assembly of Dol-

phate donor.

P-P-oligosaccharide are as follows:

Dolichol-P-P-GlcNAc (Dol-P-P-GlcNAc) is the

key lipid that acts as an acceptor for other sugars in the

(1) A second GlcNAc residue is added to the first,

assembly of Dol-P-P-oligosaccharide. It is synthesized

again using UDP-GlcNAc as the donor.

(2) Five Man residues are added, using GDP-man-

nose as the donor.

Endoglycosidase F

Man

(3) Four additional Man residues are next added,

α1,6

using Dol-P-Man as the donor. Dol-P-Man is

β1,4

Man

GlcNAc

Asn

formed by the following reaction:

α1,3

Man

Endoglycosidase H

Dol - P + GDP - Man → Dol - P - Man + GDP

Figure 47-5. Schematic diagram of the pentasac-

charide core common to all N-linked glycoproteins and

(4) Finally, the three peripheral glucose residues are

to which various outer chains of oligosaccharides may

donated by Dol-P-Glc, which is formed in a reac-

be attached. The sites of action of endoglycosidases F

tion analogous to that just presented except that

and H are also indicated.

Dol-P and UDP-Glc are the substrates.

GLYCOPROTEINS

523

Figure 47-6. The structure of dolichol.

CH₃

The phosphate in dolichol phosphate is at-

tached to the primary alcohol group at the HO CH₂

CH₂

CH C

CH₂

CH C

CH₃

left-hand end of the molecule. The group

CH₃

within the brackets is an isoprene unit (n =

17-20 isoprenoid units).

It should be noted that the first seven sugars (two

preference for the Dol-P-P-GlcNAc₂Man₉Glc₃ struc-

GlcNAc and five Man residues) are donated by nu-

ture. Glycosylation occurs at the Asn residue of an Asn-

cleotide sugars, whereas the last seven sugars (four Man

X-Ser/Thr tripeptide sequence, where X is any amino

and three Glc residues) added are donated by dolichol-

acid except proline, aspartic acid, or glutamic acid. A

P-sugars. The net result is assembly of the compound

tripeptide site contained within a β turn is favored.

illustrated in Figure 47-8 and referred to in shorthand

Only about one-third of the Asn residues that are po-

as Dol-P-P-GlcNAc₂Man₉Glc₃.

tential acceptor sites are actually glycosylated, suggest-

The oligosaccharide linked to dolichol-P-P is trans-

ing that factors other than the tripeptide are also im-

ferred en bloc to form an N-glycosidic bond with one

portant. The acceptor proteins are of both the secretory

or more specific Asn residues of an acceptor protein

and integral membrane class. Cytosolic proteins are

emerging from the luminal surface of the membrane of

rarely glycosylated. The transfer reaction and subse-

the endoplasmic reticulum. The reaction is catalyzed by

quent processes in the glycosylation of N-linked glyco-

oligosaccharide:protein transferase, a membrane-

proteins, along with their subcellular locations, are de-

associated enzyme complex. The transferase will recog-

picted in Figure

47-9. The other product of the

nize and transfer any substrate with the general struc-

oligosaccharide:protein transferase reaction is dolichol-

ture Dol-P-P-(GlcNAc)₂-R, but it has a strong

P-P, which is subsequently converted to dolichol-P by a

UDP-GIcNAc

Dol-P

Tunicamycin

UMP

M M

GIcNAc P P Dol

UDP-GIcNAc

M M

(GIcNAc)

P P Dol

UDP

G G G

M M M

P Dol

GIcNAc GIcNAc P P Dol

GDP-M

M P

Dol and G P Dol

(M)₆

(GIcNAc)

P P Dol

GDP

P Dol

M GIcNAc GIcNAc P P Dol

M P

Dol

(GIcNAc)

P P Dol

(GDP-M)₄

(GDP)₄

M M M

Figure 47-7. Pathway of biosynthesis of dolichol-P-P-oligosaccharide. The specific linkages formed are indi-

cated in Figure 47-8. Note that the first five internal mannose residues are donated by GDP-mannose, whereas

the more external mannose residues and the glucose residues are donated by dolichol-P-mannose and dolichol-

P-glucose. (UDP, uridine diphosphate; Dol, dolichol; P, phosphate; UMP, uridine monophosphate; GDP, guanosine

diphosphate; M, mannose; G, glucose.)

524

CHAPTER 47

α1,2

Man

α1,6

Man

α1,6

α1,3

α1,2

Man

β1,4

Man

GlcNAc

GlcNAc P P Dolichol

α1,2

α1,3

α1,2

α1,3

Glc

Man

Figure 47-8. Structure of dolichol-P-P-oligosaccharide. (Reproduced, with permission, from Lennarz WJ:

The Biochemistry of Glycoproteins and Proteoglycans. Plenum Press, 1980.)

phosphatase. The dolichol-P can serve again as an ac-

PHOSPHO-

ceptor for the synthesis of another molecule of Dol-P-

DIESTERASE

P-oligosaccharide.

GlcNAc-1-P-6-Man Protein

P-6-Man Protein + GlcNAc

B. PROCESSING OF THE OLIGOSACCHARIDE CHAIN

1. Early phase—The various reactions involved are in-

Man 6-P receptors, located in the Golgi apparatus,

dicated in Figure

47-9. The oligosaccharide:protein

bind the Man 6-P residue of these enzymes and direct

transferase catalyzes reaction 1 (see above). Reactions 2

them to the lysosomes. Fibroblasts from patients with

and 3 involve the removal of the terminal Glc residue

I-cell disease (see below) are severely deficient in the

by glucosidase I and of the next two Glc residues by

activity of the GlcNAc phosphotransferase.

glucosidase II, respectively. In the case of high-

2. Late phase—To assemble a typical complex

mannose glycoproteins, the process may stop here, or

oligosaccharide chain, additional sugars must be added

up to four Man residues may also be removed. How-

to the structure formed in reaction 7. Hence, in reac-

ever, to form complex chains, additional steps are nec-

tion 8, a second GlcNAc is added to the peripheral

essary, as follows. Four external Man residues are re-

Man residue of the other arm of the bi-antennary struc-

moved in reactions 4 and 5 by at least two different

ture shown in Figure 47-9; the enzyme catalyzing this

mannosidases. In reaction 6, a GlcNAc residue is added

step is GlcNAc transferase II. Reactions 9, 10, and 11

to the Man residue of the Manα1-3 arm by GlcNAc

involve the addition of Fuc, Gal, and NeuAc residues at

transferase I. The action of this latter enzyme permits

the sites indicated, in reactions catalyzed by fucosyl,

the occurrence of reaction 7, a reaction catalyzed by yet

galactosyl, and sialyl transferases, respectively. The as-

another mannosidase (Golgi α-mannosidase II) and

sembly of poly-N-acetyllactosamine chains requires ad-

which results in a reduction of the Man residues to the

ditional GlcNAc transferases.

core number of three (Figure 47-5).

An important additional pathway is indicated in re-

The Endoplasmic Reticulum

actions I and II of Figure 47-9. This involves enzymes

& Golgi Apparatus Are the

destined for lysosomes. Such enzymes are targeted to

Major Sites of Glycosylation

the lysosomes by a specific chemical marker. In reaction

I, a residue of GlcNAc-1-P is added to carbon 6 of one

As indicated in Figure 47-9, the endoplasmic reticulum

or more specific Man residues of these enzymes. The

and the Golgi apparatus are the major sites involved in

reaction is catalyzed by a GlcNAc phosphotransferase,

glycosylation processes. The assembly of Dol-P-P-

which uses UDP-GlcNAc as the donor and generates

oligosaccharide occurs on both the cytoplasmic and lu-

UMP as the other product:

minal surfaces of the ER membranes. Addition of the

oligosaccharide to protein occurs in the rough endo-

GlcNAc

PHOSPHO-

plasmic reticulum during or after translation. Removal

TRANSFERASE

of the Glc and some of the peripheral Man residues also

UDP-GlcNAc + Man Protein

occurs in the endoplasmic reticulum. The Golgi appa-

GlcNAc-1-P-6-Man Protein + UMP

ratus is composed of cis, medial, and trans cisternae;

these can be separated by appropriate centrifugation

In reaction II, the GlcNAc is removed by the action of

procedures. Vesicles containing glycoproteins appear to

a phosphodiesterase, leaving the Man residues phos-

bud off in the endoplasmic reticulum and are trans-

phorylated in the 6 position:

ported to the cis Golgi. Various studies have shown

GLYCOPROTEINS

525

ROUGH ENDOPLASMIC RETICULUM

Dol

GOLGI APPARATUS

P-

-P

-P-

cis

UDP-

medial

UDP-

GDP-

Exit

trans

UDP-

CMP-

Figure 47-9. Schematic pathway of oligosaccharide processing. The reactions are cat-

alyzed by the following enzymes: 1 , oligosaccharide:protein transferase; 2 , α-glucosidase I;

3 , α-glucosidase II;

4 , endoplasmic reticulum α1,2-mannosidase; l , N-acetylglu-

cosaminylphosphotransferase; ll , N-acetylglucosamine-1-phosphodiester α-N-acetylglu-

cosaminidase; 5 , Golgi apparatus α-mannosidase I; 6 , N-acetylglucosaminyltransferase I;

7 , Golgi apparatus α-mannosidase II; 8 , N-acetylglucosaminyltransferase II; 9 , fucosyltrans-

ferase; 10, galactosyltransferase; 11 , sialyltransferase. The thick arrows indicate various nu-

cleotide sugars involved in the oveall scheme. (Solid square, N-acetylglucosamine; open cir-

cle, mannose; solid triangle, glucose; open triangle, fucose; solid circle, galactose; solid

diamond, sialic acid.) (Reproduced, with permission, from Kornfeld R, Kornfeld S: Assembly of

asparagine-linked oligosaccharides. Annu Rev Biochem 1985;54:631.)

that the enzymes involved in glycoprotein processing

the fucosyl, galactosyl, and sialyl transferases (catalyzing

show differential locations in the cisternae of the Golgi.

reactions 9, 10, and 11) are located mainly in the trans

As indicated in Figure 47-9, Golgi α-mannosidase I

Golgi. The major features of the biosynthesis of N-

(catalyzing reaction

5) is located mainly in the cis

linked glycoproteins are summarized in Table 47-10

Golgi, whereas GlcNAc transferase I (catalyzing reac-

and should be contrasted with those previously listed

tion 6) appears to be located in the medial Golgi, and

(Table 47-9) for O-linked glycoproteins.

526

CHAPTER 47

Table 47-10. Summary of main features of

ing in the lumen of the ER. The soluble protein cal-

N-glycosylation.

reticulin appears to play a similar function.

Several Factors Regulate the Glycosylation

• The oligosaccharide Glc₃Man₉(GIcNAc)₂ is transferred from

dolichol-P-P-oligosaccharide in a reaction catalyzed by

of Glycoproteins

oligosaccharide:protein transferase, which is inhibited by

It is evident that glycosylation of glycoproteins is a

tunicamycin.

complex process involving a large number of enzymes.

• Transfer occurs to specific Asn residues in the sequence

One index of its complexity is that more than ten dis-

Asn-X-Ser/Thr, where X is any residue except Pro, Asp, or

tinct GlcNAc transferases involved in glycoprotein

Glu.

biosynthesis have been reported, and many others are

• Transfer can occur cotranslationally in the endoplasmic

theoretically possible. Multiple species of the other gly-

reticulum.

• The protein-bound oligosaccharide is then partially

cosyltransferases (eg, sialyltransferases) also exist. Con-

processed by glucosidases and mannosidases; if no addi-

trolling factors of the first stage of N-linked glycopro-

tional sugars are added, this results in a high-mannose

tein biosynthesis

(ie, oligosaccharide assembly and

chain.

transfer) include (1) the presence of suitable acceptor

• If processing occurs down to the core heptasaccharide

sites in proteins, (2) the tissue level of Dol-P, and (3) the

(Man₅[GlcNAc]₂), complex chains are synthesized by the ad-

activity of the oligosaccharide:protein transferase.

dition of GlcNAc, removal of two Man, and the stepwise ad-

Some factors known to be involved in the regulation

dition of individual sugars in reactions catalyzed by specific

of oligosaccharide processing are listed in Table

transferases (eg, GlcNAc, Gal, NeuAc transferases) that em-

47-11. Two of the points listed merit further com-

ploy appropriate nucleotide sugars.

ment. First, species variations among processing en-

zymes have assumed importance in relation to produc-

tion of glycoproteins of therapeutic use by means of

recombinant DNA technology. For instance, recombi-

nant erythropoietin (epoetin alfa; EPO) is sometimes

Some Glycan Intermediates

administered to patients with certain types of chronic

Formed During N-Glycosylation

anemia in order to stimulate erythropoiesis. The half-

Have Specific Functions

life of EPO in plasma is influenced by the nature of its

The following are a number of specific functions of N-

glycosylation pattern, with certain patterns being asso-

glycan chains that have been established or are under

ciated with a short half-life, appreciably limiting its pe-

investigation. (1) The involvement of the mannose 6-P

riod of therapeutic effectiveness. It is thus important to

signal in targeting of certain lysosomal enzymes is clear

harvest EPO from host cells that confer a pattern of

(see above and discussion of I-cell disease, below). (2) It

glycosylation consistent with a normal half-life in

is likely that the large N-glycan chains present on newly

plasma. Second, there is great interest in analysis of the

synthesized glycoproteins may assist in keeping these

activities of glycoprotein-processing enzymes in various

proteins in a soluble state inside the lumen of the endo-

types of cancer cells. These cells have often been found

plasmic reticulum. (3) One species of N-glycan chains

to synthesize different oligosaccharide chains (eg, they

has been shown to play a role in the folding and reten-

often exhibit greater branching) from those made in

tion of certain glycoproteins in the lumen of the endo-

control cells. This could be due to cancer cells contain-

plasmic reticulum. Calnexin is a protein present in the

ing different patterns of glycosyltransferases from those

endoplasmic reticulum membrane that acts as a “chap-

exhibited by corresponding normal cells, due to speci-

erone” (Chapter 46). It has been found that calnexin

fic gene activation or repression. The differences in

will bind specifically to a number of glycoproteins (eg,

oligosaccharide chains could affect adhesive interactions

the influenza virus hemagglutinin [HA]) that possess the

between cancer cells and their normal parent tissue

monoglycosylated core structure. This species is the

cells, contributing to metastasis. If a correlation could

product of reaction 2 shown in Figure 47-9 but from

be found between the activity of particular processing

which the terminal glucose residue has been removed,

enzymes and the metastatic properties of cancer cells,

leaving only the innermost glucose attached. The re-

this could be important as it might permit synthesis of

lease of fully folded HA from calnexin requires the en-

drugs to inhibit these enzymes and, secondarily, metas-

zymatic removal of this last glucosyl residue by α-glu-

tasis.

cosidase II. In this way, calnexin retains certain partly

The genes encoding many glycosyltransferases have

folded (or misfolded) glycoproteins and releases them

already been cloned, and others are under study.

when proper folding has occurred; it is thus an impor-

Cloning has revealed new information on both protein

tant component of the quality control systems operat-

and gene structures. The latter should also cast light on

GLYCOPROTEINS

527

Table 47-11. Some factors affecting the activities

Table 47-12. Three inhibitors of enzymes

of glycoprotein processing enzymes.

involved in the glycosylation of glycoproteins and

their sites of action.

Factor

Comment

Inhibitor

Site of Action

Cell type

Different cell types contain different pro-

files of processing enzymes.

Tunicamycin

Inhibits GlcNAc-P transferase, the enzyme

catalyzing addition of GlcNAc to dolichol-

Previous enzyme

Certain glycosyltransferases will only act

P, the first step in the biosynthesis of

on an oligosaccharide chain if it has al-

oligosaccharide-P-P-dolichol

ready been acted upon by another pro-

cessing enzyme.¹

Deoxynojirimycin

Inhibitor of glucosidases I and II

Development

The cellular profile of processing enzymes

Swainsonine

Inhibitor of mannosidase II

may change during development if their

genes are turned on or off.

Intracellular

For instance, if an enzyme is destined for

location

insertion into the membrane of the ER

increase the susceptibility of these proteins to proteoly-

(eg, HMG-CoA reductase), it may never

sis. Inhibition of glycosylation does not appear to have

encounter Golgi-located processing

a consistent effect upon the secretion of glycoproteins

enzymes.

that are normally secreted. The inhibitors of glycopro-

Protein

Differences in conformation of different

tein processing listed in Table 47-12 do not affect the

conformation

proteins may facilitate or hinder access of

biosynthesis of O-linked glycoproteins. The extension

processing enzymes to identical oligosac-

of O-linked chains can be prevented by GalNAc-

charide chains.

benzyl. This compound competes with natural glyco-

Species

Same cells (eg, fibroblasts) from different

protein substrates and thus prevents chain growth be-

species may exhibit different patterns of

yond GalNAc.

processing enzymes.

Cancer

Cancer cells may exhibit processing en-

SOME PROTEINS ARE ANCHORED

zymes different from those of correspond-

TO THE PLASMA MEMBRANE

ing normal cells.

BY GLYCOSYLPHOSPHATIDYL-

¹For example, prior action of GlcNAc transferase I is necessary for

INOSITOL STRUCTURES

the action of Golgi α-mannosidase II.

Glycosylphosphatidylinositol

(GPI)-linked glycopro-

teins comprise the third major class of glycoprotein.

the mechanisms involved in their transcriptional con-

The GPI structure (sometimes called a “sticky foot”)

trol, and gene knockout studies are being used to evalu-

involved in linkage of the enzyme acetylcholinesterase

ate the biologic importance of various glycosyltrans-

(ACh esterase) to the plasma membrane of the red

ferases.

blood cell is shown in Figure 47-1. GPI-linked pro-

teins are anchored to the outer leaflet of the plasma

membrane by the fatty acids of phosphatidylinositol

(PI). The PI is linked via a GlcNH₂ moiety to a glycan

Tunicamycin Inhibits

chain that contains various sugars (eg, Man, GlcNH₂).

N- but Not O-Glycosylation

In turn, the oligosaccharide chain is linked via phos-

A number of compounds are known to inhibit various

phorylethanolamine in an amide linkage to the car-

reactions involved in glycoprotein processing. Tuni-

boxyl terminal amino acid of the attached protein. The

camycin, deoxynojirimycin, and swainsonine are

core of most GPI structures contains one molecule of

three such agents. The reactions they inhibit are indi-

phosphorylethanolamine, three Man residues, one mol-

cated in Table 47-12. These agents can be used experi-

ecule of GlcNH₂, and one molecule of phosphatidyl-

mentally to inhibit various stages of glycoprotein

inositol, as follows:

biosynthesis and to study the effects of specific alter-

ations upon the process. For instance, if cells are grown

Ethanolamine - phospho

6Manα1

→

in the presence of tunicamycin, no glycosylation of

2Manα1→

6Manα1→

GlcNα1→

their normally N-linked glycoproteins will occur. In

certain cases, lack of glycosylation has been shown to

6—

myo

- inositol -1- phospholipid

528

CHAPTER 47

Additional constituents are found in many GPI struc-

text (eg, transport molecules, immunologic molecules,

tures; for example, that shown in Figure 47-1 contains

and hormones). Here, their involvement in two specific

an extra phosphorylethanolamine attached to the mid-

processes—fertilization and inflammation—will be

dle of the three Man moieties of the glycan and an extra

briefly described. In addition, the bases of a number of

fatty acid attached to GlcNH₂. The functional signifi-

diseases that are due to abnormalities in the synthesis

cance of these variations among structures is not under-

and degradation of glycoproteins will be summarized.

stood. This type of linkage was first detected by the use

of bacterial PI-specific phospholipase C

(PI-PLC),

Glycoproteins Are Important

which was found to release certain proteins from the

in Fertilization

plasma membrane of cells by splitting the bond indi-

cated in Figure 47-1. Examples of some proteins that

To reach the plasma membrane of an oocyte, a sperm

are anchored by this type of linkage are given in Table

has to traverse the zona pellucida (ZP), a thick, trans-

47-13. At least three possible functions of this type of

parent, noncellular envelope that surrounds the oocyte.

linkage have been suggested: (1) The GPI anchor may

The zona pellucida contains three glycoproteins of inter-

allow greatly enhanced mobility of a protein in the

est, ZP1-3. Of particular note is ZP3, an O-linked gly-

plasma membrane compared with that observed for a

coprotein that functions as a receptor for the sperm. A

protein that contains transmembrane sequences. This is

protein on the sperm surface, possibly galactosyl trans-

perhaps not surprising, as the GPI anchor is attached

ferase, interacts specifically with oligosaccharide chains of

only to the outer leaflet of the lipid bilayer, so that it is

ZP3; in at least certain species (eg, the mouse), this inter-

freer to diffuse than a protein anchored via both leaflets

action, by transmembrane signaling, induces the acroso-

of the bilayer. Increased mobility may be important in fa-

mal reaction, in which enzymes such as proteases and

cilitating rapid responses to appropriate stimuli. (2) Some

hyaluronidase and other contents of the acrosome of the

GPI anchors may connect with signal transduction

sperm are released. Liberation of these enzymes helps

pathways. (3) It has been shown that GPI structures can

the sperm to pass through the zona pellucida and reach

target certain proteins to apical domains of the plasma

the plasma membrane (PM) of the oocyte. In hamsters,

membrane of certain epithelial cells. The biosynthesis of

it has been shown that another glycoprotein, PH-30, is

GPI anchors is complex and begins in the endoplasmic

important in both the binding of the PM of the sperm to

reticulum. The GPI anchor is assembled independently

the PM of the oocyte and also in the subsequent fusion

by a series of enzyme-catalyzed reactions and then trans-

of the two membranes. These interactions enable the

ferred to the carboxyl terminal end of its acceptor pro-

sperm to enter and thus fertilize the oocyte. It may be

tein, accompanied by cleavage of the preexisting car-

possible to inhibit fertilization by developing drugs or

boxyl terminal hydrophobic peptide from that protein.

antibodies that interfere with the normal functions of

This process is sometimes called glypiation. An ac-

ZP3 and PH-30 and which would thus act as contracep-

quired defect in an early stage of the biosynthesis of the

tive agents.

GPI structure has been implicated in the causation of

paroxysmal nocturnal hemoglobinuria (see below).

Selectins Play Key Roles in Inflammation

& in Lymphocyte Homing

GLYCOPROTEINS ARE INVOLVED

Leukocytes play important roles in many inflammatory

IN MANY BIOLOGIC PROCESSES

and immunologic phenomena. The first steps in many

& IN MANY DISEASES

of these phenomena are interactions between circulat-

As listed in Table 47-1, glycoproteins have many dif-

ing leukocytes and endothelial cells prior to passage of

ferent functions; some have already been addressed in

the former out of the circulation. Work done to iden-

this chapter and others are described elsewhere in this

tify specific molecules on the surfaces of the cells in-

volved in such interactions has revealed that leukocytes

and endothelial cells contain on their surfaces specific

lectins, called selectins, that participate in their inter-

Table 47-13. Some GPI-linked proteins.

cellular adhesion. Features of the three major classes of

selectins are summarized in Table 47-14. Selectins are

• Acetylcholinesterase (red cell membrane)

single-chain Ca2+-binding transmembrane proteins that

• Alkaline phosphatase (intestinal, placental)

contain a number of domains (Figure 47-10). Their

• Decay-accelerating factor (red cell membrane)

amino terminal ends contain the lectin domain, which

•

5′-Nucleotidase (T lymphocytes, other cells)

is involved in binding to specific carbohydrate ligands.

• Thy-1 antigen (brain, T lymphocytes)

The adhesion of neutrophils to endothelial cells of

• Variable surface glycoprotein (Trypanosoma brucei)

postcapillary venules can be considered to occur in four

GLYCOPROTEINS

529

Table 47-14. Some molecules involved in leukocyte-endothelial cell interactions.¹

Molecule

Cell

Ligands

Selectins

L-selectin

PMN, lymphs

CD34, Gly-CAM-1²

Sialyl-Lewis^x and others

P-selectin

EC, platelets

P-selectin glycoprotein ligand-1 (PSGL-1)

Sialyl-Lewis^x and others

E-selectin

Sialyl-Lewis^x and others

Integrins

LFA-1

PMN, lymphs

ICAM-1, ICAM-2 (CD11a/CD18)

Mac-1

PMN

ICAM-1 and others (CD11b/CD18)

Immunoglobulin superfamily

ICAM-1

Lymphs, EC

LFA-1, Mac-1

ICAM-2

Lymphs, EC

LFA-1

PECAM-1

EC, PMN, lymphs

Various platelets

¹Modified from Albelda SM, Smith CW, Ward PA: Adhesion molecules and inflammatory injury. FASEB J 1994;8:504.

²These are ligands for lymphocyte L-selectin; the ligands for neutrophil L-selectin have not been identified.

Key: PMN, polymorphonuclear leukocytes; EC, endothelial cell; lymphs, lymphocytes; CD, cluster of differentiation; ICAM, intercellular ad-

hesion molecule; LFA-1, lymphocyte function-associated antigen-1; PECAM-1, platelet endothelial cell adhesion cell molecule-1.

stages, as shown in Figure 47-11. The initial baseline

this stage, activation of the neutrophils by various

stage is succeeded by slowing or rolling of the neu-

chemical mediators (discussed below) occurs, resulting

trophils, mediated by selectins. Interactions between

in a change of shape of the neutrophils and firm adhe-

L-selectin on the neutrophil surface and CD34 and

sion of these cells to the endothelium. An additional set

GlyCAM-1 or other glycoproteins on the endothelial

of adhesion molecules is involved in firm adhesion,

surface are involved. These particular interactions are

namely, LFA-1 and Mac-1 on the neutrophils and

initially short-lived, and the overall binding is of rela-

ICAM-1 and ICAM-2 on endothelial cells. LFA-1 and

tively low affinity, permitting rolling. However, during

Mac-1 are CD11/CD18 integrins (see Chapter 52 for a

discussion of integrins), whereas ICAM-1 and ICAM-2

are members of the immunoglobulin superfamily. The

L-selectin

fourth stage is transmigration of the neutrophils across

the endothelial wall. For this to occur, the neutrophils

NH₂

Lectin

EGF

COOH

insert pseudopods into the junctions between endothe-

lial cells, squeeze through these junctions, cross the

Figure 47-10. Schematic diagram of the structure

basement membrane, and then are free to migrate in

of human L-selectin. The extracellular portion contains

the extravascular space. Platelet-endothelial cell adhe-

an amino terminal domain homologous to C-type

sion molecule-1 (PECAM-1) has been found to be lo-

lectins and an adjacent epidermal growth factor-like

calized at the junctions of endothelial cells and thus

domain. These are followed by a variable number of

may have a role in transmigration. A variety of biomol-

complement regulatory-like modules (numbered cir-

ecules have been found to be involved in activation of

cles) and a transmembrane sequence (black diamond).

neutrophil and endothelial cells, including tumor

A short cytoplasmic sequence (open rectangle) is at the

necrosis factor α, various interleukins, platelet activat-

carboxyl terminal. The structures of P- and E-selectin

ing factor (PAF), leukotriene B₄, and certain comple-

are similar to that shown except that they contain more

ment fragments. These compounds stimulate various

complement-regulatory modules. The numbers of

signaling pathways, resulting in changes in cell shape

amino acids in L-, P-, and E- selectins, as deduced from

and function, and some are also chemotactic. One im-

the cDNA sequences, are 385, 789, and 589, respec-

portant functional change is recruitment of selectins to

tively. (Reproduced, with permission, from Bevilacqua MP,

the cell surface, as in some cases selectins are stored in

Nelson RM: Selectins. J Clin Invest 1993;91:370.)

granules (eg, in endothelial cells and platelets).

530

CHAPTER 47

lished. Sulfated molecules, such as the sulfatides (Chap-

ter 14), may be ligands in certain instances. This basic

Baseline

knowledge is being used in attempts to synthesize com-

pounds that block selectin-ligand interactions and thus

may inhibit the inflammatory response. Approaches in-

clude administration of specific monoclonal antibodies

or of chemically synthesized analogs of sialyl-Lewis^X,

Rolling

both of which bind selectins. Cancer cells often exhibit

sialyl-Lewis^X and other selectin ligands on their sur-

faces. It is thought that these ligands play a role in the

invasion and metastasis of cancer cells.

Activation

and

Abnormalities in the Synthesis of

firm adhesion

Glycoproteins Underlie Certain Diseases

Table 47-15 lists a number of conditions in which ab-

normalities in the synthesis of glycoproteins are of im-

Transmigration

Table 47-15. Some diseases due to or involving

Figure 47-11. Schematic diagram of neutrophil-

abnormalities in the biosynthesis of

endothelial cell interactions. A: Baseline conditions:

glycoproteins.

Neutrophils do not adhere to the vessel wall. B: The first

event is the slowing or rolling of the neutrophils within

Disease

Abnormality

the vessel (venule) mediated by selectins. C: Activation

Cancer

Increased branching of cell surface

occurs, resulting in neutrophils firmly adhering to the

glycans or presentation of selectin li-

surfaces of endothelial cells and also assuming a flat-

gands may be important in metastasis.

tened shape. This requires interaction of activated

CD18 integrins on neutrophils with ICAM-1 on the en-

Congenital disorders

See Table 47-16.

dothelium. D: The neutrophils then migrate through

of glycosylation¹

the junctions of endothelial cells into the interstitial tis-

HEMPAS² (MIM

Abnormalities in certain enzymes (eg,

sue; this requires involvement of PECAM-1. Chemotaxis

224100)

mannosidase II and others) involved in

is also involved in this latter stage. (Reproduced, with

the biosynthesis of N-glycans, particu-

permission, from Albelda SM, Smith CW, Ward PA: Adhe-

larly affecting the red blood cell mem-

sion molecules and inflammatory injury. FASEB J

brane.

1994;8;504.)

Leukocyte adhesion

Probably mutations affecting a Golgi-

deficiency, type II

located GDP-fucose transporter, re-

(MIM 266265)

sulting in defective fucosylation.

The precise chemical nature of some of the ligands

Paroxysmal nocturnal

Acquired defect in biosynthesis of the

involved in selectin-ligand interactions has been deter-

hemoglobinuria

GPI³ structures of decay accelerating

mined. All three selectins bind sialylated and fucosy-

(MIM 311770)

factor (DAF) and CD59.

lated oligosaccharides, and in particular all three bind

I-cell disease

Deficiency of GlcNAc phosphotrans-

sialyl-Lewis^X (Figure 47-12), a structure present on

(MIM 252500)

ferase, resulting in abnormal targeting

both glycoproteins and glycolipids. Whether this com-

of certain lysosomal enzymes.

pound is the actual ligand involved in vivo is not estab-

¹The MIM number for congenital disorder of glycosylation type Ia

is 212065.

²Hereditary erythroblastic multinuclearity with a positive acidified

NeuAcα2

3Galβ1

4GlcNAc

serum lysis test (congenital dyserythropoietic anemia type II). This

α 1-3

is a relatively mild form of anemia. It reflects at least in part the

Fuc

presence in the red cell membranes of various glycoproteins with

abnormal N-glycan chains, which contribute to the susceptibility

Figure 47-12. Schematic representation of the

to lysis.

structure of sialyl-Lewis^X.

³Glycosylphosphatidylinositol.

GLYCOPROTEINS

531

portance. As mentioned above, many cancer cells ex-

of somatic mutations in the PIG-A (for phosphatidyl-

hibit different profiles of oligosaccharide chains on

inositol glycan class A) gene of certain hematopoietic

their surfaces, some of which may contribute to metas-

cells. The product of this gene appears to be the en-

tasis. The congenital disorders of glycosylation

zyme that links glucosamine to phosphatidylinositol in

(CDG) are a group of disorders of considerable current

the GPI structure (Figure 47-1). Thus, proteins that

interest. The major features of these conditions are

are anchored by a GPI linkage are deficient in the red

summarized in Table 47-16. Leukocyte adhesion de-

cell membrane. Two proteins are of particular interest:

ficiency (LAD) II is a rare condition probably due to

decay accelerating factor (DAF) and another protein

mutations affecting the activity of a Golgi-located

designated CD59. They normally interact with certain

GDP-fucose transporter. It can be considered a congen-

components of the complement system (Chapter 50) to

ital disorder of glycosylation. The absence of fucosy-

prevent the hemolytic actions of the latter. However,

lated ligands for selectins leads to a marked decrease in

when they are deficient, the complement system can act

neutrophil rolling. Subjects suffer life-threatening, re-

on the red cell membrane to cause hemolysis. Paroxys-

current bacterial infections and also psychomotor and

mal nocturnal hemoglobinuria can be diagnosed rela-

mental retardation. The condition appears to respond

tively simply, as the red cells are much more sensitive to

to oral fucose. Hereditary erythroblastic multinuclear-

hemolysis in normal serum acidified to pH 6.2 (Ham’s

ity with a positive acidified lysis test (HEMPAS)—

test); the complement system is activated under these

congenital dyserythropoietic anemia type II—is an-

conditions, but normal cells are not affected. Figure

other disorder due to abnormalities in the processing of

47-13 summarizes the etiology of paroxysmal noctur-

N-glycans. Some cases have been claimed to be due to

nal hemoglobinuria.

defects in alpha-mannosidase II. I-cell disease is dis-

cussed further below. Paroxysmal nocturnal hemo-

I-Cell Disease Results From Faulty

globinuria is an acquired mild anemia characterized by

Targeting of Lysosomal Enzymes

the presence of hemoglobin in urine due to hemolysis

of red cells, particularly during sleep. This latter phe-

As indicated above, Man

6-P serves as a chemical

nomenon may reflect a slight drop in plasma pH dur-

marker to target certain lysosomal enzymes to that or-

ing sleep, which increases susceptibility to lysis by the

ganelle. Analysis of cultured fibroblasts derived from

complement system (Chapter 50). The basic defect in

patients with I-cell (inclusion cell) disease played a large

paroxysmal nocturnal hemoglobinuria is the acquisition

part in revealing the above role of Man 6-P. I-cell dis-

ease is an uncommon condition characterized by severe

progressive psychomotor retardation and a variety of

physical signs, with death often occurring in the first

Table 47-16. Major features of the congenital

decade. Cultured cells from patients with I-cell disease

were found to lack almost all of the normal lysosomal

disorders of glycosylation.

enzymes; the lysosomes thus accumulate many different

• Autosomal recessive disorders

• Multisystem disorders that have probably not been recog-

nized in the past

Acquired mutations in the PIG-A gene

• Generally affect the central nervous system, resulting in

of certain hematopoietic cells

psychomotor retardation and other features

• Type I disorders are due to mutations in genes encoding en-

zymes (eg, phosphomannomutase-2 [PMM-2], causing CDG

Defective synthesis of the GlcNH₂-PI

Ia) involved in the synthesis of dolichol-P-P-oligo-

linkage of GPI anchors

saccharide

• Type II disorders are due to mutations in genes encoding

enzymes (eg, GlcNAc transferase-2, causing CDG IIa) in-

Decreased amounts in the red blood membrane of

volved in the processing of N-glycan chains

GPI-anchored proteins, with decay accelerating factor

• About 11 distinct disorders have been recognized

(DAF) and CD59 being of especial importance

• Isoelectric focusing of transferrin is a useful biochemical test

for assisting in the diagnosis of these conditions; trun-

Certain components of the complement system

cation of the oligosaccharide chains of this protein alters its

are not opposed by DAF and CD59, resulting

isolectric focusing pattern

in complement-mediated lysis of red cells

• Oral mannose has proved of benefit in the treatment of

CDG Ia

Figure 47-13. Scheme of causation of paroxysmal

Key: CDG, congenital disorder of glycosylation.

nocturnal hemoglobinuria (MIM 311770).

532

CHAPTER 47

types of undegraded molecules, forming inclusion bod-

Mutations in DNA

ies. Samples of plasma from patients with the disease

were observed to contain very high activities of lysoso-

Mutant GlcNAc phosphotransferase

mal enzymes; this suggested that the enzymes were

being synthesized but were failing to reach their proper

intracellular destination and were instead being se-

Lack of normal transfer of GlcNAc 1-P

creted. Cultured cells from patients with the disease

to specific mannose residues of certain enzymes

were noted to take up exogenously added lysosomal en-

destined for lysosomes

zymes obtained from normal subjects, indicating that

the cells contained a normal receptor on their surfaces

These enzymes consequently lack Man 6-P

for endocytic uptake of lysosomal enzymes. In addition,

and are secreted from cells (eg, into the plasma)

this finding suggested that lysosomal enzymes from pa-

rather than targeted to lysosomes

tients with I-cell disease might lack a recognition

marker. Further studies revealed that lysosomal en-

Lysosomes are thus deficient in certain hydrolases, do

zymes from normal individuals carried the Man 6-P

not function properly, and accumulate partly digested

recognition marker described above, which interacted

cellular material, manifesting as inclusion bodies

with a specific intracellular protein, the Man 6-P recep-

tor. Cultured cells from patients with I-cell disease were

Figure 47-14. Summary of the causation of I-cell

then found to be deficient in the activity of the cis

disease (MIM 252500).

Golgi-located GlcNAc phosphotransferase, explaining

how their lysosomal enzymes failed to acquire the Man

6-P marker. It is now known that there are two Man

6-P receptor proteins, one of high (275 kDa) and one

cally recognizes and interacts with lysosomal enzymes.

of low (46 kDa) molecular mass. These proteins are

It has been proposed that the defect in pseudo-Hurler

lectins, recognizing Man 6-P. The former is cation-

polydystrophy lies in the latter domain, and the reten-

independent and also binds IGF-II (hence it is named

tion of some catalytic activity results in a milder condi-

the Man 6-P-IGF-II receptor), whereas the latter is

tion.

cation-dependent in some species and does not bind

IGF-II. It appears that both receptors function in the

Genetic Deficiencies of Glycoprotein

intracellular sorting of lysosomal enzymes into clathrin-

Lysosomal Hydrolases Cause Diseases

coated vesicles, which occurs in the trans Golgi subse-

Such as α-Mannosidosis

quent to synthesis of Man 6-P in the cis Golgi. These

vesicles then leave the Golgi and fuse with a prelysoso-

Glycoproteins, like most other biomolecules, undergo

mal compartment. The low pH in this compartment

both synthesis and degradation (ie, turnover). Degrada-

causes the lysosomal enzymes to dissociate from their

tion of the oligosaccharide chains of glycoproteins in-

receptors and subsequently enter into lysosomes. The

volves a battery of lysosomal hydrolases, including

receptors are recycled and reused. Only the smaller re-

α-neuraminidase, β-galactosidase, β-hexosaminidase,

ceptor functions in the endocytosis of extracellular lyso-

α- and β-mannosidases, α-N-acetylgalactosaminidase,

somal enzymes, which is a minor pathway for lysosomal

α-fucosidase, endo-β-N-acetylglucosaminidase, and as-

location. Not all cells employ the Man 6-P receptor to

partylglucosaminidase. The sites of action of the last

target their lysosomal enzymes (eg, hepatocytes use a

two enzymes are indicated in the legend to Figure

different but undefined pathway); furthermore, not all

47-5. Genetically determined defects of the activities of

lysosomal enzymes are targeted by this mechanism.

these enzymes can occur, resulting in abnormal degra-

Thus, biochemical investigations of I-cell disease not

dation of glycoproteins. The accumulation in tissues of

only led to elucidation of its basis but also contributed

such abnormally degraded glycoproteins can lead to

significantly to knowledge of how newly synthesized

various diseases. Among the best-recognized of these

proteins are targeted to specific organelles, in this case

diseases are mannosidosis, fucosidosis, sialidosis, as-

the lysosome. Figure 47-14 summarizes the causation

partylglycosaminuria, and Schindler disease, due re-

of I-cell disease.

spectively to deficiencies of α-mannosidase, α-fucosi-

Pseudo-Hurler polydystrophy is another genetic

dase, α-neuraminidase, aspartylglucosaminidase, and

disease closely related to I-cell disease. It is a milder

α-N-acetyl-galactosaminidase. These diseases, which

condition, and patients may survive to adulthood.

are relatively uncommon, have a variety of manifesta-

Studies have revealed that the GlcNAc phosphotrans-

tions; some of their major features are listed in Table

ferase involved in I-cell disease has several domains, in-

47-17. The fact that patients affected by these disor-

cluding a catalytic domain and a domain that specifi-

ders all show signs referable to the central nervous sys-

GLYCOPROTEINS

533

Table 47-17. Major features of some diseases

It can thus bind the agalactosyl IgG molecules, which

(eg, α-mannosidosis, β-mannosidosis, fucosidosis,

subsequently activate the complement system, con-

tributing to chronic inflammation in the synovial mem-

sialidosis, aspartylglycosaminuria, and Schindler

branes of joints. This protein can also bind the above

disease) due to deficiencies of glycoprotein

sugars when they are present on the surfaces of certain

hydrolases.¹

bacteria, fungi, and viruses, preparing these pathogens

for opsonization or for destruction by the complement

• Usually exhibit mental retardation or other neurologic ab-

system. This is an example of innate immunity, not in-

normalities, and in some disorders coarse features or vis-

volving immunoglobulins. Deficiency of this protein in

ceromegaly (or both)

young infants, due to mutation, renders them very sus-

• Variations in severity from mild to rapidly progressive

ceptible to recurrent infections.

• Autosomal recessive inheritance

• May show ethnic distribution (eg, aspartylglycosaminuria is

Other disorders in which glycoproteins have been

common in Finland)

implicated include hepatitis B and C, Creutzfeldt-

• Vacuolization of cells observed by microscopy in some

Jakob disease, and diarrheas due to a number of bacter-

disorders

ial enterotoxins. It is hoped that basic studies of glyco-

• Presence of abnormal degradation products (eg, oligo-

proteins and other glycoconjugates

(ie, the field of

saccharides that accumulate because of the enzyme

glycobiology) will lead to effective treatments for dis-

deficiency) in urine, detectable by TLC and characterizable

eases in which these molecules are involved. Already, at

by GLC-MS

least two disorders have been found to respond to oral

• Definitive diagnosis made by assay of appropriate enzyme,

supplements of sugars.

often using leukocytes

The fantastic progress made in relation to the

• Possibility of prenatal diagnosis by appropriate enzyme

human genome has stimulated intense interest in both

assays

genomics and proteomics. It is anticipated that the pace

• No definitive treatment at present

of research in glycomics—characterization of the entire

¹MIM numbers: α-mannosidosis,

248500; β-mannosidosis,

complement of sugar chains found in cells (the gly-

248510; fucosidosis,

230000; sialidosis,

256550; aspartylgly-

come)—will also accelerate markedly. For a number of

cosaminuria, 208400; Schindler disease, 104170.

reasons, this field will prove more challenging than ei-

ther genomics or proteomics. These reasons include the

complexity of the structures of oligosaccharide chains

due to linkage variations—in contrast to the generally

tem reflects the importance of glycoproteins in the de-

uniform nature of the linkages between nucleotides and

velopment and normal function of that system.

between amino acids. There are also significant varia-

From the above, it should be apparent that glyco-

tions in oligosaccharide structures among cells and at

proteins are involved in a wide variety of biologic

different stages of development. In addition, no simple

processes and diseases. Glycoproteins play direct or in-

technique exists for amplifying oligosaccharides, com-

direct roles in a number of other diseases, as shown in

parable to the PCR reaction. Despite these and other

the following examples.

problems, it seems certain that research in this area will

uncover many new important biologic interactions that

(1) The influenza virus possesses a neuraminidase

are sugar-dependent and will provide targets for drug

that plays a key role in elution of newly synthesized

and other therapies.

progeny from infected cells. If this process is inhibited,

spread of the virus is markedly diminished. Inhibitors

of this enzyme are now available for use in treating pa-

SUMMARY

tients with influenza.

• Glycoproteins are widely distributed proteins—with

(2) HIV-1, thought by many to be the causative

diverse functions—that contain one or more cova-

agent of AIDS, attaches to cells via one of its surface

lently linked carbohydrate chains.

glycoproteins, gp120.

(3) Rheumatoid arthritis is associated with an al-

• The carbohydrate components of a glycoprotein

range from 1% to more than 85% of its weight and

teration in the glycosylation of circulating im-

munoglobulin-γ (IgG) molecules (Chapter 50), such

may be simple or very complex in structure.

that they lack galactose in their Fc regions and termi-

• At least certain of the oligosaccharide chains of glyco-

nate in GlcNAc. Mannose-binding protein (not to be

proteins encode biologic information; they are also

confused with the mannose-6-P receptor), a C-lectin

important to glycoproteins in modulating their solu-

synthesized by liver cells and secreted into the circula-

bility and viscosity, in protecting them against prote-

tion, binds mannose, GlcNAc, and certain other sugars.

olysis, and in their biologic actions.

534

CHAPTER 47

•

The structures of many oligosaccharide chains can be

• Developments in the new field of glycomics are likely

elucidated by gas-liquid chromatography, mass spec-

to provide much new information on the roles of

trometry, and high-resolution NMR spectrometry.

sugars in health and disease and also indicate targets

•

Glycosidases hydrolyze specific linkages in oligosac-

for drug and other types of therapies.

charides and are used to explore both the structures

and functions of glycoproteins.

•

Lectins are carbohydrate-binding proteins involved

in cell adhesion and other biologic processes.

REFERENCES

•

The major classes of glycoproteins are O-linked (in-

Brockhausen I, Kuhns W: Glycoproteins and Human Disease. Chap-

volving an OH of serine or threonine), N-linked (in-

man & Hall, 1997.

volving the N of the amide group of asparagine), and

Kornfeld R, Kornfeld S: Assembly of asparagine-linked oligosaccha-

glycosylphosphatidylinositol (GPI)-linked.

rides. Annu Rev Biochem 1985;54:631.

•

Mucins are a class of O-linked glycoproteins that are

Lehrman MA Oligosaccharide-based information in endoplasmic

distributed on the surfaces of epithelial cells of the

reticulum quality control and other biological systems. J Biol

Chem. 2001;276:8623.

respiratory, gastrointestinal, and reproductive tracts.

Perkel JM: Glycobiology goes to the ball. The Scientist 2002;

•

The Golgi apparatus plays a major role in glycosyla-

16:32.

tion reactions involved in the biosynthesis of glyco-

Roseman S: Reflections on glycobiology. J Biol Chem 2001;276:

proteins.

41527.

•

The oligosaccharide chains of O-linked glycoproteins

Schachter H: The clinical relevance of glycobiology. J Clin Invest

are synthesized by the stepwise addition of sugars do-

2001;108:1579.

nated by nucleotide sugars in reactions catalyzed by

Schwartz NB, Domowicz M: Chondrodysplasias due to proteogly-

individual specific glycoprotein glycosyltransferases.

can defects. Glycobiology 2002;12:57R.

Science 2001;21(5512):2263. (This issue contains a special section

•

In contrast, the biosynthesis of N-linked glycopro-

entitled Carbohydrates and Glycobiology. It contains articles

teins involves a specific dolichol-P-P-oligosaccharide

on the synthesis, structural determination, and functions of

and various glycosidases. Depending on the glycosi-

sugar-containing molecules and the roles of glycosylation in

dases and precursor proteins synthesized by a tissue,

the immune system).

it can synthesize complex, hybrid, or high-mannose

Scriver CR et al (editors): The Metabolic and Molecular Bases of In-

types of N-linked oligosaccharides.

herited Disease, 8th ed. McGraw-Hill, 2001.(Various chapters

in this text give in-depth coverage of topics such as I-cell dis-

•

Glycoproteins are implicated in many biologic

ease and disorders of glycoprotein degradation.)

processes. For instance, they have been found to play

Spiro RG: Protein glycosylation: nature, distribution, enzymatic

key roles in fertilization and inflammation.

formation, and disease implications of glycopeptide bonds.

•

A number of diseases involving abnormalities in the

Glycobiology 2002;12:43R.

synthesis and degradation of glycoproteins have been

Varki A et al (editors): Essentials of Glycobiology. Cold Spring Har-

recognized. Glycoproteins are also involved in many

bor Laboratory Press, 1999.

other diseases, including influenza, AIDS, and

Vestweber W, Blanks JE: Mechanisms that regulate the function of

rheumatoid arthritis.

the selectins and their ligands. Physiol Rev 1999;79:181.

The Extracellular Matrix

Robert K. Murray, MD, PhD, & Frederick W. Keeley, PhD

BIOMEDICAL IMPORTANCE

collagen-like domains in their structures; these proteins

are sometimes referred to as “noncollagen collagens.”

Most mammalian cells are located in tissues where they

Table 48-1 summarizes the types of collagens found

are surrounded by a complex extracellular matrix

in human tissues; the nomenclature used to designate

(ECM) often referred to as “connective tissue.” The

types of collagen and their genes is described in the

ECM contains three major classes of biomolecules: (1)

footnote.

the structural proteins, collagen, elastin, and fibrillin;

The 19 types of collagen mentioned above can be

(2) certain specialized proteins such as fibrillin, fi-

subdivided into a number of classes based primarily on

bronectin, and laminin; and (3) proteoglycans, whose

the structures they form (Table 48-2). In this chapter,

chemical natures are described below. The ECM has

we shall be primarily concerned with the fibril-forming

been found to be involved in many normal and patho-

collagens I and II, the major collagens of skin and bone

logic processes—eg, it plays important roles in develop-

and of cartilage, respectively. However, mention will be

ment, in inflammatory states, and in the spread of can-

made of some of the other collagens.

cer cells. Involvement of certain components of the

ECM has been documented in both rheumatoid arthri-

tis and osteoarthritis. Several diseases (eg, osteogenesis

COLLAGEN TYPE I IS COMPOSED

imperfecta and a number of types of the Ehlers-Danlos

OF A TRIPLE HELIX STRUCTURE

syndrome) are due to genetic disturbances of the syn-

& FORMS FIBRILS

thesis of collagen. Specific components of proteogly-

cans (the glycosaminoglycans; GAGs) are affected in

All collagen types have a triple helical structure. In

the group of genetic disorders known as the mu-

some collagens, the entire molecule is triple helical,

copolysaccharidoses. Changes occur in the ECM dur-

whereas in others the triple helix may involve only a

ing the aging process. This chapter describes the basic

fraction of the structure. Mature collagen type I, con-

biochemistry of the three major classes of biomolecules

taining approximately 1000 amino acids, belongs to the

found in the ECM and illustrates their biomedical sig-

former type; in it, each polypeptide subunit or alpha

nificance. Major biochemical features of two specialized

chain is twisted into a left-handed helix of three

forms of ECM—bone and cartilage—and of a number

residues per turn (Figure 48-1). Three of these alpha

of diseases involving them are also briefly considered.

chains are then wound into a right-handed superhelix,

forming a rod-like molecule 1.4 nm in diameter and

about 300 nm long. A striking characteristic of collagen

COLLAGEN IS THE MOST ABUNDANT

is the occurrence of glycine residues at every third posi-

PROTEIN IN THE ANIMAL WORLD

tion of the triple helical portion of the alpha chain.

Collagen, the major component of most connective tis-

This is necessary because glycine is the only amino acid

sues, constitutes approximately 25% of the protein of

small enough to be accommodated in the limited space

mammals. It provides an extracellular framework for all

available down the central core of the triple helix. This

metazoan animals and exists in virtually every animal

repeating structure, represented as (Gly-X-Y)_n, is an ab-

tissue. At least 19 distinct types of collagen made up of

solute requirement for the formation of the triple helix.

30 distinct polypeptide chains (each encoded by a sepa-

While X and Y can be any other amino acids, about

rate gene) have been identified in human tissues. Al-

100 of the X positions are proline and about 100 of the

though several of these are present only in small pro-

Y positions are hydroxyproline. Proline and hydroxy-

portions, they may play important roles in determining

proline confer rigidity on the collagen molecule. Hy-

the physical properties of specific tissues. In addition, a

droxyproline is formed by the posttranslational hy-

number of proteins (eg, the C1q component of the

droxylation of peptide-bound proline residues catalyzed

complement system, pulmonary surfactant proteins

by the enzyme prolyl hydroxylase, whose cofactors are

SP-A and SP-D) that are not classified as collagens have

ascorbic acid (vitamin C) and α-ketoglutarate. Lysines

535

536

CHAPTER 48

Table 48-1. Types of collagen and their genes.^1,2

Table 48-2. Classification of collagens, based

primarily on the structures that they form.¹

Type

Genes

Tissue

Class

Type

COL1A1, COL1A2

Most connective tissues,

including bone

Fibril-forming

I, II, III, V, and XI

COL2A1

Cartilage, vitreous humor

Network-like

IV, VIII, X

III

COL3A1

Extensible connective tissues

FACITs²

IX, XII, XIV, XVI, XIX

such as skin, lung, and the

Beaded filaments

vascular system

Anchoring fibrils

VII

COL4A1-COL4A6

Basement membranes

Transmembrane domain

XIII, XVII

COL5A1-COL5A3

Minor component in tissues

containing collagen I

Others

XV, XVIII

COL6A1-COL6A3

Most connective tissues

¹Based on Prockop DJ, Kivirrikko KI: Collagens: molecular biology,

diseases, and potentials for therapy. Annu Rev Biochem

VII

COL7A1

Anchoring fibrils

1995;64:403.

VIII

COL8A1-COL8A2

Endothelium, other tissues

²FACITs

= fibril-associated

collagens

with

interrupted triple

helices.

COL9A1-COL9A3

Tissues containing collagen II

COL10A1

Hypertrophic cartilage

COL11A1, COL11A2,

Tissues containing collagen II

COL2A1

XII

COL12A1

Tissues containing collagen I

XIII

COL13A1

Many tissues

67 nm

XIV

COL14A1

Tissues containing collagen I

Fibril

COL15A1

Many tissues

XVI

COL16A1

Many tissues

XVII

COL17A1

Skin hemidesmosomes

300 nm

XVIII

COL18A1

Many tissues (eg, liver, kidney)

XIX

COL19A1

Rhabdomyosarcoma cells

Molecule

¹Adapted slightly from Prockop DJ, Kivirrikko KI: Collagens: mole-

cular biology, diseases, and potentials for therapy. Annu Rev

Biochem 1995;64:403.

²The types of collagen are designated by Roman numerals. Con-

stituent procollagen chains, called proα chains, are numbered

Triple helix

1.4 nm

using Arabic numerals, followed by the collagen type in paren-

theses. For instance, type I procollagen is assembled from two

proα1(I) and one proα2(I) chain. It is thus a heterotrimer, whereas

type 2 procollagen is assembled from three proα1(II) chains and

Alpha chain

is thus a homotrimer. The collagen genes are named according to

the collagen type, written in Arabic numerals for the gene sym-

Amino acid

bol, followed by an A and the number of the proα chain that they

- Gly - X - Y - Gly - X - Y - Gly - X - Y -

sequence

encode. Thus, the COL1A1 and COL1A2 genes encode the α1 and

α2 chains of type I collagen, respectively.

Figure 48-1. Molecular features of collagen struc-

ture from primary sequence up to the fibril. (Slightly

modified and reproduced, with permission, from Eyre DR:

Collagen: Molecular diversity in the body’s protein scaf-

American Association for the Advancement of Science.)

THE EXTRACELLULAR MATRIX

537

in the Y position may also be posttranslationally modi-

Table 48-3. Order and location of processing of

fied to hydroxylysine through the action of lysyl hy-

the fibrillar collagen precursor.

droxylase, an enzyme with similar cofactors. Some of

these hydroxylysines may be further modified by the

Intracellular

addition of galactose or galactosyl-glucose through an

1. Cleavage of signal peptide

O-glycosidic linkage, a glycosylation site that is

2. Hydroxylation of prolyl residues and some lysyl

unique to collagen.

residues; glycosylation of some hydroxylysyl residues

Collagen types that form long rod-like fibers in tis-

3. Formation of intrachain and interchain S-S bonds in ex-

sues are assembled by lateral association of these triple

tension peptides

helical units into a “quarter staggered” alignment such

4. Formation of triple helix

that each is displaced longitudinally from its neighbor

Extracellular

by slightly less than one-quarter of its length (Figure

1. Cleavage of amino and carboxyl terminal propeptides

48-1, upper part). This arrangement is responsible for

2. Assembly of collagen fibers in quarter-staggered align-

the banded appearance of these fibers in connective tis-

ment

sues. Collagen fibers are further stabilized by the forma-

3. Oxidative deamination of ε-amino groups of lysyl and

tion of covalent cross-links, both within and between

hydroxylysyl residues to aldehydes

the triple helical units. These cross-links form through

4. Formation of intra- and interchain cross-links via Schiff

the action of lysyl oxidase, a copper-dependent en-

bases and aldol condensation products

zyme that oxidatively deaminates the ε-amino groups of

certain lysine and hydroxylysine residues, yielding reac-

tive aldehydes. Such aldehydes can form aldol conden-

sation products with other lysine- or hydroxylysine-

polypeptide extensions (extension peptides) of 20-35

derived aldehydes or form Schiff bases with the

kDa at both its amino and carboxyl terminal ends, nei-

ε-amino groups of unoxidized lysines or hydroxy-

ther of which is present in mature collagen. Both exten-

lysines. These reactions, after further chemical re-

sion peptides contain cysteine residues. While the

arrangements, result in the stable covalent cross-links

amino terminal propeptide forms only intrachain disul-

that are important for the tensile strength of the fibers.

fide bonds, the carboxyl terminal propeptides form

Histidine may also be involved in certain cross-links.

both intrachain and interchain disulfide bonds. Forma-

Several collagen types do not form fibrils in tissues

tion of these disulfide bonds assists in the registration of

(Table 48-2). They are characterized by interruptions

the three collagen molecules to form the triple helix,

of the triple helix with stretches of protein lacking Gly-

winding from the carboxyl terminal end. After forma-

X-Y repeat sequences. These non-Gly-X-Y sequences

tion of the triple helix, no further hydroxylation of pro-

result in areas of globular structure interspersed in the

line or lysine or glycosylation of hydroxylysines can

triple helical structure.

take place. Self-assembly is a cardinal principle in the

Type IV collagen, the best-characterized example of

biosynthesis of collagen.

a collagen with discontinuous triple helices, is an im-

Following secretion from the cell by way of the

portant component of basement membranes, where it

Golgi apparatus, extracellular enzymes called procolla-

forms a mesh-like network.

gen aminoproteinase and procollagen carboxypro-

teinase remove the extension peptides at the amino and

carboxyl terminal ends, respectively. Cleavage of these

Collagen Undergoes Extensive

propeptides may occur within crypts or folds in the cell

Posttranslational Modifications

membrane. Once the propeptides are removed, the

Newly synthesized collagen undergoes extensive post-

triple helical collagen molecules, containing approxi-

translational modification before becoming part of a

mately 1000 amino acids per chain, spontaneously as-

mature extracellular collagen fiber (Table 48-3). Like

semble into collagen fibers. These are further stabilized

most secreted proteins, collagen is synthesized on ribo-

by the formation of inter- and intrachain cross-links

somes in a precursor form, preprocollagen, which con-

through the action of lysyl oxidase, as described previ-

tains a leader or signal sequence that directs the

ously.

polypeptide chain into the lumen of the endoplasmic

The same cells that secrete collagen also secrete fi-

reticulum. As it enters the endoplasmic reticulum, this

bronectin, a large glycoprotein present on cell surfaces,

leader sequence is enzymatically removed. Hydroxyla-

in the extracellular matrix, and in blood (see below). Fi-

tion of proline and lysine residues and glycosylation of

bronectin binds to aggregating precollagen fibers and

hydroxylysines in the procollagen molecule also take

alters the kinetics of fiber formation in the pericellular

place at this site. The procollagen molecule contains

matrix. Associated with fibronectin and procollagen in

538

CHAPTER 48

this matrix are the proteoglycans heparan sulfate and

Table 48-4. Diseases caused by mutations in

chondroitin sulfate (see below). In fact, type IX colla-

collagen genes or by deficiencies in the activities

gen, a minor collagen type from cartilage, contains at-

of posttranslational enzymes involved in the

tached proteoglycan chains. Such interactions may

biosynthesis of collagen.¹

serve to regulate the formation of collagen fibers and to

determine their orientation in tissues.

Gene or Enzyme

Disease²

Once formed, collagen is relatively metabolically sta-

ble. However, its breakdown is increased during starva-

COL1A1, COL1A2

Osteogenesis imperfecta, type 1³(MIM

tion and various inflammatory states. Excessive produc-

1566200)

tion of collagen occurs in a number of conditions, eg,

Osteoporosis⁴ (MIM 166710)

hepatic cirrhosis.

Ehlers-Danlos syndrome type VII auto-

somal dominant (130060)

A Number of Genetic Diseases Result From

COL2A1

Severe chondrodysplasias

Abnormalities in the Synthesis of Collagen

Osteoarthritis⁴ (MIM 120140)

COL3A1

Ehlers-Danlos syndrome type IV (MIM

About 30 genes encode collagen, and its pathway of

130050)

biosynthesis is complex, involving at least eight en-

zyme-catalyzed posttranslational steps. Thus, it is not

COL4A3-COL4A6

Alport syndrome (including both auto-

surprising that a number of diseases (Table 48-4) are

somal and X-linked forms) (MIM 104200)

due to mutations in collagen genes or in genes en-

COL7A1

Epidermolysis bullosa, dystrophic (MIM

coding some of the enzymes involved in these post-

131750)

translational modifications. The diseases affecting bone

(eg, osteogenesis imperfecta) and cartilage

(eg, the

COL10A1

Schmid metaphysial chondrodysplasia

chondrodysplasias) will be discussed later in this chap-

(MIM 156500)

ter.

Lysyl hydroxylase

Ehlers-Danlos syndrome type VI (MIM

Ehlers-Danlos syndrome comprises a group of in-

225400)

herited disorders whose principal clinical features are

Procollagen

Ehlers-Danlos syndrome type VII auto-

hyperextensibility of the skin, abnormal tissue fragility,

N-proteinase

somal recessive (MIM 225410)

and increased joint mobility. The clinical picture is

variable, reflecting underlying extensive genetic hetero-

Lysyl hydroxylase

Menkes disease⁵ (MIM 309400)

geneity. At least 10 types have been recognized, most

¹Adapted from Prockop DJ, Kivirrikko KI: Collagens: molecular bi-

but not all of which reflect a variety of lesions in the

ology, diseases, and potentials for therapy. Annu Rev Biochem

synthesis of collagen. Type IV is the most serious be-

1995;64:403.

cause of its tendency for spontaneous rupture of arteries

²Genetic linkage to collagen genes has been shown for a few

or the bowel, reflecting abnormalities in type III colla-

other conditions not listed here.

gen. Patients with type VI, due to a deficiency of lysyl

³At least four types of osteogenesis imperfecta are recognized;

the great majority of mutations in all types are in the COL1A1 and

hydroxylase, exhibit marked joint hypermobility and a

COL1A2 genes.

tendency to ocular rupture. A deficiency of procollagen

⁴At present applies to only a relatively small number of such

N-proteinase, causing formation of abnormal thin, ir-

patients.

regular collagen fibrils, results in type VIIC, manifested

⁵Secondary to a deficiency of copper (Chapter 50).

by marked joint hypermobility and soft skin.

Alport syndrome is the designation applied to a

number of genetic disorders (both X-linked and autoso-

mal) affecting the structure of type IV collagen fibers,

due to mutations in COL7A1, affecting the structure of

the major collagen found in the basement membranes

type VII collagen. This collagen forms delicate fibrils

of the renal glomeruli

(see discussion of laminin,

that anchor the basal lamina to collagen fibrils in the

below). Mutations in several genes encoding type IV

dermis. These anchoring fibrils have been shown to be

collagen fibers have been demonstrated. The presenting

markedly reduced in this form of the disease, probably

sign is hematuria, and patients may eventually develop

resulting in the blistering. Epidermolysis bullosa sim-

end-stage renal disease. Electron microscopy reveals

plex, another variant, is due to mutations in keratin 5

characteristic abnormalities of the structure of the base-

(Chapter 49).

ment membrane and lamina densa.

Scurvy affects the structure of collagen. However, it

In epidermolysis bullosa, the skin breaks and blis-

is due to a deficiency of ascorbic acid (Chapter 45) and

ters as a result of minor trauma. The dystrophic form is

is not a genetic disease. Its major signs are bleeding

THE EXTRACELLULAR MATRIX

539

gums, subcutaneous hemorrhages, and poor wound

Table 48-5. Major differences between collagen

healing. These signs reflect impaired synthesis of colla-

and elastin.

gen due to deficiencies of prolyl and lysyl hydroxylases,

both of which require ascorbic acid as a cofactor.

Collagen

Elastin

1. Many different genetic

One genetic type

ELASTIN CONFERS EXTENSIBILITY

types

& RECOIL ON LUNG, BLOOD

2. Triple helix

No triple helix; random coil

VESSELS, & LIGAMENTS

conformations permitting

stretching

Elastin is a connective tissue protein that is responsible

(Gly-X-Y)_n repeating

No (Gly-X-Y)_n repeating

for properties of extensibility and elastic recoil in tis-

structure

sues. Although not as widespread as collagen, elastin is

4. Presence of hydroxylysine

No hydroxylysine

present in large amounts, particularly in tissues that re-

5. Carbohydrate-containing

No carbohydrate

quire these physical properties, eg, lung, large arterial

6. Intramolecular aldol

Intramolecular desmosine

blood vessels, and some elastic ligaments. Smaller quan-

cross-links

tities of elastin are also found in skin, ear cartilage, and

7. Presence of extension

No extension peptides present

several other tissues. In contrast to collagen, there ap-

peptides during bio-

during biosynthesis

pears to be only one genetic type of elastin, although

synthesis

variants arise by alternative splicing (Chapter 37) of the

hnRNA for elastin. Elastin is synthesized as a soluble

monomer of 70 kDa called tropoelastin. Some of the

prolines of tropoelastin are hydroxylated to hydroxy-

MARFAN SYNDROME IS DUE TO

proline by prolyl hydroxylase, though hydroxylysine

MUTATIONS IN THE GENE FOR FIBRILLIN,

and glycosylated hydroxylysine are not present. Unlike

A PROTEIN PRESENT IN MICROFIBRILS

collagen, tropoelastin is not synthesized in a pro- form

with extension peptides. Furthermore, elastin does not

Marfan syndrome is a relatively prevalent inherited dis-

contain repeat Gly-X-Y sequences, triple helical struc-

ease affecting connective tissue; it is inherited as an au-

ture, or carbohydrate moieties.

tosomal dominant trait. It affects the eyes (eg, causing

After secretion from the cell, certain lysyl residues of

dislocation of the lens, known as ectopia lentis), the

tropoelastin are oxidatively deaminated to aldehydes by

skeletal system (most patients are tall and exhibit long

lysyl oxidase, the same enzyme involved in this process

digits

[arachnodactyly] and hyperextensibility of the

in collagen. However, the major cross-links formed in

joints), and the cardiovascular system

(eg, causing

elastin are the desmosines, which result from the con-

weakness of the aortic media, leading to dilation of the

densation of three of these lysine-derived aldehydes with

ascending aorta). Abraham Lincoln may have had this

an unmodified lysine to form a tetrafunctional cross-

condition. Most cases are caused by mutations in the

link unique to elastin. Once cross-linked in its mature,

gene (on chromosome 15) for fibrillin; missense muta-

extracellular form, elastin is highly insoluble and ex-

tions have been detected in several patients with Mar-

tremely stable and has a very low turnover rate. Elastin

fan syndrome.

exhibits a variety of random coil conformations that per-

Fibrillin is a large glycoprotein (about 350 kDa)

mit the protein to stretch and subsequently recoil during

that is a structural component of microfibrils, 10- to

the performance of its physiologic functions.

12-nm fibers found in many tissues. Fibrillin is secreted

Table 48-5 summarizes the main differences be-

(subsequent to a proteolytic cleavage) into the extracel-

tween collagen and elastin.

lular matrix by fibroblasts and becomes incorporated

Deletions in the elastin gene (located at 7q11.23)

into the insoluble microfibrils, which appear to provide

have been found in approximately 90% of subjects with

a scaffold for deposition of elastin. Of special relevance

Williams syndrome, a developmental disorder affect-

to Marfan syndrome, fibrillin is found in the zonular

ing connective tissue and the central nervous system.

fibers of the lens, in the periosteum, and associated

The mutations, by affecting synthesis of elastin, proba-

with elastin fibers in the aorta (and elsewhere); these lo-

bly play a causative role in the supravalvular aortic

cations respectively explain the ectopia lentis, arach-

stenosis often found in this condition. A number of

nodactyly, and cardiovascular problems found in the

skin diseases (eg, scleroderma) are associated with accu-

syndrome. Other proteins (eg, emelin and two mi-

mulation of elastin. Fragmentation or, alternatively, a

crofibril-associated proteins) are also present in these

decrease of elastin is found in conditions such as pul-

microfibrils, and it appears likely that abnormalities of

monary emphysema, cutis laxa, and aging of the skin.

them may cause other connective tissue disorders. An-

540

CHAPTER 48

other gene for fibrillin exists on chromosome 5; muta-

ECM, with a typical cell (eg, fibroblast) present in the

tions in this gene are linked to causation of congenital

matrix.

contractural arachnodactyly but not to Marfan syn-

The fibronectin receptor interacts indirectly with

drome. The probable sequence of events leading to

actin microfilaments (Chapter 49) present in the cy-

Marfan syndrome is summarized in Figure 48-2.

tosol (Figure 48-5). A number of proteins, collectively

known as attachment proteins, are involved; these in-

clude talin, vinculin, an actin-filament capping protein,

FIBRONECTIN IS AN IMPORTANT

and α-actinin. Talin interacts with the receptor and

GLYCOPROTEIN INVOLVED IN CELL

vinculin, whereas the latter two interact with actin. The

ADHESION & MIGRATION

interaction of fibronectin with its receptor provides one

Fibronectin is a major glycoprotein of the extracellular

route whereby the exterior of the cell can communicate

matrix, also found in a soluble form in plasma. It con-

with the interior and thus affect cell behavior. Via the

sists of two identical subunits, each of about 230 kDa,

interaction with its cell receptor, fibronectin plays an

joined by two disulfide bridges near their carboxyl ter-

important role in the adhesion of cells to the ECM. It is

minals. The gene encoding fibronectin is very large,

also involved in cell migration by providing a binding

containing some 50 exons; the RNA produced by its

site for cells and thus helping them to steer their way

transcription is subject to considerable alternative splic-

through the ECM. The amount of fibronectin around

ing, and as many as 20 different mRNAs have been de-

many transformed cells is sharply reduced, partly ex-

tected in various tissues. Fibronectin contains three

plaining their faulty interaction with the ECM.

types of repeating motifs (I, II, and III), which are orga-

nized into functional domains (at least seven); func-

tions of these domains include binding heparin (see

LAMININ IS A MAJOR PROTEIN

below) and fibrin, collagen, DNA, and cell surfaces

COMPONENT OF RENAL GLOMERULAR

(Figure

48-3). The amino acid sequence of the fi-

& OTHER BASAL LAMINAS

bronectin receptor of fibroblasts has been derived, and

the protein is a member of the transmembrane integrin

Basal laminas are specialized areas of the ECM that sur-

round epithelial and some other cells (eg, muscle cells);

class of proteins (Chapter 51). The integrins are het-

erodimers, containing various types of α and β

here we discuss only the laminas found in the renal

glomerulus. In that structure, the basal lamina is con-

polypeptide chains. Fibronectin contains an Arg-Gly-

Asp (RGD) sequence that binds to the receptor. The

tributed by two separate sheets of cells (one endothelial

and one epithelial), each disposed on opposite sides of

RGD sequence is shared by a number of other proteins

present in the ECM that bind to integrins present in

the lamina; these three layers make up the glomerular

membrane. The primary components of the basal lam-

cell surfaces. Synthetic peptides containing the RGD

sequence inhibit the binding of fibronectin to cell sur-

ina are three proteins—laminin, entactin, and type IV

collagen—and the GAG heparin or heparan sulfate.

faces. Figure 48-4 illustrates the interaction of collagen,

fibronectin, and laminin, all major proteins of the

These components are synthesized by the underlying

cells.

Laminin (about 850 kDa, 70 nm long) consists of

three distinct elongated polypeptide chains (A, B₁, and

Mutations in gene (on chromosome 15)

B₂) linked together to form an elongated cruciform

for fibrillin, a large glycoprotein present in

shape. It has binding sites for type IV collagen, heparin,

elastin-associated microfibrils

and integrins on cell surfaces. The collagen interacts

with laminin (rather than directly with the cell surface),

Abnormalities of the structure of fibrillin

which in turn interacts with integrins or other laminin

receptor proteins, thus anchoring the lamina to the

cells. Entactin, also known as “nidogen,” is a glycopro-

Structures of the suspensory ligament of the eye,

tein containing an RGD sequence; it binds to laminin

the periosteum, and the media of the aorta affected

and is a major cell attachment factor. The relatively

thick basal lamina of the renal glomerulus has an im-

Ectopia lentis, arachnodactyly,

portant role in glomerular filtration, regulating the

and dilation of the ascending aorta

passage of large molecules (most plasma proteins) across

the glomerulus into the renal tubule. The glomerular

Figure 48-2. Probable sequence of events in the

membrane allows small molecules, such as inulin (5.2

causation of the major signs exhibited by patients with

kDa), to pass through as easily as water. On the other

Marfan syndrome (MIM 154700).

hand, only a small amount of the protein albumin (69

THE EXTRACELLULAR MATRIX

541

RGD

Heparin A

Collagen

DNA

Cell A

Heparin B

Cell B

Fibrin B

Fibrin A

Figure 48-3. Schematic representation of fibronectin. Seven functional

domains of fibronectin are represented; two different types of domain for

heparin, cell-binding, and fibrin are shown. The domains are composed of

various combinations of three structural motifs (I, II, and III), not depicted

in the figure. Also not shown is the fact that fibronectin is a dimer joined

by disulfide bridges near the carboxyl terminals of the monomers. The ap-

proximate location of the RGD sequence of fibronectin, which interacts

with a variety of fibronectin integrin receptors on cell surfaces, is indicated

by the arrow. (Redrawn after Yamada KM: Adhesive recognition sequences.

J Biol Chem 1991;266:12809.)

kDa), the major plasma protein, passes through the

normal glomerulus. This is explained by two sets of

facts: (1) The pores in the glomerular membrane are

large enough to allow molecules up to about 8 nm to

pass through. (2) Albumin is smaller than this pore size,

but it is prevented from passing through easily by the

Collagen

negative charges of heparan sulfate and of certain sialic

acid-containing glycoproteins present in the lamina.

These negative charges repel albumin and most plasma

proteins, which are negatively charged at the pH of

Heparin

Fibronectin

blood. The normal structure of the glomerulus may be

severely damaged in certain types of glomerulonephri-

OUTSIDE

S-S

tis

(eg, caused by antibodies directed against various

S-S

components of the glomerular membrane). This alters

the pores and the amounts and dispositions of the nega-

tively charged macromolecules referred to above, and

relatively massive amounts of albumin (and of certain

Integrin

receptor

Plasma membrane

Collagen

Fibronectin

Talin

Vinculin

INSIDE

Capping protein

α-Actin

Actin

Laminin

Figure 48-5.

Schematic representation of fibro-

Figure 48-4. Schematic representation of a cell in-

nectin interacting with an integrin fibronectin receptor

teracting through various integrin receptors with colla-

situated in the exterior of the plasma membrane of a

gen, fibronectin, and laminin present in the ECM. (Spe-

cell of the ECM and of various attachment proteins in-

cific subunits are not indicated.) (Redrawn after Yamada

teracting indirectly or directly with an actin microfila-

KM: Adhesive recognition sequences. J Biol Chem

ment in the cytosol. For simplicity, the attachment pro-

1991;266:12809.)

teins are represented as a complex.

542

CHAPTER 48

other plasma proteins) can pass through into the urine,

resulting in severe albuminuria.

PROTEOGLYCANS

& GLYCOSAMINOGLYCANS

The Glycosaminoglycans Found

in Proteoglycans Are Built Up

of Repeating Disaccharides

Proteoglycans are proteins that contain covalently

linked glycosaminoglycans. A number of them have

been characterized and given names such as syndecan,

betaglycan, serglycin, perlecan, aggrecan, versican,

decorin, biglycan, and fibromodulin. They vary in tis-

sue distribution, nature of the core protein, attached

glycosaminoglycans, and function. The proteins bound

covalently to glycosaminoglycans are called “core pro-

teins”; they have proved difficult to isolate and charac-

terize, but the use of recombinant DNA technology is

beginning to yield important information about their

structures. The amount of carbohydrate in a proteogly-

can is usually much greater than is found in a glycopro-

tein and may comprise up to 95% of its weight. Figures

48-6 and 48-7 show the general structure of one par-

ticular proteoglycan, aggrecan, the major type found in

cartilage. It is very large (about 2 × 10³ kDa), with its

overall structure resembling that of a bottle brush. It

contains a long strand of hyaluronic acid (one type of

Figure 48-6.

Dark field electron micrograph of a

GAG) to which link proteins are attached noncova-

proteoglycan aggregate in which the proteoglycan

lently. In turn, these latter interact noncovalently with

subunits and filamentous backbone are particularly

core protein molecules from which chains of other

well extended. (Reproduced, with permission, from

GAGs (keratan sulfate and chondroitin sulfate in this

Rosenberg L, Hellman W, Kleinschmidt AK: Electron micro-

case) project. More details on this macromolecule are

scopic studies of proteoglycan aggregates from bovine

given when cartilage is discussed below.

articular cartilage. J Biol Chem 1975;250:1877.)

There are at least seven glycosaminoglycans

(GAGs): hyaluronic acid, chondroitin sulfate, keratan

sulfates I and II, heparin, heparan sulfate, and der-

matan sulfate. A GAG is an unbranched polysaccharide

biologic roles; and they are involved in a number of dis-

made up of repeating disaccharides, one component of

ease processes—so that interest in them is increasing

which is always an amino sugar

(hence the name

rapidly.

GAG), either D-glucosamine or D-galactosamine. The

other component of the repeating disaccharide (except

in the case of keratan sulfate) is a uronic acid, either

Biosynthesis of Glycosaminoglycans

L-glucuronic acid (GlcUA) or its 5′-epimer, L-iduronic

Involves Attachment to Core Proteins,

acid (IdUA). With the exception of hyaluronic acid, all

Chain Elongation, & Chain Termination

the GAGs contain sulfate groups, either as O-esters or

A. ATTACHMENT TO CORE PROTEINS

as N-sulfate

(in heparin and heparan sulfate).

Hyaluronic acid affords another exception because

The linkage between GAGs and their core proteins is

there is no clear evidence that it is attached covalently

generally one of three types.

to protein, as the definition of a proteoglycan given

above specifies. Both GAGs and proteoglycans have

1. An O-glycosidic bond between xylose (Xyl) and

proved difficult to work with, partly because of their

Ser, a bond that is unique to proteoglycans. This link-

complexity. However, they are major components of

age is formed by transfer of a Xyl residue to Ser from

the ground substance; they have a number of important

UDP-xylose. Two residues of Gal are then added to the

THE EXTRACELLULAR MATRIX

543

as in the case of certain types of linkages found in gly-

coproteins. The enzyme systems involved in chain elon-

Hyaluronic acid

gation are capable of high-fidelity reproduction of com-

Link protein

plex GAGs.

Keratan sulfate

C. CHAIN TERMINATION

Chondroitin sulfate

This appears to result from (1) sulfation, particularly at

certain positions of the sugars, and (2) the progression

Core protein

of the growing GAG chain away from the membrane

site where catalysis occurs.

D. FURTHER MODIFICATIONS

After formation of the GAG chain, numerous chemical

Subunits

modifications occur, such as the introduction of sulfate

groups onto GalNAc and other moieties and the

epimerization of GlcUA to IdUA residues. The enzymes

catalyzing sulfation are designated sulfotransferases and

use 3′-phosphoadenosine-5′-phosphosulfate (PAPS; ac-

tive sulfate) as the sulfate donor. These Golgi-located

enzymes are highly specific, and distinct enzymes cat-

alyze sulfation at different positions (eg, carbons 2, 3, 4,

and 6) on the acceptor sugars. An epimerase catalyzes

conversions of glucuronyl to iduronyl residues.

Figure 48-7.

Schematic representation of the pro-

The Various Glycosaminoglycans Exhibit

teoglycan aggrecan. (Reproduced, with permission, from

Differences in Structure & Have

Lennarz WJ:The Biochemistry of Glycoproteins and Proteo-

Characteristic Distributions

glycans. Plenum Press, 1980.)

The seven GAGs named above differ from each other in

a number of the following properties: amino sugar com-

position, uronic acid composition, linkages between

these components, chain length of the disaccharides, the

Xyl residue, forming a link trisaccharide, Gal-Gal-Xyl-

presence or absence of sulfate groups and their positions

Ser. Further chain growth of the GAG occurs on the

of attachment to the constituent sugars, the nature of

terminal Gal.

the core proteins to which they are attached, the nature

2. An O-glycosidic bond forms between GalNAc

of the linkage to core protein, their tissue and subcellu-

(N-acetylgalactosamine) and Ser

(Thr)

(Figure

47-

lar distribution, and their biologic functions.

1[a]), present in keratan sulfate II. This bond is formed

The structures (Figure 48-8) and the distributions

by donation to Ser (or Thr) of a GalNAc residue, em-

of each of the GAGs will now be briefly discussed. The

ploying UDP-GalNAc as its donor.

major features of the seven GAGs are summarized in

3. An N-glycosylamine bond between GlcNAc

Table 48-6.

(N-acetylglucosamine) and the amide nitrogen of Asn,

as found in N-linked glycoproteins (Figure 47-1[b]).

A. HYALURONIC ACID

Its synthesis is believed to involve dolichol-P-P-

Hyaluronic acid consists of an unbranched chain of re-

oligosaccharide.

peating disaccharide units containing GlcUA and Glc-

The synthesis of the core proteins occurs in the en-

NAc. Hyaluronic acid is present in bacteria and is

doplasmic reticulum, and formation of at least some

widely distributed among various animals and tissues,

of the above linkages also occurs there. Most of the later

including synovial fluid, the vitreous body of the eye,

steps in the biosynthesis of GAG chains and their sub-

cartilage, and loose connective tissues.

sequent modifications occur in the Golgi apparatus.

B. CHONDROITIN SULFATES (CHONDROITIN

B. CHAIN ELONGATION

4-SULFATE & CHONDROITIN 6-SULFATE)

Appropriate nucleotide sugars and highly specific

Proteoglycans linked to chondroitin sulfate by the Xyl-

Golgi-located glycosyltransferases are employed to syn-

Ser O-glycosidic bond are prominent components of

thesize the oligosaccharide chains of GAGs. The “one

cartilage

(see below). The repeating disaccharide is

enzyme, one linkage” relationship appears to hold here,

similar to that found in hyaluronic acid, containing

544

CHAPTER 48

β1,4

β1,3

β1,4

β1,3

β1,4

Hyaluronic acid:

GlcUA

GlcNAc

GlcUA

GlcNAc

β1,4

β1,3

β1,4

β1,3

β1,4

Chondroitin sulfates:

GlcUA

GalNAc

GlcUA

Gal

Xyl

Ser

4- or 6-Sulfate

GlcNAc

Asn (keratan sulfate I)

Keratan sulfates

β1,4

β1,3

β1,4

β1,3

I and II:

GlcNAc

Gal

GlcNAc

Gal

6-Sulfate

GalNAc

Thr (Ser) (keratan sulfate II)

Gal-NeuAc

6-Sulfate

Heparin and

α1,4

β1,4

α1,4

β1,3

β1,4

heparan sulfate:

IdUA

GlcN

GlcUA

GlcNAc

GlcUA

Gal

Xyl

Ser

2-Sulfate

SO3- or Ac

β1,4

α1,3

β1,4

β1,3

β1,4

β1,3

β1,4

Dermatan sulfate:

IdUA

GalNAc

GlcUA

GalNAc

GlcUA

Gal

Xyl

Ser

2-Sulfate

4-Sulfate

Figure 48-8. Summary of structures of glycosaminoglycans and their attachments to core proteins. (GlcUA,

D-glucuronic acid; IdUA, L-iduronic acid; GlcN, D-glucosamine; GalN, D-galactosamine; Ac, acetyl; Gal, D-galac-

tose; Xyl, D-xylose; Ser, L-serine; Thr, L-threonine; Asn, L-asparagine; Man, D-mannose; NeuAc, N-acetylneu-

raminic acid.) The summary structures are qualitative representations only and do not reflect, for example, the

uronic acid composition of hybrid glycosaminoglycans such as heparin and dermatan sulfate, which contain

both L-iduronic and D-glucuronic acid. Neither should it be assumed that the indicated substituents are always

present, eg, whereas most iduronic acid residues in heparin carry a 2′-sulfate group, a much smaller proportion

of these residues are sulfated in dermatan sulfate. The presence of link trisaccharides (Gal-Gal-Xyl) in the chon-

droitin sulfates, heparin, and heparan and dermatan sulfates is shown. (Slightly modified and reproduced, with

permission, from Lennarz WJ: The Biochemistry of Glycoproteins and Proteoglycans. Plenum Press, 1980.)

Table 48-6. Major properties of the glycosaminoglycans.

GAG

Sugars

Sulfate¹

Linkage of Protein

Location

GIcNAc, GlcUA

Nil

No firm evidence

Synovial fluid, vitreous humor, loose connective tissue

GaINAc, GlcUA

GalNAc

Xyl-Ser; associated with

HA via link proteins

Cartilage, bone, cornea

KS I

GlcNAc, Gal

GlcNAc

GlcNAc-Asn

Cornea

KS II

GlcNAc, Gal

Same as KS I

GalNAc-Thr

Loose connective tissue

Heparin

GlcN, IdUA

GlcN

Ser

Mast cells

GlcN

IdUA

Heparan sulfate

GlcN, GlcUA

GlcN

Xyl-Ser

Skin fibroblasts, aortic wall

Dermatan

GalNAc, IdUA,

GaINAc

Xyl-Ser

Wide distribution

sulfate

(GlcUA)

IdUa

¹The sulfate is attached to various positions of the sugars indicated (see Figure 48-7).

THE EXTRACELLULAR MATRIX

545

GlcUA but with GalNAc replacing GlcNAc. The

cept that in place of a GlcUA in β-1,3 linkage to

GalNAc is substituted with sulfate at either its 4′ or its

GalNAc it contains an IdUA in an α-1,3 linkage to

6′ position, with approximately one sulfate being pre-

GalNAc. Formation of the IdUA occurs, as in heparin

sent per disaccharide unit.

and heparan sulfate, by 5′-epimerization of GlcUA. Be-

cause this is regulated by the degree of sulfation and be-

C. KERATAN SULFATES I & II

cause sulfation is incomplete, dermatan sulfate contains

As shown in Figure 48-8, the keratan sulfates consist of

both IdUA-GalNAc and GlcUA-GalNAc disaccha-

repeating Gal-GlcNAc disaccharide units containing

rides.

sulfate attached to the 6′ position of GlcNAc or occa-

sionally of Gal. Type I is abundant in cornea, and type

II is found along with chondroitin sulfate attached to

hyaluronic acid in loose connective tissue. Types I and

Deficiencies of Enzymes That Degrade

II have different attachments to protein (Figure 48-8).

Glycosaminoglycans Result in

Mucopolysaccharidoses

D. HEPARIN

The repeating disaccharide contains glucosamine

Both exo- and endoglycosidases degrade GAGs. Like

most other biomolecules, GAGs are subject to

(GlcN) and either of the two uronic acids

(Figure

48-9). Most of the amino groups of the GlcN residues

turnover, being both synthesized and degraded. In

adult tissues, GAGs generally exhibit relatively slow

are N-sulfated, but a few are acetylated. The GlcN also

carries a C₆ sulfate ester.

turnover, their half-lives being days to weeks.

Understanding of the degradative pathways for

Approximately 90% of the uronic acid residues are

IdUA. Initially, all of the uronic acids are GlcUA, but a

GAGs, as in the case of glycoproteins (Chapter 47) and

glycosphingolipids (Chapter 24), has been greatly aided

5′-epimerase converts approximately

90% of the

GlcUA residues to IdUA after the polysaccharide chain

by elucidation of the specific enzyme deficiencies that

occur in certain inborn errors of metabolism. When

is formed. The protein molecule of the heparin proteo-

glycan is unique, consisting exclusively of serine and

GAGs are involved, these inborn errors are called mu-

copolysaccharidoses (Table 48-7).

glycine residues. Approximately two-thirds of the serine

residues contain GAG chains, usually of 5-15 kDa but

Degradation of GAGs is carried out by a battery of

lysosomal hydrolases. These include certain endogly-

occasionally much larger. Heparin is found in the gran-

ules of mast cells and also in liver, lung, and skin.

cosidases, various exoglycosidases, and sulfatases, gener-

ally acting in sequence to degrade the various GAGs. A

E. HEPARAN SULFATE

number of them are indicated in Table 48-7.

This molecule is present on many cell surfaces as a

The mucopolysaccharidoses share a common

proteoglycan and is extracellular. It contains GlcN with

mechanism of causation, as illustrated in Figure 48-10.

fewer N-sulfates than heparin, and, unlike heparin, its

They are inherited in an autosomal recessive manner,

predominant uronic acid is GlcUA.

with Hurler and Hunter syndromes being perhaps the

most widely studied. None are common. In some cases,

F. DERMATAN SULFATE

a family history of a mucopolysaccharidosis is obtained.

This substance is widely distributed in animal tissues.

Specific laboratory investigations of help in their diag-

Its structure is similar to that of chondroitin sulfate, ex-

nosis are urine testing for the presence of increased

CH₂OSO3-

CH₂OSO

CH₂OSO₃

CO2-

HNSO₃

–

OSO₃

–

HNSO₃

–

HNSO

HNAc

GlcN

IdUA

GlcN

IdUA

GlcN

GlcUA

GlcNAc

Figure 48-9. Structure of heparin. The polymer section illustrates structural features typical of heparin;

however, the sequence of variously substituted repeating disaccharide units has been arbitrarily selected. In

addition, non-O-sulfated or 3-O-sulfated glucosamine residues may also occur. (Modified, redrawn, and repro-

duced, with permission, from Lindahl U et al: Structure and biosynthesis of heparin-like polysaccharides. Fed Proc

1977;36:19.)

546

CHAPTER 48

Table 48-7. Biochemical defects and diagnostic tests in mucopolysaccharidoses (MPS) and

mucolipidoses (ML).¹

Alternative

Urinary

Name

Designation^2,3

Enzymatic Defect

Metabolites

Mucopolysaccharidoses

Hurler, Scheie,

MPS I

α-L-Iduronidase

Dermatan sulfate, heparan sulfate

Hurler-Scheie

(MIM 252800)

Hunter (MIM 309900)

MPS II

Iduronate sulfatase

Dermatan sulfate, heparan sulfate

Sanfilippo A

MPS IIIA

Heparan sulfate N-sulfatase

Heparan sulfate

(MIM 252900)

(sulfamidase)

Sanfilippo B

MPS IIIB

α-N-Acetylglucosaminidase

Heparan sulfate

(MIM 252920)

Sanfilippo C

MPS IIIC

Acetyltransferase

Heparan sulfate

(MIM 252930)

Sanfilippo D

MPS IIID

N-Acetylglucosamine

Heparan sulfate

(MIM 252940)

6-sulfatase

Morquio A

MPS IVA

Galactosamine 6-sulfatase

Keratan sulfate, chondroitin 6-sulfate

(MIM 253000)

Morquio B

MPS IVB

β-Galactosidase

Keratan sulfate

(MIM 253010)

Maroteaux-Lamy

MPS VI

N-Acetylgalactosamine 4-

Dermatan sulfate

(MIM 253200)

sulfatase (arylsulfatase B)

Sly (MIM 253220)

MPS VII

β-Glucuronidase

Dermatan sulfate, heparan sulfate, chondroitin

4-sulfate, chondroitin 6-sulfate

Mucolipidoses

Sialidosis

M LI

Sialidase (neuraminidase)

Glycoprotein fragments

(MIM 256550)

I-cell disease

ML II

UDP-N-acetylglucosamine:

Glycoprotein fragments

(MIM 252500)

glycoprotein N-acetylglu-

cosamininylphosphotrans-

ferase. (Acid hydrolases

thus lack phosphoman-

nosyl residues.)

Pseudo-Hurler

ML III

As for ML II but deficiency

Glycoprotein fragments

polydystrophy

is incomplete

(MIM 252600)

¹Modified and reproduced, with permission, from DiNatale P, Neufeld EF: The biochemical diagnosis of mucopolysaccharidoses,

mucolipidoses and related disorders. In: Perspectives in Inherited Metabolic Diseases, vol 2. Barr B et al (editors). Editiones Ermes

(Milan), 1979.

²Fibroblasts, leukocytes, tissues, amniotic fluid cells, or serum can be used for the assay of many of the above enzymes. Patients

with these disorders exhibit a variety of clinical findings that may include cloudy corneas, mental retardation, stiff joints, cardiac ab-

normalities, hepatosplenomegaly, and short stature, depending on the specific disease and its severity.

³The term MPS V is no longer used. The existence of MPS VIII (suspected glucosamine 6-sulfatase deficiency: MIM 253230) has not

been confirmed. At least one case of hyaluronidase deficiency (MPS IX; MIM 601492) has been reported.

amounts of GAGs and assays of suspected enzymes in

The term “mucolipidosis” was introduced to de-

white cells, fibroblasts, or sometimes in serum. In cer-

note diseases that combined features common to both

tain cases, a tissue biopsy is performed and the GAG

mucopolysaccharidoses and sphingolipidoses (Chapter

that has accumulated can be determined by elec-

24). Three mucolipidoses are listed in Table 48-7. In

trophoresis. DNA tests are increasingly available. Pre-

sialidosis (mucolipidosis I, ML-I), various oligosaccha-

natal diagnosis can be made using amniotic cells or

rides derived from glycoproteins and certain ganglio-

chorionic villus biopsy.

sides can accumulate in tissues. I-cell disease (ML-II)

THE EXTRACELLULAR MATRIX

547

TGF-β, modulating their effects on cells. In addition,

Mutation(s) in a gene encoding a lysosomal hydrolase

some of them interact with certain adhesive proteins

involved in the degradation of one or more GAGs

such as fibronectin and laminin (see above), also found

in the matrix. The GAGs present in the proteoglycans

Defective lysosomal hydrolase

are polyanions and hence bind polycations and cations

such as Na⁺ and K⁺. This latter ability attracts water by

Accumulation of substrate in various tissues, including

osmotic pressure into the extracellular matrix and con-

liver, spleen, bone, skin, and central nervous system

tributes to its turgor. GAGs also gel at relatively low

concentrations. Because of the long extended nature of

Figure 48-10. Simplified scheme of causation of a

the polysaccharide chains of GAGs and their ability to

mucopolysaccharidosis, such as Hurler syndrome (MIM

gel, the proteoglycans can act as sieves, restricting the

252800), in which the affected enzyme is α-L-iduroni-

passage of large macromolecules into the ECM but al-

dase. Marked accumulation of the GAGs in the tissues

lowing relatively free diffusion of small molecules.

mentioned in the figure could cause hepatomegaly,

Again, because of their extended structures and the

splenomegaly, disturbances of growth, coarse facies,

huge macromolecular aggregates they often form, they

occupy a large volume of the matrix relative to proteins.

and mental retardation, respectively.

A. SOME FUNCTIONS OF SPECIFIC

GAGS & PROTEOGLYCANS

Hyaluronic acid is especially high in concentration in

and pseudo-Hurler polydystrophy (ML-III) are de-

embryonic tissues and is thought to play an important

scribed in Chapter 47. The term “mucolipidosis” is re-

role in permitting cell migration during morphogenesis

tained because it is still in relatively widespread clinical

and wound repair. Its ability to attract water into the

usage, but it is not appropriate for these two latter dis-

extracellular matrix and thereby “loosen it up” may be

eases since the mechanism of their causation involves

important in this regard. The high concentrations of

mislocation of certain lysosomal enzymes. Genetic de-

hyaluronic acid and chondroitin sulfates present in car-

fects of the catabolism of the oligosaccharide chains of

tilage contribute to its compressibility (see below).

glycoproteins (eg, mannosidosis, fucosidosis) are also

Chondroitin sulfates are located at sites of calcifica-

described in Chapter 47. Most of these defects are char-

tion in endochondral bone and are also found in carti-

acterized by increased excretion of various fragments of

lage. They are also located inside certain neurons and

glycoproteins in the urine, which accumulate because

may provide an endoskeletal structure, helping to

of the metabolic block, as in the case of the mucolipi-

maintain their shape.

doses.

Both keratan sulfate I and dermatan sulfate are

Hyaluronidase is one important enzyme involved

present in the cornea. They lie between collagen fibrils

in the catabolism of both hyaluronic acid and chondro-

and play a critical role in corneal transparency. Changes

itin sulfate. It is a widely distributed endoglycosidase

in proteoglycan composition found in corneal scars dis-

that cleaves hexosaminidic linkages. From hyaluronic

appear when the cornea heals. The presence of der-

acid, the enzyme will generate a tetrasaccharide with

matan sulfate in the sclera may also play a role in main-

the structure

(GlcUA-β-1,3-GlcNAc-β-1,4)₂, which

taining the overall shape of the eye. Keratan sulfate I is

can be degraded further by a β-glucuronidase and β-N-

also present in cartilage.

acetylhexosaminidase. Surprisingly, only one case of an

Heparin is an important anticoagulant. It binds

apparent genetic deficiency of this enzyme appears to

with factors IX and XI, but its most important interac-

have been reported.

tion is with plasma antithrombin III (discussed in

Chapter 51). Heparin can also bind specifically to

Proteoglycans Have Numerous Functions

lipoprotein lipase present in capillary walls, causing a

As indicated above, proteoglycans are remarkably com-

release of this enzyme into the circulation.

plex molecules and are found in every tissue of the

Certain proteoglycans (eg, heparan sulfate) are as-

body, mainly in the ECM or “ground substance.”

sociated with the plasma membrane of cells, with their

There they are associated with each other and also with

core proteins actually spanning that membrane. In it

the other major structural components of the matrix,

they may act as receptors and may also participate in

collagen and elastin, in quite specific manners. Some

the mediation of cell growth and cell-cell communica-

proteoglycans bind to collagen and others to elastin.

tion. The attachment of cells to their substratum in cul-

These interactions are important in determining the

ture is mediated at least in part by heparan sulfate. This

structural organization of the matrix. Some proteogly-

proteoglycan is also found in the basement membrane

cans (eg, decorin) can also bind growth factors such as

of the kidney along with type IV collagen and laminin

548

CHAPTER 48

(see above), where it plays a major role in determining

In various types of arthritis, proteoglycans may act

the charge selectiveness of glomerular filtration.

as autoantigens, thus contributing to the pathologic

Proteoglycans are also found in intracellular loca-

features of these conditions. The amount of chon-

tions such as the nucleus; their function in this or-

droitin sulfate in cartilage diminishes with age, whereas

ganelle has not been elucidated. They are present in

the amounts of keratan sulfate and hyaluronic acid in-

some storage or secretory granules, such as the chromaf-

crease. These changes may contribute to the develop-

fin granules of the adrenal medulla. It has been postu-

ment of osteoarthritis. Changes in the amounts of cer-

lated that they play a role in release of the contents of

such granules. The various functions of GAGs are sum-

marized in Table 48-8.

B. ASSOCIATIONS WITH MAJOR DISEASES

Table 48-9. The principal proteins found

& WITH AGING

in bone.¹

Hyaluronic acid may be important in permitting

tumor cells to migrate through the ECM. Tumor cells

Proteins

Comments

can induce fibroblasts to synthesize greatly increased

Collagens

amounts of this GAG, thereby perhaps facilitating their

Collagen type I

Approximately 90% of total bone

own spread. Some tumor cells have less heparan sulfate

protein. Composed of two α1(I)

at their surfaces, and this may play a role in the lack of

and one α2(I) chains.

adhesiveness that these cells display.

Collagen type V

Minor component.

The intima of the arterial wall contains hyaluronic

acid and chondroitin sulfate, dermatan sulfate, and he-

Noncollagen proteins

paran sulfate proteoglycans. Of these proteoglycans,

Plasma proteins

Mixture of various plasma proteins.

dermatan sulfate binds plasma low-density lipopro-

Proteoglycans²

teins. In addition, dermatan sulfate appears to be the

CS-PG I (biglycan)

Contains two GAG chains; found in

major GAG synthesized by arterial smooth muscle

other tissues.

cells. Because it is these cells that proliferate in athero-

CS-PG II (decorin)

Contains one GAG chain; found in

sclerotic lesions in arteries, dermatan sulfate may play

other tissues.

an important role in development of the atheroscle-

rotic plaque.

CS-PG III

Bone-specific.

Bone SPARC³ protein

Not bone-specific.

(osteonectin)

Table 48-8. Some functions of

Osteocalcin (bone Gla

Contains γ-carboxyglutamate

glycosaminoglycans and proteoglycans.¹

protein)

residues that bind to hydroxyap-

atite. Bone-specific.

• Act as structural components of the ECM

Osteopontin

Not bone-specific. Glycosylated

• Have specific interactions with collagen, elastin, fibronectin,

and phosphorylated.

laminin, and other proteins such as growth factors

• As polyanions, bind polycations and cations

Bone sialoprotein

Bone-specific. Heavily glycosylated,

• Contribute to the characteristic turgor of various tissues

and sulfated on tyrosine.

• Act as sieves in the ECM

Bone morphogenetic

A family (eight or more) of secreted

• Facilitate cell migration (HA)

proteins (BMPs)

proteins with a variety of actions

• Have role in compressibility of cartilage in weight-bearing

on bone; many induce ectopic

(HA, CS)

bone growth.

• Play role in corneal transparency (KS I and DS)

• Have structural role in sclera (DS)

¹Various functions have been ascribed to the noncollagen

• Act as anticoagulant (heparin)

proteins, including roles in mineralization; however, most of

them are still speculative. It is considered unlikely that the

• Are components of plasma membranes, where they may

noncollagen proteins that are not bone-specific play a key

act as receptors and participate in cell adhesion and cell-cell

role in mineralization. A number of other proteins are also

interactions (eg, HS)

present in bone, including a tyrosine-rich acidic matrix pro-

• Determine charge-selectiveness of renal glomerulus (HS)

tein (TRAMP), some growth factors (eg, TGFβ), and enzymes

• Are components of synaptic and other vesicles (eg, HS)

involved in collagen synthesis (eg, lysyl oxidase).

¹ECM, extracellular matrix; HA, hyaluronic acid; CS, chondroitin

²CS-PG, chondroitin sulfate-proteoglycan; these are similar to

sulfate; KS I, keratan sulfate I; DS, dermatan sulfate; HS, heparan

the dermatan sulfate PGs (DS-PGs) of cartilage (Table 48-11).

sulfate.

³SPARC, secreted protein acidic and rich in cysteine.

THE EXTRACELLULAR MATRIX

549

Osteoclast

Mesenchyme

Newly formed matrix (osteoid)

Osteoblast

Osteocyte

Bone matrix

Figure 48-11. Schematic illustration of the major cells present in membranous

bone. Osteoblasts (lighter color) are synthesizing type I collagen, which forms a matrix

that traps cells. As this occurs, osteoblasts gradually differentiate to become osteo-

cytes. (Reproduced, with permission, from Junqueira LC, Carneiro J: Basic Histology: Text &

Atlas, 10th ed. McGraw-Hill, 2003.)

tain GAGs in the skin are also observed with aging and

bone (Chapter 45). Hydroxyapatite confers on bone

help to account for the characteristic changes noted in

the strength and resilience required by its physiologic

this organ in the elderly.

roles.

An exciting new phase in proteoglycan research is

Bone is a dynamic structure that undergoes continu-

opening up with the findings that mutations that affect

ing cycles of remodeling, consisting of resorption fol-

individual proteoglycans or the enzymes needed for

lowed by deposition of new bone tissue. This remodel-

their synthesis alter the regulation of specific signaling

ing permits bone to adapt to both physical

(eg,

pathways in drosophila and Caenorhabditis elegans, thus

increases in weight-bearing) and hormonal signals.

affecting development; it already seems likely that simi-

The major cell types involved in bone resorption

lar effects exist in mice and humans.

and deposition are osteoclasts and osteoblasts (Figure

48-11). The former are associated with resorption and

the latter with deposition of bone. Osteocytes are de-

scended from osteoblasts; they also appear to be in-

BONE IS A MINERALIZED

volved in maintenance of bone matrix but will not be

CONNECTIVE TISSUE

discussed further here.

Bone contains both organic and inorganic material.

Osteoclasts are multinucleated cells derived from

The organic matter is mainly protein. The principal

pluripotent hematopoietic stem cells. Osteoclasts pos-

proteins of bone are listed in Table 48-9; type I colla-

sess an apical membrane domain, exhibiting a ruffled

gen is the major protein, comprising 90-95% of the

border that plays a key role in bone resorption (Figure

organic material. Type V collagen is also present in

48-12). A proton-translocating ATPase expels protons

small amounts, as are a number of noncollagen pro-

across the ruffled border into the resorption area, which

teins, some of which are relatively specific to bone.

is the microenvironment of low pH shown in the fig-

The inorganic or mineral component is mainly crys-

ure. This lowers the local pH to 4.0 or less, thus in-

talline

hydroxyapatite—Ca₁₀(PO₄)₆(OH)₂—along

creasing the solubility of hydroxyapatite and allowing

with sodium, magnesium, carbonate, and fluoride; ap-

demineralization to occur. Lysosomal acid proteases are

proximately 99% of the body’s calcium is contained in

released that digest the now accessible matrix proteins.

550

CHAPTER 48

Blood capillary

Nucleus

Osteoclast

Golgi

Nucleus

Lysosomes

CO₂ + H₂O

H⁺

+ HCO₃

Section of

circumferential

clear zone

Ruffled

border

Microenvironment of low pH

Bone matrix

and lysosomal enzymes

Figure 48-12. Schematic illustration of some aspects of the role of the osteoclast in

bone resorption. Lysosomal enzymes and hydrogen ions are released into the confined

microenvironment created by the attachment between bone matrix and the peripheral

clear zone of the osteoclast. The acidification of this confined space facilitates the dis-

solution of calcium phosphate from bone and is the optimal pH for the activity of lyso-

somal hydrolases. Bone matrix is thus removed, and the products of bone resorption

are taken up into the cytoplasm of the osteoclast, probably digested further, and trans-

ferred into capillaries. The chemical equation shown in the figure refers to the action of

carbonic anhydrase II, described in the text. (Reproduced, with permission, from Jun-

queira LC, Carneiro J: Basic Histology: Text & Atlas, 10th ed. McGraw-Hill, 2003.)

Osteoblasts—mononuclear cells derived from pluripo-

Recent interest has focused on acidic phosphoproteins,

tent mesenchymal precursors—synthesize most of the

such as bone sialoprotein, acting as sites of nucleation.

proteins found in bone (Table 48-9) as well as various

These proteins contain motifs (eg, poly-Asp and poly-

growth factors and cytokines. They are responsible for

Glu stretches) that bind calcium and may provide an

the deposition of new bone matrix (osteoid) and its

initial scaffold for mineralization. Some macromole-

subsequent mineralization. Osteoblasts control miner-

cules, such as certain proteoglycans and glycoproteins,

alization by regulating the passage of calcium and phos-

can also act as inhibitors of nucleation.

phate ions across their surface membranes. The latter

It is estimated that approximately 4% of compact

contain alkaline phosphatase, which is used to generate

bone is renewed annually in the typical healthy adult,

phosphate ions from organic phosphates. The mecha-

whereas approximately 20% of trabecular bone is re-

nisms involved in mineralization are not fully under-

placed.

stood, but several factors have been implicated. Alkaline

Many factors are involved in the regulation of bone

phosphatase contributes to mineralization but in itself

metabolism, only a few of which will be mentioned

is not sufficient. Small vesicles (matrix vesicles) contain-

here. Some stimulate osteoblasts (eg, parathyroid hor-

ing calcium and phosphate have been described at sites

mone and 1,25-dihydroxycholecalciferol) and others

of mineralization, but their role is not clear. Type I col-

inhibit them (eg, corticosteroids). Parathyroid hormone

lagen appears to be necessary, with mineralization being

and 1,25-dihydroxycholecalciferol also stimulate osteo-

first evident in the gaps between successive molecules.

clasts, whereas calcitonin and estrogens inhibit them.

THE EXTRACELLULAR MATRIX

551

Table 48-10. Some metabolic and genetic

MANY METABOLIC & GENETIC

diseases affecting bone and cartilage.

DISORDERS INVOLVE BONE

A number of the more important examples of meta-

Disease

Comments

bolic and genetic disorders that affect bone are listed in

Dwarfism

Often due to a deficiency of growth

Table 48-10.

hormone, but has many other causes.

Osteogenesis imperfecta (brittle bones) is charac-

terized by abnormal fragility of bones. The scleras are

Rickets

Due to a deficiency of vitamin D

often abnormally thin and translucent and may appear

during childhood.

blue owing to a deficiency of connective tissue. Four

Osteomalacia

Due to a deficiency of vitamin D

types of this condition (mild, extensive, severe, and

during adulthood.

variable) have been recognized, of which the extensive

Hyperparathyroidism

Excess parathormone causes bone

type occurring in the newborn is the most ominous.

resorption.

Affected infants may be born with multiple fractures

and not survive. Over 90% of patients with osteogene-

Osteogenesis

Due to a variety of mutations in the

sis imperfecta have mutations in the COL1A1

and

imperfecta (eg,

COL1A1 and COL1A2 genes affecting

COL1A2

genes, encoding proα1(I) and proα2(I)

MIM 166200)

the synthesis and structure of type I

chains, respectively. Over 100 mutations in these two

collagen.

genes have been documented and include partial gene

Osteoporosis

Commonly postmenopausal or in

deletions and duplications. Other mutations affect

other cases is more gradual and re-

RNA splicing, and the most frequent type results in the

lated to age; a small number of cases

replacement of glycine by another bulkier amino acid,

are due to mutations in the COL1A1

affecting formation of the triple helix. In general, these

and COL1A2 genes and possibly in the

mutations result in decreased expression of collagen or

vitamin D receptor gene (MIM 166710)

Osteoarthritis

A small number of cases are due to

mutations in the COL1A genes.

Table 48-11. The principal proteins found

Several chondro-

Due to mutations in COL2A1 genes.

in cartilage.

dysplasias

Pfeiffer syndrome¹

Mutations in the gene encoding fi-

Proteins

Comments

(MIM 100600)

broblast growth receptor 1 (FGFR1).

Collagen proteins

Jackson-Weiss

Mutations in the gene encoding

Collagen type II

90-98% of total articular cartilage

(MIM 123150)

FGFR2.

collagen. Composed of three

and Crouzon

α1(II) chains.

(MIM 123500)

Collagens V, VI, IX,

Type IX cross-links to type II colla-

syndromes¹

X, XI

gen. Type XI may help control di-

Achondroplasia

Mutations in the gene encoding

ameter of type II fibrils.

(MIM 100800)

FGFR3.

Noncollagen proteins

and thanatophoric

Proteoglycans

dysplasia²

Aggrecan

The major proteoglycan of cartilage.

(MIM 187600)

Large non-

Found in some types of cartilage.

¹The Pfeiffer, Jackson-Weiss, and Crouzon syndromes are cran-

aggregating

iosynostosis syndromes; craniosynostosis is a term signifying pre-

proteoglycan

mature fusion of sutures in the skull.

DS-PG I (biglycan)¹

Similar to CS-PG I of bone.

²Thanatophoric (Gk thanatos “death” + phoros “bearing”) dyspla-

DS-PG II (decorin)

Similar to CS-PG II of bone.

sia is the most common neonatal lethal skeletal dysplasia, dis-

Chondronectin

May play role in binding type II colla-

playing features similar to those of homozygous achondroplasia.

gen to surface of cartilage.

Anchorin C II

May bind type II collagen to surface

of chondrocyte.

¹The core proteins of DS-PG I and DS-PG II are homologous to

those of CS-PG I and CS-PG II found in bone (Table 48-9). A possi-

ble explanation is that osteoblasts lack the epimerase required to

convert glucuronic acid to iduronic acid, the latter of which is

found in dermatan sulfate.

552

CHAPTER 48

in structurally abnormal proα chains that assemble into

ions are pumped across their ruffled borders

(see

abnormal fibrils, weakening the overall structure of

above). Thus, if CA II is deficient in activity in osteo-

bone. When one abnormal chain is present, it may in-

clasts, normal bone resorption does not occur, and os-

teract with two normal chains, but folding may be pre-

teopetrosis results. The mechanism of the cerebral calci-

vented, resulting in enzymatic degradation of all of the

fication is not clear, whereas the renal tubular acidosis

chains. This is called “procollagen suicide” and is an ex-

reflects deficient activity of CA II in the renal tubules.

ample of a dominant negative mutation, a result often

Osteoporosis is a generalized progressive reduction

seen when a protein consists of multiple different sub-

in bone tissue mass per unit volume causing skeletal

units.

weakness. The ratio of mineral to organic elements is

Osteopetrosis (marble bone disease), characterized

unchanged in the remaining normal bone. Fractures of

by increased bone density, is due to inability to resorb

various bones, such as the head of the femur, occur very

bone. One form occurs along with renal tubular acido-

easily and represent a huge burden to both the affected

sis and cerebral calcification. It is due to mutations in

patients and to the health care budget of society.

the gene (located on chromosome 8q22) encoding car-

Among other factors, estrogens and interleukins-1 and

bonic anhydrase II (CA II), one of four isozymes of car-

-6 appear to be intimately involved in the causation of

bonic anhydrase present in human tissues. The reaction

osteoporosis.

catalyzed by carbonic anhydrase is shown below:

THE MAJOR COMPONENTS OF

67 nm

CARTILAGE ARE TYPE II COLLAGEN

Fibril

& CERTAIN PROTEOGLYCANS

The principal proteins of hyaline cartilage (the major

Reaction II is spontaneous. In osteoclasts involved in

type of cartilage) are listed in Table 48-11. Type II colla-

bone resorption, CA II apparently provides protons to

gen is the principal protein (Figure 48-13), and a num-

neutralize the OH⁻ ions left inside the cell when H⁺

ber of other minor types of collagen are also present. In

Hyaluronic acid

Type II collagen fibril

Hyaluronic acid

Link protein

Chondroitin sulfate

Proteoglycan

Core protein

Collagen (type II)

Figure 48-13. Schematic representation of the molecular organization in cartilage

matrix. Link proteins noncovalently bind the core protein (lighter color) of proteogly-

cans to the linear hyaluronic acid molecules (darker color). The chondroitin sulfate side

chains of the proteoglycan electrostatically bind to the collagen fibrils, forming a

cross-linked matrix. The oval outlines the area enlarged in the lower part of the figure.

(Reproduced, with permission, from Junqueira LC, Carneiro J: Basic Histology: Text & Atlas,

10th ed. McGraw-Hill, 2003.)

THE EXTRACELLULAR MATRIX

553

addition to these components, elastic cartilage contains

degrade collagen and the other proteins found in carti-

elastin and fibroelastic cartilage contains type I collagen.

lage. Interleukin-1 (IL-1) and tumor necrosis factor α

Cartilage contains a number of proteoglycans, which

(TNFα) appear to stimulate the production of such

play an important role in its compressibility. Aggrecan

proteases, whereas transforming growth factor β

(about 2 × 10³ kDa) is the major proteoglycan. As shown

(TGFβ) and insulin-like growth factor 1 (IGF-I) gener-

in Figure 48-14, it has a very complex structure, con-

ally exert an anabolic influence on cartilage.

taining several GAGs (hyaluronic acid, chondroitin sul-

fate, and keratan sulfate) and both link and core proteins.

THE MOLECULAR BASES OF THE

The core protein contains three domains: A, B, and C.

The hyaluronic acid binds noncovalently to domain A of

CHONDRODYSPLASIAS INCLUDE

the core protein as well as to the link protein, which sta-

MUTATIONS IN GENES ENCODING

bilizes the hyaluronate-core protein interactions. The

TYPE II COLLAGEN & FIBROBLAST

keratan sulfate chains are located in domain B, whereas

GROWTH FACTOR RECEPTORS

the chondroitin sulfate chains are located in domain C;

both of these types of GAGs are bound covalently to the

Chondrodysplasias are a mixed group of hereditary dis-

core protein. The core protein also contains both O- and

orders affecting cartilage. They are manifested by short-

N-linked oligosaccharide chains.

limbed dwarfism and numerous skeletal deformities. A

The other proteoglycans found in cartilage have

number of them are due to a variety of mutations in the

simpler structures than aggrecan.

COL2A1 gene, leading to abnormal forms of type II

Chondronectin is involved in the attachment of

collagen. One example is Stickler syndrome, mani-

type II collagen to chondrocytes.

fested by degeneration of joint cartilage and of the vit-

Cartilage is an avascular tissue and obtains most of

reous body of the eye.

its nutrients from synovial fluid. It exhibits slow but

The best-known of the chondrodysplasias is achon-

continuous turnover. Various proteases (eg, collage-

droplasia, the commonest cause of short-limbed

nases and stromalysin) synthesized by chondrocytes can

dwarfism. Affected individuals have short limbs, nor-

Domain A

Domain B

Domain C

Hyaluronate-

binding

region

Core

N-linked

protein

oligosaccharide

Link

protein

Keratan

Chondroitin

O-linked

Hyaluronic acid

sulfate

oligosaccharide

Figure 48-14. Schematic diagram of the aggrecan from bovine nasal cartilage. A

strand of hyaluronic acid is shown on the left. The core protein (about 210 kDa) has

three major domains. Domain A, at its amino terminal end, interacts with approxi-

mately five repeating disaccharides in hyaluronate. The link protein interacts with

both hyaluronate and domain A, stabilizing their interactions. Approximately 30 ker-

atan sulfate chains are attached, via GalNAc-Ser linkages, to domain B. Domain C

contains about 100 chondroitin sulfate chains attached via Gal-Gal-Xyl-Ser linkages

and about 40 O-linked oligosaccharide chains. One or more N-linked glycan chains

are also found near the carboxyl terminal of the core protein. (Reproduced, with per-

mission, from Moran LA et al: Biochemistry, 2nd ed. Neil Patterson Publishers, 1994.)

554

CHAPTER 48

mal trunk size, macrocephaly, and a variety of other

SUMMARY

skeletal abnormalities. The condition is often inherited

•

The major components of the ECM are the struc-

as an autosomal dominant trait, but many cases are due

tural proteins collagen, elastin, and fibrillin; a num-

to new mutations. The molecular basis of achondropla-

ber of specialized proteins

(eg, fibronectin and

sia is outlined in Figure 48-15. Achondroplasia is not a

laminin); and various proteoglycans.

collagen disorder but is due to mutations in the gene

encoding fibroblast growth factor receptor

•

Collagen is the most abundant protein in the animal

(FGFR3). Fibroblast growth factors are a family of at

kingdom; approximately 19 types have been isolated.

least nine proteins that affect the growth and differenti-

All collagens contain greater or lesser stretches of

ation of cells of mesenchymal and neuroectodermal ori-

triple helix and the repeating structure (Gly-X-Y)_n.

gin. Their receptors are transmembrane proteins and

•

The biosynthesis of collagen is complex, featuring

form a subgroup of the family of receptor tyrosine ki-

many posttranslational events, including hydroxyla-

nases. FGFR3 is one member of this subgroup and me-

tion of proline and lysine.

diates the actions of FGF3 on cartilage. In almost all

•

Diseases associated with impaired synthesis of colla-

cases of achondroplasia that have been investigated, the

gen include scurvy, osteogenesis imperfecta, Ehlers-

mutations were found to involve nucleotide 1138 and

Danlos syndrome (many types), and Menkes disease.

resulted in substitution of arginine for glycine (residue

•

Elastin confers extensibility and elastic recoil on tis-

number 380) in the transmembrane domain of the pro-

sues. Elastin lacks hydroxylysine, Gly-X-Y sequences,

tein, rendering it inactive. No such mutation was found

triple helical structure, and sugars but contains

in unaffected individuals. As indicated in Table 48-10,

desmosine and isodesmosine cross-links not found in

other skeletal dysplasias (including certain craniosynos-

collagen.

tosis syndromes) are also due to mutations in genes en-

•

Fibrillin is located in microfibrils. Mutations in the

coding FGF receptors. Another type of skeletal dyspla-

gene for fibrillin cause Marfan syndrome.

sia (diastrophic dysplasia) has been found to be due to

•

The glycosaminoglycans (GAGs) are made up of re-

mutation in a sulfate transporter. Thus, thanks to re-

combinant DNA technology, a new era in understand-

peating disaccharides containing a uronic acid (glu-

curonic or iduronic) or hexose (galactose) and a hex-

ing of skeletal dysplasias has begun.

osamine (galactosamine or glucosamine). Sulfate is

also frequently present.

•

The major GAGs are hyaluronic acid, chondroitin

4- and 6-sulfates, keratan sulfates I and II, heparin,

Mutations of nucleotide 1138 in the gene

heparan sulfate, and dermatan sulfate.

encoding FGFR3 on chromosome 4

•

The GAGs are synthesized by the sequential actions

of a battery of specific enzymes (glycosyltransferases,

Replacement in FGFR3 of Gly (codon 380) by Arg

epimerases, sulfotransferases, etc) and are degraded

by the sequential action of lysosomal hydrolases. Ge-

Defective function of FGFR3

netic deficiencies of the latter result in mucopolysac-

charidoses (eg, Hurler syndrome).

Abnormal development and growth of cartilage

•

GAGs occur in tissues bound to various proteins

leading to short-limbed dwarfism and other features

(linker proteins and core proteins), constituting pro-

teoglycans. These structures are often of very high

Figure 48-15. Simplified scheme of the causation of

molecular weight and serve many functions in tis-

achondroplasia (MIM 100800). In most cases studied so

sues.

far, the mutation has been a G to A transition at nu-

•

Many components of the ECM bind to proteins of

cleotide 1138. In a few cases, the mutation was a G to C

the cell surface named integrins; this constitutes one

transversion at the same nucleotide. This particular nu-

pathway by which the exteriors of cells can commu-

cleotide is a real “hot spot” for mutation. Both muta-

nicate with their interiors.

tions result in replacement of a Gly residue by an Arg

•

Bone and cartilage are specialized forms of the ECM.

residue in the transmembrane segment of the receptor.

Collagen I and hydroxyapatite are the major con-

A few cases involving replacement of Gly by Cys at

stituents of bone. Collagen II and certain proteogly-

codon 375 have also been reported.

cans are major constituents of cartilage.

THE EXTRACELLULAR MATRIX

555

• The molecular causes of a number of heritable dis-

Prockop DJ, Kivirikko KI: Collagens: molecular biology, diseases,

and potential therapy. Annu Rev Biochem 1995;64:403.

eases of bone (eg, osteogenesis imperfecta) and of car-

Pyeritz RE: Ehlers-Danlos syndrome. N Engl J Med 2000;342:730.

tilage (eg, the chondrodystrophies) are being revealed

by the application of recombinant DNA technology.

Sage E: Regulation of interactions between cells and extracellular

matrix: a command performance on several stages. J Clin In-

vest 2001;107:781. (This article introduces a series of six arti-

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Muscle & the Cytoskeleton

Robert K. Murray, MD, PhD

BIOMEDICAL IMPORTANCE

shall discuss aspects of the three types of muscle found

in vertebrates: skeletal, cardiac, and smooth. Both

Proteins play an important role in movement at both

skeletal and cardiac muscle appear striated upon micro-

the organ (eg, skeletal muscle, heart, and gut) and cellu-

scopic observation; smooth muscle is nonstriated. Al-

lar levels. In this chapter, the roles of specific proteins

though skeletal muscle is under voluntary nervous con-

and certain other key molecules (eg, Ca2+) in muscular

trol, the control of both cardiac and smooth muscle is

contraction are described. A brief coverage of cyto-

involuntary.

skeletal proteins is also presented.

Knowledge of the molecular bases of a number of

The Sarcoplasm of Muscle Cells

conditions that affect muscle has advanced greatly in re-

cent years. Understanding of the molecular basis of

Contains ATP, Phosphocreatine,

Duchenne-type muscular dystrophy was greatly en-

& Glycolytic Enzymes

hanced when it was found that it was due to mutations

Striated muscle is composed of multinucleated muscle

in the gene encoding dystrophin. Significant progress

fiber cells surrounded by an electrically excitable plasma

has also been made in understanding the molecular

membrane, the sarcolemma. An individual muscle

basis of malignant hyperthermia, a serious complica-

fiber cell, which may extend the entire length of the

tion for some patients undergoing certain types of anes-

muscle, contains a bundle of many myofibrils arranged

thesia. Heart failure is a very common medical condi-

in parallel, embedded in intracellular fluid termed sar-

tion, with a variety of causes; its rational therapy

coplasm. Within this fluid is contained glycogen, the

requires understanding of the biochemistry of heart

high-energy compounds ATP and phosphocreatine,

muscle. One group of conditions that cause heart fail-

and the enzymes of glycolysis.

ure are the cardiomyopathies, some of which are ge-

netically determined. Nitric oxide

(NO) has been

The Sarcomere Is the Functional

found to be a major regulator of smooth muscle tone.

Many widely used vasodilators—such as nitroglycerin,

Unit of Muscle

used in the treatment of angina pectoris—act by in-

An overall view of voluntary muscle at several levels of

creasing the formation of NO. Muscle, partly because

organization is presented in Figure 49-1.

of its mass, plays major roles in the overall metabolism

When the myofibril is examined by electron mi-

of the body.

croscopy, alternating dark and light bands (anisotropic

bands, meaning birefringent in polarized light; and

isotropic bands, meaning not altered by polarized light)

MUSCLE TRANSDUCES CHEMICAL

can be observed. These bands are thus referred to as A

and I bands, respectively. The central region of the A

ENERGY INTO MECHANICAL ENERGY

band (the H band) appears less dense than the rest of

Muscle is the major biochemical transducer (machine)

the band. The I band is bisected by a very dense and

that converts potential (chemical) energy into kinetic

narrow Z line (Figure 49-2).

(mechanical) energy. Muscle, the largest single tissue in

The sarcomere is defined as the region between two

the human body, makes up somewhat less than 25% of

Z lines (Figures 49-1 and 49-2) and is repeated along

body mass at birth, more than 40% in the young adult,

the axis of a fibril at distances of 1500-2300 nm de-

and somewhat less than 30% in the aged adult. We

pending upon the state of contraction.

556

MUSCLE & THE CYTOSKELETON

557

Muscle

Muscle fasciculus

20-100 µm

Muscle fiber

band line

band band

1-2 µm

Myofibril

Z - Sarcomere - Z

Figure 49-1. The structure of voluntary muscle. The sarcomere is the region between the

Z lines. (Drawing by Sylvia Colard Keene. Reproduced, with permission, from Bloom W, Fawcett

DW: A Textbook of Histology, 10th ed. Saunders, 1975.)

The striated appearance of voluntary and cardiac

section), and each thick filament is surrounded sym-

muscle in light microscopic studies results from their

metrically by six thin filaments.

high degree of organization, in which most muscle fiber

The thick and thin filaments interact via cross-

cells are aligned so that their sarcomeres are in parallel

bridges that emerge at intervals of 14 nm along the

thick filaments. As depicted in Figure 49-2, the cross-

bridges (drawn as arrowheads at each end of the myosin

filaments, but not shown extending fully across to the

Thick Filaments Contain Myosin;

thin filaments) have opposite polarities at the two ends

Thin Filaments Contain Actin,

of the thick filaments. The two poles of the thick fila-

Tropomyosin, & Troponin

ments are separated by a 150-nm segment (the M band,

When myofibrils are examined by electron microscopy,

not labeled in the figure) that is free of projections.

it appears that each one is constructed of two types of

longitudinal filaments. One type, the thick filament,

confined to the A band, contains chiefly the protein

The Sliding Filament Cross-Bridge

myosin. These filaments are about 16 nm in diameter

Model Is the Foundation on Which

and arranged in cross-section as a hexagonal array (Fig-

Current Thinking About Muscle

ure 49-2, center; right-hand cross-section).

Contraction Is Built

The thin filament (about 7 nm in diameter) lies in

the I band and extends into the A band but not into its

This model was proposed independently in the 1950s

H zone (Figure 49-2). Thin filaments contain the pro-

by Henry Huxley and Andrew Huxley and their col-

teins actin, tropomyosin, and troponin (Figure 49-3).

leagues. It was largely based on careful morphologic ob-

In the A band, the thin filaments are arranged around

servations on resting, extended, and contracting mus-

the thick (myosin) filament as a secondary hexagonal

cle. Basically, when muscle contracts, there is no change

array. Each thin filament lies symmetrically between

in the lengths of the thick and thin filaments, but the

three thick filaments (Figure 49-2, center; mid cross-

H zones and the I bands shorten (see legend to Fig-

558

CHAPTER 49

H band

A. Extended

I band

A band

Z line

2300 nm

α-Actinin

Actin filaments

6-nm diameter

Myosin filaments

16-nm diameter

Cross section:

B. Contracted

Thin

6-nm diameter

filament

Thick

16-nm diameter

filament

1500 nm

Figure 49-2. Arrangement of filaments in striated muscle. A: Extended. The positions of the

I, A, and H bands in the extended state are shown. The thin filaments partly overlap the ends of the

thick filaments, and the thin filaments are shown anchored in the Z lines (often called Z disks). In

the lower part of Figure 49-2A, “arrowheads,” pointing in opposite directions, are shown emanat-

ing from the myosin (thick) filaments. Four actin (thin) filaments are shown attached to two Z lines

via α-actinin. The central region of the three myosin filaments, free of arrowheads, is called the

M band (not labeled). Cross-sections through the M bands, through an area where myosin and

actin filaments overlap and through an area in which solely actin filaments are present, are shown.

B: Contracted. The actin filaments are seen to have slipped along the sides of the myosin fibers to-

ward each other. The lengths of the thick filaments (indicated by the A bands) and the thin fila-

ments (distance between Z lines and the adjacent edges of the H bands) have not changed. How-

ever, the lengths of the sarcomeres have been reduced (from 2300 nm to 1500 nm), and the

lengths of the H and I bands are also reduced because of the overlap between the thick and thin

filaments. These morphologic observations provided part of the basis for the sliding filament

model of muscle contraction.

MUSCLE & THE CYTOSKELETON

559

G-actin

F-actin

6-7 nm

Tropomyosin

Troponin

TpC

38.5 nm

TpI

TpT

35.5 nm

The assembled thin filament

Figure 49-3. Schematic representation of the thin filament, showing the spatial configuration of its three

major protein components: actin, myosin, and tropomyosin. The upper panel shows individual molecules of

G-actin. The middle panel shows actin monomers assembled into F-actin. Individual molecules of tropomyosin

(two strands wound around one another) and of troponin (made up of its three subunits) are also shown. The

lower panel shows the assembled thin filament, consisting of F-actin, tropomyosin, and the three subunits of

troponin (TpC, TpI, and TpT).

ure 49-2). Thus, the arrays of interdigitating filaments

ACTIN & MYOSIN ARE THE MAJOR

must slide past one another during contraction. Cross-

PROTEINS OF MUSCLE

bridges that link thick and thin filaments at certain

stages in the contraction cycle generate and sustain the

The mass of a muscle is made up of 75% water and

tension. The tension developed during muscle contrac-

more than 20% protein. The two major proteins are

tion is proportionate to the filament overlap and to the

actin and myosin.

number of cross-bridges. Each cross-bridge head is con-

Monomeric G-actin (43 kDa; G, globular) makes

nected to the thick filament via a flexible fibrous seg-

up 25% of muscle protein by weight. At physiologic

ment that can bend outward from the thick filament.

ionic strength and in the presence of Mg2+, G-actin

This flexible segment facilitates contact of the head

polymerizes noncovalently to form an insoluble double

with the thin filament when necessary but is also suffi-

helical filament called F-actin

(Figure

49-3). The

ciently pliant to be accommodated in the interfilament

F-actin fiber is 6-7 nm thick and has a pitch or repeat-

spacing.

ing structure every 35.5 nm.

560

CHAPTER 49

Myosins constitute a family of proteins, with at

light chain. Skeletal muscle myosin binds actin to form

least 15 members having been identified. The myosin

actomyosin (actin-myosin), and its intrinsic ATPase ac-

discussed in this chapter is myosin-II, and when myosin

tivity is markedly enhanced in this complex. Isoforms

is referred to in this text, it is this species that is meant

of myosin exist whose amounts can vary in different

unless otherwise indicated. Myosin-I is a monomeric

anatomic, physiologic, and pathologic situations.

species that binds to cell membranes. It may serve as a

The structures of actin and of the head of myosin

linkage between microfilaments and the cell membrane

have been determined by x-ray crystallography; these

in certain locations.

studies have confirmed a number of earlier findings

Myosin contributes

55% of muscle protein by

concerning their structures and have also given rise to

weight and forms the thick filaments. It is an asymmet-

much new information.

ric hexamer with a molecular mass of approximately

460 kDa. Myosin has a fibrous tail consisting of two in-

Limited Digestion of Myosin With

tertwined helices. Each helix has a globular head por-

Proteases Has Helped to Elucidate

tion attached at one end (Figure 49-4). The hexamer

Its Structure & Function

consists of one pair of heavy (H) chains each of ap-

proximately 200 kDA molecular mass, and two pairs of

When myosin is digested with trypsin, two myosin

light (L) chains each with a molecular mass of approxi-

fragments (meromyosins) are generated. Light mero-

mately 20 kDa. The L chains differ, one being called

myosin (LMM) consists of aggregated, insoluble α-he-

the essential light chain and the other the regulatory

lical fibers from the tail of myosin (Figure 49-4). LMM

HMM S-1

9 nm

PAPAIN

HMM

TRYPSIN

HMM S-2

134 nm

LMM

85 nm

Figure 49-4. Diagram of a myosin molecule showing the two intertwined α-helices (fibrous portion), the

globular region or head (G), the light chains (L), and the effects of proteolytic cleavage by trypsin and papain.

The globular region (myosin head) contains an actin-binding site and an L chain-binding site and also attaches

to the remainder of the myosin molecule.

MUSCLE & THE CYTOSKELETON

561

exhibits no ATPase activity and does not bind to

in the power stroke, which drives movement of actin

F-actin.

filaments past myosin filaments. The energy for the

Heavy meromyosin (HMM; molecular mass about

power stroke is ultimately supplied by ATP, which is

340 kDa) is a soluble protein that has both a fibrous

hydrolyzed to ADP and P_i. However, the power stroke

portion and a globular portion (Figure 49-4). It ex-

itself occurs as a result of conformational changes in

hibits ATPase activity and binds to F-actin. Digestion

the myosin head when ADP leaves it.

of HMM with papain generates two subfragments, S-1

The major biochemical events occurring during one

and S-2. The S-2 fragment is fibrous in character, has

cycle of muscle contraction and relaxation can be repre-

no ATPase activity, and does not bind to F-actin.

sented in the five steps shown in Figure 49-6:

S-1 (molecular mass approximately 115 kDa) does

exhibit ATPase activity, binds L chains, and in the ab-

(1) In the relaxation phase of muscle contraction,

sence of ATP will bind to and decorate actin with “ar-

the S-1 head of myosin hydrolyzes ATP to ADP and P_i,

rowheads” (Figure 49-5). Both S-1 and HMM exhibit

but these products remain bound. The resultant ADP-

ATPase activity, which is accelerated 100- to 200-fold by

Pi-myosin complex has been energized and is in a so-

complexing with F-actin. As discussed below, F-actin

called high-energy conformation.

greatly enhances the rate at which myosin ATPase re-

(2) When contraction of muscle is stimulated (via

leases its products, ADP and P_i. Thus, although F-actin

events involving Ca2+, troponin, tropomyosin, and

does not affect the hydrolysis step per se, its ability to

actin, which are described below), actin becomes acces-

promote release of the products produced by the ATPase

sible and the S-1 head of myosin finds it, binds it, and

activity greatly accelerates the overall rate of catalysis.

forms the actin-myosin-ADP-P_i complex indicated.

(3) Formation of this complex promotes the re-

CHANGES IN THE CONFORMATION

lease of P_i, which initiates the power stroke. This is fol-

lowed by release of ADP and is accompanied by a large

OF THE HEAD OF MYOSIN DRIVE

conformational change in the head of myosin in rela-

MUSCLE CONTRACTION

tion to its tail (Figure 49-7), pulling actin about 10 nm

How can hydrolysis of ATP produce macroscopic

toward the center of the sarcomere. This is the power

movement? Muscle contraction essentially consists of

stroke. The myosin is now in a so-called low-energy

the cyclic attachment and detachment of the S-1 head of

state, indicated as actin-myosin.

myosin to the F-actin filaments. This process can also be

(4) Another molecule of ATP binds to the S-1 head,

referred to as the making and breaking of cross-bridges.

forming an actin-myosin-ATP complex.

The attachment of actin to myosin is followed by con-

(5) Myosin-ATP has a low affinity for actin, and

formational changes which are of particular importance

actin is thus released. This last step is a key compo-

in the S-1 head and are dependent upon which nu-

nent of relaxation and is dependent upon the binding

cleotide is present (ADP or ATP). These changes result

of ATP to the actin-myosin complex.

Actin

ATP-Myosin

H₂O

Actin-Myosin

ATP

ADP-P_i-Myosin

ATP

Actin

Actin-Myosin

ADP

Actin-Myosin

ADP-P_i

Figure 49-6. The hydrolysis of ATP drives the cyclic

Figure 49-5. The decoration of actin filaments with

association and dissociation of actin and myosin in five

the S-1 fragments of myosin to form “arrowheads.”

reactions described in the text. (Modified from Stryer L:

(Courtesy of JA Spudich.)

Biochemistry, 2nd ed. Freeman, 1981.)

562

CHAPTER 49

ADP. The hinge regions of myosin (referred to as flexi-

ble points at each end of S-2 in the legend to Figure

49-7) permit the large range of movement of S-1 and

also allow S-1 to find actin filaments.

If intracellular levels of ATP drop (eg, after death),

Thick filament

ATP is not available to bind the S-1 head (step 4

LMM

above), actin does not dissociate, and relaxation (step 5)

S-2

S-1

does not occur. This is the explanation for rigor mor-

tis, the stiffening of the body that occurs after death.

Calculations have indicated that the efficiency of

Thin filament

contraction is about 50%; that of the internal combus-

tion engine is less than 20%.

Tropomyosin & the Troponin Complex

Present in Thin Filaments Perform Key

Figure 49-7. Representation of the active cross-

Functions in Striated Muscle

bridges between thick and thin filaments. This diagram

In striated muscle, there are two other proteins that are

was adapted by AF Huxley from HE Huxley: The

minor in terms of their mass but important in terms of

mechanism of muscular contraction. Science

their function. Tropomyosin is a fibrous molecule that

1969;164:1356. The latter proposed that the force in-

consists of two chains, alpha and beta, that attach to

volved in muscular contraction originates in a tendency

F-actin in the groove between its filaments (Figure 49-3).

for the myosin head (S-1) to rotate relative to the thin

Tropomyosin is present in all muscular and muscle-like

filament and is transmitted to the thick filament by the

structures. The troponin complex is unique to striated

S-2 portion of the myosin molecule acting as an inex-

muscle and consists of three polypeptides. Troponin T

tensible link. Flexible points at each end of S-2 permit

(TpT) binds to tropomyosin as well as to the other two

S-1 to rotate and allow for variations in the separation

troponin components. Troponin I (TpI) inhibits the

between filaments. The present figure is based on HE

F-actin-myosin interaction and also binds to the other

Huxley’s proposal but also incorporates elastic (the coils

components of troponin. Troponin C (TpC) is a cal-

in the S-2 portion) and stepwise-shortening elements

cium-binding polypeptide that is structurally and func-

(depicted here as four sites of interaction between the

tionally analogous to calmodulin, an important cal-

S-1 portion and the thin filament). (See Huxley AF, Sim-

cium-binding protein widely distributed in nature.

mons RM: Proposed mechanism of force generation in

Four molecules of calcium ion are bound per molecule

striated muscle. Nature [Lond] 1971;233:533.) The

of troponin C or calmodulin, and both molecules have

strengths of binding of the attached sites are higher in

a molecular mass of 17 kDa.

position 2 than in position 1 and higher in position 3

than position 2. The myosin head can be detached from

Ca2+ Plays a Central Role in Regulation

position 3 with the utilization of a molecule of ATP; this

of Muscle Contraction

is the predominant process during shortening. The

The contraction of muscles from all sources occurs by

myosin head is seen to vary in its position from about

the general mechanism described above. Muscles from

90° to about 45°, as indicated in the text. (S-1, myosin

different organisms and from different cells and tissues

head; S-2, portion of the myosin molecule; LMM, light

within the same organism may have different molecular

meromyosin) (see legend to Figure 49-4). (Reproduced

mechanisms responsible for the regulation of their con-

from Huxley AF: Muscular contraction. J Physiol 1974;

traction and relaxation. In all systems, Ca2+ plays a key

243:1. By kind permission of the author and the Journal of

regulatory role. There are two general mechanisms of

Physiology.)

regulation of muscle contraction: actin-based and

myosin-based. The former operates in skeletal and car-

diac muscle, the latter in smooth muscle.

Another cycle then commences with the hydrolysis

of ATP (step 1 of Figure 49-6), re-forming the high-

Actin-Based Regulation Occurs

energy conformation.

in Striated Muscle

Thus, hydrolysis of ATP is used to drive the cycle,

with the actual power stroke being the conformational

Actin-based regulation of muscle occurs in vertebrate

change in the S-1 head that occurs upon the release of

skeletal and cardiac muscles, both striated. In the gen-

MUSCLE & THE CYTOSKELETON

563

eral mechanism described above

(Figure

49-6), the

T tubule

only potentially limiting factor in the cycle of muscle

Sarcolemma

contraction might be ATP. The skeletal muscle system

Dihydropyridine

is inhibited at rest; this inhibition is relieved to activate

receptor

contraction. The inhibitor of striated muscle is the tro-

Ca2+ release

ponin system, which is bound to tropomyosin and

Calsequestrin

channel

F-actin in the thin filament (Figure 49-3). In striated

muscle, there is no control of contraction unless the

Ca2+

tropomyosin-troponin systems are present along with

Ca2+

the actin and myosin filaments. As described above,

Cister na

Ca2+

tropomyosin lies along the groove of F-actin, and

Ca2+

Cister na

the three components of troponin—TpT, TpI, and

TpC—are bound to the F-actin-tropomyosin complex.

Ca2+ ATPase

TpI prevents binding of the myosin head to its F-actin

Calsequestrin

Ca2+

attachment site either by altering the conformation of

F-actin via the tropomyosin molecules or by simply

Ca2+

rolling tropomyosin into a position that directly blocks

the sites on F-actin to which the myosin heads attach.

Either way prevents activation of the myosin ATPase

Sarcomere

that is mediated by binding of the myosin head to

F-actin. Hence, the TpI system blocks the contraction

Figure 49-8. Diagram of the relationships among

cycle at step 2 of Figure 49-6. This accounts for the in-

the sarcolemma (plasma membrane), a T tubule, and

hibited state of relaxed striated muscle.

two cisternae of the sarcoplasmic reticulum of skeletal

muscle (not to scale). The T tubule extends inward from

The Sarcoplasmic Reticulum

the sarcolemma. A wave of depolarization, initiated by

Regulates Intracellular Levels

a nerve impulse, is transmitted from the sarcolemma

in Skeletal Muscle

of Ca2+

down the T tubule. It is then conveyed to the Ca²⁺ re-

In the sarcoplasm of resting muscle, the concentration

lease channel (ryanodine receptor), perhaps by interac-

of Ca2+ is 10−8 to 10−7 mol/L. The resting state is

tion between it and the dihydropyridine receptor (slow

achieved because Ca2+ is pumped into the sarcoplasmic

Ca²⁺ voltage channel), which are shown in close prox-

reticulum through the action of an active transport sys-

imity. Release of Ca²⁺ from the Ca²⁺ release channel into

tem, called the Ca2+ ATPase (Figure 49-8), initiating

the cytosol initiates contraction. Subsequently, Ca²⁺ is

relaxation. The sarcoplasmic reticulum is a network of

pumped back into the cisternae of the sarcoplasmic

fine membranous sacs. Inside the sarcoplasmic reticu-

reticulum by the Ca²⁺ ATPase (Ca²⁺ pump) and stored

lum, Ca2+ is bound to a specific Ca2+-binding protein

there, in part bound to calsequestrin.

designated calsequestrin. The sarcomere is surrounded

by an excitable membrane (the T tubule system) com-

posed of transverse (T) channels closely associated with

the sarcoplasmic reticulum.

receptor, RYR1 and RYR2, the former being present in

When the sarcolemma is excited by a nerve impulse,

skeletal muscle and the latter in heart muscle and brain.

the signal is transmitted into the T tubule system and a

Ryanodine is a plant alkaloid that binds to RYR1 and

Ca2+ release channel in the nearby sarcoplasmic reticu-

RYR2 specifically and modulates their activities. The

lum opens, releasing Ca2+ from the sarcoplasmic reticu-

Ca2+ release channel is a homotetramer made up of four

lum into the sarcoplasm. The concentration of Ca2+ in

subunits of kDa 565. It has transmembrane sequences

the sarcoplasm rises rapidly to 10−5 mol/L. The Ca2+-

at its carboxyl terminal, and these probably form the

binding sites on TpC in the thin filament are quickly

Ca2+ channel. The remainder of the protein protrudes

occupied by Ca2+. The TpC-4Ca2+ interacts with TpI

into the cytosol, bridging the gap between the sar-

and TpT to alter their interaction with tropomyosin.

coplasmic reticulum and the transverse tubular mem-

Accordingly, tropomyosin moves out of the way or al-

brane. The channel is ligand-gated, Ca2+ and ATP

ters the conformation of F-actin so that the myosin

working synergistically in vitro, although how it oper-

head-ADP-P_i (Figure 49-6) can interact with F-actin to

ates in vivo is not clear. A possible sequence of events

start the contraction cycle.

leading to opening of the channel is shown in Figure

The Ca2+ release channel is also known as the ryan-

49-9. The channel lies very close to the dihydropyri-

odine receptor (RYR). There are two isoforms of this

dine receptor (DHPR; a voltage-gated slow K type

564

CHAPTER 49

Depolarization of nerve

Table 49-1 summarizes the overall events in con-

traction and relaxation of skeletal muscle.

Depolarization of skeletal muscle

Mutations in the Gene Encoding the Ca2+

Release Channel Are One Cause of Human

Depolarization of the transverse tubular membrane

Malignant Hyperthermia

Some genetically predisposed patients experience a se-

Charge movement of the slow Ca2+ voltage

vere reaction, designated malignant hyperthermia, on

channel (DHPR) of the transverse tubular membrane

exposure to certain anesthetics (eg, halothane) and de-

polarizing skeletal muscle relaxants

(eg, succinyl-

Opening of the Ca2+ release channel (RYR1)

choline). The reaction consists primarily of rigidity of

skeletal muscles, hypermetabolism, and high fever. A

Figure 49-9. Possible chain of events leading to

high cytosolic concentration of Ca2+ in skeletal mus-

opening of the Ca²⁺ release channel. As indicated in the

cle is a major factor in its causation. Unless malignant

text, the Ca²⁺ voltage channel and the Ca²⁺ release

hyperthermia is recognized and treated immediately,

channel have been shown to interact with each other in

patients may die acutely of ventricular fibrillation or

vitro via specific regions in their polypeptide chains.

survive to succumb subsequently from other serious

(DHPR, dihydropyridine receptor; RYR1, ryanodine re-

complications. Appropriate treatment is to stop the

ceptor 1.)

anesthetic and administer the drug dantrolene intra-

venously. Dantrolene is a skeletal muscle relaxant that

acts to inhibit release of Ca2+ from the sarcoplasmic

reticulum into the cytosol, thus preventing the increase

Ca2+ channel) of the transverse tubule system (Figure

of cytosolic Ca2+ found in malignant hyperthermia.

49-8). Experiments in vitro employing an affinity col-

umn chromatography approach have indicated that a

37-amino-acid stretch in RYR1 interacts with one spe-

cific loop of DHPR.

Table 49-1. Sequence of events in contraction

Relaxation occurs when sarcoplasmic Ca2+ falls

and relaxation of skeletal muscle.¹

below 10−7 mol/L owing to its resequestration into the

sarcoplasmic reticulum by Ca2+ ATPase. TpC.4Ca2+

Steps in contraction

thus loses its Ca2+. Consequently, troponin, via interac-

(1) Discharge of motor neuron

tion with tropomyosin, inhibits further myosin head

(2) Release of transmitter (acetylcholine) at motor end-

and F-actin interaction, and in the presence of ATP the

plate

myosin head detaches from the F-actin.

(3) Binding of acetylcholine to nicotinic acetylcholine re-

Thus, Ca2+ controls skeletal muscle contraction and

ceptors

relaxation by an allosteric mechanism mediated by

(4) Increased Na⁺ and K⁺ conductance in endplate mem-

TpC, TpI, TpT, tropomyosin, and F-actin.

brane

A decrease in the concentration of ATP in the sar-

(5) Generation of endplate potential

coplasm (eg, by excessive usage during the cycle of con-

(6) Generation of action potential in muscle fibers

traction-relaxation or by diminished formation, such as

(7) Inward spread of depolarization along T tubules

might occur in ischemia) has two major effects: (1) The

(8) Release of Ca²⁺ from terminal cisterns of sarcoplasmic

Ca2+ ATPase (Ca2+ pump) in the sarcoplasmic reticu-

reticulum and diffusion to thick and thin filaments

lum ceases to maintain the low concentration of Ca2+

(9) Binding of Ca²⁺ to troponin C, uncovering myosin

in the sarcoplasm. Thus, the interaction of the myosin

binding sites of actin

heads with F-actin is promoted. (2) The ATP-depen-

(10) Formation of cross-linkages between actin and

dent detachment of myosin heads from F-actin cannot

myosin and sliding of thin on thick filaments, produc-

ing shortening

occur, and rigidity (contracture) sets in. The condition

Steps in relaxation

of rigor mortis, following death, is an extension of

(1) Ca²⁺ pumped back into sarcoplasmic reticulum

these events.

(2) Release of Ca²⁺ from troponin

Muscle contraction is a delicate dynamic balance of

(3) Cessation of interaction between actin and myosin

the attachment and detachment of myosin heads to

F-actin, subject to fine regulation via the nervous

¹Reproduced, with permission, from Ganong WF: Review of Med-

system.

ical Physiology, 21st ed. McGraw-Hill, 2003.

MUSCLE & THE CYTOSKELETON

565

Malignant hyperthermia also occurs in swine. Sus-

Mutations in the RYR1 gene

ceptible animals homozygous for malignant hyperther-

mia respond to stress with a fatal reaction (porcine

Altered Ca2+ release channel protein (RYR1)

stress syndrome) similar to that exhibited by humans.

(eg, substitution of Cys for Arg⁶¹⁵)

If the reaction occurs prior to slaughter, it affects the

quality of the pork adversely, resulting in an inferior

product. Both events can result in considerable eco-

Mutated channel opens more easily and stays open

nomic losses for the swine industry.

longer, thus flooding the cytosol with Ca2+

The finding of a high level of cytosolic Ca2+ in mus-

cle in malignant hyperthermia suggested that the con-

High intracellular levels of Ca2+ stimulate sustained

dition might be caused by abnormalities of the Ca2+

muscle contraction (rigidity); high Ca2+ also stimulates

ATPase or of the Ca2+ release channel. No abnormali-

breakdown of glycogen, glycolysis, and aerobic

ties were detected in the former, but sequencing of

metabolism (resulting in excessive production of heat)

cDNAs for the latter protein proved insightful, particu-

larly in swine. All cDNAs from swine with malignant

Figure 49-10. Simplified scheme of the causation of

hyperthermia so far examined have shown a substitu-

malignant hyperthermia (MIM 145600). At least 17 dif-

tion of T for C1843, resulting in the substitution of

ferent point mutations have been detected in the RYR1

release channel. The muta-

Cys for Arg⁶¹⁵ in the Ca2+

gene, some of which are associated with central core

tion affects the function of the channel in that it opens

disease (MIM 117000). It is estimated that at least 50%

more easily and remains open longer; the net result is

of families with members who have malignant hyper-

massive release of Ca2+ into the cytosol, ultimately caus-

thermia are linked to the RYR1 gene. Some individuals

ing sustained muscle contraction.

with mutations in the gene encoding DHPR have also

The picture is more complex in humans, since ma-

been detected; it is possible that mutations in other

lignant hyperthermia exhibits genetic heterogeneity.

genes for proteins involved in certain aspects of muscle

Members of a number of families who suffer from ma-

metabolism will also be found.

lignant hyperthermia have not shown genetic linkage

to the RYR1 gene. Some humans susceptible to malig-

nant hyperthermia have been found to exhibit the

same mutation found in swine, and others have a vari-

ety of point mutations at different loci in the RYR1

MUTATIONS IN THE GENE ENCODING

gene. Certain families with malignant hypertension

DYSTROPHIN CAUSE DUCHENNE

have been found to have mutations affecting the

MUSCULAR DYSTROPHY

DHPR. Figure 49-10 summarizes the probable chain

of events in malignant hyperthermia. The major

A number of additional proteins play various roles in

promise of these findings is that, once additional mu-

the structure and function of muscle. They include titin

tations are detected, it will be possible to screen, using

(the largest protein known), nebulin, α-actinin, desmin,

suitable DNA probes, for individuals at risk of devel-

dystrophin, and calcineurin. Some properties of these

oping malignant hyperthermia during anesthesia. Cur-

proteins are summarized in Table 49-2.

rent screening tests (eg, the in vitro caffeine-halothane

Dystrophin is of special interest. Mutations in the

test) are relatively unreliable. Affected individuals

gene encoding this protein have been shown to be the

could then be given alternative anesthetics, which

cause of Duchenne muscular dystrophy and the milder

would not endanger their lives. It should also be possi-

Becker muscular dystrophy (see Figure 49-11). They

ble, if desired, to eliminate malignant hyperthermia

are also implicated in some cases of dilated cardiomy-

from swine populations using suitable breeding prac-

opathy (see below). The gene encoding dystrophin is

tices.

the largest gene known (≈ 2300 kb) and is situated on

Another condition due to mutations in the RYR1

the X chromosome, accounting for the maternal inheri-

gene is central core disease. This is a rare myopathy

tance pattern of Duchenne and Becker muscular dys-

presenting in infancy with hypotonia and proximal

trophies. As shown in Figure 49-12, dystrophin forms

muscle weakness. Electron microscopy reveals an ab-

part of a large complex of proteins that attach to or in-

sence of mitochondria in the center of many type I (see

teract with the plasmalemma. Dystrophin links the

below) muscle fibers. Damage to mitochondria induced

actin cytoskeleton to the ECM and appears to be

by high intracellular levels of Ca2+ secondary to abnor-

needed for assembly of the synaptic junction. Impair-

mal functioning of RYR1 appears to be responsible for

ment of these processes by formation of defective dys-

the morphologic findings.

trophin is presumably critical in the causation of

566

CHAPTER 49

Table 49-2. Some other important proteins

Duchenne muscular dystrophy. Mutations in the genes

of muscle.

encoding some of the components of the sarcoglycan

complex shown in Figure 49-12 are responsible for

limb-girdle and certain other congenital forms of mus-

Protein

Location

Comment or Function

cular dystrophy.

Titin

Reaches from the Z

Largest protein in body.

line to the M line

Role in relaxation of

muscle.

CARDIAC MUSCLE RESEMBLES SKELETAL

MUSCLE IN MANY RESPECTS

Nebulin

From Z line along

May regulate assembly

length of actin

and length of actin

The general picture of muscle contraction in the heart

filaments

filaments.

resembles that of skeletal muscle. Cardiac muscle, like

α-Actinin

Anchors actin to Z

Stabilizes actin

skeletal muscle, is striated and uses the actin-myosin-

lines

filaments.

tropomyosin-troponin system described above. Unlike

skeletal muscle, cardiac muscle exhibits intrinsic rhyth-

Desmin

Lies alongside actin

Attaches to plasma

micity, and individual myocytes communicate with

filaments

membrane (plasma-

each other because of its syncytial nature. The T tubu-

lemma).

lar system is more developed in cardiac muscle,

Dystrophin

Attached to plasma-

Deficient in Duchenne

whereas the sarcoplasmic reticulum is less extensive

lemma

muscular dystrophy.

and consequently the intracellular supply of Ca2+ for

Mutations of its gene

contraction is less. Cardiac muscle thus relies on extra-

can also cause dilated

cellular Ca2+ for contraction; if isolated cardiac muscle

cardiomyopathy.

is deprived of Ca2+, it ceases to beat within approxi-

Calcineurin

Cytosol

A calmodulin-regulated

mately 1 minute, whereas skeletal muscle can continue

protein phosphatase.

to contract without an extracellular source of Ca2+.

May play important

Cyclic AMP plays a more prominent role in cardiac

roles in cardiac hyper-

than in skeletal muscle. It modulates intracellular levels

trophy and in regulating

of Ca2+ through the activation of protein kinases; these

amounts of slow and

enzymes phosphorylate various transport proteins in

fast twitch muscles.

the sarcolemma and sarcoplasmic reticulum and also in

Myosin-

Arranged trans-

Binds myosin and titin.

the troponin-tropomyosin regulatory complex, affect-

binding

versely in sarcomere

Plays a role in main-

ing intracellular levels of Ca2+ or responses to it. There

protein C

A-bands

taining the structural

is a rough correlation between the phosphorylation of

integrity of the sarco-

TpI and the increased contraction of cardiac muscle in-

mere.

duced by catecholamines. This may account for the in-

otropic effects (increased contractility) of β-adrenergic

compounds on the heart. Some differences among

skeletal, cardiac, and smooth muscle are summarized in

Table 49-3.

Deletion of part of the structural gene for dystrophin,

located on the X chromosome

Ca2+ Enters Myocytes via Ca2+ Channels

& Leaves via the Na⁺-Ca2+ Exchanger

Diminished synthesis of the mRNA for dystrophin

& the Ca2+ ATPase

As stated above, extracellular Ca2+ plays an important

Low levels or absence of dystrophin

role in contraction of cardiac muscle but not in skeletal

muscle. This means that Ca2+ both enters and leaves

Muscle contraction/relaxation affected;

myocytes in a regulated manner. We shall briefly con-

precise mechanisms not elucidated

sider three transmembrane proteins that play roles in

this process.

Progressive, usually fatal muscular weakness

A. Ca²⁺ CHANNELS

Figure 49-11. Summary of the causation of

Ca2+ enters myocytes via these channels, which allow

Duchenne muscular dystrophy (MIM 310200).

entry only of Ca2+ ions. The major portal of entry is the

MUSCLE & THE CYTOSKELETON

567

Figure 49-12. Organization of dystrophin and other proteins in relation to the plasma membrane of muscle cells.

Dystrophin is part of a large oligomeric complex associated with several other protein complexes. The dystroglycan

complex consists of α-dystroglycan, which associates with the basal lamina protein merosin, and β-dystroglycan,

which binds α-dystroglycan and dystrophin. Syntrophin binds to the carboxyl terminal of dystrophin. The sarcogly-

can complex consists of four transmembrane proteins: α-, β-, γ-, and δ-sarcoglycan. The function of the sarcoglycan

complex and the nature of the interactions within the complex and between it and the other complexes are not

clear. The sarcoglycan complex is formed only in striated muscle, and its subunits preferentially associate with each

other, suggesting that the complex may function as a single unit. Mutations in the gene encoding dystrophin cause

Duchenne and Becker muscular dystrophy; mutations in the genes encoding the various sarcoglycans have been

shown to be responsible for limb-girdle dystrophies (eg, MIM 601173). (Reproduced, with permission, from Duggan DJ

et al: Mutations in the sarcoglycan genes in patients with myopathy. N Engl J Med 1997;336:618.)

L-type (long-duration current, large conductance) or

(CICR). It is estimated that approximately 10% of the

slow Ca2+ channel, which is voltage-gated, opening

Ca2+ involved in contraction enters the cytosol from

during depolarization induced by spread of the cardiac

the extracellular fluid and 90% from the sarcoplasmic

action potential and closing when the action potential

reticulum. However, the former 10% is important, as

declines. These channels are equivalent to the dihy-

the rate of increase of Ca2+ in the myoplasm is impor-

dropyridine receptors of skeletal muscle (Figure 49-8).

tant, and entry via the Ca2+ channels contributes appre-

Slow Ca2+ channels are regulated by cAMP-dependent

ciably to this.

protein kinases

(stimulatory) and cGMP-protein ki-

nases (inhibitory) and are blocked by so-called calcium

channel blockers (eg, verapamil). Fast (or T, transient)

B. Ca²⁺-Na⁺ EXCHANGER

Ca2+ channels are also present in the plasmalemma,

This is the principal route of exit of Ca2+ from myo-

though in much lower numbers; they probably con-

cytes. In resting myocytes, it helps to maintain a low

tribute to the early phase of increase of myoplasmic

level of free intracellular Ca2+ by exchanging one Ca2+

Ca2+.

for three Na⁺. The energy for the uphill movement of

The resultant increase of Ca2+ in the myoplasm acts

Ca2+ out of the cell comes from the downhill move-

on the Ca2+ release channel of the sarcoplasmic reticu-

ment of Na⁺ into the cell from the plasma. This ex-

lum to open it. This is called Ca2+-induced Ca2+ release

change contributes to relaxation but may run in the re-

568

CHAPTER 49

Table 49-3. Some differences between skeletal, cardiac, and smooth muscle.

Skeletal Muscle

Cardiac Muscle

Smooth Muscle

Striated.

1. Nonstriated.

No syncytium.

Syncytial.

2. Syncytial.

Small T tubules.

Large T tubules.

3. Generally rudimentary T tubules.

Sarcoplasmic reticulum well-

Sarcoplasmic reticulum present and

4. Sarcoplasmic reticulum often rudimen-

developed and Ca²⁺ pump acts

Ca²⁺ pump acts relatively rapidly.

tary and Ca²⁺ pump acts slowly.

rapidly.

Plasmalemma lacks many hormone

Plasmalemma contains a variety of

5. Plasmalemma contains a variety of

receptors.

receptors (eg, α- and β-adrenergic).

Nerve impulse initiates contraction.

Has intrinsic rhythmicity.

6. Contraction initiated by nerve impulses,

hormones, etc.

Extracellular fluid Ca²⁺ not important

Extracellular fluid Ca²⁺ important

7. Extracellular fluid Ca²⁺ important for

for contraction.

contraction.

Troponin system present.

8. Lacks troponin system; uses regulatory

head of myosin.

Caldesmon not involved.

9. Caldesmon is important regulatory

protein.

10.

Very rapid cycling of the

10.

Relatively rapid cycling of the cross-

10. Slow cycling of the cross-bridges per-

cross-bridges.

bridges.

mits slow prolonged contraction

and less utilization of ATP.

important in skeletal muscle. Mutations in genes en-

verse direction during excitation. Because of the Ca2+-

coding ion channels have been shown to be responsible

Na⁺ exchanger, anything that causes intracellular Na⁺

for a number of relatively rare conditions affecting mus-

(Na+i) to rise will secondarily cause Ca2+i to rise, caus-

cle. These and other diseases due to mutations of ion

ing more forceful contraction. This is referred to as a

channels have been termed channelopathies; some are

positive inotropic effect. One example is when the drug

listed in Table 49-5.

digitalis is used to treat heart failure. Digitalis inhibits

the sarcolemmal Na⁺-K⁺ ATPase, diminishing exit of

Na⁺ and thus increasing Na+i. This in turn causes Ca2+

to increase, via the Ca2+-Na⁺ exchanger. The increased

Table 49-4. Major types of ion channels found

Ca2+i results in increased force of cardiac contraction, of

in cells.

benefit in heart failure.

C. Ca2+ ATPASE

Type

Comment

This Ca2+ pump, situated in the sarcolemma, also con-

External

Open in response to a specific extracellular

tributes to Ca2+ exit but is believed to play a relatively

ligand-gated

molecule, eg, acetylcholine.

minor role as compared with the Ca2+-Na⁺ exchanger.

It should be noted that there are a variety of ion

Internal

Open or close in response to a specific intra-

channels (Chapter 41) in most cells, for Na⁺, K⁺, Ca2+,

ligand-gated

cellular molecule, eg, a cyclic nucleotide.

etc. Many of them have been cloned in recent years and

Voltage-gated

Open in response to a change in membrane

their dispositions in their respective membranes worked

potential, eg, Na⁺, K⁺, and Ca2+ channels in

out (number of times each one crosses its membrane,

heart.

location of the actual ion transport site in the protein,

Mechanically

Open in response to change in mechanical

etc). They can be classified as indicated in Table 49-4.

gated

pressure.

Cardiac muscle is rich in ion channels, and they are also

MUSCLE & THE CYTOSKELETON

569

Table 49-5. Some disorders (channelopathies)

Table 49-6. Biochemical causes of inherited

due to mutations in genes encoding polypeptide

cardiomyopathies.^1,2

constituents of ion channels.¹

Proteins or Process

Ion Channel and Major

Cause

Affected

Disorder²

Organs Involved

Inborn errors of fatty acid

Carnitine entry into cells and

Central core disease

Ca2+ release channel (RYR1)

oxidation

mitochondria

(MIM 117000)

Skeletal muscle

Certain enzymes of fatty acid

oxidation

Cystic fibrosis

CFTR (Cl⁻ channel)

(MIM 219700)

Lungs, pancreas

Disorders of mitochondrial

Proteins encoded by mito-

oxidative phosphorylation

chondrial genes

Hyperkalemic periodic

Sodium channel

Proteins encoded by nuclear

paralysis (MIM 170500)

Skeletal muscle

genes

Hypokalemic periodic

Slow Ca2+ voltage channel (DHPR)

Abnormalities of myocardial

β-Myosin heavy chains, tropo-

paralysis (MIM 114208)

Skeletal muscle

contractile and structural

nin, tropomyosin, dys-

Malignant hyperthermia

Ca2+ release channel (RYR1)

proteins

trophin

(MIM 180901)

Skeletal muscle

¹Based on Kelly DP, Strauss AW: Inherited cardiomyopathies.

N Engl J Med 1994;330:913.

Myotonia congenita

Chloride channel

²Mutations (eg, point mutations, or in some cases deletions) in

(MIM 160800)

Skeletal muscle

the genes (nuclear or mitochondrial) encoding various proteins,

¹Data in part from Ackerman NJ, Clapham DE: Ion channels—

enzymes, or tRNA molecules are the fundamental causes of the

basic science and clinical disease. N Engl J Med 1997;336:1575.

inherited cardiomyopathies. Some conditions are mild, whereas

²Other channelopathies include the long QT syndrome (MIM

others are severe and may be part of a syndrome affecting other

192500); pseudoaldosteronism (Liddle syndrome, MIM 177200);

tissues.

persistent hyperinsulinemic hypoglycemia of infancy

(MIM

601820); hereditary X-linked recessive type II nephrolithiasis of in-

fancy (Dent syndrome, MIM 300009); and generalized myotonia,

recessive (Becker disease, MIM 255700). The term “myotonia” sig-

Mutations in the Cardiac

-Myosin Heavy

nifies any condition in which muscles do not relax after contrac-

tion.

Chain Gene Are One Cause of Familial

Hypertrophic Cardiomyopathy

Familial hypertrophic cardiomyopathy is one of the

Inherited Cardiomyopathies Are Due

most frequent hereditary cardiac diseases. Patients ex-

hibit hypertrophy—often massive—of one or both ven-

to Disorders of Cardiac Energy

tricles, starting early in life, and not related to any ex-

Metabolism or to Abnormal

trinsic cause such as hypertension. Most cases are

Myocardial Proteins

transmitted in an autosomal dominant manner; the rest

An inherited cardiomyopathy is any structural or func-

are sporadic. Until recently, its cause was obscure. How-

tional abnormality of the ventricular myocardium due

ever, this situation changed when studies of one affected

to an inherited cause. There are nonheritable types of

family showed that a missense mutation (ie, substitu-

cardiomyopathy, but these will not be described here.

tion of one amino acid by another) in the β-myosin

As shown in Table 49-6, the causes of inherited car-

heavy chain gene was responsible for the condition.

diomyopathies fall into two broad classes: (1) disorders

Subsequent studies have shown a number of missense

of cardiac energy metabolism, mainly reflecting muta-

mutations in this gene, all coding for highly conserved

tions in genes encoding enzymes or proteins involved in

residues. Some individuals have shown other mutations,

fatty acid oxidation (a major source of energy for the

such as formation of an α/β-myosin heavy chain hybrid

myocardium) and oxidative phosphorylation; and

gene. Patients with familial hypertrophic cardiomyopa-

(2) mutations in genes encoding proteins involved in or

thy can show great variation in clinical picture. This in

affecting myocardial contraction, such as myosin,

part reflects genetic heterogeneity; ie, mutation in a

tropomyosin, the troponins, and cardiac myosin-

number of other genes (eg, those encoding cardiac

binding protein C. Mutations in the genes encoding

actin, tropomyosin, cardiac troponins I and T, essential

these latter proteins cause familial hypertrophic car-

and regulatory myosin light chains, and cardiac myosin-

diomyopathy, which will now be discussed.

binding protein C) may also cause familial hypertrophic

570

CHAPTER 49

cardiomyopathy. In addition, mutations at different

in the regulation of a number of genes in these cells.

sites in the gene for β-myosin heavy chain may affect the

Current research is not only elucidating the molecular

function of the protein to a greater or lesser extent. The

causes of the cardiomyopathies but is also disclosing

missense mutations are clustered in the head and head-

mutations that cause cardiac developmental disorders

rod regions of myosin heavy chain. One hypothesis is

(eg, septal defects) and arrhythmias (eg, due to muta-

that the mutant polypeptides (“poison polypeptides”)

tions affecting ion channels).

cause formation of abnormal myofibrils, eventually re-

sulting in compensatory hypertrophy. Some mutations

Ca²⁺ Also Regulates Contraction

alter the charge of the amino acid (eg, substitution of

of Smooth Muscle

arginine for glutamine), presumably affecting the con-

formation of the protein more markedly and thus affect-

While all muscles contain actin, myosin, and tropo-

ing its function. Patients with these mutations have a

myosin, only vertebrate striated muscles contain the

significantly shorter life expectancy than patients in

troponin system. Thus, the mechanisms that regulate

whom the mutation produced no alteration in charge.

contraction must differ in various contractile systems.

Thus, definition of the precise mutations involved in the

Smooth muscles have molecular structures similar to

genesis of FHC may prove to be of important prognos-

those in striated muscle, but the sarcomeres are not

tic value; it can be accomplished by appropriate use of

aligned so as to generate the striated appearance.

the polymerase chain reaction on genomic DNA ob-

Smooth muscles contain α-actinin and tropomyosin

tained from one sample of blood lymphocytes. Figure

molecules, as do skeletal muscles. They do not have the

49-13 is a simplified scheme of the events causing fa-

troponin system, and the light chains of smooth muscle

milial hypertrophic cardiomyopathy.

myosin molecules differ from those of striated muscle

Another type of cardiomyopathy is termed dilated

myosin. Regulation of smooth muscle contraction is

cardiomyopathy. Mutations in the genes encoding dys-

myosin-based, unlike striated muscle, which is actin-

trophin, muscle LIM protein (so called because it was

based. However, like striated muscle, smooth muscle

found to contain a cysteine-rich domain originally de-

contraction is regulated by Ca2+.

tected in three proteins: Lin-II, Isl-1, and Mec-3), and

the cyclic response-element binding protein (CREB)

Phosphorylation of Myosin Light Chains

have been implicated in the causation of this condition.

Initiates Contraction of Smooth Muscle

The first two proteins help organize the contractile ap-

paratus of cardiac muscle cells, and CREB is involved

When smooth muscle myosin is bound to F-actin in the

absence of other muscle proteins such as tropomyosin,

there is no detectable ATPase activity. This absence of

activity is quite unlike the situation described for stri-

Predominantly missense mutations in the β-myosin

ated muscle myosin and F-actin, which has abundant

heavy chain gene on chromosome 14

ATPase activity. Smooth muscle myosin contains light

chains that prevent the binding of the myosin head to

Mutant polypeptide chains (“poison polypeptides”)

F-actin; they must be phosphorylated before they allow

that lead to formation of defective myofibrils

F-actin to activate myosin ATPase. The ATPase activity

then attained hydrolyzes ATP about tenfold more

slowly than the corresponding activity in skeletal mus-

Compensatory hypertrophy of one

cle. The phosphate on the myosin light chains may form

or both cardiac ventricles

a chelate with the Ca2+ bound to the tropomyosin-TpC-

actin complex, leading to an increased rate of formation

Cardiomegaly and various cardiac signs and

of cross-bridges between the myosin heads and actin.

symptoms, including sudden death

The phosphorylation of light chains initiates the attach-

ment-detachment contraction cycle of smooth muscle.

Simplified scheme of the causation of

Figure 49-13.

familial hypertrophic cardiomyopathy (MIM 192600)

Myosin Light Chain Kinase Is Activated

due to mutations in the gene encoding β-myosin heavy

by Calmodulin-4Ca2+ & Then

chain. Mutations in genes encoding other proteins,

Phosphorylates the Light Chains

such as the troponins, tropomyosin, and cardiac

myosin-binding protein C can also cause this condition.

Smooth muscle sarcoplasm contains a myosin light

Mutations in genes encoding yet other proteins (eg,

chain kinase that is calcium-dependent. The Ca2+ acti-

dystrophin) are involved in the causation of dilated

vation of myosin light chain kinase requires binding of

cardiomyopathy.

calmodulin-4Ca2+ to its kinase subunit (Figure 49-14).

MUSCLE & THE CYTOSKELETON

571

Calmodulin

Table 49-7 summarizes and compares the regula-

Myosin kinase

tion of actin-myosin interactions (activation of myosin

(inactive)

10^-5 mol/L Ca2+

10^-7 mol/L Ca²

ATPase) in striated and smooth muscles.

The myosin light chain kinase is not directly af-

Ca2+ • calmodulin

fected or activated by cAMP. However, cAMP-acti-

vated protein kinase can phosphorylate the myosin

light chain kinase (not the light chains themselves). The

phosphorylated myosin light chain kinase exhibits a sig-

nificantly lower affinity for calmodulin-Ca2+ and thus is

ATP

Ca2+ • CALMODULIN-MYOSIN

less sensitive to activation. Accordingly, an increase in

KINASE (ACTIVE)

cAMP dampens the contraction response of smooth

muscle to a given elevation of sarcoplasmic Ca2+. This

L-myosin (inhibits

ADP

molecular mechanism can explain the relaxing effect of

myosin-actin interaction)

β-adrenergic stimulation on smooth muscle.

Another protein that appears to play a Ca2+-depen-

dent role in the regulation of smooth muscle contrac-

pL-myosin (does not

tion is caldesmon (87 kDa). This protein is ubiquitous

inhibit myosin-actin interaction)

in smooth muscle and is also found in nonmuscle tis-

sue. At low concentrations of Ca2+, it binds to tro-

pomyosin and actin. This prevents interaction of actin

H₂PO

with myosin, keeping muscle in a relaxed state. At

higher concentrations of Ca2+, Ca2+-calmodulin binds

PHOSPHATASE

caldesmon, releasing it from actin. The latter is then

free to bind to myosin, and contraction can occur.

Figure 49-14. Regulation of smooth muscle con-

Caldesmon is also subject to phosphorylation-dephos-

traction by Ca²⁺. pL-myosin is the phosphorylated light

phorylation; when phosphorylated, it cannot bind

chain of myosin; L-myosin is the dephosphorylated

actin, again freeing the latter to interact with myosin.

light chain. (Adapted from Adelstein RS, Eisenberg R: Reg-

Caldesmon may also participate in organizing the struc-

ulation and kinetics of actin-myosin ATP interaction. Annu

ture of the contractile apparatus in smooth muscle.

Rev Biochem 1980;49:921.)

Many of its effects have been demonstrated in vitro,

and its physiologic significance is still under investiga-

tion.

As noted in Table 49-3, slow cycling of the cross-

The calmodulin-4Ca2+-activated light chain kinase

bridges permits slow prolonged contraction of smooth

phosphorylates the light chains, which then ceases to in-

muscle (eg, in viscera and blood vessels) with less uti-

hibit the myosin-F-actin interaction. The contraction

lization of ATP compared with striated muscle. The

cycle then begins.

ability of smooth muscle to maintain force at reduced

velocities of contraction is referred to as the latch state;

this is an important feature of smooth muscle, and its

Smooth Muscle Relaxes When

precise molecular bases are under study.

the Concentration of Ca²⁺ Falls

Below 10−7 Molar

Nitric Oxide Relaxes the Smooth Muscle

Relaxation of smooth muscle occurs when sarcoplasmic

of Blood Vessels & Also Has Many Other

Ca2+ falls below 10−7 mol/L. The Ca²⁺ dissociates from

Important Biologic Functions

calmodulin, which in turn dissociates from the myosin

light chain kinase, inactivating the kinase. No new

Acetylcholine is a vasodilator that acts by causing relax-

phosphates are attached to the p-light chain, and light

ation of the smooth muscle of blood vessels. However,

chain protein phosphatase, which is continually active

it does not act directly on smooth muscle. A key obser-

and calcium-independent, removes the existing phos-

vation was that if endothelial cells were stripped away

phates from the light chains. Dephosphorylated myosin

from underlying smooth muscle cells, acetylcholine no

p-light chain then inhibits the binding of myosin heads

longer exerted its vasodilator effect. This finding indi-

to F-actin and the ATPase activity. The myosin head

cated that vasodilators such as acetylcholine initially in-

detaches from the F-actin in the presence of ATP, but

teract with the endothelial cells of small blood vessels

it cannot reattach because of the presence of dephos-

via receptors. The receptors are coupled to the phos-

phorylated p-light chain; hence, relaxation occurs.

phoinositide cycle, leading to the intracellular release of

572

CHAPTER 49

Table 49-7. Actin-myosin interactions in striated and smooth muscle.

Smooth Muscle

Striated Muscle

(and Nonmuscle Cells)

Proteins of muscle filaments

Actin

Myosin

Myosin¹

Tropomyosin

Troponin (Tpl, TpT, TpC)

Spontaneous interaction of F-actin and

Yes

myosin alone (spontaneous activation

of myosin ATPase by F-actin

Inhibitor of F-actin-myosin interaction (in-

Troponin system (Tpl)

Unphosphorylated myosin light chain

hibitor of F-actin-dependent activation

of ATPase)

Contraction activated by

Ca²⁺

Direct effect of Ca²⁺

4Ca²⁺ bind to TpC

4Ca²⁺ bind to calmodulin

Effect of protein-bound Ca²⁺

TpC ⋅ 4Ca²⁺ antagonizes Tpl inhibition

Calmodulin ⋅ 4Ca²⁺ activates myosin light

of F-actin-myosin interaction (allows

chain kinase that phosphorylates myosin

F-actin activation of ATPase)

p-light chain. The phosphorylated p-light

chain no longer inhibits F-actin-myosin

interaction (allows F-actin activation of

ATPase).

¹Light chains of myosin are different in striated and smooth muscles.

Ca2+ through the action of inositol trisphosphate. In

zyme. NO synthase is a very complex enzyme, employ-

turn, the elevation of Ca2+ leads to the liberation of en-

ing five redox cofactors: NADPH, FAD, FMN, heme,

dothelium-derived relaxing factor

(EDRF), which

and tetrahydrobiopterin. NO can also be formed from

diffuses into the adjacent smooth muscle. There, it re-

nitrite, derived from vasodilators such as glyceryl trini-

acts with the heme moiety of a soluble guanylyl cyclase,

trate during their metabolism. NO has a very short

resulting in activation of the latter, with a consequent

half-life (approximately 3-4 seconds) in tissues because

elevation of intracellular levels of cGMP

(Figure

it reacts with oxygen and superoxide. The product of

49-15). This in turn stimulates the activities of certain

the reaction with superoxide is peroxynitrite (ONOO⁻),

cGMP-dependent protein kinases, which probably

which decomposes to form the highly reactive OH^•

phosphorylate specific muscle proteins, causing relax-

radical. NO is inhibited by hemoglobin and other

ation; however, the details are still being clarified. The

heme proteins, which bind it tightly. Chemical in-

important coronary artery vasodilator nitroglycerin,

hibitors of NO synthase are now available that can

widely used to relieve angina pectoris, acts to increase

markedly decrease formation of NO. Administration of

intracellular release of EDRF and thus of cGMP.

such inhibitors to animals and humans leads to vaso-

Quite unexpectedly, EDRF was found to be the gas

constriction and a marked elevation of blood pressure,

nitric oxide (NO). NO is formed by the action of the

indicating that NO is of major importance in the main-

enzyme NO synthase, which is cytosolic. The endothe-

tenance of blood pressure in vivo. Another important

lial and neuronal forms of NO synthase are activated by

cardiovascular effect is that by increasing synthesis of

Ca2+ (Table 49-8). The substrate is arginine, and the

cGMP, it acts as an inhibitor of platelet aggregation

products are citrulline and NO:

(Chapter 51).

Since the discovery of the role of NO as a vasodila-

NO SYNTHASE

tor, there has been intense experimental interest in this

Arginine

Citrulline + NO

substance. It has turned out to have a variety of physio-

logic roles, involving virtually every tissue of the body

NO synthase catalyzes a five-electron oxidation of

(Table 49-9). Three major isoforms of NO synthase

an amidine nitrogen of arginine. L-Hydroxyarginine is

have been identified, each of which has been cloned,

an intermediate that remains tightly bound to the en-

and the chromosomal locations of their genes in hu-

MUSCLE & THE CYTOSKELETON

573

Glyceryl

Acetylcholine

phosphate, and (4) from two molecules of ADP in a re-

trinitrate

action catalyzed by adenylyl kinase (Figure 49-16). The

amount of ATP in skeletal muscle is only sufficient to

ENDOTHELIAL

provide energy for contraction for a few seconds, so

CELL

that ATP must be constantly renewed from one or

Arginine

more of the above sources, depending upon metabolic

conditions. As discussed below, there are at least two

↑Ca2+

NO synthase

distinct types of fibers in skeletal muscle, one predomi-

nantly active in aerobic conditions and the other in

NO + Citrulline

anaerobic conditions; not unexpectedly, they use each

of the above sources of energy to different extents.

Skeletal Muscle Contains Large

GTP

Supplies of Glycogen

Guanylyl

The sarcoplasm of skeletal muscle contains large stores

Nitrate

Nitrite

cyclase

of glycogen, located in granules close to the I bands.

cGMP

The release of glucose from glycogen is dependent on a

protein

specific muscle glycogen phosphorylase (Chapter 18),

kinases

which can be activated by Ca2+, epinephrine, and AMP.

To generate glucose 6-phosphate for glycolysis in skele-

tal muscle, glycogen phosphorylase b must be activated

to phosphorylase a via phosphorylation by phosphory-

Relaxation

SMOOTH MUSCLE CELL

lase b kinase (Chapter 18). Ca2+ promotes the activa-

tion of phosphorylase b kinase, also by phosphoryla-

Figure 49-15. Diagram showing formation in an en-

tion. Thus, Ca2+ both initiates muscle contraction and

dothelial cell of nitric oxide (NO) from arginine in a re-

activates a pathway to provide necessary energy. The

action catalyzed by NO synthase. Interaction of an ago-

hormone epinephrine also activates glycogenolysis in

nist (eg, acetylcholine) with a receptor (R) probably

muscle. AMP, produced by breakdown of ADP during

leads to intracellular release of Ca2+ via inositol trisphos-

muscular exercise, can also activate phosphorylase b

phate generated by the phosphoinositide pathway, re-

without causing phosphorylation. Muscle glycogen

sulting in activation of NO synthase. The NO subse-

phosphorylase b is inactive in McArdle disease, one of

quently diffuses into adjacent smooth muscle, where it

the glycogen storage diseases (Chapter 18).

leads to activation of guanylyl cyclase, formation of

cGMP, stimulation of cGMP-protein kinases, and subse-

Under Aerobic Conditions, Muscle

quent relaxation. The vasodilator nitroglycerin is shown

Generates ATP Mainly by Oxidative

entering the smooth muscle cell, where its metabolism

Phosphorylation

also leads to formation of NO.

Synthesis of ATP via oxidative phosphorylation re-

quires a supply of oxygen. Muscles that have a high de-

mand for oxygen as a result of sustained contraction

mans have been determined. Gene knockout experi-

(eg, to maintain posture) store it attached to the heme

ments have been performed on each of the three iso-

moiety of myoglobin. Because of the heme moiety,

forms and have helped establish some of the postulated

muscles containing myoglobin are red, whereas muscles

functions of NO.

with little or no myoglobin are white. Glucose, derived

To summarize, research in the past decade has

from the blood glucose or from endogenous glycogen,

shown that NO plays an important role in many physi-

and fatty acids derived from the triacylglycerols of adi-

ologic and pathologic processes.

pose tissue are the principal substrates used for aerobic

metabolism in muscle.

SEVERAL MECHANISMS REPLENISH

STORES OF ATP IN MUSCLE

Creatine Phosphate Constitutes a

Major Energy Reserve in Muscle

The ATP required as the constant energy source for the

contraction-relaxation cycle of muscle can be generated

Creatine phosphate prevents the rapid depletion of

(1) by glycolysis, using blood glucose or muscle glyco-

ATP by providing a readily available high-energy phos-

gen, (2) by oxidative phosphorylation, (3) from creatine

phate that can be used to regenerate ATP from ADP.

574

CHAPTER 49

Table 49-8. Summary of the nomenclature of the NO synthases and of the effects of knockout of their

genes in mice.¹

Result of Gene

Subtype

Name²

Comments

Knockout in Mice³

nNOS

Activity depends on elevated Ca²⁺. First

Pyloric stenosis, resistant to vascular stroke, aggressive

identified in neurons. Calmodulin-activated.

sexual behavior (males).

iNOS⁴

Independent of elevated Ca²⁺.

More susceptible to certain types of infection.

Prominent in macrophages.

eNOS

Activity depends on elevated Ca²⁺.

Elevated mean blood pressure.

First identified in endothelial cells.

¹Adapted from Snyder SH: No endothelial NO. Nature 1995;377:196.

²n, neuronal; i, inducible; e, endothelial.

³Gene knockouts were performed by homologous recombination in mice. The enzymes are characterized as neuronal, inducible

(macrophage), and endothelial because these were the sites in which they were first identified. However, all three enzymes have been

found in other sites, and the neuronal enzyme is also inducible. Each gene has been cloned, and its chromosomal location in humans has

been determined.

⁴iNOS is Ca²⁺ -independent but binds calmodulin very tightly.

Creatine phosphate is formed from ATP and creatine

idative) and type II

(fast twitch, glycolytic)

(Table

(Figure 49-16) at times when the muscle is relaxed and

49-10). The type I fibers are red because they contain

demands for ATP are not so great. The enzyme catalyz-

myoglobin and mitochondria; their metabolism is aero-

ing the phosphorylation of creatine is creatine kinase

bic, and they maintain relatively sustained contractions.

(CK), a muscle-specific enzyme with clinical utility in

The type II fibers, lacking myoglobin and containing

the detection of acute or chronic diseases of muscle.

few mitochondria, are white: they derive their energy

from anaerobic glycolysis and exhibit relatively short du-

rations of contraction. The proportion of these two

SKELETAL MUSCLE CONTAINS SLOW

types of fibers varies among the muscles of the body, de-

(RED) & FAST (WHITE) TWITCH FIBERS

pending on function (eg, whether or not a muscle is in-

Different types of fibers have been detected in skeletal

volved in sustained contraction, such as maintaining

muscle. One classification subdivides them into type I

posture). The proportion also varies with training; for

(slow twitch), type IIA (fast twitch-oxidative), and type

example, the number of type I fibers in certain leg mus-

IIB (fast twitch-glycolytic). For the sake of simplicity,

cles increases in athletes training for marathons, whereas

we shall consider only two types: type I (slow twitch, ox-

the number of type II fibers increases in sprinters.

A Sprinter Uses Creatine Phosphate

& Anaerobic Glycolysis to Make ATP,

Table 49-9. Some physiologic functions and

Whereas a Marathon Runner Uses

pathologic involvements of nitric oxide (NO).

Oxidative Phosphorylation

• Vasodilator, important in regulation of blood pressure

In view of the two types of fibers in skeletal muscle and

• Involved in penile erection; sildenafil citrate (Viagra) affects

of the various energy sources described above, it is of

this process by inhibiting a cGMP phosphodiesterase

interest to compare their involvement in a sprint (eg,

• Neurotransmitter in the brain and peripheral autonomic

100 meters) and in the marathon (42.2 km; just over

nervous system

26 miles) (Table 49-11).

• Role in long-term potentiation

The major sources of energy in the 100-m sprint

• Role in neurotoxicity

are creatine phosphate (first 4-5 seconds) and then

• Low level of NO involved in causation of pylorospasm in in-

anaerobic glycolysis, using muscle glycogen as the

fantile hypertrophic pyloric stenosis

source of glucose. The two main sites of metabolic con-

• May have role in relaxation of skeletal muscle

trol are at glycogen phosphorylase and at PFK-1. The

• May constitute part of a primitive immune system

former is activated by Ca²⁺ (released from the sarcoplas-

• Inhibits adhesion, activation, and aggregation of platelets

mic reticulum during contraction), epinephrine, and

MUSCLE & THE CYTOSKELETON

575

Creatine phosphate

Muscle glycogen

CREATINE

PHOSPHOKINASE

ADP

MUSCLE

PHOSPHORYLASE

Creatine

Glucose 6-P

GLYCOLYSIS

Muscle

ATP

contraction

MYOSIN

OXIDATIVE

ATPase

PHOSPHORYLATION

ADP + P_i

AMP

ADP

ADENYLYL

KINASE

Figure 49-16. The multiple sources of ATP in muscle.

AMP. PFK-1 is activated by AMP, P_i, and NH₃. Attest-

ergy during a marathon for 4 minutes, 18 minutes, 70

ing to the efficiency of these processes, the flux through

minutes, and approximately

4000 minutes, respec-

glycolysis can increase as much as 1000-fold during a

tively. However, the rate of oxidation of fatty acids by

sprint.

muscle is slower than that of glucose, so that oxidation

In contrast, in the marathon, aerobic metabolism is

of glucose and of fatty acids are both major sources of

the principal source of ATP. The major fuel sources are

energy in the marathon.

blood glucose and free fatty acids, largely derived from

A number of procedures have been used by athletes

the breakdown of triacylglycerols in adipose tissue,

to counteract muscle fatigue and inadequate strength.

stimulated by epinephrine. Hepatic glycogen is de-

These include carbohydrate loading, soda (sodium bi-

graded to maintain the level of blood glucose. Muscle

glycogen is also a fuel source, but it is degraded much

more gradually than in a sprint. It has been calculated

that the amounts of glucose in the blood, of glycogen in

Table 49-11. Types of muscle fibers and major

the liver, of glycogen in muscle, and of triacylglycerol in

fuel sources used by a sprinter and by a marathon

adipose tissue are sufficient to supply muscle with en-

runner.

Sprinter (100 m)

Marathon Runner

Table 49-10. Characteristics of type I and type II

Type II (glycolytic) fibers are

Type I (oxidative) fibers are

fibers of skeletal muscle.

used predominantly.

Creatine phosphate is the

ATP is the major energy

Type I

Type II

major energy source dur-

source throughout.

Slow Twitch

Fast Twitch

ing the first 4-5 seconds.

Myosin ATPase

Low

High

Glucose derived from muscle

Blood glucose and free fatty

Energy utilization

Low

High

glycogen and metabolized

acids are the major fuel

Mitochondria

Many

Few

by anaerobic glycolysis is

sources.

Color

Red

White

the major fuel source.

Myoglobin

Yes

Contraction rate

Slow

Fast

Muscle glycogen is rapidly

Muscle glycogen is slowly

Duration

Prolonged

Short

depleted.

576

CHAPTER 49

carbonate) loading, blood doping (administration of

Table 49-12. Summary of major features of

red blood cells), and ingestion of creatine and an-

the biochemistry of skeletal muscle related to

drostenedione. Their rationales and efficacies will not

its metabolism.¹

be discussed here.

•

Skeletal muscle functions under both aerobic (resting) and

SKELETAL MUSCLE CONSTITUTES

anaerobic (eg, sprinting) conditions, so both aerobic and

THE MAJOR RESERVE OF

anaerobic glycolysis operate, depending on conditions.

PROTEIN IN THE BODY

•

Skeletal muscle contains myoglobin as a reservoir of oxy-

gen.

In humans, skeletal muscle protein is the major nonfat

•

Skeletal muscle contains different types of fibers primarily

source of stored energy. This explains the very large

suited to anaerobic (fast twitch fibers) or aerobic (slow

losses of muscle mass, particularly in adults, resulting

twitch fibers) conditions.

from prolonged caloric undernutrition.

•

Actin, myosin, tropomyosin, troponin complex (TpT, Tpl,

The study of tissue protein breakdown in vivo is dif-

and TpC), ATP, and Ca²⁺ are key constituents in relation to

ficult, because amino acids released during intracellular

contraction.

breakdown of proteins can be extensively reutilized for

•

The Ca²⁺ ATPase, the Ca²⁺ release channel, and calse-

protein synthesis within the cell, or the amino acids

questrin are proteins involved in various aspects of Ca²⁺ me-

may be transported to other organs where they enter

tabolism in muscle.

•

Insulin acts on skeletal muscle to increase uptake of glu-

anabolic pathways. However, actin and myosin are

cose.

methylated by a posttranslational reaction, forming

•

In the fed state, most glucose is used to synthesize glyco-

3-methylhistidine. During intracellular breakdown of

gen, which acts as a store of glucose for use in exercise;

actin and myosin, 3-methylhistidine is released and ex-

“preloading” with glucose is used by some long-distance

creted into the urine. The urinary output of the methy-

athletes to build up stores of glycogen.

lated amino acid provides a reliable index of the rate of

•

Epinephrine stimulates glycogenolysis in skeletal muscle,

myofibrillar protein breakdown in the musculature of

whereas glucagon does not because of absence of its re-

human subjects.

ceptors.

Various features of muscle metabolism, most of

•

Skeletal muscle cannot contribute directly to blood glucose

which are dealt with in other chapters of this text, are

because it does not contain glucose-6-phosphatase.

summarized in Table 49-12.

•

Lactate produced by anaerobic metabolism in skeletal mus-

cle passes to liver, which uses it to synthesize glucose,

THE CYTOSKELETON PERFORMS

which can then return to muscle (the Cori cycle).

•

Skeletal muscle contains phosphocreatine, which acts as an

MULTIPLE CELLULAR FUNCTIONS

energy store for short-term (seconds) demands.

Nonmuscle cells perform mechanical work, including

•

Free fatty acids in plasma are a major source of energy, par-

self-propulsion, morphogenesis, cleavage, endocytosis,

ticularly under marathon conditions and in prolonged star-

exocytosis, intracellular transport, and changing cell

vation.

shape. These cellular functions are carried out by an ex-

•

Skeletal muscle can utilize ketone bodies during starvation.

tensive intracellular network of filamentous structures

•

Skeletal muscle is the principal site of metabolism of

branched-chain amino acids, which are used as an energy

constituting the cytoskeleton. The cell cytoplasm is

source.

not a sac of fluid, as once thought. Essentially all eu-

•

Proteolysis of muscle during starvation supplies amino

karyotic cells contain three types of filamentous struc-

acids for gluconeogenesis.

tures: actin filaments

(7-9.5 nm in diameter; also

•

Major amino acids emanating from muscle are alanine (des-

known as microfilaments), microtubules (25 nm), and

tined mainly for gluconeogenesis in liver and forming part

intermediate filaments (10-12 nm). Each type of fila-

of the glucose-alanine cycle) and glutamine (destined

ment can be distinguished biochemically and by the

mainly for the gut and kidneys).

electron microscope.

¹This table brings together material from various chapters in this

book.

Nonmuscle Cells Contain Actin

That Forms Microfilaments

G-actin is present in most if not all cells of the body.

With appropriate concentrations of magnesium and

potassium chloride, it spontaneously polymerizes to

form double helical F-actin filaments like those seen in

muscle. There are at least two types of actin in nonmus-

MUSCLE & THE CYTOSKELETON

577

cle cells: β-actin and γ-actin. Both types can coexist in

dynamin, and myosins are referred to as molecular

the same cell and probably even copolymerize in the

motors.

same filament. In the cytoplasm, F-actin forms micro-

An absence of dynein in cilia and flagella results in

filaments of 7-9.5 nm that frequently exist as bundles

immotile cilia and flagella, leading to male sterility and

of a tangled-appearing meshwork. These bundles are

chronic respiratory infection, a condition known as

prominent just underlying the plasma membrane of

Kartagener syndrome.

many cells and are there referred to as stress fibers. The

Certain drugs bind to microtubules and thus inter-

stress fibers disappear as cell motility increases or upon

fere with their assembly or disassembly. These include

malignant transformation of cells by chemicals or onco-

colchicine (used for treatment of acute gouty arthritis),

genic viruses.

vinblastine (a vinca alkaloid used for treating certain

Although not organized as in muscle, actin filaments

types of cancer), paclitaxel

(Taxol) (effective against

in nonmuscle cells interact with myosin to cause cellu-

ovarian cancer), and griseofulvin (an antifungal agent).

lar movements.

Intermediate Filaments Differ From

Microfilaments & Microtubules

Microtubules Contain

- &

-Tubulins

An intracellular fibrous system exists of filaments with

Microtubules, an integral component of the cellular cy-

an axial periodicity of 21 nm and a diameter of 8-10

toskeleton, consist of cytoplasmic tubes 25 nm in diam-

nm that is intermediate between that of microfilaments

eter and often of extreme length. Microtubules are nec-

(6 nm) and microtubules (23 nm). Four classes of inter-

essary for the formation and function of the mitotic

mediate filaments are found, as indicated in Table

spindle and thus are present in all eukaryotic cells.

49-13. They are all elongated, fibrous molecules, with

They are also involved in the intracellular movement of

a central rod domain, an amino terminal head, and a

endocytic and exocytic vesicles and form the major

carboxyl terminal tail. They form a structure like a

structural components of cilia and flagella. Micro-

rope, and the mature filaments are composed of

tubules are a major component of axons and dendrites,

tetramers packed together in a helical manner. They are

in which they maintain structure and participate in the

important structural components of cells, and most are

axoplasmic flow of material along these neuronal

relatively stable components of the cytoskeleton, not

processes.

undergoing rapid assembly and disassembly and not

Microtubules are cylinders of

13 longitudinally

arranged protofilaments, each consisting of dimers of

α-tubulin and β-tubulin, closely related proteins of ap-

proximately

50 kDa molecular mass. The tubulin

dimers assemble into protofilaments and subsequently

Table 49-13. Classes of intermediate filaments of

into sheets and then cylinders. A microtubule-organiz-

eukaryotic cells and their distributions.

ing center, located around a pair of centrioles, nucleates

the growth of new microtubules. A third species of

Molecular

tubulin, γ-tubulin, appears to play an important role in

Proteins

Mass

Distributions

this assembly. GTP is required for assembly. A variety

of proteins are associated with microtubules (micro-

Keratins

tubule-associated proteins [MAPs], one of which is tau)

Type I (acidic)

40-60 kDa

Epithelial cells, hair,

Type II (basic)

50-70 kDa

nails

and play important roles in microtubule assembly and

stabilization. Microtubules are in a state of dynamic

Vimentin-like

instability, constantly assembling and disassembling.

Vimentin

54 kDa

Various mesenchymal

They exhibit polarity (plus and minus ends); this is im-

cells

portant in their growth from centrioles and in their

Desmin

53 kDa

Muscle

ability to direct intracellular movement. For instance,

Glial fibrillary acid

50 kDa

Glial cells

in axonal transport, the protein kinesin, with a

protein

myosin-like ATPase activity, uses hydrolysis of ATP to

Peripherin

66 kDa

Neurons

move vesicles down the axon toward the positive end of

Neurofilaments

the microtubular formation. Flow of materials in the

Low (L), medium (M),

60-130 kDa

Neurons

opposite direction, toward the negative end, is powered

and high (H)¹

by cytosolic dynein, another protein with ATPase ac-

Lamins

tivity. Similarly, axonemal dyneins power ciliary and

A, B, and C

65-75 kDa

Nuclear lamina

flagellar movement. Another protein, dynamin, uses

GTP and is involved in endocytosis. Kinesins, dyneins,

¹Refers to their molecular masses.

578

CHAPTER 49

disappearing during mitosis, as do actin and many mi-

cle, the sarcoplasmic reticulum regulates distribution

crotubular filaments. An important exception to this is

of Ca²⁺ to the sarcomeres, whereas inflow of Ca²⁺ via

provided by the lamins, which, subsequent to phosphor-

Ca²⁺ channels in the sarcolemma is of major impor-

ylation, disassemble at mitosis and reappear when it ter-

tance in cardiac and smooth muscle.

minates.

•

Many cases of malignant hyperthermia in humans

Keratins form a large family, with about 30 mem-

are due to mutations in the gene encoding the Ca²⁺

bers being distinguished. As indicated in Table 49-13,

release channel.

two major types of keratins are found; all individual

•

A number of differences exist between skeletal and

keratins are heterodimers made up of one member of

cardiac muscle; in particular, the latter contains a va-

each class.

riety of receptors on its surface.

Vimentins are widely distributed in mesodermal

•

Some cases of familial hypertrophic cardiomyopathy

cells, and desmin, glial fibrillary acidic protein, and pe-

are due to missense mutations in the gene coding for

ripherin are related to them. All members of the vi-

β-myosin heavy chain.

mentin-like family can copolymerize with each other.

•

Smooth muscle, unlike skeletal and cardiac muscle,

Intermediate filaments are very prominent in nerve

cells; neurofilaments are classified as low, medium, and

does not contain the troponin system; instead, phos-

phorylation of myosin light chains initiates contrac-

high on the basis of their molecular masses. Lamins

form a meshwork in apposition to the inner nuclear

tion.

membrane. The distribution of intermediate filaments

•

Nitric oxide is a regulator of vascular smooth muscle;

in normal and abnormal (eg, cancer) cells can be stud-

blockage of its formation from arginine causes an

ied by the use of immunofluorescent techniques, using

acute elevation of blood pressure, indicating that reg-

antibodies of appropriate specificities. These antibodies

ulation of blood pressure is one of its many func-

to specific intermediate filaments can also be of use to

tions.

pathologists in helping to decide the origin of certain

•

Duchenne-type muscular dystrophy is due to muta-

dedifferentiated malignant tumors. These tumors may

tions in the gene, located on the X chromosome, en-

still retain the type of intermediate filaments found in

coding the protein dystrophin.

their cell of origin.

•

Two major types of muscle fibers are found in hu-

A number of skin diseases, mainly characterized by

mans: white (anaerobic) and red (aerobic). The for-

blistering, have been found to be due to mutations in

mer are particularly used in sprints and the latter in

genes encoding various keratins. Three of these disor-

prolonged aerobic exercise. During a sprint, muscle

ders are epidermolysis bullosa simplex, epidermolytic

uses creatine phosphate and glycolysis as energy

hyperkeratosis, and epidermolytic palmoplantar kerato-

sources; in the marathon, oxidation of fatty acids is

derma. The blistering probably reflects a diminished ca-

of major importance during the later phases.

pacity of various layers of the skin to resist mechanical

•

Nonmuscle cells perform various types of mechanical

stresses due to abnormalities in microfilament structure.

work carried out by the structures constituting the

cytoskeleton. These structures include actin filaments

SUMMARY

(microfilaments), microtubules (composed primarily

of α- tubulin and β-tubulin), and intermediate fila-

• The myofibrils of skeletal muscle contain thick and

ments. The latter include keratins, vimentin-like pro-

thin filaments. The thick filaments contain myosin.

teins, neurofilaments, and lamins.

The thin filaments contain actin, tropomyosin, and

the troponin complex (troponins T, I, and C).

• The sliding filament cross-bridge model is the foun-

dation of current thinking about muscle contraction.

REFERENCES

The basis of this model is that the interdigitating fila-

Ackerman MJ, Clapham DE: Ion channels—basic science and clin-

ments slide past one another during contraction and

ical disease. N Engl J Med 1997;336:1575.

cross-bridges between myosin and actin generate and

Andreoli TE: Ion transport disorders: introductory comments. Am

sustain the tension.

J Med 1998;104:85. (First of a series of articles on ion trans-

• The hydrolysis of ATP is used to drive movement of

port disorders published between January and August, 1998.

the filaments. ATP binds to myosin heads and is hy-

Topics covered were structure and function of ion channels,

arrhythmias and antiarrhythmic drugs, Liddle syndrome,

drolyzed to ADP and P_i by the ATPase activity of the

cholera, malignant hyperthermia, cystic fibrosis, the periodic

actomyosin complex.

paralyses and Bartter syndrome, and Gittelman syndrome.)

• Ca²⁺ plays a key role in the initiation of muscle con-

Fuller GM, Shields D: Molecular Basis of Medical Cell Biology. Ap-

traction by binding to troponin C. In skeletal mus-

pleton & Lange, 1998.

Plasma Proteins & Immunoglobulins

Robert K. Murray, MD, PhD

BIOMEDICAL IMPORTANCE

solvents or electrolytes (or both) to remove different

protein fractions in accordance with their solubility

The fundamental role of blood in the maintenance of

characteristics. This is the basis of the so-called salting-

homeostasis and the ease with which blood can be ob-

out methods, which find some usage in the determina-

tained have meant that the study of its constituents has

tion of protein fractions in the clinical laboratory.

been of central importance in the development of bio-

Thus, one can separate the proteins of the plasma into

chemistry and clinical biochemistry. The basic proper-

three major groups—fibrinogen, albumin, and globu-

ties of a number of plasma proteins, including the

lins—by the use of varying concentrations of sodium

immunoglobulins

(antibodies), are described in this

or ammonium sulfate.

chapter. Changes in the amounts of various plasma pro-

The most common method of analyzing plasma

teins and immunoglobulins occur in many diseases and

proteins is by electrophoresis. There are many types of

can be monitored by electrophoresis or other suitable

electrophoresis, each using a different supporting

procedures. As indicated in an earlier chapter, alterations

medium. In clinical laboratories, cellulose acetate is

of the activities of certain enzymes found in plasma are

widely used as a supporting medium. Its use permits

of diagnostic use in a number of pathologic conditions.

resolution, after staining, of plasma proteins into five

bands, designated albumin, α₁, α₂, β, and γ fractions,

THE BLOOD HAS MANY FUNCTIONS

respectively (Figure 50-2). The stained strip of cellu-

lose acetate (or other supporting medium) is called an

The functions of blood—except for specific cellular

electrophoretogram. The amounts of these five bands

ones such as oxygen transport and cell-mediated im-

can be conveniently quantified by use of densitomet-

munologic defense—are carried out by plasma and its

ric scanning machines. Characteristic changes in the

constituents (Table 50-1).

amounts of one or more of these five bands are found

Plasma consists of water, electrolytes, metabolites,

in many diseases.

nutrients, proteins, and hormones. The water and elec-

trolyte composition of plasma is practically the same as

that of all extracellular fluids. Laboratory determina-

The Concentration of Protein in Plasma Is

tions of levels of Na⁺, K⁺, Ca2+, Cl⁻, HCO3−, PaCO₂,

Important in Determining the Distribution

and of blood pH are important in the management of

of Fluid Between Blood & Tissues

many patients.

In arterioles, the hydrostatic pressure is about 37 mm

PLASMA CONTAINS A COMPLEX

Hg, with an interstitial (tissue) pressure of 1 mm Hg

opposing it. The osmotic pressure (oncotic pressure)

MIXTURE OF PROTEINS

exerted by the plasma proteins is approximately 25 mm

The concentration of total protein in human plasma is

Hg. Thus, a net outward force of about 11 mm Hg

approximately 7.0-7.5 g/dL and comprises the major

drives fluid out into the interstitial spaces. In venules,

part of the solids of the plasma. The proteins of the

the hydrostatic pressure is about 17 mm Hg, with the

plasma are actually a complex mixture that includes not

oncotic and interstitial pressures as described above;

only simple proteins but also conjugated proteins such

thus, a net force of about 9 mm Hg attracts water back

as glycoproteins and various types of lipoproteins.

into the circulation. The above pressures are often re-

Thousands of antibodies are present in human plasma,

ferred to as the Starling forces. If the concentration of

though the amount of any one antibody is usually quite

plasma proteins is markedly diminished (eg, due to se-

low under normal circumstances. The relative dimen-

vere protein malnutrition), fluid is not attracted back

sions and molecular masses of some of the most impor-

into the intravascular compartment and accumulates in

tant plasma proteins are shown in Figure 50-1.

the extravascular tissue spaces, a condition known as

The separation of individual proteins from a com-

edema. Edema has many causes; protein deficiency is

plex mixture is frequently accomplished by the use of

one of them.

580

PLASMA PROTEINS & IMMUNOGLOBULINS

581

Table 50-1. Major functions of blood.

Plasma Proteins Have Been

Studied Extensively

(1)

Respiration—transport of oxygen from the lungs to the

Because of the relative ease with which they can be ob-

tissues and of CO₂ from the tissues to the lungs

tained, plasma proteins have been studied extensively in

(2)

Nutrition—transport of absorbed food materials

both humans and animals. Considerable information is

(3)

Excretion—transport of metabolic waste to the kidneys,

available about the biosynthesis, turnover, structure,

lungs, skin, and intestines for removal

and functions of the major plasma proteins. Alterations

(4)

Maintenance of the normal acid-base balance in the

of their amounts and of their metabolism in many dis-

body

ease states have also been investigated. In recent years,

(5)

Regulation of water balance through the effects of

many of the genes for plasma proteins have been cloned

blood on the exchange of water between the circulating

fluid and the tissue fluid

and their structures determined.

(6)

Regulation of body temperature by the distribution of

The preparation of antibodies specific for the indi-

body heat

vidual plasma proteins has greatly facilitated their

(7)

Defense against infection by the white blood cells and

study, allowing the precipitation and isolation of pure

circulating antibodies

proteins from the complex mixture present in tissues or

(8)

Transport of hormones and regulation of metabolism

plasma. In addition, the use of isotopes has made pos-

(9)

Transport of metabolites

sible the determination of their pathways of biosynthe-

(10)

Coagulation

sis and of their turnover rates in plasma.

The following generalizations have emerged from

studies of plasma proteins.

A. MOST PLASMA PROTEINS ARE

SYNTHESIZED IN THE LIVER

This has been established by experiments at the whole-

Scale

animal level (eg, hepatectomy) and by use of the iso-

lated perfused liver preparation, of liver slices, of liver

homogenates, and of in vitro translation systems using

10 nm

Na⁺

CI^-

Glucose

preparations of mRNA extracted from liver. However,

the γ-globulins are synthesized in plasma cells and cer-

tain plasma proteins are synthesized in other sites, such

as endothelial cells.

Albumin

Hemoglobin

69,000

64,500

B. PLASMA PROTEINS ARE GENERALLY SYNTHESIZED

ON MEMBRANE-BOUND POLYRIBOSOMES

They then traverse the major secretory route in the cell

β₁-Globulin

γ-Globulin

90,000

156,000

(rough endoplasmic membrane → smooth endoplasmic

membrane → Golgi apparatus → secretory vesicles) prior

to entering the plasma. Thus, most plasma proteins are

synthesized as preproteins and initially contain amino

terminal signal peptides (Chapter 46). They are usually

subjected to various posttranslational modifications (pro-

-Lipoprotein

α₁

teolysis, glycosylation, phosphorylation, etc) as they travel

200,000

through the cell. Transit times through the hepatocyte

β₁-Lipoprotein

1,300,000

from the site of synthesis to the plasma vary from 30 min-

utes to several hours or more for individual proteins.

C. MOST PLASMA PROTEINS ARE GLYCOPROTEINS

Fibrinogen

340,000

Accordingly, they generally contain either N- or O-

linked oligosaccharide chains, or both (Chapter 47). Al-

Figure 50-1. Relative dimensions and approximate

bumin is the major exception; it does not contain sugar

molecular masses of protein molecules in the blood

residues. The oligosaccharide chains have various func-

(Oncley).

tions

(Table 47-2). Removal of terminal sialic acid

582

CHAPTER 50

Albumin α₁

α₂

Albumin α₁

α₂

Figure 50-2. Technique of cellulose acetate zone electrophoresis. A: A small amount of serum or other

fluid is applied to a cellulose acetate strip. B: Electrophoresis of sample in electrolyte buffer is performed.

C: Separated protein bands are visualized in characteristic positions after being stained. D: Densitometer

scanning from cellulose acetate strip converts bands to characteristic peaks of albumin, α₁-globulin, α₂-glob-

ulin, β-globulin, and γ-globulin. (Reproduced, with permission, from Parslow TG et al [editors]: Medical Immunol-

ogy, 10th ed. McGraw-Hill, 2001.)

residues from certain plasma proteins (eg, ceruloplas-

nondenaturing conditions. This isotope unites covalently

min) by exposure to neuraminidase can markedly

with tyrosine residues in the protein. The labeled protein

shorten their half-lives in plasma (Chapter 47).

is freed of unbound ¹³¹I and its specific activity (disinte-

grations per minute per milligram of protein) deter-

D. MANY PLASMA PROTEINS EXHIBIT POLYMORPHISM

mined. A known amount of the radioactive protein is

A polymorphism is a mendelian or monogenic trait that

then injected into a normal adult subject, and samples of

exists in the population in at least two phenotypes, nei-

blood are taken at various time intervals for determina-

ther of which is rare (ie, neither of which occurs with

tions of radioactivity. The values for radioactivity are

frequency of less than 1-2%). The ABO blood group

plotted against time, and the half-life of the protein (the

substances (Chapter 52) are the best-known examples

time for the radioactivity to decline from its peak value

of human polymorphisms. Human plasma proteins

to one-half of its peak value) can be calculated from the

that exhibit polymorphism include α₁-antitrypsin, hap-

resulting graph, discounting the times for the injected

toglobin, transferrin, ceruloplasmin, and immunoglob-

protein to equilibrate (mix) in the blood and in the ex-

ulins. The polymorphic forms of these proteins can be

travascular spaces. The half-lives obtained for albumin

distinguished by different procedures (eg, various types

and haptoglobin in normal healthy adults are approxi-

of electrophoresis or isoelectric focusing), in which each

mately 20 and 5 days, respectively. In certain diseases,

form may show a characteristic migration. Analyses of

the half-life of a protein may be markedly altered. For in-

these human polymorphisms have proved to be of ge-

stance, in some gastrointestinal diseases such as regional

netic, anthropologic, and clinical interest.

ileitis

(Crohn disease), considerable amounts of plasma

proteins, including albumin, may be lost into the bowel

E. EACH PLASMA PROTEIN HAS A CHARACTERISTIC

through the inflamed intestinal mucosa. Patients with

HALF-LIFE IN THE CIRCULATION

this condition have a protein-losing gastroenteropathy,

The half-life of a plasma protein can be determined by

and the half-life of injected iodinated albumin in these

labeling the isolated pure protein with ¹³¹I under mild,

subjects may be reduced to as little as 1 day.

PLASMA PROTEINS & IMMUNOGLOBULINS

583

Table 50-2. Some functions of plasma proteins.

F. THE LEVELS OF CERTAIN PROTEINS IN PLASMA

INCREASE DURING ACUTE INFLAMMATORY STATES OR

SECONDARY TO CERTAIN TYPES OF TISSUE DAMAGE

Function

Plasma Proteins

These proteins are called “acute phase proteins” (or re-

Antiproteases

Antichymotrypsin

actants) and include C-reactive protein (CRP, so-named

α₁-Antitrypsin (α₁-antiproteinase)

because it reacts with the C polysaccharide of pneumo-

α₂-Macroglobulin

cocci), α₁-antitrypsin, haptoglobin, α₁-acid glycopro-

Antithrombin

tein, and fibrinogen. The elevations of the levels of these

Blood clotting

Various coagulation factors, fibrinogen

proteins vary from as little as 50% to as much as 1000-

fold in the case of CRP. Their levels are also usually ele-

Enzymes

Function in blood, eg, coagulation

vated during chronic inflammatory states and in pa-

factors, cholinesterase

tients with cancer. These proteins are believed to play a

Leakage from cells or tissues, eg, amino-

role in the body’s response to inflammation. For exam-

transferases

ple, C-reactive protein can stimulate the classic comple-

Hormones

Erythropoietin¹

ment pathway, and α₁-antitrypsin can neutralize certain

proteases released during the acute inflammatory state.

Immune defense

Immunoglobulins, complement proteins,

β₂-microglobulin

CRP is used as a marker of tissue injury, infection, and

inflammation, and there is considerable interest in its

Involvement in

Acute phase response proteins (eg,

use as a predictor of certain types of cardiovascular con-

inflammatory

C-reactive protein, α₁-acid glyco-

ditions secondary to atherosclerosis. Interleukin-1

responses

protein [orosomucoid])

(IL-1), a polypeptide released from mononuclear phago-

Oncofetal

α₁-Fetoprotein (AFP)

cytic cells, is the principal—but not the sole—stimula-

tor of the synthesis of the majority of acute phase reac-

Transport or

Albumin (various ligands, including bili-

tants by hepatocytes. Additional molecules such as IL-6

binding

rubin, free fatty acids, ions [Ca2+],

are involved, and they as well as IL-1 appear to work at

proteins

metals [eg, Cu2+, Zn2+], metheme,

the level of gene transcription.

steroids, other hormones, and a vari-

ety of drugs

Table 50-2 summarizes the functions of many of

Ceruloplasmin (contains Cu2+; albumin

the plasma proteins. The remainder of the material in

probably more important in physio-

this chapter presents basic information regarding se-

logic transport of Cu2+)

lected plasma proteins: albumin, haptoglobin, transfer-

Corticosteroid-binding globulin (trans-

rin, ceruloplasmin, α₁-antitrypsin, α₂-macroglobulin,

cortin) (binds cortisol)

the immunoglobulins, and the complement system.

Haptoglobin (binds extracorpuscular

The lipoproteins are discussed in Chapter 25.

hemoglobin)

Lipoproteins (chylomicrons, VLDL, LDL,

Albumin Is the Major Protein

HDL)

in Human Plasma

Hemopexin (binds heme)

Retinol-binding protein (binds retinol)

Albumin (69 kDa) is the major protein of human

Sex hormone-binding globulin (binds

plasma (3.4-4.7 g/dL) and makes up approximately

testosterone, estradiol)

60% of the total plasma protein. About 40% of albu-

Thyroid-binding globulin (binds T₄, T₃)

min is present in the plasma, and the other 60% is pre-

Transferrin (transport iron)

sent in the extracellular space. The liver produces about

Transthyretin (formerly prealbumin;

12 g of albumin per day, representing about 25% of

binds T₄ and forms a complex with

total hepatic protein synthesis and half its secreted pro-

retinol-binding protein)

tein. Albumin is initially synthesized as a prepropro-

¹Various other protein hormones circulate in the blood but are

tein. Its signal peptide is removed as it passes into the

not usually designated as plasma proteins. Similarly, ferritin is also

cisternae of the rough endoplasmic reticulum, and a

found in plasma in small amounts, but it too is not usually charac-

hexapeptide at the resulting amino terminal is subse-

terized as a plasma protein.

quently cleaved off farther along the secretory pathway.

The synthesis of albumin is depressed in a variety of

diseases, particularly those of the liver. The plasma of

patients with liver disease often shows a decrease in the

ratio of albumin to globulins

(decreased albumin-

globulin ratio). The synthesis of albumin decreases rela-

584

CHAPTER 50

tively early in conditions of protein malnutrition, such

of the kidney, enters the tubules, and tends to precipi-

as kwashiorkor.

tate therein (as can happen after a massive incompatible

Mature human albumin consists of one polypeptide

blood transfusion, when the capacity of haptoglobin to

chain of 585 amino acids and contains 17 disulfide

bind hemoglobin is grossly exceeded) (Figure 50-3).

bonds. By the use of proteases, albumin can be subdi-

However, the Hb-Hp complex is too large to pass

vided into three domains, which have different func-

through the glomerulus. The function of Hp thus ap-

tions. Albumin has an ellipsoidal shape, which means

pears to be to prevent loss of free hemoglobin into the

that it does not increase the viscosity of the plasma as

kidney. This conserves the valuable iron present in he-

much as an elongated molecule such as fibrinogen does.

moglobin, which would otherwise be lost to the body.

Because of its relatively low molecular mass (about 69

Human haptoglobin exists in three polymorphic

kDa) and high concentration, albumin is thought to be

forms, known as Hp 1-1, Hp 2-1, and Hp 2-2. Hp 1-1

responsible for 75-80% of the osmotic pressure of

migrates in starch gel electrophoresis as a single band,

human plasma. Electrophoretic studies have shown that

whereas Hp 2-1 and Hp 2-2 exhibit much more com-

the plasma of certain humans lacks albumin. These

plex band patterns. Two genes, designated Hp¹ and Hp²,

subjects are said to exhibit analbuminemia. One cause

direct these three phenotypes, with Hp 2-1 being the

of this condition is a mutation that affects splicing.

heterozygous phenotype. It has been suggested that the

Subjects with analbuminemia show only moderate

haptoglobin polymorphism may be associated with

edema, despite the fact that albumin is the major deter-

the prevalence of many inflammatory diseases.

minant of plasma osmotic pressure. It is thought that

The levels of haptoglobin in human plasma vary and

the amounts of the other plasma proteins increase and

are of some diagnostic use. Low levels of haptoglobin are

compensate for the lack of albumin.

found in patients with hemolytic anemias. This is ex-

Another important function of albumin is its ability

plained by the fact that whereas the half-life of haptoglo-

to bind various ligands. These include free fatty acids

bin is approximately 5 days, the half-life of the Hb-Hp

(FFA), calcium, certain steroid hormones, bilirubin,

complex is about 90 minutes, the complex being rapidly

and some of the plasma tryptophan. In addition, albu-

removed from plasma by hepatocytes. Thus, when hap-

min appears to play an important role in transport of

toglobin is bound to hemoglobin, it is cleared from the

copper in the human body (see below). A variety of

plasma about 80 times faster than normally. Accord-

drugs, including sulfonamides, penicillin G, dicumarol,

ingly, the level of haptoglobin falls rapidly in situations

and aspirin, are bound to albumin; this finding has im-

where hemoglobin is constantly being released from red

portant pharmacologic implications.

blood cells, such as occurs in hemolytic anemias. Hapto-

Preparations of human albumin have been widely

globin is an acute phase protein, and its plasma level is

used in the treatment of hemorrhagic shock and of

elevated in a variety of inflammatory states.

burns. However, this treatment is under review because

Certain other plasma proteins bind heme but not

some recent studies have suggested that administration of

hemoglobin. Hemopexin is a β₁-globulin that binds

albumin in these conditions may increase mortality rates.

free heme. Albumin will bind some metheme (ferric

heme) to form methemalbumin, which then transfers

the metheme to hemopexin.

Haptoglobin Binds Extracorpuscular

Hemoglobin, Preventing Free Hemoglobin

Absorption of Iron From the Small

From Entering the Kidney

Intestine Is Tightly Regulated

Haptoglobin (Hp) is a plasma glycoprotein that binds

Transferrin (Tf) is a plasma protein that plays a central

extracorpuscular hemoglobin (Hb) in a tight noncova-

role in transporting iron around the body to sites where

lent complex (Hb-Hp). The amount of haptoglobin in

human plasma ranges from 40 mg to 180 mg of hemo-

globin-binding capacity per deciliter. Approximately

10% of the hemoglobin that is degraded each day is re-

Hb → Kidney → Excreted in urine or precipitates in tubules;

leased into the circulation and is thus extracorpuscular.

(MW 65,000)

iron is lost to body

The other 90% is present in old, damaged red blood

+ Hp → Hb : Hp complex → Kidney

cells, which are degraded by cells of the histiocytic sys-

(MW 65,000)

(MW 90,000)

(MW 155,000)

tem. The molecular mass of hemoglobin is approxi-

mately 65 kDa, whereas the molecular mass of the sim-

Catabolized by liver cells;

plest polymorphic form of haptoglobin (Hp 1-1) found

iron is conserved and reused

in humans is approximately 90 kDa. Thus, the Hb-Hp

complex has a molecular mass of approximately 155

Figure 50-3. Different fates of free hemoglobin and

kDa. Free hemoglobin passes through the glomerulus

of the hemoglobin-haptoglobin complex.

PLASMA PROTEINS & IMMUNOGLOBULINS

585

it is needed. Before we discuss it further, certain aspects

Table 50-3. Distribution of iron in a 70-kg

of iron metabolism will be reviewed.

adult male.¹

Iron is important in the human body because of its

occurrence in many hemoproteins such as hemoglobin,

Transferrin

3-4 mg

myoglobin, and the cytochromes. It is ingested in the

Hemoglobin in red blood cells

2500 mg

diet either as heme or nonheme iron (Figure 50-4); as

In myoglobin and various enzymes

300 mg

shown, these different forms involve separate pathways.

In stores (ferritin and hemosiderin)

1000 mg

Absorption of iron in the proximal duodenum is tightly

Absorption

1 mg/d

regulated, as there is no physiologic pathway for its ex-

Losses

1 mg/d

cretion from the body. Under normal circumstances,

¹In an adult female of similar weight, the amount in stores would

the body guards its content of iron zealously, so that a

generally be less (100-400 mg) and the losses would be greater

healthy adult male loses only about 1 mg/d, which is re-

(1.5-2 mg/d).

placed by absorption. Adult females are more prone to

states of iron deficiency because some may lose excessive

blood during menstruation. The amounts of iron in var-

ious body compartments are shown in Table 50-3.

brane into the plasma, where it is carried by transferrin

Enterocytes in the proximal duodenum are responsi-

(see below). Passage across the basolateral membrane

ble for absorption of iron. Incoming iron in the Fe3+

appears to be carried out by another protein, possibly

state is reduced to Fe2+ by a ferrireductase present on

iron regulatory protein 1 (IREG1). This protein may

the surface of enterocytes. Vitamin C in food also favors

interact with the copper-containing protein hephaestin,

reduction of ferric iron to ferrous iron. The transfer of

a protein similar to ceruloplasmin (see below). Hepha-

iron from the apical surfaces of enterocytes into their in-

estin is thought to have a ferroxidase activity, which is

teriors is performed by a proton-coupled divalent metal

important in the release of iron from cells. Thus, Fe2+ is

transporter

(DMT1). This protein is not specific for

converted back to Fe3+, the form in which it is trans-

iron, as it can transport a wide variety of divalent cations.

ported in the plasma by transferrin.

Once inside an enterocyte, iron can either be stored

Overall regulation of iron absorption is complex

as ferritin or transferred across the basolateral mem-

and not well understood mechanistically. It occurs at

Brush

border

Intestinal

Enterocyte

Blood

lumen

Heme

Fe3+

Fe2⁺

reductase

Fe2⁺

DMT1

Fe2⁺

Fe3+-ferritin

Fe3⁺

Shed

Fe3⁺−TF

Figure 50-4. Absorption of iron. Fe3+ is converted to Fe2+ by ferric reductase,

and Fe2+ is transported into the enterocyte by the apical membrane iron trans-

porter DMT1. Heme is transported into the enterocyte by a separate heme

transporter (HT), and heme oxidase (HO) releases Fe2+ from the heme. Some of

the intracellular Fe2+ is converted to Fe3+ and bound by ferritin. The remainder

binds to the basolateral Fe2+ transporter (FP) and is transported into the blood-

stream, aided by hephaestin (HP). In plasma, Fe3+ is bound to the iron transport

protein transferrin (TF). (Reproduced, with permission, from Ganong WF: Review of

Medical Physiology, 21st ed. McGraw-Hill, 2003.)

586

CHAPTER 50

the level of the enterocyte, where further absorption of

the protein is normally only one-third saturated with

iron is blocked if a sufficient amount has been taken up

iron. In iron deficiency anemia, the protein is even less

(so-called dietary regulation exerted by

“mucosal

saturated with iron, whereas in conditions of storage of

block”). It also appears to be responsive to the overall

excess iron in the body (eg, hemochromatosis) the satu-

requirement of erythropoiesis for iron (erythropoietic

ration with iron is much greater than one-third.

regulation). Absorption is excessive in hereditary he-

mochromatosis (see below).

Ferritin Stores Iron in Cells

Ferritin is another protein that is important in the me-

Transferrin Shuttles Iron to Sites

tabolism of iron. Under normal conditions, it stores

Where It Is Needed

iron that can be called upon for use as conditions re-

Transferrin (Tf ) is a β₁-globulin with a molecular mass

quire. In conditions of excess iron (eg, hemochromato-

of approximately 76 kDa. It is a glycoprotein and is

sis), body stores of iron are greatly increased and much

synthesized in the liver. About 20 polymorphic forms

more ferritin is present in the tissues, such as the liver

of transferrin have been found. It plays a central role in

and spleen. Ferritin contains approximately 23% iron,

the body’s metabolism of iron because it transports iron

and apoferritin (the protein moiety free of iron) has a

(2 mol of Fe3+ per mole of Tf) in the circulation to sites

molecular mass of approximately 440 kDa. Ferritin is

where iron is required, eg, from the gut to the bone

composed of 24 subunits of 18.5 kDa, which surround

marrow and other organs. Approximately 200 billion

in a micellar form some 3000-4500 ferric atoms. Nor-

red blood cells (about 20 mL) are catabolized per day,

mally, there is a little ferritin in human plasma. How-

releasing about 25 mg of iron into the body—most of

ever, in patients with excess iron, the amount of ferritin

which will be transported by transferrin.

in plasma is markedly elevated. The amount of ferritin

There are receptors (TfRs) on the surfaces of many

in plasma can be conveniently measured by a sensitive

cells for transferrin. It binds to these receptors and is in-

and specific radioimmunoassay and serves as an index

ternalized by receptor-mediated endocytosis (compare

of body iron stores.

the fate of LDL; Chapter 25). The acid pH inside the

Synthesis of the transferrin receptor (TfR) and that

lysosome causes the iron to dissociate from the protein.

of ferritin are reciprocally linked to cellular iron con-

The dissociated iron leaves the endosome via DMT1 to

tent. Specific untranslated sequences of the mRNAs for

enter the cytoplasm. Unlike the protein component of

both proteins (named iron response elements) interact

LDL, apoTf is not degraded within the lysosome. In-

with a cytosolic protein sensitive to variations in levels

stead, it remains associated with its receptor, returns to

of cellular iron (iron-responsive element-binding pro-

the plasma membrane, dissociates from its receptor,

tein). When iron levels are high, cells use stored ferritin

reenters the plasma, picks up more iron, and again de-

mRNA to synthesize ferritin, and the TfR mRNA is de-

livers the iron to needy cells.

graded. In contrast, when iron levels are low, the TfR

Abnormalities of the glycosylation of transferrin

mRNA is stabilized and increased synthesis of receptors

occur in the congenital disorders of glycosylation

occurs, while ferritin mRNA is apparently stored in an

(Chapter 47) and in chronic alcohol abuse. Their detec-

inactive form. This is an important example of control

tion by, for example, isoelectric focusing is used to help

of expression of proteins at the translational level.

diagnose these conditions.

Hemosiderin is a somewhat ill-defined molecule; it

appears to be a partly degraded form of ferritin but still

containing iron. It can be detected by histologic stains

Iron Deficiency Anemia

(eg, Prussian blue) for iron, and its presence is deter-

Is Extremely Prevalent

mined histologically when excessive storage of iron

Attention to iron metabolism is particularly impor-

occurs.

tant in women for the reason mentioned above. Addi-

tionally, in pregnancy, allowances must be made for

Hereditary Hemochromatosis Is Due

the growing fetus. Older people with poor dietary

to Mutations in the HFE Gene

habits (“tea and toasters”) may develop iron deficiency.

Iron deficiency anemia due to inadequate intake, inade-

Hereditary (primary) hemochromatosis is a very preva-

quate utilization, or excessive loss of iron is one of the

lent autosomal recessive disorder in certain parts of the

most prevalent conditions seen in medical practice.

world (eg, Scotland, Ireland, and North America). It is

The concentration of transferrin in plasma is approx-

characterized by excessive storage of iron in tissues, lead-

imately 300 mg/dL. This amount of transferrin can

ing to tissue damage. Total body iron ranges between

bind 300 µg of iron per deciliter, so that this represents

2.5 g and 3.5 g in normal adults; in primary hemochro-

the total iron-binding capacity of plasma. However,

matosis it usually exceeds 15 g. The accumulated iron

PLASMA PROTEINS & IMMUNOGLOBULINS

587

damages organs and tissues such as the liver, pancreatic

Mutations in HFE, located on chromosome 6p21.3,

islets, and heart, perhaps in part due to effects on free

leading to abnormalities in the structure

radical production (Chapter 52). Melanin and various

of its protein product

amounts of iron accumulate in the skin, accounting for

the slate-gray color often seen. The precise cause of

Loss of regulation of absorption of iron

melanin accumulation is not clear. The frequent coexis-

in the small intestine

tence of diabetes mellitus (due to islet damage) and the

skin pigmentation led to use of the term bronze dia-

betes for this condition. In 1995, Feder and colleagues

Accumulation of iron in various tissues, but particularly

isolated a gene, now known as HFE, located on chromo-

liver, pancreatic islets, skin, and heart muscle

some 6 close to the major histocompatibility complex

genes. The encoded protein (HFE) was found to be re-

Iron directly or indirectly causes damage to the

lated to MHC class 1 antigens. Initially, two different

above tissues, resulting in hepatic cirrhosis, diabetes

missense mutations were found in HFE in individuals

mellitus, skin pigmentation, and cardiac problems

with hereditary hemochromatosis. The more frequent

mutation was one that changed cysteinyl residue 282 to

Figure 50-5. Tentative scheme of the main events

a tyrosyl residue (CY282Y), disrupting the structure of

in causation of primary hemochromatosis (MIM

the protein. The other mutation changed histidyl resi-

235200). The two principal mutations are CY282Y and

due 63 to an aspartyl residue (H63D). Some patients

H63D (see text). Mutations in genes other than HFE are

with hereditary hemochromatosis have neither muta-

also involved in some cases.

tion, perhaps due to other mutations in HFE or because

one or more other genes may be involved in its causa-

tion. Genetic screening for this condition has been eval-

uated but is not presently recommended. However, test-

carries 90% of the copper present in plasma. Each mol-

ing for HFE mutations in individuals with elevated

ecule of ceruloplasmin binds six atoms of copper very

serum iron concentrations may be useful.

tightly, so that the copper is not readily exchangeable.

HFE has been shown to be located in cells in the

Albumin carries the other 10% of the plasma copper

crypts of the small intestine, the site of iron absorption.

but binds the metal less tightly than does ceruloplas-

There is evidence that it associates with β₂-microglobu-

min. Albumin thus donates its copper to tissues more

lin, an association that may be necessary for its stability,

readily than ceruloplasmin and appears to be more im-

intracellular processing, and cell surface expression. The

portant than ceruloplasmin in copper transport in the

complex interacts with the transferrin receptor (TfR);

human body. Ceruloplasmin exhibits a copper-depen-

how this leads to excessive storage of iron when HFE is

dent oxidase activity, but its physiologic significance

altered by mutation is under close study. The mouse

has not been clarified. The amount of ceruloplasmin in

homolog of HFE has been knocked out, resulting in a

plasma is decreased in liver disease. In particular, low

potentially useful animal model of hemochromatosis.

levels of ceruloplasmin are found in Wilson disease

A scheme of the likely main events in the causation of

(hepatolenticular degeneration), a disease due to abnor-

hereditary hemochromatosis is set forth in Figure 50-5.

mal metabolism of copper. In order to clarify the de-

Secondary hemochromatosis can occur after re-

scription of Wilson disease, we shall first consider the

peated transfusions (eg, for treatment of sickle cell ane-

metabolism of copper in the human body and then

mia), excessive oral intake of iron (eg, by African Bantu

Menkes disease, another condition involving abnormal

peoples who consume alcoholic beverages fermented in

copper metabolism.

containers made of iron), or a number of other condi-

tions.

Table 50-4 summarizes laboratory tests useful in the

assessment of patients with abnormalities of iron me-

Table 50-4. Laboratory tests for assessing

tabolism.

patients with disorders of iron metabolism.

• Red blood cell count and estimation of hemoglobin

Ceruloplasmin Binds Copper, & Low Levels

• Determinations of plasma iron, total iron-binding capacity

of This Plasma Protein Are Associated

(TIBC), and % transferrin saturation

With Wilson Disease

• Determination of ferritin in plasma by radioimmunoassay

• Prussian blue stain of tissue sections

Ceruloplasmin (about 160 kDa) is an α₂-globulin. It

• Determination of amount of iron (µg/g) in a tissue biopsy

has a blue color because of its high copper content and

588

CHAPTER 50

Copper Is a Cofactor for Certain Enzymes

only male infants, involves the nervous system, connec-

tive tissue, and vasculature, and is usually fatal in in-

Copper is an essential trace element. It is required in

fancy. In 1993, it was reported that the basis of Menkes

the diet because it is the metal cofactor for a variety of

disease was mutations in the gene for a copper-binding

enzymes (see Table 50-5). Copper accepts and donates

P-type ATPase. Interestingly, the enzyme showed struc-

electrons and is involved in reactions involving dismu-

tural similarity to certain metal-binding proteins in mi-

tation, hydroxylation, and oxygenation. However, ex-

croorganisms. This ATPase is thought to be responsible

cess copper can cause problems because it can oxidize

for directing the efflux of copper from cells. When al-

proteins and lipids, bind to nucleic acids, and enhance

tered by mutation, copper is not mobilized normally

the production of free radicals. It is thus important to

from the intestine, in which it accumulates, as it does in

have mechanisms that will maintain the amount of

a variety of other cells and tissues, from which it cannot

copper in the body within normal limits. The body of

exit. Despite the accumulation of copper, the activities

the normal adult contains about 100 mg of copper, lo-

of many copper-dependent enzymes are decreased, per-

cated mostly in bone, liver, kidney, and muscle. The

haps because of a defect of its incorporation into the

daily intake of copper is about 2-4 mg, with about

apoenzymes. Normal liver expresses very little of the

50% being absorbed in the stomach and upper small

ATPase, which explains the absence of hepatic involve-

intestine and the remainder excreted in the feces. Cop-

ment in Menkes disease. This work led to the sugges-

per is carried to the liver bound to albumin, taken up

tion that liver might contain a different copper-binding

by liver cells, and part of it is excreted in the bile. Cop-

ATPase, which could be involved in the causation of

per also leaves the liver attached to ceruloplasmin,

Wilson disease. As described below, this turned out to

which is synthesized in that organ.

be the case.

The Tissue Levels of Copper & of Certain

Wilson Disease Is Also Due to Mutations

Other Metals Are Regulated in

in a Gene Encoding a Copper-Binding

Part by Metallothioneins

P-Type ATPase

Metallothioneins are a group of small proteins (about

Wilson disease is a genetic disease in which copper fails

6.5 kDa), found in the cytosol of cells, particularly of

to be excreted in the bile and accumulates in liver,

liver, kidney, and intestine. They have a high content of

brain, kidney, and red blood cells. It can be regarded as

cysteine and can bind copper, zinc, cadmium, and mer-

an inability to maintain a near-zero copper balance, re-

cury. The SH groups of cysteine are involved in binding

sulting in copper toxicosis. The increase of copper in

the metals. Acute intake (eg, by injection) of copper and

liver cells appears to inhibit the coupling of copper to

of certain other metals increases the amount (induction)

apoceruloplasmin and leads to low levels of ceruloplas-

of these proteins in tissues, as does administration of

min in plasma. As the amount of copper accumulates,

certain hormones or cytokines. These proteins may

patients may develop a hemolytic anemia, chronic liver

function to store the above metals in a nontoxic form

disease (cirrhosis, hepatitis), and a neurologic syndrome

and are involved in their overall metabolism in the

owing to accumulation of copper in the basal ganglia

body. Sequestration of copper also diminishes the

and other centers. A frequent clinical finding is the

amount of this metal available to generate free radicals.

Kayser-Fleischer ring. This is a green or golden pig-

ment ring around the cornea due to deposition of cop-

Menkes Disease Is Due to Mutations

per in Descemet’s membrane. The major laboratory

in the Gene Encoding a Copper-

tests of copper metabolism are listed in Table 50-6. If

Binding P-Type ATPase

Wilson disease is suspected, a liver biopsy should be

Menkes disease (“kinky” or “steely” hair disease) is a

performed; a value for liver copper of over 250 µg per

disorder of copper metabolism. It is X-linked, affects

gram dry weight along with a plasma level of cerulo-

plasmin of under 20 mg/dL is diagnostic.

The cause of Wilson disease was also revealed in

1993, when it was reported that a variety of mutations

Table 50-5. Some important enzymes that

in a gene encoding a copper-binding P-type ATPase

contain copper.

were responsible. The gene is estimated to encode a

protein of 1411 amino acids, which is highly homolo-

• Amine oxidase

gous to the product of the gene affected in Menkes dis-

• Copper-dependent superoxide dismutase

ease. In a manner not yet fully explained, a nonfunc-

• Cytochrome oxidase

tional ATPase causes defective excretion of copper into

• Tyrosinase

the bile, a reduction of incorporation of copper into

PLASMA PROTEINS & IMMUNOGLOBULINS

589

Table 50-6. Major laboratory tests used in the

other proteases by forming complexes with them. At

investigation of diseases of copper metabolism.¹

least 75 polymorphic forms occur, many of which can

be separated by electrophoresis. The major genotype is

MM, and its phenotypic product is PiM. There are two

Normal Adult

areas of clinical interest concerning α₁-antitrypsin. A de-

Test

Range

ficiency of this protein has a role in certain cases (ap-

Serum copper

10-22 µmol/L

proximately 5%) of emphysema. This occurs mainly in

subjects with the ZZ genotype, who synthesize PiZ, and

Ceruloplasmin

200-600 mg/L

also in PiSZ heterozygotes, both of whom secrete con-

Urinary copper

< 1 µmol/24 h

siderably less protein than PiMM individuals. Consider-

Liver copper

20-50 µg/g dry weight

ably less of this protein is secreted as compared with

PiM. When the amount of α₁-antitrypsin is deficient

¹Based on Gaw A et al: Clinical Biochemistry. Churchill Livingstone,

and polymorphonuclear white blood cells increase in the

1995.

lung (eg, during pneumonia), the affected individual

lacks a countercheck to proteolytic damage of the lung

by proteases such as elastase (Figure 50-6). It is of con-

apoceruloplasmin, and the accumulation of copper in

siderable interest that a particular methionine (residue

liver and subsequently in other organs such as brain.

358) of α₁-antitrypsin is involved in its binding to pro-

Treatment for Wilson disease consists of a diet low

teases. Smoking oxidizes this methionine to methionine

in copper along with lifelong administration of penicil-

sulfoxide and thus inactivates it. As a result, affected

lamine, which chelates copper, is excreted in the urine,

molecules of α₁-antitrypsin no longer neutralize

and thus depletes the body of the excess of this mineral.

proteases. This is particularly devastating in patients

Another condition involving ceruloplasmin is aceru-

(eg, PiZZ phenotype) who already have low levels of

loplasminemia. In this genetic disorder, levels of cerulo-

α₁-antitrypsin. The further diminution in α₁-antitrypsin

plasmin are very low and consequently its ferroxidase ac-

brought about by smoking results in increased proteolytic

tivity is markedly deficient. This leads to failure of release

destruction of lung tissue, accelerating the development

of iron from cells, and iron accumulates in certain brain

of emphysema. Intravenous administration of α₁-anti-

cells, hepatocytes, and pancreatic islet cells. Affected indi-

trypsin (augmentation therapy) has been used as an ad-

viduals show severe neurologic signs and have diabetes

junct in the treatment of patients with emphysema due

mellitus. Use of a chelating agent or administration of

to α₁-antitrypsin deficiency. Attempts are being made,

plasma or ceruloplasmin concentrate may be beneficial.

using the techniques of protein engineering, to replace

methionine 358 by another residue that would not be

subject to oxidation. The resulting “mutant” α₁-anti-

Deficiency of

trypsin would thus afford protection against proteases

1-Antiproteinase

for a much longer period of time than would native

(

1-Antitrypsin) Is Associated

α₁-antitrypsin. Attempts are also being made to develop

With Emphysema & One Type

gene therapy for this condition. One approach is to use

of Liver Disease

a modified adenovirus (a pathogen of the respiratory

α₁-Antiproteinase (about 52 kDa) was formerly called

tract) into which the gene for α₁-antitrypsin has been

α₁-antitrypsin, and this name is retained here. It is a

inserted. The virus would then be introduced into the

single-chain protein of 394 amino acids, contains three

respiratory tract (eg, by an aerosol). The hope is that

oligosaccharide chains, and is the major component

pulmonary epithelial cells would express the gene and

(> 90%) of the α₁ fraction of human plasma. It is syn-

secrete α₁-antitrypsin locally. Experiments in animals

thesized by hepatocytes and macrophages and is the

have indicated the feasibility of this approach.

principal serine protease inhibitor (serpin, or Pi) of

Deficiency of α₁-antitrypsin is also implicated in

human plasma. It inhibits trypsin, elastase, and certain

one type of liver disease (α₁-antitrypsin deficiency liver

A. Active elastase + α₁-AT → Inactive elastase: α₁-AT complex → No proteolysis of lung → No tissue damage

B. Active elastase + or no α₁-AT → Active elastase → Proteolysis of lung → Tissue damage

Figure 50-6. Scheme illustrating (A) normal inactivation of elastase by α₁-antitrypsin and

(B) situation in which the amount of α₁-antitrypsin is substantially reduced, resulting in pro-

teolysis by elastase and leading to tissue damage.

590

CHAPTER 50

disease). In this condition, molecules of the ZZ pheno-

comprises 8-10% of the total plasma protein in hu-

type accumulate and aggregate in the cisternae of the

mans. Approximately 10% of the zinc in plasma is

endoplasmic reticulum of hepatocytes. Aggregation is

transported by α₂-macroglobulin, the remainder being

due to formation of polymers of mutant α₁-antitrypsin,

transported by albumin. The protein is synthesized by a

the polymers forming via a strong interaction between a

variety of cell types, including monocytes, hepatocytes,

specific loop in one molecule and a prominent β-

and astrocytes. It is the major member of a group of

pleated sheet in another (loop-sheet polymerization).

plasma proteins that include complement proteins C3

By mechanisms that are not understood, hepatitis re-

and C4. These proteins contain a unique internal cyclic

sults with consequent cirrhosis (accumulation of mas-

thiol ester bond (formed between a cysteine and a glut-

sive amounts of collagen, resulting in fibrosis). It is pos-

amine residue) and for this reason have been designated

sible that administration of a synthetic peptide

as the thiol ester plasma protein family.

resembling the loop sequence could inhibit loop-sheet

α₂-Macroglobulin binds many proteinases and is

polymerization. Diseases such as α₁-antitrypsin defi-

thus an important panproteinase inhibitor. The α₂-

ciency, in which cellular pathology is primarily caused

macroglobulin-proteinase complexes are rapidly cleared

by the presence of aggregates of aberrant forms of indi-

from the plasma by a receptor located on many cell

vidual proteins, have been named conformational dis-

types. In addition, α₂-macroglobulin binds many cy-

eases. Most appear to be due to the formation by con-

tokines (platelet-derived growth factor, transforming

formationally unstable proteins of β sheets, which in

growth factor-β, etc) and appears to be involved in tar-

turn leads to formation of aggregates. Other members

geting them toward particular tissues or cells. Once

of this group of conditions include Alzheimer disease,

taken up by cells, the cytokines can dissociate from α₂-

Parkinson disease, and Huntington disease.

macroglobulin and subsequently exert a variety of ef-

At present, severe α₁-antitrypsin deficiency liver dis-

fects on cell growth and function. The binding of pro-

ease can be successfully treated by liver transplantation.

teinases and cytokines by α₂-macroglobulin involves

In the future, introduction of the gene for normal α₁-

different mechanisms that will not be considered here.

antitrypsin into hepatocytes may become possible, but

this would not stop production of the PiZ protein. Fig-

Amyloidosis Occurs by the Deposition

ure 50-7 is a scheme of the causation of this disease.

of Fragments of Various Plasma

Proteins in Tissues

2-Macroglobulin Neutralizes Many

Amyloidosis is the accumulation of various insoluble

Proteases & Targets Certain

fibrillar proteins between the cells of tissues to an extent

Cytokines to Tissues

that affects function. The fibrils generally represent

α₂-Macroglobulin is a large plasma glycoprotein (720

proteolytic fragments of various plasma proteins and

kDa) made up of four identical subunits of 180 kDa. It

possess a β-pleated sheet structure. The term “amyloi-

dosis” is a misnomer, as it was originally thought that

the fibrils were starch-like in nature. Among the most

common precursor proteins are immunoglobulin light

chains (see below), amyloid-associated protein derived

GAG to AAG mutation in exon 5 of gene for α₁-AT

from serum amyloid-associated protein (a plasma glyco-

on chromosome 14

protein), and transthyretin (Table 50-2). The precursor

proteins in plasma are generally either increased in

amount (eg, immunoglobulin light chains in multiple

Results in Glu³⁴² to Lys³⁴² substitution in α₁-AT,

myeloma or β₂-microglobulin in patients being main-

causing formation of PiZZ

tained on chronic dialysis) or mutant forms (eg, of

transthyretin in familial amyloidotic neuropathies).

PiZZ accumulates in cisternae

The precise factors that determine the deposition of

of endoplasmic reticulum and aggregates

proteolytic fragments in tissues await elucidation.

via loop-sheet polymerization

Other proteins have been found in amyloid fibrils, such

as calcitonin and amyloid β protein (not derived from a

Leads to hepatitis (mechanism unknown)

plasma protein) in Alzheimer disease; a total of about

and cirrhosis in ~10% of ZZ homozygotes

15 different proteins have been found. All fibrils have a

P component associated with them, which is derived

Figure 50-7. Scheme of causation of α₁-antitrypsin-

from serum amyloid P component, a plasma protein

deficiency liver disease. The mutation shown causes

closely related to C-reactive protein. Tissue sections

formation of PiZZ (MIM 107400). (α₁-AT, α₁-antitrypsin.)

containing amyloid fibrils interact with Congo red stain

PLASMA PROTEINS & IMMUNOGLOBULINS

591

and display striking green birefringence when viewed

munoglobulins other than direct binding of antigens.

by polarizing microscopy. Deposition of amyloid oc-

Because there are two Fab regions, IgG molecules bind

curs in patients with a variety of disorders; treatment of

two molecules of antigen and are termed divalent. The

the underlying disorder should be provided if possible.

site on the antigen to which an antibody binds is

termed an antigenic determinant, or epitope. The

area in which papain cleaves the immunoglobulin mol-

PLASMA IMMUNOGLOBULINS PLAY

ecule—ie, the region between the C_H1 and C_H2 do-

A MAJOR ROLE IN THE BODY’S

mains—is referred to as the “hinge region.” The hinge

DEFENSE MECHANISMS

region confers flexibility and allows both Fab arms to

The immune system of the body consists of two major

move independently, thus helping them to bind to

components: B lymphocytes and T lymphocytes. The

antigenic sites that may be variable distances apart (eg,

B lymphocytes are mainly derived from bone marrow

on bacterial surfaces). Fc and hinge regions differ in the

cells in higher animals and from the bursa of Fabricius

different classes of antibodies, but the overall model of

in birds. The T lymphocytes are of thymic origin. The

antibody structure for each class is similar to that

B cells are responsible for the synthesis of circulating,

shown in Figure 50-8 for IgG.

humoral antibodies, also known as immunoglobulins.

The T cells are involved in a variety of important cell-

All Light Chains Are Either Kappa

mediated immunologic processes such as graft rejec-

or Lambda in Type

tion, hypersensitivity reactions, and defense against ma-

lignant cells and many viruses. This section considers

There are two general types of light chains, kappa (κ)

only the plasma immunoglobulins, which are synthe-

and lambda (λ), which can be distinguished on the

sized mainly in plasma cells. These are specialized cells

basis of structural differences in their C_L regions. A

of B cell lineage that synthesize and secrete immuno-

given immunoglobulin molecule always contains two κ

globulins into the plasma in response to exposure to a

or two λ light chains—never a mixture of κ and λ. In

variety of antigens.

humans, the κ chains are more frequent than λ chains

in immunoglobulin molecules.

All Immunoglobulins Contain a Minimum

of Two Light & Two Heavy Chains

The Five Types of Heavy Chain Determine

Immunoglobulin Class

Immunoglobulins contain a minimum of two iden-

tical light (L) chains (23 kDa) and two identical heavy

Five classes of H chain have been found in humans

(H) chains (53-75 kDa), held together as a tetramer

(Table 50-7), distinguished by differences in their C_H

(L₂H₂) by disulfide bonds The structure of IgG is

regions. They are designated γ, α, µ δ, and ε. The µ

shown in Figure 50-8; it is Y-shaped, with binding of

and ε chains each have four C_H domains rather than

antigen occurring at both tips of the Y. Each chain can

the usual three. The type of H chain determines the

be divided conceptually into specific domains, or re-

class of immunoglobulin and thus its effector function.

gions, that have structural and functional significance.

There are thus five immunoglobulin classes: IgG, IgA,

The half of the light (L) chain toward the carboxyl ter-

IgM, IgD, and IgE. The biologic functions of these

minal is referred to as the constant region (C_L), while

five classes are summarized in Table 50-8.

the amino terminal half is the variable region of the

light chain

(V_L). Approximately one-quarter of the

No Two Variable Regions Are Identical

heavy (H) chain at the amino terminals is referred to as

its variable region (V_H), and the other three-quarters of

The variable regions of immunoglobulin molecules

the heavy chain are referred to as the constant regions

consist of the V_L and V_H domains and are quite hetero-

(C_H1, C_H2, C_H3) of that H chain. The portion of the

geneous. In fact, no two variable regions from different

immunoglobulin molecule that binds the specific anti-

humans have been found to have identical amino acid

gen is formed by the amino terminal portions (variable

sequences. However, amino acid analyses have shown

regions) of both the H and L chains—ie, the V_H and

that the variable regions are comprised of relatively in-

VL domains. The domains of the protein chains consist

variable regions and other hypervariable regions (Figure

of two sheets of antiparallel distinct stretches of amino

50-9). L chains have three hypervariable regions (in

acids that bind antigen.

VL) and H chains have four (in VH). These hypervari-

As depicted in Figure 50-8, digestion of an im-

able regions comprise the antigen-binding site (located

munoglobulin by the enzyme papain produces two

at the tips of the Y shown in Figure 50-8) and dictate

antigen-binding fragments (Fab) and one crystallizable

the amazing specificity of antibodies. For this reason,

fragment (Fc), which is responsible for functions of im-

hypervariable regions are also termed complementar-

592

CHAPTER 50

⁺H3N

V_L

Fab

⁺H₃N

C_L

Hinge region

V_H

C_H2

C_H3

C_H1

S S

COO—

S S

H chain

COO—

S S

Pepsin

Cleavage sites

Papain

⁺H₃N

Fab

⁺H₃N

Figure 50-8. Structure of IgG. The molecule consists of two light (L) chains and

two heavy (H) chains. Each light chain consists of a variable (V_L) and a constant (C_L)

region. Each heavy chain consists of a variable region (V_H) and a constant region

that is divided into three domains (C_H1, C_H2, and C_H3). The C_H2 domain contains

the complement-binding site and the C_H3 domain contains a site that attaches to

receptors on neutrophils and macrophages. The antigen-binding site is formed by

the hypervariable regions of both the light and heavy chains, which are located in

the variable regions of these chains (see Figure 50-9). The light and heavy chains

are linked by disulfide bonds, and the heavy chains are also linked to each other

by disulfide bonds. (Reproduced, with permission, from Parslow TG et al [editors]:

Medical Immunology, 10th ed. McGraw-Hill, 2001.)

ity-determining regions (CDRs). About five to ten

specificities, a feature that contributes to the tremen-

amino acids in each hypervariable region (CDR) con-

dous diversity of antibody molecules and is termed

tribute to the antigen-binding site. CDRs are located

combinatorial diversity. Large antigens interact with

on small loops of the variable domains, the surrounding

all of the CDRs of an antibody, whereas small ligands

polypeptide regions between the hypervariable regions

may interact with only one or a few CDRs that form a

being termed framework regions. CDRs from both

pocket or groove in the antibody molecule. The essence

VH and VL domains, brought together by folding of the

of antigen-antibody interactions is mutual complemen-

polypeptide chains in which they are contained, form a

tarity between the surfaces of CDRs and epitopes. The

single hypervariable surface comprising the antigen-

interactions between antibodies and antigens involve

binding site. Various combinations of H and L chain

noncovalent forces and bonds (electrostatic and van der

CDRs can give rise to many antibodies of different

Waals forces and hydrogen and hydrophobic bonds).

PLASMA PROTEINS & IMMUNOGLOBULINS

593

Table 50-7. Properties of human immunoglobulins.¹

Property

IgG

IgA

IgM

IgD

IgE

Percentage of total immunoglo-

0.2

0.004

bulin in serum (approximate)

Serum concentration

1000

200

120

0.05

(mg/dL) (approximate)

Sedimentation coefficient

7S or 11S²

19S

Molecular weight

150

170 or

900

180

190

(× 1000)

400²

Structure

Monomer

Monomer or dimer

Monomer

H-chain symbol

Complement fixation

−

Transplacental passage

−

Mediation of allergic responses

−

Found in secretions

−

Opsonization

−

−³

−

Antigen receptor on B cell

−

Polymeric form contains J chain

−

¹Reproduced, with permission, from Levinson W, Jawetz E: Medical Microbiology and Immunology, 7th ed. McGraw-Hill,

2002.

²The 11S form is found in secretions (eg, saliva, milk, tears) and fluids of the respiratory, intestinal, and genital tracts.

³IgM opsonizes indirectly by activating complement. This produces C3b, which is an opsonin.

The Constant Regions Determine

(V_L) gene, a joining region (J) gene (bearing no rela-

Class-Specific Effector Functions

tionship to the J chain of IgA or IgM), and a constant

region (C_L) gene. Each heavy chain is the product of at

The constant regions of the immunoglobulin molecules,

least four different genes: a variable region (V_H) gene, a

particularly the C_H2 and C_H3 (and C_H4 of IgM and

diversity region (D) gene, a joining region (J) gene, and

IgE), which constitute the Fc fragment, are responsible

a constant region (C_H) gene. Thus, the “one gene, one

for the class-specific effector functions of the different

protein” concept is not valid. The molecular mecha-

immunoglobulin molecules (Table 50-7, bottom part),

nisms responsible for the generation of the single im-

eg, complement fixation or transplacental passage.

munoglobulin chains from multiple structural genes are

Some immunoglobulins such as immune IgG exist

discussed in Chapters 36 and 39.

only in the basic tetrameric structure, while others such

as IgA and IgM can exist as higher order polymers of

two, three (IgA), or five (IgM) tetrameric units (Figure

Antibody Diversity Depends

50-10).

on Gene Rearrangements

The L chains and H chains are synthesized as sepa-

rate molecules and are subsequently assembled within

Each person is capable of generating antibodies directed

the B cell or plasma cell into mature immunoglobulin

against perhaps 1 million different antigens. The gener-

molecules, all of which are glycoproteins.

ation of such immense antibody diversity depends

upon a number of factors including the existence of

multiple gene segments (V, C, J, and D segments),

Both Light & Heavy Chains Are Products

their recombinations (see Chapters 36 and 39), the

of Multiple Genes

combinations of different L and H chains, a high fre-

Each immunoglobulin light chain is the product of at

quency of somatic mutations in immunoglobulin genes,

least three separate structural genes: a variable region

and junctional diversity. The latter reflects the addi-

594

CHAPTER 50

Table 50-8. Major functions of

Light chain

hypervariable

immunoglobulins.

regions

Immunoglobulin

Major Functions

IgG

Main antibody in the secondary re-

sponse. Opsonizes bacteria, making

Interchain

them easier to phagocytose. Fixes com-

disulfide

plement, which enhances bacterial

bonds

killing. Neutralizes bacterial toxins and

viruses. Crosses the placenta.

Heavy chain

hypervariable

IgA

Secretory IgA prevents attachment of

regions

bacteria and viruses to mucous mem-

Intrachain

branes. Does not fix complement.

disulfide

bonds

IgM

Produced in the primary response to an

antigen. Fixes complement. Does not

cross the placenta. Antigen receptor on

the surface of B cells.

IgD

Uncertain. Found on the surface of

many B cells as well as in serum.

IgE

Mediates immediate hypersensitivity by

causing release of mediators from mast

cells and basophils upon exposure to

Figure 50-9.

Schematic model of an IgG molecule

antigen (allergen). Defends against

showing approximate positions of the hypervariable re-

worm infections by causing release of

gions in heavy and light chains. The antigen-binding

enzymes from eosinophils. Does not fix

site is formed by these hypervariable regions. The hy-

complement. Main host defense against

pervariable regions are also called complementarity-

helminthic infections.

determining regions (CDRs). (Modified and reproduced,

¹Reproduced, with permission, from Levinson W, Jawetz E: Med-

with permission, from Parslow TG et al [editors]: Medical

ical Microbiology and Immunology, 7th ed. McGraw-Hill, 2002.

Immunology, 10th ed. McGraw-Hill, 2001.)

tion or deletion of a random number of nucleotides

generate an IgG molecule with antigen specificity iden-

when certain gene segments are joined together, and in-

tical to that of the original IgM molecule. The same

troduces an additional degree of diversity. Thus, the

light chain can also combine with an α heavy chain,

above factors ensure that a vast number of antibodies

again containing the identical V_H region, to form an IgA

can be synthesized from several hundred gene segments.

molecule with identical antigen specificity. These three

classes (IgM, IgG, and IgA) of immunoglobulin mole-

Class (Isotype) Switching Occurs

cules against the same antigen have identical variable do-

mains of both their light (V_L ) chains and heavy (V_H)

During Immune Responses

chains and are said to share an idiotype. (Idiotypes are

In most humoral immune responses, antibodies with

the antigenic determinants formed by the specific amino

identical specificity but of different classes are generated

acids in the hypervariable regions.) The different classes

in a specific chronologic order in response to the im-

of these three immunoglobulins (called isotypes) are

munogen (immunizing antigen). For instance, antibod-

thus determined by their different C_H regions, which are

ies of the IgM class normally precede molecules of the

combined with the same antigen-specific V_H regions.

IgG class. The switch from one class to another is desig-

nated “class or isotype switching,” and its molecular

Both Over- & Underproduction

basis has been investigated extensively. A single type of

of Immunoglobulins May Result

immunoglobulin light chain can combine with an anti-

in Disease States

gen-specific µ chain to generate a specific IgM molecule.

Subsequently, the same antigen-specific light chain

Disorders of immunoglobulins include increased pro-

combines with a γ chain with an identical V_H region to

duction of specific classes of immunoglobulins or even

PLASMA PROTEINS & IMMUNOGLOBULINS

595

Monomer

Dimer

A. Serum lgA

J chain

B. Secretory lgA

(dimer)

J chain

Secretory

component

J chain

Figure 50-10. Schematic repre-

sentation of serum IgA, secretory

IgA, and IgM. Both IgA and IgM have C. lgM

(pentamer)

a J chain, but only secretory IgA has

a secretory component. Polypeptide

chains are represented by thick lines;

disulfide bonds linking different

polypeptide chains are represented

by thin lines. (Reproduced, with per-

mission, from Parslow TG et al [edi-

tors]: Medical Immunology, 10th ed.

McGraw-Hill, 2001.)

specific immunoglobulin molecules, the latter by clonal

Hybridomas Provide Long-Term Sources

tumors of plasma cells called myelomas. Multiple

of Highly Useful Monoclonal Antibodies

myeloma is a neoplastic condition; electrophoresis of

serum or urine will usually reveal a large increase of one

When an antigen is injected into an animal, the result-

particular immunoglobulin or one particular light chain

ing antibodies are polyclonal, being synthesized by a

(the latter termed a Bence Jones protein). Decreased

mixture of B cells. Polyclonal antibodies are directed

production may be restricted to a single class of im-

against a number of different sites (epitopes or determi-

munoglobulin molecules (eg, IgA or IgG) or may in-

nants) on the antigen and thus are not monospecific.

volve underproduction of all classes of immunoglobu-

However, by means of a method developed by Kohler

lins (IgA, IgD, IgE, IgG, and IgM). A severe reduction

and Milstein, large amounts of a single monoclonal an-

in synthesis of an immunoglobulin class due to a ge-

tibody specific for one epitope can be obtained.

netic abnormality can result in a serious immunodefi-

The method involves cell fusion, and the resulting

ciency disease—eg, agammaglobulinemia, in which

permanent cell line is called a hybridoma. Typically, B

production of IgG is markedly affected—because of

cells are obtained from the spleen of a mouse (or other

impairment of the body’s defense against microorgan-

suitable animal) previously injected with an antigen or

isms.

mixture of antigens (eg, foreign cells). The B cells are

596

CHAPTER 50

mixed with mouse myeloma cells and exposed to poly-

tery of monoclonal antibodies can be obtained, many of

ethylene glycol, which causes cell fusion. A summary of

which are specific for individual components of the im-

the principles involved in generating hybridoma cells is

munogenic mixture. The hybridoma cells can be frozen

given in Figure 50-11. Under the conditions used, only

and stored and subsequently thawed when more of the

the hybridoma cells multiply in cell culture. This in-

antibody is required; this ensures its long-term supply.

volves plating the hybrid cells into hypoxanthine-

The hybridoma cells can also be grown in the abdomen

aminopterin-thymidine (HAT)-containing medium at

of mice, providing relatively large supplies of anti-

a concentration such that each dish contains approxi-

bodies.

mately one cell. Thus, a clone of hybridoma cells mul-

Because of their specificity, monoclonal antibodies

tiplies in each dish. The culture medium is harvested

have become useful reagents in many areas of biology

and screened for antibodies that react with the original

and medicine. For example, they can be used to mea-

antigen or antigens. If the immunogen is a mixture of

sure the amounts of many individual proteins

(eg,

many antigens (eg, a cell membrane preparation), an

plasma proteins), to determine the nature of infectious

individual culture dish will contain a clone of hy-

agents (eg, types of bacteria), and to subclassify both

bridoma cells synthesizing a monoclonal antibody to

normal (eg, lymphocytes) and tumor cells (eg, leukemic

one specific antigenic determinant of the mixture. By

cells). In addition, they are being used to direct thera-

harvesting the media from many culture dishes, a bat-

peutic agents to tumor cells and also to accelerate re-

moval of drugs from the circulation when they reach

toxic levels (eg, digoxin).

Myeloma cell

B cell

The Complement System Comprises About

20 Plasma Proteins & Is Involved in Cell

Lysis, Inflammation, & Other Processes

Plasma contains approximately 20 proteins that are

Fused in presence of PEG

members of the complement system. This system was

discovered when it was observed that addition of fresh

Hybridoma cell

serum containing antibodies directed to a bacterium

caused its lysis. Unlike antibodies, the factor was labile

Grown in presence of HAT medium

when heated at 56 °C. Subsequent work has resolved

Hybridoma multiplies; myeloma and B cells die

the proteins of the system and how they function; most

have been cloned and sequenced. The major protein

components are designated C1-9, with C9 associated

Hybridoma cell

with the C5-8 complex

(together constituting the

Figure 50-11. Scheme of production of a hy-

membrane attack complex) being involved in generat-

bridoma cell. The myeloma cells are immortalized, do

ing a lipid-soluble pore in the cell membrane that

not produce antibody, and are HGPRT^- (rendering the

causes osmotic lysis.

The details of this system are relatively complex, and

salvage pathway of purine synthesis [Chapter 34] inac-

a textbook of immunology should be consulted. The

tive). The B cells are not immortalized, each produces a

basic concept is that the normally inactive proteins of

specific antibody, and they are HGPRT⁺. Polyethylene

the system, when triggered by a stimulus, become acti-

glycol (PEG) stimulates cell fusion. The resulting hy-

vated by proteolysis and interact in a specific sequence

bridoma cells are immortalized (via the parental

with one or more of the other proteins of the system.

myeloma cells), produce antibody, and are HGPRT⁺

This results in cell lysis and generation of peptide or

(both latter properties gained from the parental B cells).

polypeptide fragments that are involved in various as-

The B cells will die in the medium because they are not

pects of inflammation (chemotaxis, phagocytosis, etc).

immortalized. In the presence of HAT, the myeloma

The system has other functions, such as clearance of

cells will also die, since the aminopterin in HAT sup-

antigen-antibody complexes from the circulation. Acti-

presses purine synthesis by the de novo pathway by in-

vation of the complement system is triggered by one of

hibiting reutilization of tetrahydrofolate (Chapter 34).

two routes, called the classic and the alternative path-

However, the hybridoma cells will survive, grow (be-

ways. The first involves interaction of C1 with antigen-

cause they are HGPRT⁺), and—if cloned—produce

antibody complexes, and the second (not involving an-

monoclonal antibody. (HAT, hypoxanthine,

tibody) involves direct interaction of bacterial cell

aminopterin, and thymidine; HGPRT, hypoxanthine-

surfaces or polysaccharides with a component desig-

guanine phosphoribosyl transferase.)

nated C3b.

PLASMA PROTEINS & IMMUNOGLOBULINS

597

The complement system resembles blood coagula-

trophils. Genetic deficiency of this protein is a cause

tion (Chapter 51) in that it involves both conversion of

of emphysema and can also lead to liver disease.

inactive precursors to active products by proteases and a

• α₂-Macroglobulin is a major plasma protein that

cascade with amplification.

neutralizes many proteases and targets certain cy-

tokines to specific organs.

SUMMARY

• Immunoglobulins play a key role in the defense

mechanisms of the body, as do proteins of the com-

•

Plasma contains many proteins with a variety of

plement system. Some of the principal features of

functions. Most are synthesized in the liver and are

these proteins are described.

glycosylated.

•

Albumin, which is not glycosylated, is the major pro-

tein and is the principal determinant of intravascular

osmotic pressure; it also binds many ligands, such as

REFERENCES

drugs and bilirubin.

•

Haptoglobin binds extracorpuscular hemoglobin,

Andrews NC: Disorders of iron metabolism. N Engl J Med

1999;341:1986.

prevents its loss into the kidney and urine, and hence

Carrell RW, Lomas DA: Alpha₁-antitrypsin deficiency—a model

preserves its iron for reutilization.

for conformational diseases. N Engl J Med 2002;346:45.

•

Transferrin binds iron, transporting it to sites where

Gabay C, Kushner I: Acute-phase proteins and other systemic re-

it is required. Ferritin provides an intracellular store

sponses to inflammation. New Engl J Med 1999;340:448.

of iron. Iron deficiency anemia is a very prevalent

Harris ED: Cellular copper transport and metabolism. Annu Rev

disorder. Hereditary hemochromatosis has been

Nutr 2000;20:291.

shown to be due to mutations in HFE, a gene encod-

Langlois MR, Delanghe JR: Biological and clinical significance of

ing the protein HFE, which appears to play an im-

haptoglobin polymorphism in humans. Clin Chem 1996;

portant role in absorption of iron.

2:1589.

•

Ceruloplasmin contains substantial amounts of cop-

Levinson W, Jawetz E: Medical Microbiology and Immunology, 6th

ed. Appleton & Lange, 2000.

per, but albumin appears to be more important with

Parslow TG et al (editors): Medical Immunology, 10th ed. Appleton

regard to its transport. Both Wilson disease and

& Lange, 2001.

Menkes disease, which reflect abnormalities of copper

Pepys MB, Berger A: The renaissance of C reactive protein. BMJ

metabolism, have been found to be due to mutations

2001;322:4.

in genes encoding copper-binding P-type ATPases.

Waheed A et al: Regulation of transferrin-mediated iron uptake by

•

α₁-Antitrypsin is the major serine protease inhibitor

HFE, the protein defective in hereditary hemochromatosis.

of plasma, in particular inhibiting the elastase of neu-

Proc Natl Acad U S A 2002;99:3117.

Hemostasis & Thrombosis

Margaret L. Rand, PhD, & Robert K. Murray, MD, PhD

BIOMEDICAL IMPORTANCE

There Are Three Types of Thrombi

Basic aspects of the proteins of the blood coagulation

Three types of thrombi or clots are distinguished. All

system and of fibrinolysis are described in this chapter.

three contain fibrin in various proportions.

Some fundamental aspects of platelet biology are also

(1) The white thrombus is composed of platelets and

presented. Hemorrhagic and thrombotic states can

fibrin and is relatively poor in erythrocytes. It

cause serious medical emergencies, and thromboses in

forms at the site of an injury or abnormal vessel

the coronary and cerebral arteries are major causes of

wall, particularly in areas where blood flow is

death in many parts of the world. Rational manage-

rapid (arteries).

ment of these conditions requires a clear understanding

of the bases of blood clotting and fibrinolysis.

(2) The red thrombus consists primarily of red cells

and fibrin. It morphologically resembles the clot

formed in a test tube and may form in vivo in

HEMOSTASIS & THROMBOSIS HAVE

areas of retarded blood flow or stasis (eg, veins)

THREE COMMON PHASES

with or without vascular injury, or it may form at

a site of injury or in an abnormal vessel in con-

Hemostasis is the cessation of bleeding from a cut or

junction with an initiating platelet plug.

severed vessel, whereas thrombosis occurs when the en-

(3) A third type is a disseminated fibrin deposit in

dothelium lining blood vessels is damaged or removed

very small blood vessels or capillaries.

(eg, upon rupture of an atherosclerotic plaque). These

processes encompass blood clotting (coagulation) and

We shall first describe the coagulation pathway lead-

involve blood vessels, platelet aggregation, and plasma

ing to the formation of fibrin. Then we shall briefly de-

proteins that cause formation or dissolution of platelet

scribe some aspects of the involvement of platelets and

aggregates.

blood vessel walls in the overall process. This separation

In hemostasis, there is initial vasoconstriction of the

of clotting factors and platelets is artificial, since both

injured vessel, causing diminished blood flow distal to

play intimate and often mutually interdependent roles

the injury. Then hemostasis and thrombosis share three

in hemostasis and thrombosis, but it facilitates descrip-

phases:

tion of the overall processes involved.

(1) Formation of a loose and temporary platelet ag-

gregate at the site of injury. Platelets bind to col-

Both Intrinsic & Extrinsic Pathways Result

lagen at the site of vessel wall injury and are acti-

in the Formation of Fibrin

vated by thrombin (the mechanism of activation

Two pathways lead to fibrin clot formation: the intrin-

of platelets is described below), formed in the co-

sic and the extrinsic pathways. These pathways are not

agulation cascade at the same site, or by ADP re-

independent, as previously thought. However, this arti-

leased from other activated platelets. Upon acti-

ficial distinction is retained in the following text to fa-

vation, platelets change shape and, in the

cilitate their description.

presence of fibrinogen, aggregate to form the he-

Initiation of the fibrin clot in response to tissue in-

mostatic plug (in hemostasis) or thrombus (in

jury is carried out by the extrinsic pathway. How the

thrombosis).

intrinsic pathway is activated in vivo is unclear, but it

(2) Formation of a fibrin mesh that binds to the

involves a negatively charged surface. The intrinsic and

platelet aggregate, forming a more stable hemosta-

extrinsic pathways converge in a final common path-

tic plug or thrombus.

way involving the activation of prothrombin to throm-

(3) Partial or complete dissolution of the hemostatic

bin and the thrombin-catalyzed cleavage of fibrinogen

plug or thrombus by plasmin.

to form the fibrin clot. The intrinsic, extrinsic, and

final common pathways are complex and involve many

different proteins (Figure 51-1 and Table 51-1). In

598

HEMOSTASIS & THROMBOSIS

599

Intrinsic pathway

XII

XIIa

Ca2+

Extrinsic pathway

XIa

VII

Ca2+

IXa

VIIa/Tissue factor

VIII

VIIIa

Figure 51-1. The pathways of

blood coagulation. The intrinsic

Ca2+

and extrinsic pathways are indi-

cated. The events depicted below

factor Xa are designated the final

common pathway, culminating in

Prothrombin

Thrombin

the formation of cross-linked fibrin.

New observations (dotted arrow)

include the finding that complexes

of tissue factor and factor VIIa acti-

Fibrinogen

XIII

vate not only factor X (in the classic

extrinsic pathway) but also factor

IX in the intrinsic pathway. In ad-

Classic coagulation

cascade

dition, thrombin and factor Xa

Fibrin monomer

XIIIa

Positive feedback

feedback-activate at the two sites

(hypothesized)

indicated (dashed arrows). (PK,

Extrinsic-to-intrinsic

prekallikrein; HK, HMW kininogen;

activation

Fibrin polymer

PL, phospholipids.) (Reproduced,

with permission, from Roberts HR,

Lozier JN: New perspectives on the

coagulation cascade. Hosp Pract [Off

Cross-linked

Ed] 1992 Jan;27:97.)

fibrin polymer

600

CHAPTER 51

Table 51-1. Numerical system for nomenclature

Table 51-2. The functions of the proteins

of blood clotting factors. The numbers indicate

involved in blood coagulation.

the order in which the factors have been

discovered and bear no relationship to the order

Zymogens of serine proteases

in which they act.

Factor XII

Binds to negatively charged surface at site of

vessel wall injury; activated by high-MW

kininogen and kallikrein.

Factor

Common Name

Factor XI

Activated by factor XIIa.

Fibrinogen

These factors are usually referred

Factor IX

Activated by factor Xla in presence of Ca2+.

Prothrombin

to by their common names.

Factor VII

Activated thrombin in presence of Ca2+.

III

Tissue factor

These factors are usually not re-

Factor X

Activated on surface of activated platelets by

Ca2+

ferred to as coagulation factors.

tenase complex (Ca2+, factors VIIIa and IXa)

Proaccelerin, labile factor, accelerator (Ac-)

and by factor VIIa in presence of tissue fac-

globulin

tor and Ca2+.

VII¹

Proconvertin, serum prothrombin conversion

Factor II

Activated on surface of activated platelets by

accelerator (SPCA), cothromboplastin

prothrombinase complex (Ca2+, factors Va

VIII

Antihemophilic factor A, antihemophilic globulin

and Xa).

(AHG)

[Factors II, VII, IX, and X are Gla-containing

Antihemophilic factor B, Christmas factor, plasma

zymogens.] (Gla = γ-carboxyglutamate.)

thromboplastin component (PTC)

Cofactors

Stuart-Prower factor

Factor VIII

Activated by thrombin; factor VIIIa is a co-

Plasma thromboplastin antecedent (PTA)

factor in the activation of factor X by

XII

Hageman factor

factor IXa.

XIII

Fibrin stabilizing factor (FSF), fibrinoligase

Factor V

Activated by thrombin; factor Va is a co-

¹There is no factor VI.

factor in the activation of prothrombin by

factor Xa.

Tissue factor

A glycoprotein expressed on the surface of

(factor III)

injured or stimulated endothelial cells to

general, as shown in Table 51-2, these proteins can be

act as a cofactor for factor VIIa.

classified into five types: (1) zymogens of serine-depen-

dent proteases, which become activated during the

Fibrinogen

process of coagulation; (2) cofactors;

(3) fibrinogen;

Factor I

Cleaved by thrombin to form fibrin clot.

(4) a transglutaminase, which stabilizes the fibrin clot;

Thiol-dependent transglutaminase

and (5) regulatory and other proteins.

Factor XIII

Activated by thrombin in presence of Ca2+;

stabilizes fibrin clot by covalent cross-

The Intrinsic Pathway Leads

linking.

to Activation of Factor X

Regulatory and other proteins

Protein C

Activated to protein Ca by thrombin bound

The intrinsic pathway (Figure 51-1) involves factors

to thrombomodulin; then degrades fac-

XII, XI, IX, VIII, and X as well as prekallikrein, high-

tors VIIIa and Va.

molecular-weight (HMW) kininogen, Ca2+, and plate-

Protein S

Acts as a cofactor of protein C; both proteins

let phospholipids. It results in the production of fac-

contain Gla (γ-carboxyglutamate)

tor Xa (by convention, activated clotting factors are

residues.

referred to by use of the suffix a).

Thrombo-

Protein on the surface of endothelial

This pathway commences with the “contact phase”

modulin

cells; binds thrombin, which then acti-

in which prekallikrein, HMW kininogen, factor XII,

vates protein C.

and factor XI are exposed to a negatively charged acti-

vating surface. In vivo, the proteins probably assemble

XIa and also releases bradykinin (a nonapeptide with

on endothelial cell membranes, whereas glass or kaolin

potent vasodilator action) from HMW kininogen.

can be used for in vitro tests of the intrinsic pathway.

Factor XIa in the presence of Ca2+ activates factor IX

When the components of the contact phase assemble

(55 kDa, a zymogen containing vitamin K-dependent

on the activating surface, factor XII is activated to fac-

γ-carboxyglutamate [Gla] residues; see Chapter 45), to

tor XIIa upon proteolysis by kallikrein. This factor

the serine protease, factor IXa. This in turn cleaves an

XIIa, generated by kallikrein, attacks prekallikrein to

Arg-Ile bond in factor X (56 kDa) to produce the two-

generate more kallikrein, setting up a reciprocal activa-

chain serine protease, factor Xa. This latter reaction re-

tion. Factor XIIa, once formed, activates factor XI to

quires the assembly of components, called the tenase

HEMOSTASIS & THROMBOSIS

601

cleave plasminogen and kallikrein can activate single-

complex, on the surface of activated platelets: Ca2+ and

chain urokinase.

factor VIIIa, as well as factors IXa and X. It should be

Tissue factor pathway inhibitor (TFPI) is a major

noted that in all reactions involving the Gla-containing

physiologic inhibitor of coagulation. It is a protein that

zymogens (factors II, VII, IX, and X), the Gla residues

circulates in the blood associated with lipoproteins.

in the amino terminal regions of the molecules serve as

TFPI directly inhibits factor Xa by binding to the en-

high-affinity binding sites for Ca2+. For assembly of the

zyme near its active site. This factor Xa-TFPI complex

tenase complex, the platelets must first be activated to

then inhibits the factor VIIa-tissue factor complex.

expose the acidic

(anionic) phospholipids, phos-

phatidylserine and phosphatidylinositol, that are

normally on the internal side of the plasma membrane

The Final Common Pathway of Blood

of resting, nonactivated platelets. Factor VIII

(330

Clotting Involves Activation of

kDa), a glycoprotein, is not a protease precursor but a

Prothrombin to Thrombin

cofactor that serves as a receptor for factors IXa and X

on the platelet surface. Factor VIII is activated by

In the final common pathway, factor Xa, produced by

minute quantities of thrombin to form factor VIIIa,

either the intrinsic or the extrinsic pathway, activates

which is in turn inactivated upon further cleavage by

prothrombin (factor II) to thrombin

(factor IIa),

thrombin.

which then converts fibrinogen to fibrin (Figure 51-1).

The activation of prothrombin, like that of factor X,

occurs on the surface of activated platelets and requires

The Extrinsic Pathway Also Leads

the assembly of a prothrombinase complex, consisting

to Activation of Factor X But

of platelet anionic phospholipids, Ca2+, factor Va, fac-

by a Different Mechanism

tor Xa, and prothrombin.

Factor Xa occurs at the site where the intrinsic and ex-

Factor V (330 kDa), a glycoprotein with homology

trinsic pathways converge (Figure 51-1) and lead into

to factor VIII and ceruloplasmin, is synthesized in the

the final common pathway of blood coagulation. The

liver, spleen, and kidney and is found in platelets as

extrinsic pathway involves tissue factor, factors VII and

well as in plasma. It functions as a cofactor in a manner

X, and Ca2+ and results in the production of factor Xa.

similar to that of factor VIII in the tenase complex.

It is initiated at the site of tissue injury with the expo-

When activated to factor Va by traces of thrombin, it

sure of tissue factor (Figure 51-1) on subendothelial

binds to specific receptors on the platelet membrane

cells. Tissue factor interacts with and activates factor

(Figure 51-2) and forms a complex with factor Xa and

VII (53 kDa), a circulating Gla-containing glycoprotein

prothrombin. It is subsequently inactivated by further

synthesized in the liver. Tissue factor acts as a cofactor

action of thrombin, thereby providing a means of limit-

for factor VIIa, enhancing its enzymatic activity to acti-

ing the activation of prothrombin to thrombin. Pro-

vate factor X. The association of tissue factor and factor

thrombin (72 kDa; Figure 51-3) is a single-chain gly-

VIIa is called tissue factor complex. Factor VIIa

coprotein synthesized in the liver. The amino terminal

cleaves the same Arg-Ile bond in factor X that is cleaved

region of prothrombin (1 in Figure 51-3) contains ten

by the tenase complex of the intrinsic pathway. Activa-

Gla residues, and the serine-dependent active protease

tion of factor X provides an important link between the

site (indicated by the arrowhead) is in the carboxyl ter-

intrinsic and extrinsic pathways.

minal region of the molecule. Upon binding to the

Another important interaction between the extrinsic

complex of factors Va and Xa on the platelet mem-

and intrinsic pathways is that complexes of tissue factor

brane, prothrombin is cleaved by factor Xa at two sites

and factor VIIa also activate factor IX in the intrinsic

(Figure 51-2) to generate the active, two-chain throm-

pathway. Indeed, the formation of complexes be-

bin molecule, which is then released from the platelet

tween tissue factor and factor VIIa is now consid-

surface. The A and B chains of thrombin are held to-

ered to be the key process involved in initiation of

gether by a disulfide bond.

blood coagulation in vivo. The physiologic signifi-

cance of the initial steps of the intrinsic pathway, in

Conversion of Fibrinogen to Fibrin

which factor XII, prekallikrein, and HMW kininogen

Is Catalyzed by Thrombin

are involved, has been called into question because pa-

tients with a hereditary deficiency of these components

Fibrinogen (factor I, 340 kDa; see Figures 51-1 and

do not exhibit bleeding problems. Similarly, patients

51-4 and Tables 51-1 and 51-2) is a soluble plasma

with a deficiency of factor XI may not have bleeding

glycoprotein that consists of three nonidentical pairs of

problems. The intrinsic pathway may actually be more

polypeptide chains

(Aα,Bβγ)₂

covalently linked by

important in fibrinolysis (see below) than in coagula-

disulfide bonds. The Bβ and γ chains contain as-

tion, since kallikrein, factor XIIa, and factor XIa can

paragine-linked complex oligosaccharides. All three

602

CHAPTER 51

Prethrombin

Figure 51-2. Diagrammatic representation

(not to scale) of the binding of factors Va, Xa,

F-1.2

Ca2+, and prothrombin to the plasma membrane

S-S

COO^-

of the activated platelet. The sites of cleavage of

prothrombin by factor Xa are indicated by two

Ca2+ Ca2+

Platelet

arrows. The part of prothrombin destined to

plasma

form thrombin is labeled prethrombin. The Ca2+

membrane

Indicates negative charges

is bound to anionic phospholipids of the plasma

to which Ca2+ binds.

membrane of the activated platelet.

chains are synthesized in the liver; the three structural

ture (α, β, γ)₂. Since FPA and FPB contain only 16 and

genes involved are on the same chromosome, and their

14 residues, respectively, the fibrin molecule retains

expression is coordinately regulated in humans. The

98% of the residues present in fibrinogen. The removal

amino terminal regions of the six chains are held in

of the fibrinopeptides exposes binding sites that allow

close proximity by a number of disulfide bonds, while

the molecules of fibrin monomers to aggregate sponta-

the carboxyl terminal regions are spread apart, giving

neously in a regularly staggered array, forming an insol-

rise to a highly asymmetric, elongated molecule (Figure

uble fibrin clot. It is the formation of this insoluble fib-

51-4). The A and B portions of the Aα and Bβ chains,

rin polymer that traps platelets, red cells, and other

designated fibrinopeptides A (FPA) and B (FPB), re-

components to form the white or red thrombi. This

spectively, at the amino terminal ends of the chains,

initial fibrin clot is rather weak, held together only by

bear excess negative charges as a result of the presence

the noncovalent association of fibrin monomers.

of aspartate and glutamate residues, as well as an un-

In addition to converting fibrinogen to fibrin,

usual tyrosine O-sulfate in FPB. These negative charges

thrombin also converts factor XIII to factor XIIIa. This

contribute to the solubility of fibrinogen in plasma and

factor is a highly specific transglutaminase that cova-

also serve to prevent aggregation by causing electrosta-

lently cross-links fibrin molecules by forming peptide

tic repulsion between fibrinogen molecules.

bonds between the amide groups of glutamine and the

Thrombin (34 kDa), a serine protease formed by

ε-amino groups of lysine residues

(Figure

51-5B),

the prothrombinase complex, hydrolyzes the four Arg-

yielding a more stable fibrin clot with increased resis-

Gly bonds between the fibrinopeptides and the α and β

tance to proteolysis.

portions of the Aα and Bβ chains of fibrinogen (Figure

51-5A). The release of the fibrinopeptides by thrombin

generates fibrin monomer, which has the subunit struc-

Levels of Circulating Thrombin Must Be

Carefully Controlled or Clots May Form

X_a

Once active thrombin is formed in the course of hemo-

S - S

Gla

stasis or thrombosis, its concentration must be carefully

controlled to prevent further fibrin formation or

platelet activation. This is achieved in two ways.

Thrombin circulates as its inactive precursor, pro-

X_a

thrombin, which is activated as the result of a cascade

of enzymatic reactions, each converting an inactive zy-

F-1

Prethrombin

(before Xa cleavage)

mogen to an active enzyme and leading finally to the

Thrombin (after Xa cleavage)

conversion of prothrombin to thrombin (Figure 51-1).

At each point in the cascade, feedback mechanisms

Diagrammatic representation (not to

Figure 51-3.

produce a delicate balance of activation and inhibition.

scale) of prothrombin. The amino terminal is to the left;

The concentration of factor XII in plasma is approxi-

region 1 contains all ten Gla residues. The sites of cleav-

mately 30 µg/mL, while that of fibrinogen is 3 mg/mL,

age by factor Xa are shown and the products named.

with intermediate clotting factors increasing in concen-

The site of the catalytically active serine residue is indi-

tration as one proceeds down the cascade, showing that

cated by the solid triangle. The A and B chains of active

the clotting cascade provides amplification. The second

thrombin (shaded) are held together by the disulfide

means of controlling thrombin activity is the inactiva-

bridge.

tion of any thrombin formed by circulating inhibi-

HEMOSTASIS & THROMBOSIS

603

FPA

FPB

Aα chain

Figure 51-4. Diagrammatic representation

(not to scale) of fibrinogen showing pairs of Aα,

Bβ chain

γ chain

Bβ, and γ chains linked by disulfide bonds. (FPA,

COO^-

NH₃

COO^-

fibrinopeptide A; FPB, fibrinopeptide B.)

tors, the most important of which is antithrombin III

thrombin as well as to its other substrates. This is the

(see below).

basis for the use of heparin in clinical medicine to in-

hibit coagulation. The anticoagulant effects of heparin

can be antagonized by strongly cationic polypeptides

The Activity of Antithrombin III,

such as protamine, which bind strongly to heparin,

an Inhibitor of Thrombin,

thus inhibiting its binding to antithrombin III. Individ-

Is Increased by Heparin

uals with inherited deficiencies of antithrombin III are

Four naturally occurring thrombin inhibitors exist in

prone to develop venous thrombosis, providing evi-

normal plasma. The most important is antithrombin

dence that antithrombin III has a physiologic function

III

(often called simply antithrombin), which con-

and that the coagulation system in humans is normally

tributes approximately 75% of the antithrombin activ-

in a dynamic state.

ity. Antithrombin III can also inhibit the activities of

Thrombin is involved in an additional regulatory

factors IXa, Xa, XIa, XIIa, and VIIa complexed with

mechanism that operates in coagulation. It combines

tissue factor.

with thrombomodulin, a glycoprotein present on the

2-Macroglobulin contributes most of

the remainder of the antithrombin activity, with hep-

surfaces of endothelial cells. The complex activates pro-

arin cofactor II and

tein C. In combination with protein S, activated pro-

1-antitrypsin acting as minor in-

hibitors under physiologic conditions.

tein C (APC) degrades factors Va and VIIIa, limiting

The endogenous activity of antithrombin III is

their actions in coagulation. A genetic deficiency of ei-

greatly potentiated by the presence of acidic proteogly-

ther protein C or protein S can cause venous thrombo-

cans such as heparin (Chapter 48). These bind to a

sis. Furthermore, patients with factor V Leiden (which

specific cationic site of antithrombin III, inducing a

has a glutamine residue in place of an arginine at posi-

conformational change and promoting its binding to

tion 506) have an increased risk of venous thrombotic

Thrombin

Arg

Gly

COO^-

NH₃

–

Fibrinopeptide

Fibrin chain

(A or B)

(α or

β)

Figure 51-5. Formation of a fibrin

clot. A: Thrombin-induced cleavage

Fibrin

CH₂

NH3+

H₂N

CH₂

Fibrin

of Arg-Gly bonds of the Aα and Bβ

(Lysyl)

(Glutaminyl)

chains of fibrinogen to produce fi-

brinopeptides (left-hand side) and

the α and β chains of fibrin mono-

NH4+

Factor XIIIa (Transglutaminase)

mer (right-hand side). B: Cross-

linking of fibrin molecules by acti-

Fibrin

CH₂

Fibrin

vated factor XIII (factor XIIIa).

604

CHAPTER 51

disease because factor V Leiden is resistant to inactiva-

In past years, treatment for patients with hemophilia

tion by APC. This condition is termed APC resistance.

A has consisted of administration of cryoprecipitates

(enriched in factor VIII) prepared from individual

donors or lyophilized factor VIII concentrates prepared

from plasma pools of up to 5000 donors. It is now pos-

Coumarin Anticoagulants Inhibit the

sible to prepare factor VIII by recombinant DNA

Vitamin K-Dependent Carboxylation of

technology. Such preparations are free of contaminat-

Factors II, VII, IX, & X

ing viruses (eg, hepatitis A, B, C, or HIV-1) found in

The coumarin drugs (eg, warfarin), which are used as

human plasma but are at present expensive; their use

anticoagulants, inhibit the vitamin K-dependent car-

may increase if cost of production decreases.

boxylation of Glu to Gla residues (see Chapter 45) in

the amino terminal regions of factors II, VII, IX, and X

Fibrin Clots Are Dissolved by Plasmin

and also proteins C and S. These proteins, all of which

are synthesized in the liver, are dependent on the Ca2+-

As stated above, the coagulation system is normally in a

binding properties of the Gla residues for their normal

state of dynamic equilibrium in which fibrin clots are

function in the coagulation pathways. The coumarins

constantly being laid down and dissolved. This latter

act by inhibiting the reduction of the quinone deriva-

process is termed fibrinolysis. Plasmin, the serine pro-

tives of vitamin K to the active hydroquinone forms

tease mainly responsible for degrading fibrin and fi-

(Chapter 45). Thus, the administration of vitamin K

brinogen, circulates in the form of its inactive zymogen,

will bypass the coumarin-induced inhibition and allow

plasminogen (90 kDa), and any small amounts of plas-

maturation of the Gla-containing factors. Reversal of

min that are formed in the fluid phase under physio-

coumarin inhibition by vitamin K requires

12-24

logic conditions are rapidly inactivated by the fast-

hours, whereas reversal of the anticoagulant effects of

acting plasmin inhibitor, α₂-antiplasmin. Plasminogen

heparin by protamine is almost instantaneous.

binds to fibrin and thus becomes incorporated in clots

Heparin and warfarin are widely used in the treat-

as they are produced; since plasmin that is formed

ment of thrombotic and thromboembolic conditions,

when bound to fibrin is protected from α₂-antiplasmin,

such as deep vein thrombosis and pulmonary embolus.

it remains active. Activators of plasminogen of various

Heparin is administered first, because of its prompt

types are found in most body tissues, and all cleave the

onset of action, whereas warfarin takes several days to

same Arg-Val bond in plasminogen to produce the two-

reach full effect. Their effects are closely monitored by

chain serine protease, plasmin (Figure 51-6).

use of appropriate tests of coagulation (see below) be-

Tissue plasminogen activator (alteplase; t-PA) is a

cause of the risk of producing hemorrhage.

serine protease that is released into the circulation from

vascular endothelium under conditions of injury or

Hemophilia A Is Due to a Genetically

Plasminogen

PLASMINOGEN

Determined Deficiency of Factor VIII

activators

Inherited deficiencies of the clotting system that result

in bleeding are found in humans. The most common is

NH3+

Arg -Val

COO

deficiency of factor VIII, causing hemophilia A, an X

S S

chromosome-linked disease that has played a major role

in the history of the royal families of Europe. Hemo-

philia B is due to a deficiency of factor IX; its clinical

features are almost identical to those of hemophilia A,

but the conditions can be separated on the basis of spe-

NH3+

Arg Val

COO

cific assays that distinguish between the two factors.

The gene for human factor VIII has been cloned

S S

and is one of the largest so far studied, measuring 186

PLASMIN

kb in length and containing 26 exons. A variety of mu-

tations have been detected leading to diminished activ-

Figure 51-6. Activation of plasminogen. The same

Arg-Val bond is cleaved by all plasminogen activators

ity of factor VIII; these include partial gene deletions

and point mutations resulting in premature chain ter-

to give the two-chain plasmin molecule. The solid trian-

mination. Prenatal diagnosis by DNA analysis after

gle indicates the serine residue of the active site. The

chorionic villus sampling is now possible.

two chains of plasmin are held together by a disulfide

bridge.

HEMOSTASIS & THROMBOSIS

605

stress and is catalytically inactive unless bound to fibrin.

slightly better survival rate. Table 51-3 compares some

Upon binding to fibrin, t-PA cleaves plasminogen

thrombolytic features of streptokinase and t-PA.

within the clot to generate plasmin, which in turn di-

There are a number of disorders, including cancer

gests the fibrin to form soluble degradation products

and shock, in which the concentrations of plasmino-

and thus dissolves the clot. Neither plasmin nor the

gen activators increase. In addition, the antiplasmin

plasminogen activator can remain bound to these

activities contributed by α₁-antitrypsin and α₂-antiplas-

degradation products, and so they are released into the

min may be impaired in diseases such as cirrhosis. Since

fluid phase, where they are inactivated by their natural

certain bacterial products, such as streptokinase, are ca-

inhibitors. Prourokinase is the precursor of a second ac-

pable of activating plasminogen, they may be responsi-

tivator of plasminogen, urokinase. Originally isolated

ble for the diffuse hemorrhage sometimes observed in

from urine, it is now known to be synthesized by cell

patients with disseminated bacterial infections.

types such as monocytes and macrophages, fibroblasts,

and epithelial cells. Its main action is probably in the

degradation of extracellular matrix. Figure 51-7 indi-

Activation of Platelets Involves

cates the sites of action of five proteins that influence

Stimulation of the

the formation and action of plasmin.

Polyphosphoinositide Pathway

Platelets normally circulate in an unstimulated disk-

Recombinant t-PA & Streptokinase Are

shaped form. During hemostasis or thrombosis, they

Used as Clot Busters

become activated and help form hemostatic plugs or

Alteplase (t-PA), produced by recombinant DNA tech-

thrombi. Three major steps are involved: (1) adhesion

nology, is used therapeutically as a fibrinolytic agent, as

to exposed collagen in blood vessels, (2) release of the

is streptokinase. However, the latter is less selective

contents of their granules, and (3) aggregation.

than t-PA, activating plasminogen in the fluid phase

Platelets adhere to collagen via specific receptors on

(where it can degrade circulating fibrinogen) as well as

the platelet surface, including the glycoprotein complex

plasminogen that is bound to a fibrin clot. The amount

GPIa-IIa (α₂β₁ integrin; Chapter 52), in a reaction

of plasmin produced by therapeutic doses of streptoki-

that involves von Willebrand factor. This is a glyco-

nase may exceed the capacity of the circulating α₂-

protein, secreted by endothelial cells into the plasma,

antiplasmin, causing fibrinogen as well as fibrin to be

which stabilizes factor VIII and binds to collagen and

degraded and resulting in the bleeding often encoun-

the subendothelium. Platelets bind to von Willebrand

tered during fibrinolytic therapy. Because of its selec-

factor via a glycoprotein complex (GPIb-V-IX) on the

tivity for degrading fibrin, there is considerable thera-

platelet surface; this interaction is especially important

peutic interest in the use of recombinant t-PA to restore

in platelet adherence to the subendothelium under con-

the patency of coronary arteries following thrombosis.

ditions of high shear stress that occur in small vessels

If administered early enough, before irreversible dam-

and stenosed arteries.

age of heart muscle occurs (about 6 hours after onset of

Platelets adherent to collagen change shape and

thrombosis), t-PA can significantly reduce the mortality

spread out on the subendothelium. They release the

rate from myocardial damage following coronary

contents of their storage granules (the dense granules

thrombosis. t-PA is more effective than streptokinase at

and the alpha granules); secretion is also stimulated by

restoring full patency and also appears to result in a

thrombin.

Streptokinase

Plasminogen

Streptokinase-plasminogen

complex

Figure 51-7. Scheme of sites of action

Plasminogen

t-PA

Urokinase

of streptokinase, tissue plasminogen acti-

activator

vator (t-PA), urokinase, plasminogen acti-

inhibitor

vator inhibitor, and α₂-antiplasmin (the

last two proteins exert inhibitory actions).

Streptokinase forms a complex with plas-

Plasmin

α₂-Antiplasmin

minogen, which exhibits proteolytic activ-

ity; this cleaves some plasminogen to plas-

Fibrin

Fibrin degradation products

min, initiating fibrinolysis.

606

CHAPTER 51

Table 51-3. Comparison of some properties of

Thrombin, formed from the coagulation cascade, is

streptokinase (SK) and tissue plasminogen

the most potent activator of platelets and initiates

platelet activation by interacting with its receptor on

activator (t-PA) with regard to their use as

the plasma membrane

(Figure

51-8). The further

thrombolytic agents.¹

events leading to platelet activation are examples of

transmembrane signaling, in which a chemical mes-

t-PA

senger outside the cell generates effector molecules in-

Selective for fibrin clot

−

side the cell. In this instance, thrombin acts as the ex-

Produces plasminemia

−

ternal chemical messenger (stimulus or agonist). The

Reduces mortality

interaction of thrombin with its receptor stimulates the

Causes allergic reaction

−

activity of an intracellular phospholipase C

. This en-

Causes hypotension

−

zyme hydrolyzes the membrane phospholipid phos-

Cost per treatment

Relatively low

Relatively high

phatidylinositol 4,5-bisphosphate (PIP₂, a polyphospho-

(approximate)

inositide) to form the two internal effector molecules,

¹Data from Webb J, Thompson C: Thrombolysis for acute myocar-

1,2-diacylglycerol and 1,4,5-inositol trisphosphate.

dial infarction. Can Fam Physician 1992;38:1415.

Hydrolysis of PIP₂ is also involved in the action of

many hormones and drugs. Diacylglycerol stimulates

Collagen

Prostacyclin

TxA₂

Thrombin

ADP

Aggregation

Fibrinogen

GPllb-llla

R¹

R²

R³

R⁴

R⁵

Plasma

membrane

PLCβ

PIP₂

PLA₂

PKC

Signaling

cAMP

Arachidonic acid

events

DAG

Phosphorylation

TxA₂

of pleckstrin

Ca2+

Release of contents

of platelet granules

Phosphorylation

(dense and alpha),

of light chain of

including ADP;

Actin

myosin

signaling events

Actomyosin

Change of shape

Figure 51-8.

Diagrammatic representation of platelet activation. The external environ-

ment, the plasma membrane, and the inside of a platelet are depicted from top to bottom.

Thrombin and collagen are the two most important platelet activators. ADP is considered

a weak agonist; it causes aggregation but not granule release. (GP, glycoprotein; R¹-R⁵,

various receptors; AC, adenylyl cyclase; PLA₂, phospholipase A₂; PL, phospholipids; PLCβ,

phospholipase Cβ; PIP₂, phosphatidylinositol 4,5-bisphosphate; cAMP, cyclic AMP; PKC,

protein kinase C; TxA₂, thromboxane A₂; IP₃, inositol 1,4,5-trisphosphate; DAG, 1,2-diacyl-

glycerol. The G proteins that are involved are not shown.)

HEMOSTASIS & THROMBOSIS

607

protein kinase C, which phosphorylates the protein

tors, which may help dissolve thrombi. Table 51-4 lists

pleckstrin (47 kDa). This results in aggregation and re-

some molecules produced by endothelial cells that af-

lease of the contents of the storage granules. ADP re-

fect thrombosis and fibrinolysis. Endothelium-derived

leased from dense granules can also activate platelets,

relaxing factor (nitric oxide) is discussed in Chapter 49.

resulting in aggregation of additional platelets. IP₃

Analysis of the mechanisms of uptake of atherogenic

causes release of Ca2+

into the cytosol mainly from the

lipoproteins, such as LDL, by endothelial, smooth mus-

dense tubular system (or residual smooth endoplasmic

cle, and monocytic cells of arteries, along with detailed

reticulum from the megakaryocyte), which then inter-

studies of how these lipoproteins damage such cells is a

acts with calmodulin and myosin light chain kinase,

key area of study in elucidating the mechanisms of ath-

leading to phosphorylation of the light chains of

erosclerosis (Chapter 26).

myosin. These chains then interact with actin, causing

changes of platelet shape.

Aspirin Is an Effective Antiplatelet Drug

Collagen-induced activation of a platelet phospholi-

pase A₂ by increased levels of cytosolic Ca2+ results in

Certain drugs (antiplatelet drugs) modify the behavior

of platelets. The most important is aspirin (acetylsali-

liberation of arachidonic acid from platelet phospho-

lipids, leading to the formation of thromboxane A₂

cylic acid), which irreversibly acetylates and thus in-

hibits the platelet cyclooxygenase system involved in

(Chapter 23), which in turn, in a receptor-mediated

fashion, can further activate phospholipase C, promot-

formation of thromboxane A₂ (Chapter 14), a potent

aggregator of platelets and also a vasoconstrictor.

ing platelet aggregation.

Activated platelets, besides forming a platelet aggre-

Platelets are very sensitive to aspirin; as little as 30 mg/d

(one aspirin tablet usually contains 325 mg) effectively

gate, are required, via newly expressed anionic phos-

pholipids on the membrane surface, for acceleration of

eliminates the synthesis of thromboxane A₂. Aspirin

also inhibits production of prostacyclin (PGI₂, which

the activation of factors X and II in the coagulation cas-

cade (Figure 51-1).

opposes platelet aggregation and is a vasodilator) by en-

All of the aggregating agents, including thrombin,

collagen, ADP, and others such as platelet-activating

factor, modify the platelet surface so that fibrinogen

Table 51-4. Molecules synthesized by

can bind to a glycoprotein complex, GPIIb-IIIa

(α_IIbβ₃ integrin; Chapter 52), on the activated platelet

endothelial cells that play a role in the regulation

surface. Molecules of divalent fibrinogen then link adja-

of thrombosis and fibrinolysis.¹

cent activated platelets to each other, forming a platelet

aggregate. Some agents, including epinephrine, sero-

Molecule

Action

tonin, and vasopressin, exert synergistic effects with

ADPase (an ectoenzyme)

Degrades ADP (an aggregating

other aggregating agents.

agent of platelets) to AMP + P_i

Endothelium-derived relax-

Inhibits platelet adhesion and

Endothelial Cells Synthesize Prostacyclin

ing factor (nitric oxide)

aggregation by elevating lev-

& Other Compounds That Affect

els of cGMP

Clotting & Thrombosis

Heparan sulfate (a glycos-

Anticoagulant; combines with

aminoglycan)

antithrombin III to inhibit

The endothelial cells in the walls of blood vessels make

thrombin

important contributions to the overall regulation of he-

Prostacyclin (PGI₂, a prosta-

Inhibits platelet aggregation by

mostasis and thrombosis. As described in Chapter 23,

glandin)

increasing levels of cAMP

these cells synthesize prostacyclin (PGI₂), a potent in-

Thrombomodulin (a glyco-

Binds protein C, which is then

hibitor of platelet aggregation, opposing the action of

protein)

cleaved by thrombin to yield

thromboxane A₂. Prostacyclin acts by stimulating the

activated protein C; this in

activity of adenylyl cyclase in the surface membranes of

combination with protein S

platelets. The resulting increase of intraplatelet cAMP

degrades factors Va and VIIIa,

opposes the increase in the level of intracellular Ca2+

limiting their actions

produced by IP₃ and thus inhibits platelet activation

Tissue plasminogen activa-

Activates plasminogen to plas-

(Figure 51-8). Endothelial cells play other roles in the

tor (t-PA, a protease)

min, which digests fibrin; the

action of t-PA is opposed by

regulation of thrombosis. For instance, these cells pos-

plasminogen activator in-

sess an ADPase, which hydrolyzes ADP, and thus op-

hibitor-1 (PAI-1)

poses its aggregating effect on platelets. In addition,

these cells appear to synthesize heparan sulfate, an anti-

¹Adapted from Wu KK: Endothelial cells in hemostasis, thrombosis

coagulant, and they also synthesize plasminogen activa-

and inflammation. Hosp Pract (Off Ed) 1992 Apr; 27:145.

608

CHAPTER 51

dothelial cells, but unlike platelets, these cells regenerate

• For activity, factors II, VII, IX, and X and proteins C

cyclooxygenase within a few hours. Thus, the overall

and S require vitamin K-dependent γ-carboxylation

balance between thromboxane A₂ and prostacyclin can

of certain glutamate residues, a process that is inhib-

be shifted in favor of the latter, opposing platelet aggre-

ited by the anticoagulant warfarin.

gation. Indications for treatment with aspirin thus in-

• Fibrin is dissolved by plasmin. Plasmin exists as an

clude management of angina and evolving myocardial

inactive precursor, plasminogen, which can be acti-

infarction and also prevention of stroke and death in

vated by tissue plasminogen activator (t-PA). Both

patients with transient cerebral ischemic attacks.

t-PA and streptokinase are widely used to treat early

thrombosis in the coronary arteries.

Laboratory Tests Measure Coagulation

• Thrombin and other agents cause platelet aggrega-

& Thrombolysis

tion, which involves a variety of biochemical and

morphologic events. Stimulation of phospholipase C

A number of laboratory tests are available to measure

and the polyphosphoinositide pathway is a key event

the phases of hemostasis described above. The tests in-

in platelet activation, but other processes are also in-

clude platelet count, bleeding time, activated partial

volved.

thromboplastin time (aPTT or PTT), prothrombin time

• Aspirin is an important antiplatelet drug that acts by

(PT), thrombin time (TT), concentration of fibrin-

ogen, fibrin clot stability, and measurement of fibrin

inhibiting production of thromboxane A₂.

degradation products. The platelet count quantitates

the number of platelets, and the bleeding time is an

REFERENCES

overall test of platelet function. aPTT is a measure of

the intrinsic pathway and PT of the extrinsic pathway.

Bennett JS: Mechanisms of platelet adhesion and aggregation: an

PT is used to measure the effectiveness of oral anticoag-

update. Hosp Pract (Off Ed) 1992;27:124.

ulants such as warfarin, and aPTT is used to monitor

Broze GJ: Tissue factor pathway inhibitor and the revised theory of

heparin therapy. The reader is referred to a textbook of

coagulation. Annu Rev Med 1995;46:103.

hematology for a discussion of these tests.

Clemetson KJ: Platelet activation: signal transduction via mem-

brane receptors. Thromb Haemost 1995;74:111.

Collen D, Lijnen HR: Basic and clinical aspects of fibrinolysis and

SUMMARY

thrombolysis. Blood 1991;78:3114.

• Hemostasis and thrombosis are complex processes

Handin RI: Anticoagulant, fibrinolytic and antiplatelet therapy.

involving coagulation factors, platelets, and blood

In: Harrison’s Principles of Internal Medicine, 15th ed. Braun-

wald E et al (editors). McGraw-Hill, 2001.

vessels.

Handin RI: Disorders of coagulation and thrombosis. In: Harri-

• Many coagulation factors are zymogens of serine pro-

son’s Principles of Internal Medicine, 15th ed. Braunwald E et

teases, becoming activated during the overall process.

al (editors). McGraw-Hill, 2001.

• Both intrinsic and extrinsic pathways of coagulation

Handin RI: Disorders of the platelet and vessel wall. In: Harrison’s

exist, the latter initiated by tissue factor. The path-

Principles of Internal Medicine, 15th ed. Braunwald E et al

ways converge at factor Xa, embarking on the com-

(editors). McGraw-Hill, 2001.

mon final pathway resulting in thrombin-catalyzed

Kroll MH, Schafer AI: Biochemical mechanisms of platelet activa-

tion. Blood 1989;74:1181.

conversion of fibrinogen to fibrin, which is strength-

ened by cross-linking, catalyzed by factor XIII.

Roberts HR, Lozier JN: New perspectives on the coagulation cas-

cade. Hosp Pract (Off Ed) 1992;27:97.

• Genetic disorders of coagulation factors occur, and

Roth GJ, Calverley DC: Aspirin, platelets, and thrombosis: theory

the two most common involve factors VIII (hemo-

and practice. Blood 1994;83:885.

philia A) and IX (hemophilia B).

Schmaier AH: Contact activation: a revision. Thromb Haemost

• An important natural inhibitor of coagulation is an-

1997;78:101.

tithrombin III; genetic deficiency of this protein can

Wu KK: Endothelial cells in hemostasis, thrombosis and inflamma-

result in thrombosis.

tion. Hosp Pract (Off Ed) 1992;27:145.

Red & White Blood Cells

Robert K. Murray, MD, PhD

BIOMEDICAL IMPORTANCE

intracellular organelles, such as mitochondria, lyso-

somes, or Golgi apparatus. Human red blood cells, like

Blood cells have been studied intensively because they

most red cells of animals, are nonnucleated. However,

are obtained easily, because of their functional impor-

the red cell is not metabolically inert. ATP is synthe-

tance, and because of their involvement in many disease

sized from glycolysis and is important in processes that

processes. The structure and function of hemoglobin,

help the red blood cell maintain its biconcave shape

the porphyrias, jaundice, and aspects of iron metabo-

and also in the regulation of the transport of ions (eg,

lism are discussed in previous chapters. Reduction of

by the Na⁺-K⁺ ATPase and the anion exchange protein

the number of red blood cells and of their content of

[see below]) and of water in and out of the cell. The bi-

hemoglobin is the cause of the anemias, a diverse and

concave shape increases the surface-to-volume ratio of

important group of conditions, some of which are seen

the red blood cell, thus facilitating gas exchange. The

very commonly in clinical practice. Certain of the

red cell contains cytoskeletal components (see below)

blood group systems, present on the membranes of

that play an important role in determining its shape.

erythrocytes and other blood cells, are of extreme im-

portance in relation to blood transfusion and tissue

About Two Million Red Blood Cells Enter

transplantation. Table 52-1 summarizes the causes of a

the Circulation per Second

number of important diseases affecting red blood cells;

some are discussed in this chapter, and the remainder

The life span of the normal red blood cell is 120 days;

are discussed elsewhere in this text. Every organ in the

this means that slightly less than 1% of the population

body can be affected by inflammation; neutrophils play

of red cells (200 billion cells, or 2 million per second) is

a central role in acute inflammation, and other white

replaced daily. The new red cells that appear in the cir-

blood cells, such as lymphocytes, play important roles

culation still contain ribosomes and elements of the en-

in chronic inflammation. Leukemias, defined as malig-

doplasmic reticulum. The RNA of the ribosomes can be

nant neoplasms of blood-forming tissues, can affect

detected by suitable stains (such as cresyl blue), and cells

precursor cells of any of the major classes of white

containing it are termed reticulocytes; they normally

blood cells; common types are acute and chronic my-

number about 1% of the total red blood cell count. The

elocytic leukemia, affecting precursors of the neu-

life span of the red blood cell can be dramatically short-

trophils; and acute and chronic lymphocytic leukemias.

ened in a variety of hemolytic anemias. The number of

Combination chemotherapy, using combinations of

reticulocytes is markedly increased in these conditions,

various chemotherapeutic agents, all of which act at one

as the bone marrow attempts to compensate for rapid

or more biochemical loci, has been remarkably effective

breakdown of red blood cells by increasing the amount

in the treatment of certain of these types of leukemias.

of new, young red cells in the circulation.

Understanding the role of red and white cells in health

and disease requires a knowledge of certain fundamen-

Erythropoietin Regulates Production

tal aspects of their biochemistry.

of Red Blood Cells

Human erythropoietin is a glycoprotein of 166 amino

THE RED BLOOD CELL IS SIMPLE IN

acids (molecular mass about 34 kDa). Its amount in

TERMS OF ITS STRUCTURE & FUNCTION

plasma can be measured by radioimmunoassay. It is the

The major functions of the red blood cell are relatively

major regulator of human erythropoiesis. Erythropoietin

simple, consisting of delivering oxygen to the tissues

is synthesized mainly by the kidney and is released in re-

and of helping in the disposal of carbon dioxide and

sponse to hypoxia into the bloodstream, in which it

protons formed by tissue metabolism. Thus, it has a

travels to the bone marrow. There it interacts with pro-

much simpler structure than most human cells, being

genitors of red blood cells via a specific receptor. The re-

essentially composed of a membrane surrounding a so-

ceptor is a transmembrane protein consisting of two dif-

lution of hemoglobin (this protein forms about 95% of

ferent subunits and a number of domains. It is not a

the intracellular protein of the red cell). There are no

tyrosine kinase, but it stimulates the activities of specific

609

610

CHAPTER 52

Table 52-1. Summary of the causes of some

members of this class of enzymes involved in down-

important disorders affecting red blood cells.

stream signal transduction. Erythropoietin interacts

with a red cell progenitor, known as the burst-forming

unit-erythroid (BFU-E), causing it to proliferate and

Disorder

Sole or Major Cause

differentiate. In addition, it interacts with a later pro-

Iron deficiency anemia

Inadequate intake or excessive loss

genitor of the red blood cell, called the colony-forming

of iron

unit-erythroid (CFU-E), also causing it to proliferate

and further differentiate. For these effects, erythropoi-

Methemoglobinemia

Intake of excess oxidants (various

chemicals and drugs)

etin requires the cooperation of other factors (eg, inter-

Genetic deficiency in the NADH-

leukin-3 and insulin-like growth factor; Figure 52-1).

dependent methemoglobin re-

The availability of a cDNA for erythropoietin has

ductase system (MIM 250800)

made it possible to produce substantial amounts of this

Inheritance of HbM (MIM 141800)

hormone for analysis and for therapeutic purposes; pre-

viously the isolation of erythropoietin from human

Sickle cell anemia

Sequence of codon 6 of the β chain

urine yielded very small amounts of the protein. The

(MIM 141900)

changed from GAG in the normal

major use of recombinant erythropoietin has been in

gene to GTG in the sickle cell

the treatment of a small number of anemic states, such

gene, resulting in substitution of

as that due to renal failure.

valine for glutamic acid

α-Thalassemias

Mutations in the α-globin genes,

MANY GROWTH FACTORS REGULATE

(MIM 141800)

mainly unequal crossing-over and

large deletions and less com-

PRODUCTION OF WHITE BLOOD CELLS

monly nonsense and frameshift

A large number of hematopoietic growth factors have

mutations

been identified in recent years in addition to erythro-

β-Thalassemia

A very wide variety of mutations in

poietin. This area of study adds to knowledge about the

(MIM 141900)

the β-globin gene, including dele-

differentiation of blood cells, provides factors that may

tions, nonsense and frameshift

be useful in treatment, and also has implications for un-

mutations, and others affecting

derstanding of the abnormal growth of blood cells (eg,

every aspect of its structure (eg,

the leukemias). Like erythropoietin, most of the growth

splice sites, promoter mutants)

factors isolated have been glycoproteins, are very active

Megaloblastic anemias

Decreased absorption of B₁₂, often

in vivo and in vitro, interact with their target cells via

Deficiency of

due to a deficiency of intrinsic fac-

specific cell surface receptors, and ultimately (via intra-

vitamin B₁₂

tor, normally secreted by gastric

cellular signals) affect gene expression, thereby promot-

parietal cells

ing differentiation. Many have been cloned, permitting

their production in relatively large amounts. Two of

Deficiency of folic

Decreased intake, defective absorp-

particular interest are granulocyte- and granulocyte-

acid

tion, or increased demand (eg, in

macrophage colony-stimulating factors (G-CSF and

pregnancy) for folate

GM-CSF, respectively). G-CSF is relatively specific, in-

Hereditary

Deficiencies in the amount or in the

ducing mainly granulocytes. GM-CSF affects a variety

spherocytosis¹

structure of α or β spectrin,

of progenitor cells and induces granulocytes, macro-

ankyrin, band 3 or band 4.1

phages, and eosinophils. When the production of neu-

Glucose-6-phosphate

A variety of mutations in the gene

trophils is severely depressed, this condition is referred

dehydrogenase

(X-linked) for G6PD, mostly single

to as neutropenia. It is particularly likely to occur in

(G6PD) deficiency¹

point mutations

patients treated with certain chemotherapeutic regi-

(MIM 305900)

mens and after bone marrow transplantation. These pa-

tients are liable to develop overwhelming infections.

Pyruvate kinase (PK)

Presumably a variety of mutations

G-CSF has been administered to such patients to boost

deficiency¹

in the gene for the R (red cell) iso-

production of neutrophils.

(MIM 255200)

zyme of PK

Paroxysmal nocturnal

Mutations in the PIG-A gene, affect-

THE RED BLOOD CELL HAS A UNIQUE &

hemoglobinemia¹

ing synthesis of GPI-anchored

(MIM 311770)

proteins

RELATIVELY SIMPLE METABOLISM

¹The last four disorders cause hemolytic anemias, as do a number

Various aspects of the metabolism of the red cell, many

of the other disorders listed. Most of the above conditions are dis-

of which are discussed in other chapters of this text, are

cussed in other chapters of this text. MIM numbers apply only to

summarized in Table 52-2.

disorders with a genetic basis.

RED & WHITE BLOOD CELLS

611

Interleukins

GM-CSF

Epo

Pluripotent

Erythroid

Mature

CFU-GEMM

BFU-E

stem cell

precursor

RBCs

(early

and late)

Figure 52-1. Greatly simplified scheme of differentiation of stem cells to red

blood cells. Various interleukins (ILs), such as IL-3, IL-4, IL-9, and IL-11, are involved

at different steps of the overall process. Erythroid precursors include the pronor-

moblast, basophilic, polychromatophilic, and orthochromatophilic normoblasts,

and the reticulocyte. Epo acts on basophilic normoblasts but not on later ery-

throid cells. (CFU-GEMM, colony-forming unit whose cells give rise to granulo-

cytes, erythrocytes, macrophages, and megakaryocytes; BFU-E, burst-forming

unit-erythroid; GM-CSF, granulocyte-macrophage colony-stimulating factor; Epo,

erythropoietin; RBC, red blood cell.)

The Red Blood Cell Has a Glucose

grammed by adding purified mRNAs or whole-cell ex-

Transporter in Its Membrane

tracts of mRNAs, and radioactive proteins are synthe-

sized in the presence of ³⁵S-labeled L-methionine or

The entry rate of glucose into red blood cells is far

other radiolabeled amino acids. The radioactive pro-

greater than would be calculated for simple diffusion.

teins synthesized are separated by SDS-PAGE and de-

Rather, it is an example of facilitated diffusion (Chap-

tected by radioautography.

ter 41). The specific protein involved in this process is

called the glucose transporter or glucose permease.

Some of its properties are summarized in Table 52-3.

Superoxide Dismutase, Catalase,

The process of entry of glucose into red blood cells is of

& Glutathione Protect Blood Cells

major importance because it is the major fuel supply for

From Oxidative Stress & Damage

these cells. About seven different but related glucose

transporters have been isolated from various tissues; un-

Several powerful oxidants are produced during the

like the red cell transporter, some of these are insulin-

course of metabolism, in both blood cells and most

dependent (eg, in muscle and adipose tissue). There is

other cells of the body. These include superoxide (O₂⋅ ),

considerable interest in the latter types of transporter

hydrogen peroxide (H₂O₂), peroxyl radicals (ROO^•),

because defects in their recruitment from intracellular

and hydroxyl radicals (OH^•). The last is a particularly

sites to the surface of skeletal muscle cells may help ex-

reactive molecule and can react with proteins, nucleic

plain the insulin resistance displayed by patients with

acids, lipids, and other molecules to alter their structure

type 2 diabetes mellitus.

and produce tissue damage. The reactions listed in

Table 52-4 play an important role in forming these ox-

idants and in disposing of them; each of these reactions

Reticulocytes Are Active

will now be considered in turn.

in Protein Synthesis

Superoxide is formed (reaction 1) in the red blood

The mature red blood cell cannot synthesize protein.

cell by the auto-oxidation of hemoglobin to methemo-

Reticulocytes are active in protein synthesis. Once retic-

globin (approximately 3% of hemoglobin in human red

ulocytes enter the circulation, they lose their intracellu-

blood cells has been calculated to auto-oxidize per day);

lar organelles

(ribosomes, mitochondria, etc) within

in other tissues, it is formed by the action of enzymes

about 24 hours, becoming young red blood cells and

such as cytochrome P450 reductase and xanthine oxi-

concomitantly losing their ability to synthesize protein.

dase. When stimulated by contact with bacteria, neu-

Extracts of rabbit reticulocytes (obtained by injecting

trophils exhibit a respiratory burst (see below) and pro-

rabbits with a chemical—phenylhydrazine—that causes

duce superoxide in a reaction catalyzed by NADPH

a severe hemolytic anemia, so that the red cells are al-

oxidase (reaction 2). Superoxide spontaneously dismu-

most completely replaced by reticulocytes) are widely

tates to form H₂O₂ and O₂; however, the rate of this

used as an in vitro system for synthesizing proteins. En-

same reaction is speeded up tremendously by the action

dogenous mRNAs present in these reticulocytes are

of the enzyme superoxide dismutase (reaction 3). Hy-

destroyed by use of a nuclease, whose activity can be in-

drogen peroxide is subject to a number of fates. The en-

hibited by addition of Ca2+. The system is then pro-

zyme catalase, present in many types of cells, converts

612

CHAPTER 52

Table 52-2. Summary of important aspects of

Table 52-3. Some properties of the glucose

the metabolism of the red blood cell.

transporter of the membrane of the red

blood cell.

•

The RBC is highly dependent upon glucose as its energy

source; its membrane contains high affinity glucose trans-

• It accounts for about 2% of the protein of the membrane of

porters.

the RBC.

•

Glycolysis, producing lactate, is the site of production of

• It exhibits specificity for glucose and related D-hexoses

ATP.

(L-hexoses are not transported).

•

Because there are no mitochondria in RBCs, there is no pro-

• The transporter functions at approximately 75% of its V_max

duction of ATP by oxidative phosphorylation.

at the physiologic concentration of blood glucose, is sat-

•

The RBC has a variety of transporters that maintain ionic

urable and can be inhibited by certain analogs of glucose.

and water balance.

• At least seven similar but distinct glucose transporters have

•

Production of 2,3-bisphosphoglycerate, by reactions closely

been detected to date in mammalian tissues, of which the

associated with glycolysis, is important in regulating the

red cell transporter is one.

ability of Hb to transport oxygen.

• It is not dependent upon insulin, unlike the corresponding

•

The pentose phosphate pathway is operative in the RBC (it

carrier in muscle and adipose tissue.

metabolizes about 5-10% of the total flux of glucose) and

• Its complete amino acid sequence (492 amino acids) has

produces NADPH; hemolytic anemia due to a deficiency of

been determined.

the activity of glucose-6-phosphate dehydrogenase is com-

• It transports glucose when inserted into artificial liposomes.

mon.

• It is estimated to contain 12 transmembrane helical seg-

•

Reduced glutathione (GSH) is important in the metabolism

ments.

of the RBC, in part to counteract the action of potentially

• It functions by generating a gated pore in the membrane to

toxic peroxides; the RBC can synthesize GSH and requires

permit passage of glucose; the pore is conformationally de-

NADPH to return oxidized glutathione (G-S-S-G) to the re-

pendent on the presence of glucose and can oscillate

duced state.

rapidly (about 900 times/s).

•

The iron of Hb must be maintained in the ferrous state; fer-

ric iron is reduced to the ferrous state by the action of an

NADH-dependent methemoglobin reductase system in-

8), which also produces OH^• and OH⁻. Superoxide can

volving cytochrome b₅ reductase and cytochrome b₅.

•

Synthesis of glycogen, fatty acids, protein, and nucleic acids

release iron ions from ferritin. Thus, production of

does not occur in the RBC; however, some lipids (eg, choles-

OH^• may be one of the mechanisms involved in tissue

terol) in the red cell membrane can exchange with corre-

injury due to iron overload

(eg, hemochromatosis;

sponding plasma lipids.

Chapter 50).

•

The RBC contains certain enzymes of nucleotide metabo-

Chemical compounds and reactions capable of gen-

lism (eg, adenosine deaminase, pyrimidine nucleotidase,

erating potential toxic oxygen species can be referred to

and adenylyl kinase); deficiencies of these enzymes are in-

as pro-oxidants. On the other hand, compounds and

volved in some cases of hemolytic anemia.

reactions disposing of these species, scavenging them,

•

When RBCs reach the end of their life span, the globin is de-

suppressing their formation, or opposing their actions

graded to amino acids (which are reutilized in the body),

are antioxidants and include compounds such as

the iron is released from heme and also reutilized, and the

NADPH, GSH, ascorbic acid, and vitamin E. In a nor-

tetrapyrrole component of heme is converted to bilirubin,

mal cell, there is an appropriate pro-oxidant:antioxi-

which is mainly excreted into the bowel via the bile.

dant balance. However, this balance can be shifted to-

ward the pro-oxidants when production of oxygen

species is increased greatly (eg, following ingestion of

it to H₂O and O₂ (reaction 4). Neutrophils possess a

certain chemicals or drugs) or when levels of antioxi-

unique enzyme, myeloperoxidase, that uses H₂O₂ and

dants are diminished (eg, by inactivation of enzymes in-

halides to produce hypohalous acids (reaction 5); this

volved in disposal of oxygen species and by conditions

subject is discussed further below. The selenium-

that cause low levels of the antioxidants mentioned

containing enzyme glutathione peroxidase (Chapter 20)

above). This state is called “oxidative stress” and can

will also act on reduced glutathione (GSH) and H₂O₂

result in serious cell damage if the stress is massive or

to produce oxidized glutathione (GSSG) and H₂O (re-

prolonged.

action 6); this enzyme can also use other peroxides as

Oxygen species are now thought to play an impor-

substrates. OH^• and OH⁻ can be formed from H₂O₂ in

tant role in many types of cellular injury (eg, resulting

a nonenzymatic reaction catalyzed by Fe2+ (the Fenton

from administration of various toxic chemicals or from

reaction, reaction 7). O₂⋅ and H2O2 are the substrates

ischemia), some of which can result in cell death. Indi-

in the iron-catalyzed Haber-Weiss reaction (reaction

rect evidence supporting a role for these species in gen-

RED & WHITE BLOOD CELLS

613

Table 52-4. Reactions of importance in relation to oxidative stress in blood cells and

various tissues.

(1) Production of superoxide (by-product of various reactions)

⋅

(2) NADPH-oxidase

2 O2 + NADPH → 2 O2−⋅ + NADP + H+

(3) Superoxide dismutase

⋅ + 2 H⁺ → H₂O₂ + O₂

(4) Catalase

H₂O₂ → 2 H₂O + O₂

(5) Myeloperoxidase

H₂O₂ + X⁻ + H⁺ → HOX + H₂O (X⁻ = Cl⁻, Br⁻, SCN⁻)

(6) Glutathione peroxidase (Se-dependent)

2 GSH + R-O-OH → GSSG + H₂O + ROH

(7) Fenton reaction

Fe2+ + H₂O₂ → Fe3+ + OH⋅ + OH⁻

(8) Iron-catalyzed Haber-Weiss reaction

⋅ + H₂O₂ → O₂ + OH⋅ + OH⁻

(9) Glucose-6-phosphate dehydrogenase (G6PD)

G6P + NADP → 6 Phosphogluconate + NADPH + H⁺

(10) Glutathione reductase

G-S-S-G + NADPH + H⁺ → 2 GSH + NADP

erating cell injury is provided if administration of an

sensitive hemolytic anemia] and sulfonamides) and

enzyme such as superoxide dismutase or catalase is

chemicals (eg, naphthalene) precipitate an attack, be-

found to protect against cell injury in the situation

cause their intake leads to generation of H₂O₂ or O₂

^⋅.

under study.

Normally, H₂O₂ is disposed of by catalase and glu-

tathione peroxidase (Table 52-4, reactions 4 and 6),

Deficiency of Glucose-6-Phosphate

the latter causing increased production of GSSG. GSH

is regenerated from GSSG by the action of the enzyme

Dehydrogenase Is Frequent in Certain

glutathione reductase, which depends on the availabil-

Areas & Is an Important Cause

ity of NADPH (reaction 10). The red blood cells of in-

of Hemolytic Anemia

dividuals who are deficient in the activity of glucose-6-

NADPH, produced in the reaction catalyzed by the

phosphate dehydrogenase cannot generate sufficient

X-linked glucose-6-phosphate dehydrogenase

(Table

NADPH to regenerate GSH from GSSG, which in

52-4, reaction 9) in the pentose phosphate pathway

turn impairs their ability to dispose of H₂O₂ and of

(Chapter 20), plays a key role in supplying reducing

oxygen radicals. These compounds can cause oxidation

equivalents in the red cell and in other cells such as the

of critical SH groups in proteins and possibly peroxida-

hepatocyte. Because the pentose phosphate pathway is

tion of lipids in the membrane of the red cell, causing

virtually its sole means of producing NADPH, the red

lysis of the red cell membrane. Some of the SH groups

blood cell is very sensitive to oxidative damage if the

of hemoglobin become oxidized, and the protein pre-

function of this pathway is impaired (eg, by enzyme de-

cipitates inside the red blood cell, forming Heinz bod-

ficiency). One function of NADPH is to reduce GSSG

ies, which stain purple with cresyl violet. The presence

to GSH, a reaction catalyzed by glutathione reductase

of Heinz bodies indicates that red blood cells have been

(reaction 10).

subjected to oxidative stress. Figure 52-2 summarizes

Deficiency of the activity of glucose-6-phosphate

the possible chain of events in hemolytic anemia due to

dehydrogenase, owing to mutation, is extremely fre-

deficiency of glucose-6-phosphate dehydrogenase.

quent in some regions of the world (eg, tropical Africa,

the Mediterranean, certain parts of Asia, and in North

Methemoglobin Is Useless

America among blacks). It is the most common of all

in Transporting Oxygen

enzymopathies (diseases caused by abnormalities of en-

zymes), and over 300 genetic variants of the enzyme

The ferrous iron of hemoglobin is susceptible to oxida-

have been distinguished; at least 100 million people are

tion by superoxide and other oxidizing agents, forming

deficient in this enzyme owing to these variants. The

methemoglobin, which cannot transport oxygen. Only

disorder resulting from deficiency of glucose-6-phos-

a very small amount of methemoglobin is present in

phate dehydrogenase is hemolytic anemia. Consump-

normal blood, as the red blood cell possesses an effec-

tion of broad beans (Vicia faba) by individuals deficient

tive system (the NADH-cytochrome b₅ methemoglobin

in activity of the enzyme can precipitate an attack of

reductase system) for reducing heme Fe3+ back to the

hemolytic anemia (most likely because the beans con-

Fe2+ state. This system consists of NADH (generated

tain potential oxidants). In addition, a number of drugs

by glycolysis), a flavoprotein named cytochrome b₅ re-

(eg, the antimalarial drug primaquine [the condition

ductase (also known as methemoglobin reductase), and

caused by intake of primaquine is called primaquine-

cytochrome b₅. The Fe3+ of methemoglobin is reduced

614

CHAPTER 52

branes due to increased amounts of deoxygenated he-

Mutations in the gene for G6PD

moglobin in arterial blood, or in this case due to in-

creased amounts of methemoglobin) is usually the pre-

Decreased activity of G6PD

senting sign in both types and is evident when over 10%

of hemoglobin is in the “met” form. Diagnosis is made

Decreased levels of NADPH

by spectroscopic analysis of blood, which reveals the

characteristic absorption spectrum of methemoglobin.

Additionally, a sample of blood containing methemo-

Decreased regeneration of GSH from GSSG by

globin cannot be fully reoxygenated by flushing oxygen

glutathione reductase (which uses NADPH)

through it, whereas normal deoxygenated blood can.

Electrophoresis can be used to confirm the presence of

Oxidation, due to decreased levels of GSH and

an abnormal hemoglobin. Ingestion of methylene blue

increased levels of intracellular oxidants (eg, O₂

•

or ascorbic acid (reducing agents) is used to treat mild

of SH groups of Hb (forming Heinz bodies), and of

methemoglobinemia due to enzyme deficiency. Acute

membrane proteins, altering membrane structure

massive methemoglobinemia (due to ingestion of chem-

and increasing susceptibility to ingestion

icals) should be treated by intravenous injection of

by macrophages (peroxidative damage to lipids

methylene blue.

in the membrane also possible)

Hemolysis

MORE IS KNOWN ABOUT THE MEMBRANE

OF THE HUMAN RED BLOOD CELL THAN

Figure 52-2. Summary of probable events causing

ABOUT THE SURFACE MEMBRANE OF

hemolytic anemia due to deficiency of the activity of

ANY OTHER HUMAN CELL

glucose-6-phosphate dehydrogenase (G6PD) (MIM

305900).

A variety of biochemical approaches have been used to

study the membrane of the red blood cell. These in-

clude analysis of membrane proteins by SDS-PAGE,

the use of specific enzymes (proteinases, glycosidases,

back to the Fe2+ state by the action of reduced cyto-

and others) to determine the location of proteins and

chrome b₅:

glycoproteins in the membrane, and various techniques

to study both the lipid composition and disposition of

Hb - Fe

Cyt

red

→Hb - Fe

Cyt

5 ox

individual lipids. Morphologic

(eg, electron micros-

copy, freeze-fracture electron microscopy) and other

Reduced cytochrome b₅ is then regenerated by the ac-

techniques (eg, use of antibodies to specific compon-

ents) have also been widely used. When red blood

tion of cytochrome b₅ reductase:

cells are lysed under specific conditions, their mem-

branes will reseal in their original orientation to form

Cyt

+NADH→Cyt

5 red

+NAD

ghosts (right-side-out ghosts). By altering the condi-

tions, ghosts can also be made to reseal with their cy-

tosolic aspect exposed on the exterior

(inside-out

Methemoglobinemia Is Inherited

ghosts). Both types of ghosts have been useful in ana-

or Acquired

lyzing the disposition of specific proteins and lipids in

the membrane. In recent years, cDNAs for many pro-

Methemoglobinemia can be classified as either inherited

teins of this membrane have become available, permit-

or acquired by ingestion of certain drugs and chemicals.

ting the deduction of their amino sequences and do-

Neither type occurs frequently, but physicians must be

mains. All in all, more is known about the membrane

aware of them. The inherited form is usually due to de-

of the red blood cell than about any other membrane of

ficient activity of methemoglobin reductase, transmitted

human cells (Table 52-5).

in an autosomal recessive manner. Certain abnormal he-

moglobins (Hb M) are also rare causes of methemoglo-

binemia. In Hb M, mutation changes the amino acid

Analysis by SDS-PAGE Resolves

residue to which heme is attached, thus altering its affin-

the Proteins of the Membrane

ity for oxygen and favoring its oxidation. Ingestion of

of the Red Blood Cell

certain drugs (eg, sulfonamides) or chemicals (eg, ani-

line) can cause acquired methemoglobinemia. Cyanosis

When the membranes of red blood cells are analyzed

(bluish discoloration of the skin and mucous mem-

by SDS-PAGE, about ten major proteins are resolved

RED & WHITE BLOOD CELLS

615

Table 52-5. Summary of biochemical

information about the membrane of the human

Spectrin

red blood cell.

2.1

Ankyrin

2.2

and

2.3

isoforms

•

The membrane is a bilayer composed of about 50% lipid

2.6

and 50% protein.

Anion exchange protein

•

The major lipid classes are phospholipids and cholesterol;

4.1

the major phospholipids are phosphatidylcholine (PC),

4.2

phosphatidylethanolamine (PE), and phosphatidylserine

(PS) along with sphingomyelin (Sph).

Glycophorins

Actin

•

The choline-containing phospholipids, PC and Sph, pre-

dominate in the outer leaflet and the amino-containing

G3PD

phospholipids (PE and PS) in the inner leaflet.

•

Glycosphingolipids (GSLs) (neutral GSLs, gangliosides, and

complex species, including the ABO blood group sub-

Globin

stances) constitute about 5-10% of the total lipid.

•

Analysis by SDS-PAGE shows that the membrane contains

about 10 major proteins and more than 100 minor species.

•

The major proteins (which include spectrin, ankyrin, the

anion exchange protein, actin, and band 4.1) have been

studied intensively, and the principal features of their dis-

position (eg, integral or peripheral), structure, and function

Coomassie

PAS stain

have been established.

blue stain

•

Many of the proteins are glycoproteins (eg, the glyco-

phorins) containing O- or N-linked (or both) oligosaccharide

Figure 52-3. Diagrammatic representation of the

chains located on the external surface of the membrane.

major proteins of the membrane of the human red

blood cell separated by SDS-PAGE. The bands detected

by staining with Coomassie blue are shown in the two

left-hand channels, and the glycoproteins detected by

(Figure 52-3), several of which have been shown to be

glycoproteins. Their migration on SDS-PAGE was

staining with periodic acid-Schiff (PAS) reagent are

used to name these proteins, with the slowest migrating

shown in the right-hand channel. (Reproduced, with

(and hence highest molecular mass) being designated

permission, from Beck WS, Tepper RI: Hemolytic anemias

band 1 or spectrin. All these major proteins have been

III: membrane disorders. In: Hematology, 5th ed. Beck WS

isolated, most of them have been identified, and con-

[editor]. The MIT Press, 1991.)

siderable insight has been obtained about their func-

tions

(Table

52-6). Many of their amino acid se-

quences also have been established. In addition, it has

been determined which are integral or peripheral mem-

bilayer at least ten times. It probably exists as a dimer in

brane proteins, which are situated on the external sur-

the membrane, in which it forms a tunnel, permitting

face, which are on the cytosolic surface, and which span

the exchange of chloride for bicarbonate. Carbon diox-

the membrane (Figure 52-4). Many minor compo-

ide, formed in the tissues, enters the red cell as bicar-

nents can also be detected in the red cell membrane by

bonate, which is exchanged for chloride in the lungs,

use of sensitive staining methods or two-dimensional

where carbon dioxide is exhaled. The amino terminal

gel electrophoresis. One of these is the glucose trans-

end binds many proteins, including hemoglobin, pro-

porter described above.

teins 4.1 and 4.2, ankyrin, and several glycolytic en-

zymes. Purified band 3 has been added to lipid vesicles

in vitro and has been shown to perform its transport

The Major Integral Proteins of the Red

functions in this reconstituted system.

Blood Cell Membrane Are the Anion

Glycophorins A, B, and C are also transmembrane

Exchange Protein & the Glycophorins

glycoproteins but of the single-pass type, extending

The anion exchange protein (band 3) is a transmem-

across the membrane only once. A is the major gly-

brane glycoprotein, with its carboxyl terminal end on

cophorin, is made up of 131 amino acids, and is heavily

the external surface of the membrane and its amino ter-

glycosylated (about 60% of its mass). Its amino terminal

minal end on the cytoplasmic surface. It is an example

end, which contains 16 oligosaccharide chains (15 of

of a multipass membrane protein, extending across the

which are O-glycans), extrudes out from the surface of

616

CHAPTER 52

Table 52-6. Principal proteins of the red cell membrane.¹

Integral (I) or

Approximate

Band Number²

Protein

Peripheral (P)

Molecular Mass (kDa)

Spectrin (α)

240

Spectrin (β)

220

2.1

Ankyrin

210

2.2

“

195

2.3

“

175

2.6

“

145

Anion exchange protein

100

4.1

Unnamed

Actin

Glyceraldehyde-3-phosphate dehydrogenase

Tropomyosin

Unnamed

Glycophorins A, B, and C

31, 23, and 28

¹Adapted from Lux DE, Becker PS: Disorders of the red cell membrane skeleton: hereditary spherocytosis and

hereditary elliptocytosis. Chapter 95 in: The Metabolic Basis of Inherited Disease, 6th ed. Scriver CR et al (editors).

McGraw-Hill, 1989.

²The band number refers to the position of migration on SDS-PAGE (see Figure 52-3). The glycophorins are de-

tected by staining with the periodic acid-Schiff reagent. A number of other components (eg, 4.2 and 4.9) are not

listed. Native spectrin is α₂β₂.

the red blood cell. Approximately 90% of the sialic acid

of the red cell membrane is located in this protein. Its

transmembrane segment (23 amino acids) is α-helical.

The carboxyl terminal end extends into the cytosol and

binds to protein 4.1, which in turn binds to spectrin.

Polymorphism of this protein is the basis of the MN

SPECTRIN-

blood group system (see below). Glycophorin A con-

ANKYRIN-3

ACTIN-4.1

tains binding sites for influenza virus and for Plasmo-

INTERACTION

dium falciparum, the cause of one form of malaria. In-

triguingly, the function of red blood cells of individuals

who lack glycophorin A does not appear to be affected.

Glycophorin

Outside

Lipid bilayer

Spectrin, Ankyrin, & Other Peripheral

Inside

Alpha

4.1

Membrane Proteins Help Determine the

Spectrin

Shape & Flexibility of the Red Blood Cell

Ankyrin

The red blood cell must be able to squeeze through

Actin

some tight spots in the microcirculation during its nu-

Beta

merous passages around the body; the sinusoids of the

SPECTRIN

spleen are of special importance in this regard. For the

SELF-

ASSOCIATION

red cell to be easily and reversibly deformable, its mem-

brane must be both fluid and flexible; it should also

Diagrammatic representation of the

Figure 52-4.

preserve its biconcave shape, since this facilitates gas ex-

interaction of cytoskeletal proteins with each other and

change. Membrane lipids help determine membrane

with certain integral proteins of the membrane of the

fluidity. Attached to the inner aspect of the membrane

red blood cell. (Reproduced, with permission, from Beck

of the red blood cell are a number of peripheral cy-

WS, Tepper RI: Hemolytic anemias III: membrane disor-

toskeletal proteins

(Table 52-6) that play important

ders. In: Hematology, 5th ed. Beck WS [editor]. The MIT

roles in respect to preserving shape and flexibility; these

Press, 1991.)

will now be described.

RED & WHITE BLOOD CELLS

617

Spectrin is the major protein of the cytoskeleton. It

0.85 g/dL. When exposed to a concentration of NaCl

is composed of two polypeptides: spectrin 1 (α chain)

of 0.5 g/dL, very few normal red blood cells are he-

and spectrin 2 (β chain). These chains, measuring ap-

molyzed, whereas approximately 50% of spherocytes

proximately 100 nm in length, are aligned in an an-

would lyse under these conditions. The explanation is

tiparallel manner and are loosely intertwined, forming a

that the spherocyte, being almost circular, has little po-

dimer. Both chains are made up of segments of 106

tential extra volume to accommodate additional water

amino acids that appear to fold into triple-stranded

and thus lyses readily when exposed to a slightly lower

α-helical coils joined by nonhelical segments. One

osmotic pressure than is normal.

dimer interacts with another, forming a head-to-head

One cause of hereditary spherocytosis (Figure 52-5)

tetramer. The overall shape confers flexibility on the

is a deficiency in the amount of spectrin or abnormali-

protein and in turn on the membrane of the red blood

ties of its structure, so that it no longer tightly binds the

cell. At least four binding sites can be defined in spec-

other proteins with which it normally interacts. This

trin: (1) for self-association, (2) for ankyrin (bands 2.1,

weakens the membrane and leads to the spherocytic

etc), (3) for actin (band 5), and (4) for protein 4.1.

shape. Abnormalities of ankyrin and of bands 3 and 4.1

Ankyrin is a pyramid-shaped protein that binds

are involved in other cases.

spectrin. In turn, ankyrin binds tightly to band 3, se-

Hereditary elliptocytosis is a genetic disorder that

curing attachment of spectrin to the membrane. Anky-

is similar to hereditary spherocytosis except that af-

rin is sensitive to proteolysis, accounting for the appear-

fected red blood cells assume an elliptic, disk-like shape,

ance of bands 2.2, 2.3, and 2.6, all of which are derived

recognizable by microscopy. It is also due to abnormali-

from band 2.1.

ties in spectrin; some cases reflect abnormalities of band

Actin (band 5) exists in red blood cells as short, dou-

4.1 or of glycophorin C.

ble-helical filaments of F-actin. The tail end of spectrin

dimers binds to actin. Actin also binds to protein 4.1.

THE BIOCHEMICAL BASES OF THE

Protein 4.1, a globular protein, binds tightly to the

ABO BLOOD GROUP SYSTEM

tail end of spectrin, near the actin-binding site of the

HAVE BEEN ESTABLISHED

latter, and thus is part of a protein 4.1-spectrin-actin

ternary complex. Protein 4.1 also binds to the integral

At least 21 human blood group systems are recognized,

proteins, glycophorins A and C, thereby attaching the

the best known of which are the ABO, Rh (Rhesus),

ternary complex to the membrane. In addition, protein

and MN systems. The term “blood group” applies to a

4.1 may interact with certain membrane phospholipids,

defined system of red blood cell antigens (blood group

thus connecting the lipid bilayer to the cytoskeleton.

substances) controlled by a genetic locus having a vari-

Certain other proteins (4.9, adducin, and tropo-

able number of alleles (eg, A, B, and O in the ABO sys-

myosin) also participate in cytoskeletal assembly.

tem). The term “blood type” refers to the antigenic

phenotype, usually recognized by the use of appropriate

Abnormalities in the Amount or Structure

of Spectrin Cause Hereditary

Spherocytosis & Elliptocytosis

Mutations in DNA affecting the amount or structure

of α or β spectrin or of certain other cytoskeletal

Hereditary spherocytosis is a genetic disease, transmitted

proteins (eg, ankyrin, band 3, band 4.1)

as an autosomal dominant, that affects about 1:5000

North Americans. It is characterized by the presence of

spherocytes (spherical red blood cells, with a low sur-

Weakens interactions among the peripheral and

face-to-volume ratio) in the peripheral blood, by a he-

integral proteins of the red cell membrane

molytic anemia, and by splenomegaly. The spherocytes

are not as deformable as are normal red blood cells, and

Weakens the structure of the red cell membrane

they are subject to destruction in the spleen, thus greatly

shortening their life in the circulation. Hereditary sphe-

Adopts spherocytic shape and is subject to

rocytosis is curable by splenectomy because the sphero-

destruction in the spleen

cytes can persist in the circulation if the spleen is absent.

The spherocytes are much more susceptible to os-

motic lysis than are normal red blood cells. This is as-

Hemolytic anemia

sessed in the osmotic fragility test, in which red blood

cells are exposed in vitro to decreasing concentrations

Figure 52-5. Summary of the causation of heredi-

of NaCl. The physiologic concentration of NaCl is

tary spherocytosis (MIM 182900).

618

CHAPTER 52

antibodies. For purposes of blood transfusion, it is par-

golipids, whereas in secretions the same oligosaccha-

ticularly important to know the basics of the ABO and

rides are present in glycoproteins. Their presence in se-

Rh systems. However, knowledge of blood group sys-

cretions is determined by a gene designated Se (for se-

tems is also of biochemical, genetic, immunologic, an-

cretor), which codes for a specific fucosyl

(Fuc)

thropologic, obstetric, pathologic, and forensic interest.

transferase in secretory organs, such as the exocrine

Here, we shall discuss only some key features of the

glands, but which is not active in red blood cells. Indi-

ABO system. From a biochemical viewpoint, the major

viduals of SeSe or Sese genotypes secrete A or B antigens

interests in the ABO substances have been in isolating

(or both), whereas individuals of the sese genotype do

and determining their structures, elucidating their

not secrete A or B substances, but their red blood cells

pathways of biosynthesis, and determining the natures

can express the A and B antigens.

of the products of the A, B, and O genes.

H Substance Is the Biosynthetic Precursor

The ABO System Is of Crucial Importance

of Both the A & B Substances

in Blood Transfusion

The ABO substances have been isolated and their struc-

This system was first discovered by Landsteiner in 1900

tures determined; simplified versions, showing only

when investigating the basis of compatible and incom-

their nonreducing ends, are presented in Figure 52-6.

patible transfusions in humans. The membranes of the

It is important to first appreciate the structure of the H

red blood cells of most individuals contain one blood

substance, since it is the precursor of both the A and B

group substance of type A, type B, type AB, or type O.

substances and is the blood group substance found in

Individuals of type A have anti-B antibodies in their

persons of type O. H substance itself is formed by the

plasma and will thus agglutinate type B or type AB

action of a fucosyltransferase, which catalyzes the ad-

blood. Individuals of type B have anti-A antibodies and

dition of the terminal fucose in α1 → 2 linkage onto

will agglutinate type A or type AB blood. Type AB

the terminal Gal residue of its precursor:

blood has neither anti-A nor anti-B antibodies and has

been designated the universal recipient. Type O blood

GDP Fuc+Gal β R→Fuc

α1,2

Gal

R+GDP

has neither A nor B substances and has been designated

ecursor

H substance

the universal donor. The explanation of these findings

is related to the fact that the body does not usually pro-

The H locus codes for this fucosyltransferase. The h al-

duce antibodies to its own constituents. Thus, individ-

lele of the H locus codes for an inactive fucosyltrans-

uals of type A do not produce antibodies to their own

ferase; therefore, individuals of the hh genotype cannot

blood group substance, A, but do possess antibodies to

generate H substance, the precursor of the A and B

the foreign blood group substance, B, possibly because

antigens. Thus, individuals of the hh genotype will have

similar structures are present in microorganisms to

red blood cells of type O, even though they may possess

which the body is exposed early in life. Since individu-

the enzymes necessary to make the A or B substances

als of type O have neither A nor B substances, they pos-

(see below).

sess antibodies to both these foreign substances. The

above description has been simplified considerably; eg,

The A Gene Encodes a GalNAc Transferase,

there are two subgroups of type A: A₁ and A₂.

the B Gene a Gal Transferase, & the O Gene

The genes responsible for production of the ABO

an Inactive Product

substances are present on the long arm of chromo-

some 9. There are three alleles, two of which are

In comparison with H substance (Figure 52-6), A sub-

codominant (A and B) and the third (O) recessive;

stance contains an additional GalNAc and B substance

these ultimately determine the four phenotypic prod-

an additional Gal, linked as indicated. Anti-A antibod-

ucts: the A, B, AB, and O substances.

ies are directed to the additional GalNAc residue found

in the A substance, and anti-B antibodies are directed

toward the additional Gal residue found in the B sub-

The ABO Substances Are

stance. Thus, GalNAc is the immunodominant sugar

Glycosphingolipids & Glycoproteins

(ie, the one determining the specificity of the antibody

Sharing Common Oligosaccharide Chains

formed) of blood group A substance, whereas Gal is the

The ABO substances are complex oligosaccharides pre-

immunodominant sugar of the B substance. In view of

sent in most cells of the body and in certain secretions.

the structural findings, it is not surprising that A sub-

On membranes of red blood cells, the oligosaccharides

stance can be synthesized in vitro from O substance in a

that determine the specific natures of the ABO sub-

reaction catalyzed by a GalNAc transferase, employing

stances appear to be mostly present in glycosphin-

UDP-GalNAc as the sugar donor. Similarly, blood

RED & WHITE BLOOD CELLS

619

Fucα1

2Galβ1

4GlcNAc - R

α1

Fucα1

2Galβ1

4GlcNAc - R

GalNAc

A substance

H (or O) substance

Fucα1

2Galβ1

4GlcNAc - R

α1

Gal

B substance

Figure 52-6. Diagrammatic representation of the structures of the H, A, and B

blood group substances. R represents a long complex oligosaccharide chain,

joined either to ceramide where the substances are glycosphingolipids, or to the

polypeptide backbone of a protein via a serine or threonine residue where the

substances are glycoproteins. Note that the blood group substances are bianten-

nary; ie, they have two arms, formed at a branch point (not indicated) between the

GlcNAc—R, and only one arm of the branch is shown. Thus, the H, A, and B sub-

stances each contain two of their respective short oligosaccharide chains shown

above. The AB substance contains one type A chain and one type B chain.

group B can be synthesized from O substance by the

Immunologic abnormalities (eg, transfusion reactions,

action of a Gal transferase, employing UDP-Gal. It is

the presence in plasma of warm and cold antibodies

crucial to appreciate that the product of the A gene is

that lyse red blood cells, and unusual sensitivity to com-

the GalNAc transferase that adds the terminal GalNAc

plement) also fall in this class, as do toxins released by

to the O substance. Similarly, the product of the B gene

various infectious agents, such as certain bacteria (eg,

is the Gal transferase adding the Gal residue to the O

clostridium). Some snakes release venoms that act to

substance. Individuals of type AB possess both enzymes

lyse the red cell membrane (eg, via the action of phos-

and thus have two oligosaccharide chains

(Figure

pholipases or proteinases).

52-6), one terminated by a GalNAc and the other by a

Causes within the membrane include abnormalities

Gal. Individuals of type O apparently synthesize an in-

of proteins. The most important conditions are heredi-

active protein, detectable by immunologic means; thus,

tary spherocytosis and hereditary elliptocytosis, princi-

H substance is their ABO blood group substance.

pally caused by abnormalities in the amount or struc-

In 1990, a study using cloning and sequencing tech-

ture of spectrin (see above).

nology described the nature of the differences between

Causes inside the red blood cell include hemoglo-

the glycosyltransferase products of the A, B, and O

binopathies and enzymopathies. Sickle cell anemia is

genes. A difference of four nucleotides is apparently re-

the most important hemoglobinopathy. Abnormalities

sponsible for the distinct specificities of the A and B

of enzymes in the pentose phosphate pathway and in

glycosyltransferases. On the other hand, the O allele has

glycolysis are the most frequent enzymopathies in-

a single base-pair mutation, causing a frameshift muta-

volved, particularly the former. Deficiency of glucose-

tion resulting in a protein lacking transferase activity.

6-phosphate dehydrogenase is prevalent in certain parts

of the world and is a frequent cause of hemolytic ane-

HEMOLYTIC ANEMIAS ARE CAUSED

mia (see above). Deficiency of pyruvate kinase is not

BY ABNORMALITIES OUTSIDE,

frequent, but it is the second commonest enzyme defi-

ciency resulting in hemolytic anemia; the mechanism

WITHIN, OR INSIDE THE RED

appears to be due to impairment of glycolysis, resulting

BLOOD CELL MEMBRANE

in decreased formation of ATP, affecting various as-

Causes outside the membrane include hypersplenism, a

pects of membrane integrity.

condition in which the spleen is enlarged from a variety

Laboratory investigations that aid in the diagnosis of

of causes and red blood cells become sequestered in it.

hemolytic anemia are listed in Table 52-7.

620

CHAPTER 52

Table 52-7. Laboratory investigations that assist

known as the “acute inflammatory response.” They in-

in the diagnosis of hemolytic anemia.

clude (1) increase of vascular permeability, (2) entry of

activated neutrophils into the tissues, (3) activation of

platelets, and (4) spontaneous subsidence (resolution) if

General tests and findings

the invading microorganisms have been dealt with suc-

Increased nonconjugated (indirect) bilirubin

cessfully.

Shortened red cell survival time as measured by injection

A variety of molecules are released from cells and

of autologous ⁵¹Cr-labeled red cells

plasma proteins during acute inflammation whose net

Reticulocytosis

Hemoglobinemia

overall effect is to increase vascular permeability, result-

Low level of plasma haptoglobin

ing in tissue edema (Table 52-10).

Specific tests and findings

In acute inflammation, neutrophils are recruited from

Hb electrophoresis (eg, HbS)

the bloodstream into the tissues to help eliminate the for-

Red cell enzymes (eg, G6PD or PK deficiency)

eign invaders. The neutrophils are attracted into the tis-

Osmotic fragility (eg, hereditary spherocytosis)

sues by chemotactic factors, including complement

Coombs test

fragment C5a, small peptides derived from bacteria (eg,

Cold agglutinins

N-formyl-methionyl-leucyl-phenylalanine), and a num-

¹The direct Coombs test detects the presence of antibodies on

ber of leukotrienes. To reach the tissues, circulating neu-

red cells, whereas the indirect test detects the presence of circu-

trophils must pass through the capillaries. To achieve

lating antibodies to antigens present on red cells.

this, they marginate along the vessel walls and then ad-

here to endothelial (lining) cells of the capillaries.

Integrins Mediate Adhesion of Neutrophils

NEUTROPHILS HAVE AN ACTIVE

to Endothelial Cells

METABOLISM & CONTAIN SEVERAL

UNIQUE ENZYMES & PROTEINS

Adhesion of neutrophils to endothelial cells employs

specific adhesive proteins (integrins) located on their

The major biochemical features of neutrophils are sum-

surface and also specific receptor proteins in the en-

marized in Table 52-8. Prominent features are active

dothelial cells. (See also the discussion of selectins in

aerobic glycolysis, active pentose phosphate pathway,

Chapter 47.)

moderately active oxidative phosphorylation (because

The integrins are a superfamily of surface proteins

mitochondria are relatively sparse), and a high content

present on a wide variety of cells. They are involved in

of lysosomal enzymes. Many of the enzymes listed in

the adhesion of cells to other cells or to specific compo-

Table 52-4 are also of importance in the oxidative me-

nents of the extracellular matrix. They are heterodimers,

tabolism of neutrophils (see below). Table 52-9 sum-

containing an α and a β subunit linked noncovalently.

marizes the functions of some proteins that are rela-

The subunits contain extracellular, transmembrane,

tively unique to neutrophils.

and intracellular segments. The extracellular segments

bind to a variety of ligands such as specific proteins of

Neutrophils Are Key Players in the Body’s

the extracellular matrix and of the surfaces of other

Defense Against Bacterial Infection

cells. These ligands often contain Arg-Gly-Asp (R-G-

Neutrophils are motile phagocytic cells that play a key

D) sequences. The intracellular domains bind to vari-

role in acute inflammation. When bacteria enter tissues,

ous proteins of the cytoskeleton, such as actin and vin-

a number of phenomena result that are collectively

culin. The integrins are proteins that link the outsides

of cells to their insides, thereby helping to integrate re-

sponses of cells

(eg, movement, phagocytosis) to

changes in the environment.

Table 52-8. Summary of major biochemical

Three subfamilies of integrins were recognized ini-

features of neutrophils.

tially. Members of each subfamily were distinguished

by containing a common β subunit, but they differed

• Active glycolysis

in their α subunits. However, more than three β sub-

• Active pentose phosphate pathway

units have now been identified, and the classification of

• Moderate oxidative phosphorylation

integrins has become rather complex. Some integrins of

• Rich in lysosomes and their degradative enzymes

specific interest with regard to neutrophils are listed in

• Contain certain unique enzymes (eg, myeloperoxidase and

Table 52-11.

NADPH-oxidase) and proteins

A deficiency of the β₂

subunit

(also designated

• Contain CD 11/CD18 integrins in plasma membrane

CD18) of LFA-1 and of two related integrins found in

RED & WHITE BLOOD CELLS

621

Table 52-9. Some important enzymes and proteins of neutrophils.¹

Enzyme or Protein

Reaction Catalyzed or Function

Comment

Myeloperoxidase

H₂O₂ +X⁻ (halide) + H⁺ →HOX + H₂O

Responsible for the green color of pus

(MPO)

(where X⁻ = Cl⁻, HOX = hypochlorous

Genetic deficiency can cause recurrent infections

acid)

NADPH-oxidase

2O2 + NADPH ^→ 2O2−⋅ + NADP + H+

Key component of the respiratory burst

Deficient in chronic granulomatous disease

Lysozyme

Hydrolyzes link between N-acetylmura-

Abundant in macrophages

mic acid and N-acetyl-D-glucosamine

found in certain bacterial cell walls

Defensins

Basic antibiotic peptides of 20-33 amino

Apparently kill bacteria by causing membrane

acids

damage

Lactoferrin

Iron-binding protein

May inhibit growth of certain bacteria by binding

iron and may be involved in regulation of prolif-

eration of myeloid cells

CD11a/CD18, CD11b/CD18,

Adhesion molecules (members of the

Deficient in leukocyte adhesion deficiency type I

CD11c/CD18²

integrin family)

(MIM 116920)

Receptors for Fc fragments of IgGs

Bind Fc fragments of IgG molecules

Target antigen-antibody complexes to myeloid

and lymphoid cells, eliciting phagocytosis and

other responses

¹The expression of many of these molecules has been studied during various stages of differentiation of normal neutrophils and also of

corresponding leukemic cells employing molecular biology techniques (eg, measurements of their specific mRNAs). For the majority,

cDNAs have been isolated and sequenced, amino acid sequences deduced, genes have been localized to specific chromosomal locations,

and exons and intron sequences have been defined. Some important proteinases of neutrophils are listed in Table 52-12.

²CD = cluster of differentiation. This refers to a uniform system of nomenclature that has been adopted to name surface markers of leuko-

cytes. A specific surface protein (marker) that identifies a particular lineage or differentiation stage of leukocytes and that is recognized by

a group of monoclonal antibodies is called a member of a cluster of differentiation. The system is particularly helpful in categorizing sub-

classes of lymphocytes. Many CD antigens are involved in cell-cell interactions, adhesion, and transmembrane signaling.

neutrophils and macrophages, Mac-1 (CD11b/CD18)

to turn on many of the metabolic processes involved in

and p150,95 (CD11c/CD18), causes type 1 leukocyte

phagocytosis and killing of bacteria.

adhesion deficiency, a disease characterized by recur-

rent bacterial and fungal infections. Among various re-

Activation of Neutrophils Is Similar

sults of this deficiency, the adhesion of affected white

to Activation of Platelets

blood cells to endothelial cells is diminished, and lower

& Involves Hydrolysis of

numbers of neutrophils thus enter the tissues to combat

Phosphatidylinositol Bisphosphate

infection.

Once having passed through the walls of small

The mechanisms involved in platelet activation are dis-

blood vessels, the neutrophils migrate toward the high-

cussed in Chapter 51 (see Figure 51-8). The process in-

est concentrations of the chemotactic factors, encounter

volves interaction of the stimulus (eg, thrombin) with a

the invading bacteria, and attempt to attack and de-

receptor, activation of G proteins, stimulation of phos-

stroy them. The neutrophils must be activated in order

pholipase C, and liberation from phosphatidylinositol

Table 52-10. Sources of biomolecules with vasoactive properties involved in acute inflammation.

Mast Cells and Basophils

Platelets

Neutrophils

Plasma Proteins

Histamine

Serotonin

Platelet-activating factor (PAF)

C3a, C4a, and C5a from the complement system

Eicosanoids (various prostaglan-

Bradykinin and fibrin split products from the coagu-

dins and leukotrienes)

lation system

622

CHAPTER 52

Table 52-11. Examples of integrins that are important in the function of neutrophils, of other white

blood cells, and of platelets.¹

Integrin

Cell

Subunit

Ligand

Function

VLA-1 (CD49a)

WBCs, others

α1β1

Collagen, laminin

Cell-ECM adhesion

VLA-5 (CD49e)

WBCs, others

α5β1

Fibronectin

Cell-ECM adhesion

VLA-6 (CD49f)

WBCs, others

α6β1

Laminin

Cell-ECM adhesion

LFA-1 (CD11a)

WBCs

αLβ2

ICAM-1

Adhesion of WBCs

Glycoprotein llb/llla

Platelets

αllbβ3

ICAM-2

Platelet adhesion and aggregation

Fibrinogen, fibronectin, von Willebrand

factor

¹LFA-1, lymphocyte function-associated antigen 1; VLA, very late antigen; CD, cluster of differentiation; ICAM, intercellular adhesion mole-

cule; ECM, extracellular matrix. A deficiency of LFA-1 and related integrins is found in type I leukocyte adhesion deficiency (MIM 116290).

A deficiency of platelet glycoprotein llb/llla complex is found in Glanzmann thrombasthenia (MIM 273800), a condition characterized by a

history of bleeding, a normal platelet count, and abnormal clot retraction. These findings illustrate how fundamental knowledge of cell

surface adhesion proteins is shedding light on the causation of a number of diseases.

bisphosphate of inositol triphosphate and diacylglyc-

22 kDa. When the system is activated (see below), two

erol. These two second messengers result in an eleva-

cytoplasmic polypeptides of 47 kDa and 67 kDa are re-

tion of intracellular Ca2+ and activation of protein ki-

cruited to the plasma membrane and, together with cy-

nase C. In addition, activation of phospholipase A₂

tochrome b₅₅₈, form the NADPH oxidase responsible

produces arachidonic acid that can be converted to a

for the respiratory burst. The reaction catalyzed by

variety of biologically active eicosanoids.

NADPH oxidase, involving formation of superoxide

The process of activation of neutrophils is essentially

anion, is shown in Table 52-4 (reaction 2). This system

similar. They are activated, via specific receptors, by in-

catalyzes the one-electron reduction of oxygen to super-

teraction with bacteria, binding of chemotactic factors,

oxide anion. The NADPH is generated mainly by the

or antibody-antigen complexes. The resultant rise in in-

pentose phosphate cycle, whose activity increases mark-

tracellular Ca2+ affects many processes in neutrophils,

edly during phagocytosis.

such as assembly of microtubules and the actin-myosin

The above reaction is followed by the spontaneous

system. These processes are respectively involved in se-

production (by spontaneous dismutation) of hydrogen

cretion of contents of granules and in motility, which

peroxide from two molecules of superoxide:

enables neutrophils to seek out the invaders. The acti-

vated neutrophils are now ready to destroy the invaders

−⋅

O ⋅+O

H+→H O +O

by mechanisms that include production of active deriv-

atives of oxygen.

The superoxide ion is discharged to the outside of

the cell or into phagolysosomes, where it encounters in-

The Respiratory Burst of Phagocytic

gested bacteria. Killing of bacteria within phagolyso-

Cells Involves NADPH Oxidase

somes appears to depend on the combined action of

& Helps Kill Bacteria

elevated pH, superoxide ion, or further oxygen deriva-

When neutrophils and other phagocytic cells engulf

tives (H₂O₂, OH^•, and HOCl [hypochlorous acid; see

bacteria, they exhibit a rapid increase in oxygen con-

below]) and on the action of certain bactericidal pep-

sumption known as the respiratory burst. This phe-

tides (defensins) and other proteins (eg, cathepsin G and

nomenon reflects the rapid utilization of oxygen (fol-

certain cationic proteins) present in phagocytic cells.

lowing a lag of 15-60 seconds) and production from it

Any superoxide that enters the cytosol of the phagocytic

of large amounts of reactive derivatives, such as O₂

^⋅,

cell is converted to H₂O₂ by the action of superoxide

H₂O₂, OH^•, and OCl⁻ (hypochlorite ion). Some of

dismutase, which catalyzes the same reaction as the

these products are potent microbicidal agents.

spontaneous dismutation shown above. In turn, H₂O₂ is

The electron transport chain system responsible

used by myeloperoxidase (see below) or disposed of by

for the respiratory burst (named NADPH oxidase) is

the action of glutathione peroxidase or catalase.

composed of several components. One is cytochrome

NADPH oxidase is inactive in resting phagocytic

b₅₅₈, located in the plasma membrane; it is a het-

cells and is activated upon contact with various ligands

erodimer, containing two polypeptides of 91 kDa and

(complement fragment C5a, chemotactic peptides, etc)

RED & WHITE BLOOD CELLS

623

with receptors in the plasma membrane. The events re-

Mutations in genes for the polypeptide components

sulting in activation of the oxidase system have been

of the NADPH oxidase system

much studied and are similar to those described above

for the process of activation of neutrophils. They in-

Diminished production of superoxide ion

volve G proteins, activation of phospholipase C, and

and other active derivatives of oxygen

generation of inositol 1,4,5-triphosphate (IP₃). The

last mediates a transient increase in the level of cytosolic

Ca2+, which is essential for induction of the respiratory

Diminished killing of certain bacteria

burst. Diacylglycerol is also generated and induces the

translocation of protein kinase C into the plasma mem-

Recurrent infections and formation of tissue

brane from the cytosol, where it catalyzes the phosphor-

granulomas in order to wall off surviving bacteria

ylation of various proteins, some of which are compo-

nents of the oxidase system. A second pathway of

Figure 52-7. Simplified scheme of the sequence of

also operates.

activation not involving Ca2+

events involved in the causation of chronic granuloma-

tous disease (MIM 306400). Mutations in any of the

Mutations in the Genes for Components

genes for the four polypeptides involved (two are com-

of the NADPH Oxidase System Cause

ponents of cytochrome b₅₅₈ and two are derived from

Chronic Granulomatous Disease

the cytoplasm) can cause the disease. The polypeptide

of 91 kDa is encoded by a gene in the X chromosome;

The importance of the NADPH oxidase system was

approximately 60% of cases of chronic granulomatous

clearly shown when it was observed that the respiratory

disease are X-linked, with the remainder being inher-

burst was defective in chronic granulomatous disease, a

ited in an autosomal recessive fashion.

relatively uncommon condition characterized by recur-

rent infections and widespread granulomas (chronic in-

flammatory lesions) in the skin, lungs, and lymph

nodes. The granulomas form as attempts to wall off

cause it reacts with primary or secondary amines pre-

bacteria that have not been killed, owing to genetic de-

sent in neutrophils and tissues to produce various nitro-

ficiencies in the NADPH oxidase system. The disorder

gen-chlorine derivatives; these chloramines are also oxi-

is due to mutations in the genes encoding the four

dants, though less powerful than HOCl, and act as

polypeptides that constitute the NADPH oxidase sys-

microbicidal agents (eg, in sterilizing wounds) without

tem. Some patients have responded to treatment with

causing tissue damage.

gamma interferon, which may increase transcription of

the 91-kDa component if it is affected. The probable

The Proteinases of Neutrophils

sequence of events involved in the causation of chronic

Can Cause Serious Tissue Damage

granulomatous disease is shown in Figure 52-7.

If Their Actions Are Not Checked

Neutrophils Contain Myeloperoxidase,

Neutrophils contain a number of proteinases (Table

52-12) that can hydrolyze elastin, various types of col-

Which Catalyzes the Production

lagens, and other proteins present in the extracellular

of Chlorinated Oxidants

matrix. Such enzymatic action, if allowed to proceed

The enzyme myeloperoxidase, present in large amounts

unopposed, can result in serious damage to tissues.

in neutrophil granules and responsible for the green color

Most of these proteinases are lysosomal enzymes and

of pus, can act on H₂O₂ to produce hypohalous acids:

exist mainly as inactive precursors in normal neu-

trophils. Small amounts of these enzymes are released

MYELOPEROXIDASE

into normal tissues, with the amounts increasing

H₂O₂ + X^— + H⁺

HOX + H₂O

markedly during inflammation. The activities of elas-

(X^— = Cl^—, Br^—, I^— or SCN^—; HOCl = hypochlorous acid)

tase and other proteinases are normally kept in check

by a number of antiproteinases (also listed in Table

The H₂O₂ used as substrate is generated by the

52-12) present in plasma and the extracellular fluid.

NADPH oxidase system. Cl^- is the halide usually em-

Each of them can combine—usually forming a nonco-

ployed, since it is present in relatively high concentra-

valent complex—with one or more specific proteinases

tion in plasma and body fluids. HOCl, the active ingre-

and thus cause inhibition. In Chapter 50 it was shown

dient of household liquid bleach, is a powerful oxidant

that a genetic deficiency of

1-antiproteinase inhi-

and is highly microbicidal. When applied to normal tis-

bitor (α₁-antitrypsin) permits elastase to act unopposed

sues, its potential for causing damage is diminished be-

and digest pulmonary tissue, thereby participating in

624

CHAPTER 52

Table 52-12. Proteinases of neutrophils and

ter

51) have been greatly clarified by investigations

antiproteinases of plasma and tissues.¹

using cloning and sequencing. The study of oncogenes

and chromosomal translocations has advanced under-

standing of the leukemias. As discussed above, cloning

Proteinases

Antiproteinases

techniques have made available therapeutic amounts of

Elastase

α₁-Antiproteinase (α₁-antitrypsin)

erythropoietin and other growth factors. Deficiency of

Collagenase

α₂-Macroglobulin

adenosine deaminase, which affects lymphocytes partic-

Gelatinase

Secretory leukoproteinase inhibitor

ularly, is the first disease to be treated by gene therapy.

Cathepsin G

α₁-Antichymotrypsin

Like many other areas of biology and medicine, hema-

Plasminogen activator

Plasminogen activator inhibitor-1

tology has been and will continue to be revolutionized

Tissue inhibitor of metalloproteinase

by this technology.

¹The table lists some of the important proteinases of neutrophils

and some of the proteins that can inhibit their actions. Most of

the proteinases listed exist inside neutrophils as precursors. Plas-

SUMMARY

minogen activator is not a proteinase, but it is included because it

•

The red blood cell is simple in terms of its structure

influences the activity of plasmin, which is a proteinase. The pro-

teinases listed can digest many proteins of the extracellular ma-

and function, consisting principally of a concentrated

trix, causing tissue damage. The overall balance of proteinase:an-

solution of hemoglobin surrounded by a membrane.

tiproteinase action can be altered by activating the precursors of

•

The production of red cells is regulated by erythro-

the proteinases, or by inactivating the antiproteinases. The latter

poietin, whereas other growth factors (eg, granulo-

can be caused by proteolytic degradation or chemical modifica-

cyte- and granulocyte-macrophage colony-stimulat-

tion, eg, Met-358 of α₁-antiproteinase inhibitor is oxidized by cig-

ing factors) regulate the production of white blood

arette smoke.

cells.

•

The red cell contains a battery of cytosolic enzymes,

such as superoxide dismutase, catalase, and glu-

the causation of emphysema.

2-Macroglobulin is a

tathione peroxidase, to dispose of powerful oxidants

plasma protein that plays an important role in the

generated during its metabolism.

body’s defense against excessive action of proteases; it

•

Genetically determined deficiency of the activity of

combines with and thus neutralizes the activities of a

glucose-6-phosphate dehydrogenase, which produces

number of important proteases (Chapter 50).

NADPH, is an important cause of hemolytic anemia.

When increased amounts of chlorinated oxidants are

•

Methemoglobin is unable to transport oxygen; both

formed during inflammation, they affect the pro-

genetic and acquired causes of methemoglobinemia

teinase:antiproteinase equilibrium, tilting it in favor of

are recognized. Considerable information has accu-

the former. For instance, certain of the proteinases

mulated concerning the proteins and lipids of the red

listed in Table 52-12 are activated by HOCl, whereas

cell membrane. A number of cytoskeletal proteins,

certain of the antiproteinases are inactivated by this

such as spectrin, ankyrin, and actin, interact with spe-

compound. In addition, the tissue inhibitor of metallo-

cific integral membrane proteins to help regulate the

proteinases and α₁-antichymotrypsin can be hydrolyzed

shape and flexibility of the membrane.

by activated elastase, and α₁-antiproteinase inhibitor

•

Deficiency of spectrin results in hereditary spherocy-

can be hydrolyzed by activated collagenase and gelati-

tosis, another important cause of hemolytic anemia.

nase. In most circumstances, an appropriate balance

of proteinases and antiproteinases is achieved. How-

•

The ABO blood group substances in the red cell

membrane are complex glycosphingolipids; the im-

ever, in certain instances, such as in the lung when

α₁-antiproteinase inhibitor is deficient or when large

munodominant sugar of A substance is N-acetyl-

galactosamine, whereas that of the B substance is

amounts of neutrophils accumulate in tissues because of

inadequate drainage, considerable tissue damage can re-

galactose.

sult from the unopposed action of proteinases.

•

Neutrophils play a major role in the body’s defense

mechanisms. Integrins on their surface membranes

determine specific interactions with various cell and

RECOMBINANT DNA TECHNOLOGY

tissue components.

HAS HAD A PROFOUND IMPACT

•

Leukocytes are activated on exposure to bacteria and

ON HEMATOLOGY

other stimuli; NADPH oxidase plays a key role in the

Recombinant DNA technology has had a major impact

process of activation (the respiratory burst). Muta-

on many aspects of hematology. The bases of the tha-

tions in this enzyme and associated proteins cause

lassemias and of many disorders of coagulation (Chap-

chronic granulomatous disease.

RED & WHITE BLOOD CELLS

625

• The proteinases of neutrophils can digest many tissue

Israels LG, Israels ED: Mechanisms in Hematology, 2nd ed. Univ

Manitoba Press, 1997. (Includes an excellent interactive CD.)

proteins; normally, this is kept in check by a battery

Jaffe ER, Hultquist DE: Cytochrome b₅ reductase deficiency and

of antiproteinases. However, this defense mechanism

enzymopenic hereditary methemoglobinemia. In: The Meta-

can be overcome in certain circumstances, resulting

bolic and Molecular Bases of Inherited Disease, 8th ed. Scriver

in extensive tissue damage.

CR et al (editors). McGraw-Hill, 2001.

• The application of recombinant DNA technology is

Lekstrom-Hunes JA, Gallin JI: Immunodeficiency diseases caused

revolutionizing the field of hematology.

by defects in granulocytes. N Engl J Med 2000;343:1703.

Luzzato L et al: Glucose-6-phosphate dehydrogenase. In: The

Metabolic and Molecular Bases of Inherited Disease, 8th ed.

REFERENCES

Scriver CR et al (editors). McGraw-Hill, 2001.

Borregaard N, Cowland JB: Granules of the human neutrophilic

Rosse WF et al: New Views of Sickle Cell Disease Pathophysiology

polymorphonuclear leukocyte. Blood 1997;89:3503.

and Treatment. The American Society of Hematology.

Daniels G: A century of human blood groups. Wien Klin Wochen-

www.asheducationbook.org

schr 2001;113:781.

Tse WT, Lux SE: Hereditary spherocytosis and hereditary ellipto-

Goodnough LT et al: Erythropoietin, iron, and erythropoiesis.

cytosis. In: The Molecular Bases of Inherited Disease, 8th ed.

Blood 2000;96:823.

Scriver CR et al (editors). McGraw-Hill, 2001.

Hirono A et al: Pyruvate kinase deficiency and other enzy-

Weatherall DJ et al: The hemoglobinopathies. In: The Metabolic

mopathies of the erythrocyte. In: The Metabolic and Molecu-

and Molecular Bases of Inherited Disease, 8th ed. Scriver CR et

lar Bases of Inherited Disease, 8th ed. Scriver CR et al (edi-

al (editors). McGraw-Hill, 2001.

tors). McGraw-Hill, 2001.

Metabolism of Xenobiotics

Robert K. Murray, MD, PhD

BIOMEDICAL IMPORTANCE

In phase 2, the hydroxylated or other compounds

produced in phase 1 are converted by specific enzymes

Increasingly, humans are subjected to exposure to vari-

to various polar metabolites by conjugation with glu-

ous foreign chemicals (xenobiotics)—drugs, food addi-

curonic acid, sulfate, acetate, glutathione, or certain

tives, pollutants, etc. The situation is well summarized

amino acids, or by methylation.

in the following quotation from Rachel Carson: “As

The overall purpose of the two phases of metabo-

crude a weapon as the cave man’s club, the chemical

lism of xenobiotics is to increase their water solubility

barrage has been hurled against the fabric of life.” Un-

(polarity) and thus excretion from the body. Very hy-

derstanding how xenobiotics are handled at the cellular

drophobic xenobiotics would persist in adipose tissue

level is important in learning how to cope with the

almost indefinitely if they were not converted to more

chemical onslaught.

polar forms. In certain cases, phase 1 metabolic reac-

Knowledge of the metabolism of xenobiotics is basic

tions convert xenobiotics from inactive to biologically

to a rational understanding of pharmacology and thera-

active compounds. In these instances, the original

peutics, pharmacy, toxicology, management of cancer,

xenobiotics are referred to as “prodrugs” or “procar-

and drug addiction. All these areas involve administra-

cinogens.” In other cases, additional phase 1 reactions

tion of, or exposure to, xenobiotics.

(eg, further hydroxylation reactions) convert the active

compounds to less active or inactive forms prior to con-

HUMANS ENCOUNTER THOUSANDS

jugation. In yet other cases, it is the conjugation reac-

OF XENOBIOTICS THAT MUST BE

tions themselves that convert the active products of

METABOLIZED BEFORE BEING EXCRETED

phase 1 reactions to less active or inactive species, which

are subsequently excreted in the urine or bile. In a very

A xenobiotic (Gk xenos “stranger”) is a compound that

few cases, conjugation may actually increase the bio-

is foreign to the body. The principal classes of xenobi-

logic activity of a xenobiotic.

otics of medical relevance are drugs, chemical carcino-

The term “detoxification” is sometimes used for

gens, and various compounds that have found their way

many of the reactions involved in the metabolism of

into our environment by one route or another, such as

xenobiotics. However, the term is not always appropri-

polychlorinated biphenyls (PCBs) and certain insecti-

ate because, as mentioned above, in some cases the reac-

cides. More than 200,000 manufactured environmental

tions to which xenobiotics are subject actually increase

chemicals exist. Most of these compounds are subject to

their biologic activity and toxicity.

metabolism (chemical alteration) in the human body,

with the liver being the main organ involved; occasion-

ally, a xenobiotic may be excreted unchanged. At least

ISOFORMS OF CYTOCHROME

30 different enzymes catalyze reactions involved in

P450 HYDROXYLATE A MYRIAD

xenobiotic metabolism; however, this chapter will only

OF XENOBIOTICS IN PHASE 1

cover a selected group of them.

It is convenient to consider the metabolism of xeno-

OF THEIR METABOLISM

biotics in two phases. In phase 1, the major reaction in-

Hydroxylation is the chief reaction involved in phase

volved is hydroxylation, catalyzed by members of a

1. The responsible enzymes are called monooxygenases

class of enzymes referred to as monooxygenases or cy-

or cytochrome P450s; the human genome encodes at

tochrome P450s. Hydroxylation may terminate the ac-

least

14 families of these enzymes. Estimates of the

tion of a drug, though this is not always the case. In ad-

number of distinct cytochrome P450s in human tissues

dition to hydroxylation, these enzymes catalyze a wide

range from approximately 35 to 60. The reaction cat-

range of reactions, including those involving deamina-

alyzed by a monooxygenase (cytochrome P450) is as

tion, dehalogenation, desulfuration, epoxidation, per-

follows:

oxygenation, and reduction. Reactions involving hy-

drolysis (eg, catalyzed by esterases) and certain other

non-P450-catalyzed reactions also occur in phase 1.

RH + O + NADPH + H

→R—OH+H O+NADP

626

METABOLISM OF XENOBIOTICS

627

RH above can represent a very wide variety of xenobi-

described above except that italics are used; thus, the

otics, including drugs, carcinogens, pesticides, petro-

gene encoding CYP1A1 is CYP1A1.

leum products, and pollutants (such as a mixture of

(2) Like hemoglobin, they are hemoproteins.

PCBs). In addition, endogenous compounds, such as

(3) They are widely distributed across species. Bac-

certain steroids, eicosanoids, fatty acids, and retinoids,

teria possess cytochrome P450s, and P450_cam (involved

are also substrates. The substrates are generally lip-

in the metabolism of camphor) of Pseudomonas putida

ophilic and are rendered more hydrophilic by hydroxy-

is the only P450 isoform whose crystal structure has

lation.

been established.

Cytochrome P450 is considered the most versatile

(4) They are present in highest amount in liver and

biocatalyst known. The actual reaction mechanism is

small intestine but are probably present in all tissues.

complex and has been briefly described previously (Fig-

In liver and most other tissues, they are present mainly

ure 11-6). It has been shown by the use of ¹⁸O₂ that

in the membranes of the smooth endoplasmic reticu-

one atom of oxygen enters ROH and one atom en-

lum, which constitute part of the microsomal fraction

ters water. This dual fate of the oxygen accounts for the

when tissue is subjected to subcellular fractionation. In

former naming of monooxygenases as

“mixed-

hepatic microsomes, cytochrome P450s can comprise

function oxidases.” The reaction catalyzed by cy-

as much as 20% of the total protein. P450s are found

tochrome P450 can also be represented as follows:

in most tissues, though often in low amounts compared

with liver. In the adrenal, they are found in mitochon-

Reduced cytochrome P450 Oxidized cytochrome P450

dria as well as in the endoplasmic reticulum; the vari-

RH + O₂ → R—OH + H₂O

ous hydroxylases present in that organ play an impor-

tant role in cholesterol and steroid biosynthesis. The

The major monooxygenases in the endoplasmic reticu-

mitochondrial cytochrome P450 system differs from

lum are cytochrome P450s—so named because the en-

the microsomal system in that it uses an NADPH-

zyme was discovered when it was noted that prepara-

linked flavoprotein, adrenodoxin reductase, and a

tions of microsomes that had been chemically reduced

nonheme iron-sulfur protein, adrenodoxin. In addi-

and then exposed to carbon monoxide exhibited a dis-

tion, the specific P450 isoforms involved in steroid

tinct peak at 450 nm. Among reasons that this enzyme

biosynthesis are generally much more restricted in their

is important is the fact that approximately 50% of the

substrate specificity.

drugs humans ingest are metabolized by isoforms of cy-

(5) At least six isoforms of cytochrome P450 are

tochrome P450; these enzymes also act on various car-

present in the endoplasmic reticulum of human liver,

cinogens and pollutants.

each with wide and somewhat overlapping substrate

specificities and acting on both xenobiotics and en-

Isoforms of Cytochrome P450 Make Up a

dogenous compounds. The genes for many isoforms of

Superfamily of Heme-Containing Enzymes

P450 (from both humans and animals such as the rat)

have been isolated and studied in detail in recent years.

The following are important points concerning cy-

(6) NADPH, not NADH, is involved in the reac-

tochrome P450s.

tion mechanism of cytochrome P450. The enzyme that

(1) Because of the large number of isoforms (about

uses NADPH to yield the reduced cytochrome P450,

150) that have been discovered, it became important to

shown at the left-hand side of the above equation, is

have a systematic nomenclature for isoforms of P450

called NADPH-cytochrome P450 reductase. Elec-

and for their genes. This is now available and in wide

trons are transferred from NADPH to NADPH-

use and is based on structural homology. The abbrevi-

cytochrome P450 reductase and then to cytochrome

ated root symbol CYP denotes a cytochrome P450.

P450. This leads to the reductive activation of molec-

This is followed by an Arabic number designating the

ular oxygen, and one atom of oxygen is subsequently

family; cytochrome P450s are included in the same

inserted into the substrate. Cytochrome b₅, another

family if they exhibit 40% or more sequence identity.

hemoprotein found in the membranes of the smooth

The Arabic number is followed by a capital letter indi-

endoplasmic reticulum (Chapter 11), may be involved

cating the subfamily, if two or more members exist;

as an electron donor in some cases.

P450s are in the same subfamily if they exhibit greater

(7) Lipids are also components of the cytochrome

than 55% sequence identity. The individual P450s

P450 system. The preferred lipid is phosphatidyl-

are then arbitrarily assigned Arabic numerals. Thus,

choline, which is the major lipid found in membranes

CYP1A1 denotes a cytochrome P450 that is a member

of the endoplasmic reticulum.

of family 1 and subfamily A and is the first individual

(8) Most isoforms of cytochrome P450 are in-

member of that subfamily. The nomenclature for the

ducible. For instance, the administration of phenobar-

genes encoding cytochrome P450s is identical to that

bital or of many other drugs causes hypertrophy of the

628

CHAPTER 53

smooth endoplasmic reticulum and a three- to fourfold

potentially altering the quantities of metabolites of

increase in the amount of cytochrome P450 within 4-5

PAHs (some of which could be harmful) to which the

days. The mechanism of induction has been studied ex-

fetus is exposed.

tensively and in most cases involves increased transcrip-

(10) Certain cytochrome P450s exist in polymor-

tion of mRNA for cytochrome P450. However, certain

phic forms (genetic isoforms), some of which exhibit

cases of induction involve stabilization of mRNA, en-

low catalytic activity. These observations are one im-

zyme stabilization, or other mechanisms (eg, an effect

portant explanation for the variations in drug responses

on translation).

noted among many patients. One P450 exhibiting

polymorphism is CYP2D6, which is involved in the

Induction of cytochrome P450 has important clini-

metabolism of debrisoquin (an antihypertensive drug;

cal implications, since it is a biochemical mechanism of

see Table 53-2) and sparteine (an antiarrhythmic and

drug interaction. A drug interaction has occurred

oxytocic drug). Certain polymorphisms of CYP2D6

when the effects of one drug are altered by prior, con-

cause poor metabolism of these and a variety of other

current, or later administration of another. As an illus-

drugs so that they can accumulate in the body, resulting

tration, consider the situation when a patient is taking

in untoward consequences. Another interesting poly-

the anticoagulant warfarin to prevent blood clotting.

morphism is that of CYP2A6, which is involved in the

This drug is metabolized by CYP2C9. Concomitantly,

metabolism of nicotine to conitine. Three CYP2A6 al-

the patient is started on phenobarbital (an inducer of

leles have been identified: a wild type and two null or

this P450) to treat a certain type of epilepsy, but the

inactive alleles. It has been reported that individuals

dose of warfarin is not changed. After 5 days or so, the

with the null alleles, who have impaired metabolism of

level of CYP2C9 in the patient’s liver will be elevated

nicotine, are apparently protected against becoming to-

three- to fourfold. This in turn means that warfarin will

bacco-dependent smokers (Table 53-2). These individ-

be metabolized much more quickly than before, and its

uals smoke less, presumably because their blood and

dosage will have become inadequate. Therefore, the

brain concentrations of nicotine remain elevated longer

dose must be increased if warfarin is to be therapeuti-

than those of individuals with the wild-type allele. It

cally effective. To pursue this example further, a prob-

has been speculated that inhibiting CYP2A6 may be a

lem could arise later on if the phenobarbital is discon-

novel way to help prevent and to treat smoking.

tinued but the increased dosage of warfarin stays the

same. The patient will be at risk of bleeding, since the

Table 53-1 summarizes some principal features of

high dose of warfarin will be even more active than be-

cytochrome P450s.

fore, because the level of CYP2C9 will decline once

phenobarbital has been stopped.

Another example of enzyme induction involves

CONJUGATION REACTIONS PREPARE

CYP2E1, which is induced by consumption of

XENOBIOTICS FOR EXCRETION IN

ethanol. This is a matter for concern, because this

PHASE 2 OF THEIR METABOLISM

P450 metabolizes certain widely used solvents and also

components found in tobacco smoke, many of which

In phase 1 reactions, xenobiotics are generally con-

are established carcinogens. Thus, if the activity of

verted to more polar, hydroxylated derivatives. In phase

CYP2E1 is elevated by induction, this may increase the

2 reactions, these derivatives are conjugated with mole-

risk of carcinogenicity developing from exposure to

cules such as glucuronic acid, sulfate, or glutathione.

such compounds.

This renders them even more water-soluble, and they

are eventually excreted in the urine or bile.

(9) Certain isoforms of cytochrome P450

(eg,

CYP1A1) are particularly involved in the metabolism of

polycyclic aromatic hydrocarbons (PAHs) and related

Five Types of Phase 2 Reactions

molecules; for this reason they were formerly called aro-

Are Described Here

matic hydrocarbon hydroxylases (AHHs). This enzyme

A. GLUCURONIDATION

is important in the metabolism of PAHs and in car-

cinogenesis produced by these agents. For example, in

The glucuronidation of bilirubin is discussed in Chap-

the lung it may be involved in the conversion of inac-

ter

32; the reactions whereby xenobiotics are glu-

tive PAHs (procarcinogens), inhaled by smoking, to ac-

curonidated are essentially similar. UDP-glucuronic

tive carcinogens by hydroxylation reactions. Smokers

acid is the glucuronyl donor, and a variety of glu-

have higher levels of this enzyme in some of their cells

curonosyltransferases, present in both the endoplasmic

and tissues than do nonsmokers. Some reports have in-

reticulum and cytosol, are the catalysts. Molecules such

dicated that the activity of this enzyme may be elevated

as 2-acetylaminofluorene (a carcinogen), aniline, ben-

(induced) in the placenta of a woman who smokes, thus

zoic acid, meprobamate (a tranquilizer), phenol, and

METABOLISM OF XENOBIOTICS

629

Table 53-1. Some properties of human

GSH (because of the sulfhydryl group of its cysteine,

cytochrome P450s.

which is the business part of the molecule). A number

of potentially toxic electrophilic xenobiotics (such as

certain carcinogens) are conjugated to the nucleophilic

•

Involved in phase I of the metabolism of innumerable

GSH in reactions that can be represented as follows:

xenobiotics, including perhaps 50% of the drugs adminis-

tered to humans

R+ GSH→R—S—G

•

Involved in the metabolism of many endogenous com-

pounds (eg, steroids)

where R = an electrophilic xenobiotic. The enzymes

•

All are hemoproteins

catalyzing these reactions are called glutathione S-

•

Often exhibit broad substrate specificity, thus acting on

transferases and are present in high amounts in liver

many compounds; consequently, different P450s may cat-

cytosol and in lower amounts in other tissues. A variety

alyze formation of the same product

of glutathione S-transferases are present in human tis-

•

Extremely versatile catalysts, perhaps catalyzing about 60

sue. They exhibit different substrate specificities and

types of reactions

can be separated by electrophoretic and other tech-

•

However, basically they catalyze reactions involving intro-

niques. If the potentially toxic xenobiotics were not

duction of one atom of oxygen into the substrate and one

conjugated to GSH, they would be free to combine co-

into water

valently with DNA, RNA, or cell protein and could

•

Their hydroxylated products are more water-soluble than

thus lead to serious cell damage. GSH is therefore an

their generally lipophilic substrates, facilitating excretion

important defense mechanism against certain toxic

•

Liver contains highest amounts, but found in most if not

compounds, such as some drugs and carcinogens. If the

all tissues, including small intestine, brain, and lung

•

Located in the smooth endoplasmic reticulum or in mito-

levels of GSH in a tissue such as liver are lowered (as

chondria (steroidogenic hormones)

can be achieved by the administration to rats of certain

•

In some cases, their products are mutagenic or carcino-

compounds that react with GSH), then that tissue can

genic

be shown to be more susceptible to injury by various

•

Many have a molecular mass of about 55 kDa

chemicals that would normally be conjugated to GSH.

•

Many are inducible, resulting in one cause of drug interac-

Glutathione conjugates are subjected to further metab-

tions

olism before excretion. The glutamyl and glycinyl

•

Many are inhibited by various drugs or their metabolic

groups belonging to glutathione are removed by spe-

products, providing another cause of drug interactions

cific enzymes, and an acetyl group (donated by acetyl-

•

Some exhibit genetic polymorphisms, which can result in

CoA) is added to the amino group of the remaining

atypical drug metabolism

cysteinyl moiety. The resulting compound is a mercap-

•

Their activities may be altered in diseased tissues (eg, cir-

turic acid, a conjugate of L-acetylcysteine, which is

rhosis), affecting drug metabolism

then excreted in the urine.

•

Genotyping the P450 profile of patients (eg, to detect

Glutathione has other important functions in

polymorphisms) may in the future permit individualization

human cells apart from its role in xenobiotic metabo-

of drug therapy

lism.

1. It participates in the decomposition of potentially

toxic hydrogen peroxide in the reaction cat-

many steroids are excreted as glucuronides. The glu-

alyzed by glutathione peroxidase (Chapter 20).

curonide may be attached to oxygen, nitrogen, or sulfur

2. It is an important intracellular reductant, help-

groups of the substrates. Glucuronidation is probably

the most frequent conjugation reaction.

ing to maintain essential SH groups of enzymes in

their reduced state. This role is discussed in Chap-

B. SULFATION

ter 20, and its involvement in the hemolytic ane-

Some alcohols, arylamines, and phenols are sulfated.

mia caused by deficiency of glucose-6-phosphate

The sulfate donor in these and other biologic sulfation

dehydrogenase is discussed in Chapters 20 and

reactions (eg, sulfation of steroids, glycosaminoglycans,

52.

glycolipids, and glycoproteins) is adenosine 3 -phos-

3. A metabolic cycle involving GSH as a carrier has

phate-5 -phosphosulfate (PAPS) (Chapter 24); this

been implicated in the transport of certain

compound is called “active sulfate.”

amino acids across membranes in the kidney.

The first reaction of the cycle is shown below.

C. CONJUGATION WITH GLUTATHIONE

Glutathione (γ-glutamyl-cysteinylglycine) is a tripep-

Amino acid + GSH

→γ

- Glutamyl amino acid+

tide consisting of glutamic acid, cysteine, and glycine

Cysteinylglycine

(Figure

3-3). Glutathione is commonly abbreviated

630

CHAPTER 53

This reaction helps transfer certain amino acids across

Again, this can affect the doses of certain drugs that are

the plasma membrane, the amino acid being subse-

administered to patients. Various diseases (eg, cirrhosis

quently hydrolyzed from its complex with GSH and

of the liver) can affect the activities of drug-metaboliz-

the GSH being resynthesized from cysteinylglycine.

ing enzymes, sometimes necessitating adjustment of

The enzyme catalyzing the above reaction is

-glu-

dosages of various drugs for patients with these disor-

tamyltransferase (GGT). It is present in the plasma

ders.

membrane of renal tubular cells and bile ductule cells,

and in the endoplasmic reticulum of hepatocytes. The

enzyme has diagnostic value because it is released into

RESPONSES TO XENOBIOTICS

the blood from hepatic cells in various hepatobiliary

INCLUDE PHARMACOLOGIC,

diseases.

TOXIC, IMMUNOLOGIC,

D. OTHER REACTIONS

& CARCINOGENIC EFFECTS

The two most important other reactions are acetylation

Xenobiotics are metabolized in the body by the reac-

and methylation.

tions described above. When the xenobiotic is a drug,

1. Acetylation—Acetylation is represented by

phase 1 reactions may produce its active form or may

diminish or terminate its action if it is pharmacologi-

X+Acetyl-CoA →Acetyl-X+ CoA

cally active in the body without prior metabolism. The

where X represents a xenobiotic. As for other acetyla-

diverse effects produced by drugs comprise the area of

tion reactions, acetyl-CoA (active acetate) is the acetyl

study of pharmacology; here it is important to appreci-

donor. These reactions are catalyzed by acetyltrans-

ate that drugs act primarily through biochemical mech-

ferases present in the cytosol of various tissues, particu-

anisms. Table 53-2 summarizes four important reac-

larly liver. The drug isoniazid, used in the treatment of

tions to drugs that reflect genetically determined

tuberculosis, is subject to acetylation. Polymorphic

differences in enzyme and protein structure among in-

types of acetyltransferases exist, resulting in individuals

dividuals—part of the field of study known as pharma-

who are classified as slow or fast acetylators, and influ-

cogenetics (see below).

ence the rate of clearance of drugs such as isoniazid

from blood. Slow acetylators are more subject to certain

toxic effects of isoniazid because the drug persists

Table 53-2. Some important drug reactions due

longer in these individuals.

to mutant or polymorphic forms of enzymes or

2. Methylation—A few xenobiotics are subject to

proteins.¹

methylation by methyltransferases, employing S-adeno-

sylmethionine (Figure 30-17) as the methyl donor.

Enzyme or Protein

THE ACTIVITIES OF XENOBIOTIC-

Affected

Reaction or Consequence

METABOLIZING ENZYMES ARE

Glucose-6-phosphate

Hemolytic anemia following in-

AFFECTED BY AGE, SEX,

dehydrogenase (G6PD)

gestion of drugs such as prim-

[mutations] (MIM 305900)

aquine

& OTHER FACTORS

Ca2+ release channel (ryan-

Malignant hyperthermia (MIM

Various factors affect the activities of the enzymes me-

odine receptor) in the

145600) following administra-

tabolizing xenobiotics. The activities of these enzymes

sarcoplasmic reticulum

tion of certain anesthetics (eg,

may differ substantially among species. Thus, for exam-

[mutations] (MIM 180901)

halothane)

ple, the possible toxicity or carcinogenicity of xenobi-

otics cannot be extrapolated freely from one species to

CYP2D6 [polymorphisms]

Slow metabolism of certain

(MIM 124030)

drugs (eg, debrisoquin), result-

another. There are significant differences in enzyme ac-

ing in their accumulation

tivities among individuals, many of which appear to be

due to genetic factors. The activities of some of these

CYP2A6 [polymorphisms]

Impaired metabolism of nico-

enzymes vary according to age and sex.

(MIM 122720)

tine, resulting in protection

Intake of various xenobiotics such as phenobarbital,

against becoming a tobacco-

PCBs, or certain hydrocarbons can cause enzyme in-

dependent smoker

duction. It is thus important to know whether or not

¹G6PD deficiency is discussed in Chapters 20 and 52 and malig-

an individual has been exposed to these inducing agents

nant hyperthermia in Chapter 49. At least one gene other than

in evaluating biochemical responses to xenobiotics.

that encoding the ryanodine receptor is involved in certain cases

Metabolites of certain xenobiotics can inhibit or stimu-

of malignant hypertension. Many other examples of drug reac-

late the activities of xenobiotic-metabolizing enzymes.

tions based on polymorphism or mutation are available.

METABOLISM OF XENOBIOTICS

631

Certain xenobiotics are very toxic even at low levels

in the endoplasmic reticulum to become carcinogenic

(eg, cyanide). On the other hand, there are few xenobi-

(they are thus called indirect carcinogens). The activi-

otics, including drugs, that do not exert some toxic ef-

ties of the monooxygenases and of other xenobiotic-

fects if sufficient amounts are administered. The toxic

metabolizing enzymes present in the endoplasmic retic-

effects of xenobiotics cover a wide spectrum, but the

ulum thus help to determine whether such compounds

major effects can be considered under three general

become carcinogenic or are “detoxified.” Other chemi-

headings (Figure 53-1).

cals (eg, various alkylating agents) can react directly (di-

The first is cell injury (cytotoxicity), which can be

rect carcinogens) with DNA without undergoing intra-

severe enough to result in cell death. There are many

cellular chemical activation.

mechanisms by which xenobiotics injure cells. The one

The enzyme epoxide hydrolase is of interest be-

considered here is covalent binding to cell macromol-

cause it can exert a protective effect against certain car-

ecules of reactive species of xenobiotics produced by

cinogens. The products of the action of certain

metabolism. These macromolecular targets include

monooxygenases on some procarcinogen substrates are

DNA, RNA, and protein. If the macromolecule to

epoxides. Epoxides are highly reactive and mutagenic

which the reactive xenobiotic binds is essential for

or carcinogenic or both. Epoxide hydrolase—like cy-

short-term cell survival, eg, a protein or enzyme in-

tochrome P450, also present in the membranes of the

volved in some critical cellular function such as oxida-

endoplasmic reticulum—acts on these compounds,

tive phosphorylation or regulation of the permeability

converting them into much less reactive dihydrodiols.

of the plasma membrane, then severe effects on cellular

The reaction catalyzed by epoxide hydrolase can be rep-

function could become evident quite rapidly.

resented as follows:

Second, the reactive species of a xenobiotic may

bind to a protein, altering its antigenicity. The xenobi-

C C

H₂O

C C

otic is said to act as a hapten, ie, a small molecule that

by itself does not stimulate antibody synthesis but will

combine with antibody once formed. The resulting an-

Epoxide

Dihydrodiol

tibodies can then damage the cell by several immuno-

logic mechanisms that grossly perturb normal cellular

PHARMACOGENOMICS WILL DRIVE THE

biochemical processes.

DEVELOPMENT OF NEW & SAFER DRUGS

Third, reactions of activated species of chemical car-

cinogens with DNA are thought to be of great impor-

As indicated above, pharmacogenetics is the study of

tance in chemical carcinogenesis. Some chemicals (eg,

the roles of genetic variations in the responses to drugs.

benzo[α]pyrene) require activation by monooxygenases

As a result of the progress made in sequencing the

GSH S-transferase or

Cytochrome P450

epoxide hydrolase

Xenobiotic

Reactive metabolite

Nontoxic metabolite

Covalent binding to

macromolecules

Cell injury

Hapten

Mutation

Antibody production

Cancer

Cell injury

Figure 53-1. Simplified scheme showing how metabolism of a xenobiotic can result in cell injury, immuno-

logic damage, or cancer. In this instance, the conversion of the xenobiotic to a reactive metabolite is catalyzed

by a cytochrome P450, and the conversion of the reactive metabolite (eg, an epoxide) to a nontoxic metabolite

is catalyzed either by a GSH S-transferase or by epoxide hydrolase.

632

CHAPTER 53

human genome, a new field of study—pharmacoge-

•

Cytochrome P450s are generally located in the endo-

nomics—has developed recently. It includes pharmaco-

plasmic reticulum of cells and are particularly en-

genetics but covers a much wider sphere of activity. In-

riched in liver.

formation from genomics, proteomics, bioinformatics,

•

Many cytochrome P450s are inducible. This has im-

and other disciplines such as biochemistry and toxicol-

portant implications in phenomena such as drug in-

ogy will be integrated to make possible the synthesis of

teraction.

newer and safer drugs. As the sequences of all our genes

•

Mitochondrial cytochrome P450s also exist and are

and their encoded proteins are determined, this will re-

involved in cholesterol and steroid biosynthesis.

veal many new targets for drug actions. It will also re-

They use a nonheme iron-containing sulfur protein,

veal polymorphisms (this term is briefly discussed in

adrenodoxin, not required by microsomal isoforms.

Chapter 50) of enzymes and proteins related to drug

•

Cytochrome P450s, because of their catalytic activi-

metabolism, action, and toxicity. DNA probes capable

ties, play major roles in the reactions of cells to

of detecting them will be synthesized, permitting

chemical compounds and in chemical carcinogenesis.

screening of individuals for potentially harmful poly-

•

Phase 2 reactions are catalyzed by enzymes such as

morphisms prior to the start of drug therapy. As the

structures of relevant proteins and their polymorphisms

glucuronosyltransferases, sulfotransferases, and glu-

tathione S-transferases, using UDP-glucuronic acid,

are revealed, model building and other techniques will

permit the design of drugs that take into account both

PAPS (active sulfate), and glutathione, respectively,

as donors.

normal protein targets and their polymorphisms. At

least to some extent, drugs will be tailor-made for indi-

•

Glutathione not only plays an important role in

viduals based on their genetic profiles. A new era of ra-

phase 2 reactions but is also an intracellular reducing

tional drug design built on information derived from

agent and is involved in the transport of certain

genomics and proteomics has already commenced.

amino acids into cells.

•

Xenobiotics can produce a variety of biologic effects,

SUMMARY

including pharmacologic responses, toxicity, immuno-

logic reactions, and cancer.

•

Xenobiotics are chemical compounds foreign to the

body, such as drugs, food additives, and environmen-

•

Catalyzed by the progress made in sequencing the

tal pollutants; more than 200,000 have been identi-

human genome, the new field of pharmacogenomics

fied.

offers the promise of being able to make available a

host of new rationally designed, safer drugs.

•

Xenobiotics are metabolized in two phases. The

major reaction of phase 1 is hydroxylation catalyzed

by a variety of monooxygenases, also known as the

REFERENCES

cytochrome P450s. In phase 2, the hydroxylated

species are conjugated with a variety of hydrophilic

Evans WE, Johnson JA: Pharmacogenomics: the inherited basis for

compounds such as glucuronic acid, sulfate, or glu-

interindividual differences in drug response. Annu Rev Ge-

nomics Hum Genet 2001;2:9.

tathione. The combined operation of these two

Guengerich FP: Common and uncommon cytochrome P450 reac-

phases renders lipophilic compounds into water-

tions related to metabolism and chemical toxicity. Chem Res

soluble compounds that can be eliminated from the

Toxicol 2001;14:611.

body.

Honkakakoski P, Negishi M: Regulation of cytochrome P450

•

Cytochrome P450s catalyze reactions that introduce

(CYP) genes by nuclear receptors. Biochem J 2000;347:321.

one atom of oxygen derived from molecular oxygen

Kalow W, Grant DM: Pharmacogenetics. In: The Metabolic and

into the substrate, yielding a hydroxylated product.

Molecular Bases of Inherited Disease, 8th ed. Scriver CR et al

NADPH and NADPH-cytochrome P450 reductase

(editors). McGraw-Hill, 2001.

are involved in the complex reaction mechanism.

Katzung BG (editor): Basic & Clinical Pharmacology, 8th ed. Mc-

Graw-Hill, 2001.

•

All cytochrome P450s are hemoproteins and gener-

McLeod HL, Evans WE: Pharmacogenomics: unlocking the

ally have a wide substrate specificity, acting on many

human genome for better drug therapy. Annu Rev Pharmacol

exogenous and endogenous substrates. They repre-

Toxicol 2001;41:101.

sent the most versatile biocatalyst known.

Nelson DR et al: P450 superfamily: update on new sequences, gene

•

Members of at least 11 families of cytochrome P450

mapping, accession numbers and nomenclature. Pharmacoge-

are found in human tissue.

netics 1996;6:1.

The Human Genome Project

Robert K. Murray, MD, PhD

BIOMEDICAL SIGNIFICANCE

rizes the differences between a genetic map, a cytoge-

netic map, and a physical map of a chromosome. These

The information deriving from determination of the se-

and other initial goals were achieved and surpassed by

quences of the human genome and those of other or-

the mid-nineties. In 1998, new goals for the United

ganisms will change biology and medicine for all time.

States wing of the HGP were announced. These in-

For example, with reference to the human genome, new

cluded the aim of completing the entire sequence by

information on our origins, on disease genes, on diag-

the end of 2003 or sooner. Other specific objectives

nosis, and possible approaches to therapy are already

concerned sequencing technology, comparative ge-

flooding in. Progress in fields such as genomics, pro-

nomics, bioinformatics, ethical considerations, and

teomics, bioinformatics, biotechnology, and pharma-

other issues. By the fall of

1998, about 6% of the

cogenomics is accelerating rapidly.

human genome sequence had been completed and the

The aims of this chapter are to briefly summarize

foundations for future work laid. Further progress was

the major findings of the Human Genome Project

catalyzed by the announcement that a second group,

(HGP) and indicate their implications for biology and

the private company Celera Genomics, led by Craig

medicine.

Venter, had undertaken the objective of sequencing the

human genome. Venter and colleagues had published

THE HUMAN GENOME PROJECT

in 1995 the entire genome sequences of Haemophilus

influenzae and Mycoplasma genitalium, the first of many

HAS A VARIETY OF GOALS

species to have their genomic sequences determined. An

The HGP, which started in 1990, is an international

important factor in the success of these workers was the

effort whose principal goals were to sequence the entire

use of a shotgun approach, ie, sonicating the DNA, se-

human genome and the genomes of several other model

quencing the fragments, and reassembling the se-

organisms that have been basic to the study of genetics

quence, based on overlaps. For comparison, a variety of

(eg, Escherichia coli, Saccharomyces cerevisiae [a yeast],

approaches that have been used at different times to

Drosophila melanogaster [the fruit fly], Caenorhabditis

study normal and disease genes are listed in Table 54-1.

elegans [the roundworm], and Mus musculus [the com-

mon house mouse]). Most of these goals have been ac-

A Draft Sequence of the Human Genome

complished. In the United States, the National Center

Was Announced in June 2000

for Human Genome Research (NCHGR) was estab-

lished in 1989, initially directed by James D. Watson

In June 2000, leaders of the IHGSC and the personnel

and subsequently by Francis Collins. The NCHGR

at Celera Genomics announced completion of working

played a leading role in directing the United States ef-

drafts of the sequence of the human genome, covering

fort in the HGP. In 1997, it became the National

more than 90% of it. The principal findings of the two

Human Genome Research Institute (NHGRI). The in-

groups were published separately in February 2001 in

ternational collaboration—involving groups from the

special issues of Nature (the IHGSC) and Science (Cel-

USA, UK, Japan, France, Germany, and China—came

era). The draft published by the Consortium was the

to be known as the International Human Genome Se-

product of at least 10 years of work involving 20 se-

quencing Consortium (IHGSC). Initially, a number of

quencing centers located in six countries. That pub-

short-term goals were established for the United States

lished by Celera and associates was the product of some

effort—eg, producing a human genetic map with mark-

3 years or less of work; it relied in part on data obtained

ers

2-5 centimorgans (cM) apart and constructing a

by the IHGSC. The combined achievement has been

physical map of all 24 human chromosomes (22 auto-

hailed, among other descriptions, as providing a Library

somal plus X and Y) with markers spaced at approxi-

of Life, supplying a Periodic Table of Life, and finding

mately 100,000 base pairs (bp). Figure 54-1 summa-

the Holy Grail of Human Genetics.

633

634

CHAPTER 54

Genetic map

Cytogenetic

map

Physical map

100

125

150 Mb

EcoRI Hind III NotI

Restriction map

STS map

Contig map

Figure 54-1. Principal methods used to identify and isolate normal

and disease genes. For the genetic map, the positions of several hypo-

thetical genetic markers are shown, along with the genetic distances in

centimorgans between them. The circle shows the position of the cen-

tromere. For the cytogenetic map, the classic banding pattern of a hypo-

thetical chromosome is shown. For the physical map, the approximate

physical positions of the above genetic markers are shown, along with

the relative physical distances in megabase pairs. Examples of a restric-

tion map, a contig mark, and an STS map are also shown. (Reproduced,

with permission, from Green ED, Waterston RH: The Human Genome

Project: Prospects and implications for clinical medicine. JAMA 1991;266:

Different Approaches Were Used

(STSs), whose locations had been already determined.

by the Two Groups

STSs are short (usually < 500 bp), unique genomic loci

for which a PCR assay is available. Clones of the BACs

We shall summarize the major findings reported in the

were then broken into small fragments (shotgunning).

two drafts and comment on their implications. While

Each fragment was then sequenced, and computer algo-

there are differences between the drafts, they will not be

rithms were used that recognized matching sequence

dwelt on here, as the areas of general agreement are

information from overlapping fragments to piece to-

much more extensive. It is worthwhile, however, to

gether the complete sequence.

summarize the different approaches used by the two

Celera used the whole genome shotgun approach,

groups. Basically, the IHGSC employed a map first,

in effect bypassing the mapping step. Shotgun frag-

sequence later approach. In part, this was because se-

ments were assembled by algorithms onto large scaf-

quencing was a slow process when the public project

folds, and the correct position of these scaffolds in the

started, and the strategy of the Consortium evolved

genome was determined using STSs. A scaffold is a se-

over time as advances were made in sequencing and

ries of “contigs” that are in the right order but not nec-

other techniques. The overall approach, referred to as

essarily connected in one continuous sequence. Contigs

hierarchical shotgun sequencing, consisted of frag-

are contiguous sequences of DNA made by assembling

menting the entire genome into pieces of approxi-

overlapping sequenced fragments of a natural chromo-

mately 100-200 kb and inserting them into bacterial

some or a BAC. The availability of high-throughput

artificial chromosomes (BACs). The BACs were then

sequenators, powerful computer programs, the ele-

positioned on individual chromosomes by looking for

ment of competition, and other factors accounted for the

marker sequences known as sequence-tagged sites

rapid progress made by both groups from 1998 onward.

THE HUMAN GENOME PROJECT

635

Table 54-1. Principal methods used to identify and isolate normal and disease genes.

Procedure

Comments

Detection of specific cytogenetic

For instance, a small deletion of band Xp21.2 was important in cloning the gene involved in

abnormalities

Duchenne muscular dystrophy.

Extensive linkage studies

Large families with defined pedigrees are desirable. Dominant genes are easier to recognize

than recessives.

Use of probes to define marker

Probes identify STSs, RFLPs, SNPs,¹ etc; thousands, covering all the chromosomes, are now

loci

available. It is desirable to flank the gene on both sides, clearly delineating it.

Radiation hybrid mapping²

Now the most rapid method of localizing a gene or DNA fragment to a subregion of a human

chromosome and constructing a physical map.

Use of rodent or human somatic

Permits assignment of a gene to one specific chromosome but not to a subregion.

cell hybrids

Fluorescence in situ hybridization

Permits localization of a gene to one chromosomal band.

Use of pulsed-field gel

Permits isolation of large DNA fragments obtained by use of restriction endonucleases (rare

electrophoresis (PFGE) to

cutters) that result in very limited cutting of DNA.

separate large DNA fragments

Chromosome walking

Involves repeated cloning of overlapping DNA segments; the procedure is laborious and can

usually cover only 100-200 kb.

Chromosome jumping

By cutting DNA into relatively large fragments and circularizing it, one can move more quickly

and cover greater lengths of DNA than with chromosomal walking.

Cloning via YACs, BACs, cosmids,

Permits isolation of fragments of varying lengths.

phages, and plasmids

Detection of expression of

The mRNA should be expressed in affected tissues.

mRNAs in tissues by Northern

blotting using one or more

fragments of the gene as a

probe

PCR

Can be used to amplify fragments of the gene; also many other applications.

DNA sequencing

Establishes the highest resolution physical map. Identifies open reading frame. Facilities with

many high throughput instruments could sequence millions of base pairs per day.

Databases

Comparison of DNA and protein sequences obtained from unknown gene with known se-

quences in databases can facilitate gene identification.

Abbreviations: STS, sequence tagged site; RFLP, restriction fragment linked polymorphism; SNP, single nucleotide polymorphism; YAC,

yeast artificial chromosome; BAC, bacterial artificial chromosome; PCR, polymerase chain reaction.

¹Many single nucleotide polymorphisms (SNPs) are being detected and catalogued. These are stable and frequent, and their detection

can be automated. It is anticipated that they will be particularly useful for mapping complex traits such as diabetes mellitus.

²Radiation hybrid mapping (consult http://compgen.rutgers.edu/rhmap/ for a detailed bibliography of this technique) makes use of a

panel of somatic cell hybrids, with each cell line containing a random set of irradiated human genomic DNA in a hamster background.

Briefly, the radiation fragments the DNA into small pieces of variable length; if a gene is located close to another known gene, it is likely

that the two will remain linked (compare genetic linkage) on the same fragment. An STS marker is typed against a radiation hybrid panel

by using its two oligonucleotide primers to perform a PCR assay against the DNA from each hybrid cell line of the panel. If enough mark-

ers are typed on one panel, continuous linkage can be established along each arm of a chromosome, and the markers can be assembled

into the map as a single linkage group.

636

CHAPTER 54

DETERMINATION OF THE SEQUENCE OF

fly (13,061). The figures suggest that the complexity of

humans compared with that of the two simpler organ-

THE HUMAN GENOME HAS PRODUCED A

isms must have explanations other than strictly gene

WEALTH OF NEW FINDINGS

number.

Only a small fraction of the findings can be covered

here. The interested reader is referred to the original ar-

Only 1.1-1.5% of the Human

ticles. Table 54-2 summarizes a number of the high-

Genome Encodes Proteins

lights, which can now be described.

Analyses of the available data reveal that 1.1-1.5% of

Most of the Human Genome

the genome consists of exons. About 24% consists of

Has Been Sequenced

introns, and 75% of sequences lying between genes (in-

tergenic). Comparisons with the data on the round-

Over 90% of the human genome had been sequenced

worm and fruit fly have shown that exon size across the

by July 2000. This is by far the largest genome se-

three species is relatively constant (mean size of 145 bp

quenced, with an estimated size of approximately 3.2

in humans). However, intron size in humans is much

gigabases (Gb). Prior to the human genome, that of the

more variable (mean size of over 3300 bp), resulting in

fruit fly had been the largest (~180 Mb) sequenced.

great variation in gene size.

Gaps still exist, small and large, and the quality of some

of the sequencing data will be refined since some of the

findings are probably not exactly right.

The Landscape of Human Chromosomes

Varies Widely

The Human Genome Is Estimated to

There are marked differences among individual chro-

Encode About 30,000-40,000 Proteins

mosomes in many features, such as gene number per

The greatest surprise provided by the results to date has

megabase, density of single nucleotide polymorphisms

been the apparently low number of genes encoding pro-

(SNPs), GC content, number of transposable elements

teins, estimated to lie between 30,000 and 40,000. The

and CpG islands, and recombination rate. To take one

higher number could increase as new data are obtained.

example, chromosome 19 has the richest gene content

This number is approximately twice that found in the

(23 genes per megabase), whereas chromosome 13 and

roundworm (19,099) and three times that of the fruit

the Y chromosome have the sparsest content (5 genes

per megabase). Explanations for these variations are not

apparent at this time.

Table 54-2. Major findings reported in the rough

drafts of the human genome.

Human Genes Do More Work Than

Those of Simpler Organisms

• More than 90% of the genome has been sequenced; gaps,

Alternative splicing appears to be more prevalent in hu-

large and small, remain to be filled in.

mans, involving at least 35% of their genes. Data indi-

• Estimated number of protein-coding genes ranges from

cate that the average number of distinct transcripts per

30,000 to 40,000.

gene for chromosomes 22 and 19 were 2.6 and 3.2, re-

• Only 1.1-1.5% of the genome codes for proteins.

spectively. These figures are higher than for the round-

• There are wide variations in features of individual chromo-

worm, where only 12.2% of genes appear to be alterna-

somes (eg, in gene number per Mb, SNP density, GC con-

tively spliced and only 1.34 splice variants per gene

tent, numbers of transposable elements and CpG islands,

were noted.

recombination rate).

• Human genes do more work than those of the roundworm

or fruit fly (eg, alternative splicing is used more frequently).

The Human Proteome Is More Complex

• The human proteome is more complex than that found in

Than That of Invertebrates

invertebrates.

• Repeat sequences probably constitute more than 50% of

Relatively few new protein domains appear to have

the genome.

emerged among vertebrates. However, the number of

• Approximately 100 coding regions have been copied and

distinct domain architectures (~1800) in human pro-

moved by RNA-based transposons.

teins is 1.8 times that of the roundworm or fruit fly.

• Approximately 200 genes may be derived from bacteria by

About 90 vertebrate-specific families of proteins have

lateral transfer.

been identified, and these have been found to be en-

• More than 3 million SNPs have been identified.

riched in proteins of the immune and nervous systems.

THE HUMAN GENOME PROJECT

637

The results of the two drafts are rich in information

Segmental duplications have been found to be much

about protein families and classes. One example is

more common than in the roundworm or fruit fly. It is

shown in Table 54-3, in which the major classes of

possible that these structures may be involved in exon

proteins encoded by human genes are listed. As can be

shuffling and the increased diversity of proteins found

seen, the largest class is “unknown.” Identification of

in humans.

these unknown proteins will be a major focus of activity

for many laboratories.

Other Findings of Interest

The last three major points of interest listed in Table

Repeat Sequences Probably Constitute

54-2 will be briefly described together.

More Than 50% of the Human Genome

Approximately 100 coding regions are estimated to

Repeat sequences probably account for at least half of

have been copied and moved by RNA-based trans-

the genome. They fall into five classes: (1) transposon-

posons (retrotransposons). It is possible that some of

derived repeats

(interspersed repeats);

(2) processed

these genes may adopt new roles in the course of time.

pseudogenes; (3) simple sequence repeats; (4) segmental

A surprising finding is that over 200 genes may be de-

duplications, made up of 10-300 kb that have been

rived from bacteria by lateral transfer. None of these

copied from one region of the genome into another;

genes are present in nonvertebrate eukaryotes. More

and (5) blocks of tandemly repeated sequences, found

than 3 million SNPs have been identified. It is likely

at centromeres, telomeres, and other areas. Consider-

that they will prove invaluable for certain aspects of

able information on most of the above classes of repeat

gene mapping.

sequences—of great value in understanding the archi-

It is stressed that the findings listed here are only a

tecture and development of the human genome—is re-

few of those reported in the drafts, and the reader is

ported in the drafts. Only two points of interest will be

urged to consult the original reports (see References,

mentioned here. It is speculated that Alu elements, the

below).

most prominent members (about 10% of the total

genome) of the short interspersed elements (SINEs),

FURTHER WORK IS PLANNED ON THE

may be present in GC-rich areas because of positive se-

HUMAN & OTHER GENOMES

lection, implying that they are of benefit to the host.

The IHGSC has indicated that it will determine the

complete sequence, it is hoped, by 2003. The task in-

Table 54-3. Major classes of proteins encoded by

volves filling in the gaps and identifying new genes,

human genes.¹

their locations, and functions. Regulatory regions will

be identified, and the sequences of other large genomes

(eg, of the house mouse; of Rattus norvegicus, the Nor-

Class of Protein

Number (%)²

way rat; of Danio rerio, the zebra fish; of Fugu rubripes,

Unknown

12,809 (41%)

the tiger puffer fish; and of one or more primates) will

be obtained; indeed, a draft version of the genome of

Nucleic acid enzymes

2,308 (7.5%)

the tiger puffer fish was published in 2002. Additional

Transcription factors

1,850 (6%)

SNPs will be identified; a complete catalog of these

Receptors

1,543 (5%)

variants is expected to be of great value in mapping

genes associated with complex traits and for other uses

Hydrolases

1,227 (4.0%)

as well. Along with the above, existing databases will be

Select regulatory molecules (eg,

988 (3.2%)

added to as new information flows in, and new data-

G proteins, cell cycle regulators)

bases will probably be established to serve specific pur-

poses. A variety of studies in functional genomics (ie,

Protooncogenes

902 (2.9%)

the study of genomes to determine the functions of all

Cytoskeletal structural proteins

876 (2.8%)

their genes and their products) will also be undertaken.

Kinases

868 (2.8%)

IMPLICATIONS FOR PROTEOMICS,

¹Data from Venter JC et al: The sequence of the human genome.

Science 2001;291:1304.

BIOTECHNOLOGY, & BIOINFORMATICS

²The percentages are derived from a total of 26,383 genes re-

ported in the rough draft by Celera Genomics. Classes containing

Many fields will be influenced by knowledge of the

more than 2.5% of the total proteins encoded by the genes iden-

human genome. Only a few are briefly discussed here.

tified when this rough draft was written are arbitrarily listed as

Proteomics (see Chapter 4) in its broadest sense is

major.

the study of all the proteins encoded in an organism (ie,

638

CHAPTER 54

the proteome), including their structures, modifica-

effects on health services and the diagnosis and treat-

tions, functions, and interactions. In a narrower sense,

ment of disease.

it involves the identification and study of multiple pro-

teins linked through cellular actions—but not necessar-

SUMMARY

ily the entire proteome. With regard to humans, many

• Determination of the complete sequence of the

individual proteins will be identified and characterized;

human genome, now almost completed, is one of the

their interactions and levels will be determined in phys-

most significant scientific achievements of all time.

iologic and pathologic states, and the resulting informa-

tion will be entered into appropriate databases. Tech-

• Many important findings have already emerged. The

niques such as two-dimensional electrophoresis, a

one to date that has generated the most discussion is

variety of modes of mass spectrometry, and antibody

that the number of human genes may be only two to

arrays will be central to expansion of this rapidly grow-

three times that estimated for the roundworm and

ing field. Overall, proteomics will greatly advance our

the fruit fly.

knowledge of proteins at the basic level and will also

• Information flowing from the Human Genome Proj-

nourish biotechnology as new proteins that are likely

ect is having major influences in fields such as pro-

to have diagnostic, therapeutic, and other uses are dis-

teomics, bioinformatics, biotechnology, and phar-

covered and methods for their economic production are

macogenomics as well as all areas of biology and

developed. Specialists in bioinformatics will be in de-

medicine.

mand, as this field rapidly gears up to manage, analyze,

• It is hoped that the knowledge derived will be used

and utilize the flood of data from genomic and pro-

wisely and fairly and that the benefits that will ensue

teomic studies.

regarding health, disease, and other matters will be

made available to all people everywhere.

IMPLICATIONS FOR MEDICINE

REFERENCES

Practically every area of medicine will be affected by the

Collins FS, McKusick VA: Implications of the Human Genome

new information accruing from knowledge of the

Project for medical science. JAMA 2001;285:540. (The Feb-

human genome. The tracking of disease genes will be

ruary 7, 2001, issue describes opportunities for medical re-

enormously facilitated. As mentioned above, SNP maps

search in the 21st century. Many articles of interest.)

should greatly assist determination of genes involved in

Hedges SB, Kumar S: Vertebrate genomes compared. Science

complex diseases. Probes for any gene will be available

2002;297:1283. (The same issue—No. 5585, August 23—

contains a draft version of the genome of the tiger puffer

if needed, leading to improved diagnostic testing for

fish.)

disease susceptibility genes and for genes directly in-

McKusick VA: The anatomy of the human genome: a neo-Vesalian

volved in the causation of specific diseases. The field of

basis for medicine in the

21st century. JAMA 2001;286:

pharmacogenomics (see Chapter 53) is already ex-

2289. (The November 14, 2001, issue contains a number of

panding greatly, and it is possible that in the future

other excellent articles—eg, on clinical proteomics, pharma-

drugs will be tailored to accommodate the variations in

cogenomics—relating to the Human Genome Project and its

enzymes and other proteins involved in drug action and

impact on medicine.)

metabolism found among individuals. Studies of genes

Nature

2001;409(6822)

(February

15), and Science

2001;291

involved in behavior may lead to new insights into the

(5507) (February 16). (These two issues present the rough

drafts prepared by the IHGSC and Celera, respectively, along

causation and possible treatment of psychiatric disor-

with many other articles analyzing the meaning and signifi-

ders. Many ethical issues—eg, privacy concerns and

cance of the findings.)

the use of genomic information for commercial pur-

Science 2001;294(5540) (October 5). (This issue contains a num-

poses—will have to be addressed. It will also be impor-

ber of articles under the title Genome: Unlocking Biology’s

tant that medical and economic benefits accrue to indi-

Storehouse. They describe new ideas, approaches, and re-

viduals in Third World countries from the anticipated

search related to genome information.)

APPENDIX

SELECTED WORLD WIDE WEB SITES

(Maintains the EMBL Nucleotide and SWISS-PROT databases as

well as other databases.)

The following is a list of Web sites that readers may

GeneCards: http://bioinformatics.weizmann.ac.il/cards/

find useful. The sites have been visited at various times

(A database of human genes, their products, and their involvements

by one of the authors (RKM). Most are located in the

in diseases. From the Weizmann Institute of Science.)

USA, but many provide extensive links to international

GeneTestsGeneClinics: http://www.geneclinics.org/

sites and to databases (eg, for protein and nucleic acid

(A medical genetics information resource with comprehensive arti-

sequences) and online journals. RKM would be grateful

cles on many genetic diseases.)

if readers who find other useful sites would notify him

Genes and Disease: http://www.ncbi.nlm.nih.gov/disease/

of their URLs by e-mail (rmurray6745@rogers. com) so

(Coverage of the genetic basis of many different types of diseases.)

that they may be considered for inclusion in future edi-

The Glycoscience Network (TGN): http://www.vei.co.uk/TGN/

tions of this text.

tgn_side.htm

Readers should note that URLs may change or cease

(TGN is an informal worldwide grouping of scientists who share an

to exist.

interest in carbohydrates. The site contains considerable in-

formation on carbohydrates and an extensive list of links to

other sites dealing with sugar-containing molecules).

Howard Hughes Medical Institute: http://www.hhmi.org/

ACCESS TO THE BIOMEDICAL

(An excellent site for following current biomedical research. Con-

tains a comprehensive Research News Archive.)

LITERATURE

The Human Gene Mutation Database: http://archive.uwcm.ac.uk/

uwcm/mg/hgmd0.html

HighWire Press: http://highwire.stanford.edu

(An extensive tabulation of mutations in human genes from the In-

stitute of Medical Genetics in Cardiff, Wales.)

(Extensive lists of various classes of journals—biology, medicine,

etc—and offers also the most extensive list of journals with

Human Genome Project Information: http://www.ornl.gov/hgmis/

free online access.)

(From the Human Genome Program of the United States Depart-

National Library of Medicine: http://www.nlm.nih.gov/

ment of Energy.)

(Free access to Medline via PubMed.)

The Institute for Genetic Research: http://www.tigr.org/

(Sequences of various bacterial genomes and other information.)

GENERAL RESOURCE SITES

Karolinska Institute Nutritional and Metabolic Diseases: http://

www.mic.ki.se/Diseases/c18.html

The Biology Project

(from the University of Arizona): http://

(Access to information on many nutritional and metabolic disor-

www.biology.arizona.edu/default.html

ders.)

Harvard Department of Molecular & Cellular Biology Links:

MITOMAP: http://www.mitomap.org/

http://mcb.harvard.edu/BioLinks.html

(A human mitochondrial genome database.)

National Center for Biotechnology Information: http://ncbi.nlm.

SITES ON SPECIFIC TOPICS

nih.gov/

American Heart Association: http://www.americanheart.org

(Information on molecular biology and how molecular processes af-

fect human health and disease.)

(Useful information on nutrition, on the role of various biomole-

cules—eg, cholesterol, lipoproteins—in heart disease, and on

National Human Genome Research Institute: http://www.genome.

the major cardiovascular diseases.)

gov/

Cancer Genome Anatomy Project (CGAP): http://www.ncbi.nlm.

(Extensive information about the Human Genome Project.)

nih.gov/ncicgap

National Institutes of Health (NIH): http://www.nih.gov/

(An interdisciplinary program to generate the information and

(Includes links to the separate Institutes and Centers that constitute

technical tools needed to decipher the molecular anatomy of

NIH, covering a wide range of biomedical research.)

the cancer cell.)

Neuroscience (Biosciences): http://neuro.med.cornell.edu/VL/

European Bioinformatics Institute: http://www.ebi.ac.uk/ebi_

(A comprehensive list of neuroscience resources; part of the World-

home.html

Wide Web Virtual Library.)

639

APPENDIX

641

BIOCHEMICAL JOURNALS AND REVIEWS

Biochimica et Biophysica Acta (Biochim Biophys

Acta)

The following is a partial list of biochemistry journals

Biochimie (Biochimie)

and review series and of some biomedical journals that

European Journal of Biochemistry (Eur J Biochem)

contain biochemical articles. Biochemistry and biology

journals now usually have Web sites, often with useful

Indian Journal of Biochemistry and Biophysics (In-

links, and some journals are fully accessible without

dian J Biochem Biophys)

charge. The reader can obtain the URLs for the follow-

Journal of Biochemistry

(Tokyo)

(J Biochem

ing by using a search engine.

[Tokyo])

Journal of Biological Chemistry (J Biol Chem)

Annual Reviews of Biochemistry, Cell and Develop-

Journal of Clinical Investigation (J Clin Invest)

mental Biology, Genetics, Genomics and Human

Journal of Lipid Research (J Lipid Res)

Genetics

Nature (Nature)

Archives of Biochemistry and Biophysics

(Arch

Biochem Biophys)

Nature Genetics (Nat Genet)

Biochemical and Biophysical Research Communica-

Proceedings of the National Academy of Sciences

tions (Biochem Biophys Res Commun)

USA (Proc Natl Acad Sci USA)

Biochemical Journal (Biochem J)

Science (Science)

Biochemistry (Biochemistry)

Trends in Biochemical Sciences (Trends Biochem

Sci)

Biochemistry (Moscow) (Biochemistry [Mosc])

Index

Note: Page numbers in bold face type indicate a major discussion. A t following a page number indicates tabular ma-

terial and an f following a page number indicates a figure.

A bands, 556, 557f, 558f

formation of, 254f, 255-259, 255f, 256f,

molecular structure affecting strength of,

A blood group substance, 618, 619f

257f, 258f, 259f

12, 12t

A cyclins, 333, 334f, 335t

lipogenesis and, 173-177, 174f, 175f

polyfunctional, nucleotides as, 290

A gene, GalNAc transferase encoded by,

as fatty acid building block, 176-177

as proton donors, 9

618-619

pyruvate dehydrogenase regulated by,

strong, 9

A (aminoacyl/acceptor) site, aminoacyl-

141-142, 142f, 178

weak. See Weak acids

tRNA binding to, in protein

pyruvate oxidation to, 134, 135f,

Aciduria

synthesis, 368, 368f

140-142, 141f, 142f, 143t

dicarboxylic, 188

ABC-1. See ATP-binding cassette

xenobiotic metabolism and, 630

methylmalonic, 155

transporter-1

Acetyl-CoA carboxylase, 156t, 179

orotic, 300, 301

Abetalipoproteinemia, 207, 228t

in lipogenesis regulation, 156t, 173,

urocanic, 250

ABO blood group system, biochemical basis

174f, 178, 178f, 179

Aconitase (aconitate hydratase), 130

of, 617-619, 619f

N-Acetylgalactosamine (GalNAc), in

ACP. See Acyl carrier protein

Absorption, 474-480

glycoproteins, 515, 516t

Acrosomal reaction, glycoproteins in, 528

Absorption chromatography, for

N-Acetylglucosamine (GlcNAc), in

ACTH. See Adrenocorticotropic hormone

protein/peptide purification, 22

glycoproteins, 516t

Actin, 557, 559, 560

Absorption spectra, of porphyrins,

N-Acetylglucosamine phosphotransferase

decoration of, 561, 561f

273-274, 277f

(GlcNAc phosphotransferase)

fibronectin receptor interacting with,

Absorptive pinocytosis, 430

in I-cell disease, 532

540, 541f

ACAT (acyl-CoA:cholesterol acyltrans-

in pseudo-Hurler polydystrophy, 532

in muscle contraction, 557-559, 558f,

ferase), 223

N-Acetylglutamate, in urea biosynthesis,

561-562, 561f, 562f

Accelerator (Ac-) globulin (factor V), 600t,

245, 246f

regulation of striated muscle and,

601, 602f

Acetyl hexosamines, in glycoproteins, 109t

562-563

Acceptor (A/aminoacyl) site, aminoacyl-

N-Acetyl lactosamines, on N-linked glycan

in nonmuscle cells, 576-577

tRNA binding to, in protein

chains, 521

in red cell membranes, 615f, 616f, 616t,

synthesis, 368, 368f

Acetyl (acyl)-malonyl enzyme, 173, 175f

617

Acceptor arm, of tRNA, 310, 312f, 360,

N-Acetyl neuraminic acid, 169, 171f

in striated vs. smooth muscle, 572t

361f

in gangliosides, 201, 203f

β-Actin, 577

Aceruloplasminemia, 589

in glycoproteins, 169, 171f, 515, 516t

F-Actin, 559, 559f, 561

ACEs. See Angiotensin-converting enzyme

in mucins, 519f, 520

in nonmuscle cells, 576, 577

inhibitors

Acetyl transacylase, 173, 174f, 175f

G-Actin, 559, 559f

Acetal links, 105-106

Acetyltransferases, xenobiotic metabolism

in nonmuscle cells, 576

Acetic acid, 112t

and, 630

γ-Actin, 577

pK/pK_a value of, 12t

Acholuric jaundice, 282

Actin-filament capping protein, 540, 541f

Acetoacetate, 183-184, 184f

Achondroplasia, 432t, 551t, 553-554, 554f

Actin (thin) filaments, 557, 558f, 559f

in tyrosine catabolism, 254f, 255

Acid anhydride bonds, 287

α-Actinin, 540, 566t

Acetoacetyl-CoA synthetase, in mevalonate

Acid anhydrides, group transfer potential

Activated protein C, in blood coagulation,

synthesis, 219, 220f

for, 289-290, 289f, 290f, 290t

603

Acetone, 183

Acid-base balance, ammonia metabolism in,

Activation energy, 61, 63

Acetone bodies. See Ketone bodies

245

Activation energy barrier, enzymes affecting,

Acetylation

Acid-base catalysis, 51-52

in covalent modification, mass increases

HIV protease in, 52, 53f

Activation function 1, 460

and, 27t

α₁-Acid glycoprotein (orosomucoid), 583t

Activation function 2, 460

of xenobiotics, 630

Acid hydrolysis, for polypeptide cleavage,

Activation reaction, 456, 458f

Acetyl-CoA, 122, 122f

26t

Activator-recruited cofactor (ARC), 472t,

carbohydrate metabolism and, 122, 122f,

Acid phosphatase, diagnostic significance

473

123f

of, 57t

Activators

catabolism of, 130-135, 131f, 132f. See

Acidemia, isovaleric, 259, 259-262

in regulation of gene expression, 374,

also Citric acid cycle

Acidosis

376. See also Enhancers

cholesterol synthesis and, 219-220, 220f,

lactic. See Lactic acidosis

transcription, 351, 351t

221f, 222f

metabolic, ammonia in, 245

Active chromatin, 316-318, 318f, 383

fatty acid oxidation to, 123-124, 123f,

Acids

Active site, 51, 51f. See also Catalytic site

181-183, 181f, 182f

conjugate, 10

∆G_F and, 63

643

644

INDEX

Active sulfate (adenosine 3′-phosphate-

metabolism in, 214-215, 214f, 235t

Alanine (alanine-pyruvate) aminotransferase

5′-phosphosulfate), 289, 289f, 629

control of, 216-217

(ALT/SGPT)

Active transport, 423, 423t, 424f, 426-427,

ADP, 287f

diagnostic significance of, 57t

427-428, 428f

free energy of hydrolysis of, 82t

in urea synthesis, 243-244, 244f

in bilirubin secretion, 280, 281f

mitochondrial respiratory rate and,

β-Alanyl dipeptides, 264, 265f

Activity (physical), energy expenditure and,

94-95, 97t, 98f

Albumin, 580, 581f, 583-584, 583t

478

myosin, muscle contraction and, 561,

conjugated bilirubin binding to, 283

Actomyosin, 560

561f

free fatty acids in combination with, 180,

ACTR coactivator, 472, 472t

in platelet activation, 606f, 607

206, 206t, 584

Acute fatty liver of pregnancy, 188

in respiratory control, 94-95, 97, 97t,

glomerular membrane permeability to,

Acute inflammatory response, neutrophils

98f, 134-135

540-541

in, 620

structure of, 83f

Albuminuria, 542

Acute phase proteins, 583, 583t

ADP/ATP transporter, 95, 98f

Alcohol, ethyl. See Ethanol

negative, vitamin A as, 483-484

ADPase, 607, 607t

Alcohol dehydrogenase, in fatty liver, 212

Acylcarnitine, 180-181, 181f

ADP-chaperone complex, 508. See also

Alcoholism

Acyl carrier protein (ACP), 173, 174f

Chaperones

cirrhosis and, 212

synthesis of, from pantothenic acid, 173,

ADP-ribose, NAD as source of, 490

fatty liver and, 212-214

495

ADP-ribosylation, 490

Aldehyde dehydrogenase, 87

Acyl-CoA:cholesterol acyltransferase

Adrenal cortical hormones. See also specific

in fatty liver, 212

(ACAT), 223

hormone and Glucocorticoids;

Aldolase A, deficiency of, 143

Acyl-CoA dehydrogenase, 87, 181, 182f

Mineralocorticoids

Aldolase B, 167, 168f

medium-chain, deficiency of, 188

synthesis of, 438-442, 440f, 441f

deficiency of, 171

Acyl-CoA synthetase (thiokinase)

Adrenal gland, cytochrome P450 isoforms

Aldolases, in glycolysis, 137, 138f

in fatty acid activation, 180, 181f

in, 627

Aldose-ketose isomerism, 103f, 104, 104f

in triacylglycerol synthesis, 199, 214f, 215

Adrenal medulla, catecholamines produced

Aldose reductase, 167, 169f, 172

Acylglycerols, 197

in, 445

Aldoses, 102, 102t, 104t

metabolism of, 197-201

Adrenergic receptors, in glycogenolysis, 148

ring structure of, 104

catabolism, 197

Adrenocorticotropic hormone (ACTH),

Aldosterone

clinical aspects of, 202

437, 438, 439f, 453, 453f

binding of, 455

synthesis, 197-201, 197f, 198f

Adrenodoxin, 627

synthesis of, 438-440, 441f

in endoplasmic reticulum, 126, 127f

Adrenodoxin reductase, 627

angiotensin affecting, 451, 452

Adapter proteins, in absorptive pinocytosis,

Adrenogenital syndrome, 442

Aldosterone synthase (18-hydroxylase), in

430

Adrenoleukodystrophy, neonatal, 503, 503t

steroid synthesis, 440, 441f

Adenine, 288f, 288t

Aerobic glycolysis, 139

Alkaline phosphatase

Adenosine, 287f, 288t

as muscle ATP source, 575, 575f, 575t

in bone mineralization, 550

base pairing of in DNA, 303, 304, 305f

Aerobic respiration

isozymes of, diagnostic significance of,

in uric acid formation, 299, 299f

citric acid cycle and, 130

57t

Adenosine deaminase

AF-1. See Activation function 1

in recombinant DNA technology, 400t

deficiency of, 300

AF-2. See Activation function 2

Alkalosis, metabolic, ammonia in, 245

localization of gene for, 407t

AF-2 domain, 470

Alkaptonuria, 255

Adenosine diphosphate. See ADP

Affinity chromatography

Allergic reactions, peptide absorption

Adenosine monophosphate. See AMP

for protein/peptide purification, 23

causing, 474

Adenosine 3′-phosphate-5′-phosphosulfate,

in recombinant fusion protein

Allopurinol, 290, 297

289, 289f, 629

purification, 58, 59f

Allosteric activators, 157

Adenosine triphosphate. See ATP

AFP. See Alpha-fetoprotein

Allosteric effectors/modifiers, 129

S-Adenosylmethionine, 258f, 259, 264,

Agammaglobulinemia, 595

in gluconeogenesis regulation, 157

266f, 289, 290f, 290t

Age, xenobiotic-metabolizing enzymes

negative, 74. See also Feedback inhibition

Adenylic acid, as second messenger, 457

affected by, 630

second messengers as, 76

Adenylyl cyclase

Aggrecan, 542, 551t, 553, 553f

Allosteric enzymes, 75, 129

in cAMP-dependent signal transduction,

Aggregates, formation of after denaturation,

aspartate transcarbamoylase as model of,

458-459, 460t

cAMP derived from, 147

Aging, glycosaminoglycans and, 549

Allosteric properties of hemoglobin, 42-46

in lipolysis, 215, 216f

Aglycone, 105, 106

Allosteric regulation, of enzymatic catalysis,

Adenylyl kinase (myokinase), 84

AHG. See Antihemophilic factor A/globulin

74, 74-76, 75f, 128f, 129

deficiencies of, 151-152

AIB1 coactivator, 472, 472t

gluconeogenesis regulation and, 157

in gluconeogenesis regulation, 157

ALA. See Aminolevulinate

Allosteric site, 74, 75

as source of ATP in muscle, 573, 575f

Alanine, 15t

Alpha-adrenergic receptors, in

Adhesion molecules, 529, 529t. See also Cell

pI of, 17

glycogenolysis, 148

adhesion

in pyruvate formation, 250, 252f

Alpha-amino acids. See also Amino acids

Adipose tissue, 111, 214-215, 214f

synthesis of, 237, 238f

genetic code specifying, 14, 15-16t

brown, 217, 217f

β-Alanine, 264, 300, 301f

in proteins, 14

INDEX

645

Alpha-amino nitrogen. See Amino acid

absorption of, 477

Aminolevulinate dehydratase, 270, 273f

nitrogen

analysis/identification of, 20

in porphyria, 274, 277t

Alpha anomers, 104

blood glucose and, 159

Aminolevulinate synthase, 270, 272-273,

Alpha₁ antiproteinase/antitrypsin. See

branched chain, catabolism of, 259,

273f, 276f

α₁-Antiproteinase

260f, 261f, 262f

in porphyria, 274, 277f, 278

Alpha- (α) carotene, 482

disorders of, 259-262

Aminopeptidases, 477

Alpha-fetoprotein, 583t

in catalysis, conservation of, 54, 55t

Aminophospholipids, membrane

Alpha-globin gene, localization of, 407t

chemical reactions of, functional groups

asymmetry and, 420

Alpha (α) helix, 31-32, 32f, 33f

dictating, 18-20

Aminoproteinase, procollagen, 537

amphipathic, 31-32

deamination of. See Deamination

Amino sugars (hexosamines), 106, 106f

in myoglobin, 40, 41f

deficiency of, 237, 480

glucose as precursor of, 169, 171f

Alpha (α) ketoglutarate. See

excitatory. See also Aspartate; Glutamate

in glycosaminoglycans, 109, 169, 171f

α-Ketoglutarate

glucogenic, 231-232

in glycosphingolipids, 169, 171f

Alpha-lipoproteins, 205. See also High-

in gluconeogenesis, 133-134, 134f

interrelationships in metabolism of,

density lipoproteins

interconvertability of, 231-232

171f

familial deficiency of, 228t

keto acid replacement of in diet, 240

Aminotransferases (transaminases),

Alpha-R groups, amino acid properties

ketogenic, 232

133-134, 134f

affected by, 18

melting point of, 18

diagnostic significance of, 57t

Alpha (α) thalassemia, 47

metabolism of, 122f, 124, 124f, 125f. See

in urea biosynthesis, 243-244, 244f

Alpha-tocopherol. See Tocopherol

also Amino acid carbon skeletons,

Ammonia

Alport syndrome, 538, 538t

catabolism of; Amino acid

in acid-base balance, 245

ALT. See Alanine aminotransferase

nitrogen, catabolism of

detoxification of, 242

Alteplase (tissue plasminogen activator/t-PA),

pyridoxal phosphate in, 491

excess of, 247

604-605, 605f, 606t, 607t

net charge of, 16-17, 17f

glutamine synthase fixing, 245, 245f

Alternative pathway, of complement

nutritionally essential, 124

nitrogen removed as, 244, 244f

activation, 596

nutritionally nonessential, 124

Ammonia intoxication, 244

Altitude, high, adaptation to, 46

synthesis of, 237-241

Ammonium ion, pK/pK_a value of, 12t

Alu family, 321-322

in peptides, 14, 19, 19f

Amobarbital, oxidative phosphorylation/

Alzheimer disease, amyloid in, 37, 590

pK/pK_a values of, 15-16t, 17, 17f

respiratory chain affected by, 92,

α-Amanitin, RNA polymerases affected by,

environment affecting, 18, 18t

95, 96f

343

products derived from, 264-269. See also

AMP, 287f, 288f, 288t, 297f

Ambiguity, genetic code and, 359

specific product

coenzyme derivatives of, 290t

Amino acid carbon skeletons, catabolism of,

properties of, 14-18

cyclic. See Cyclic AMP

249-263

α-R group determining, 18

free energy of hydrolysis of, 82t

acetyl-CoA formation and, 254f,

protein degradation and, 242, 243f

IMP conversion to, 293, 296f

255-259, 255f, 256f, 257f, 258f,

in proteins, 14

feedback-regulation of, 294, 296f

259f

removal of ammonia from, 244, 244f

PRPP glutamyl amidotransferase

branched-chain, 259, 260f, 261f, 262f

requirements for, 480

regulated by, 294

disorders of, 259-262

sequence of, primary structure

structure of, 83f, 288f

pyruvate formation and, 250-255, 252f,

determined by, 18-19

Amp resistance genes, 402, 403f

253f

solubility point of, 18

Amphibolic pathways/processes, 122

transamination in initiation of, 249-250,

substitutions of, missense mutations

citric acid cycle and, 133

249f, 250f, 251f

caused by, 362-363, 362f

Amphipathic α-helix, 31-32

Amino acid nitrogen

synthesis of, 237-241

Amphipathic lipids, 119-121, 120f

catabolism of, 242-248

in carbohydrate metabolism, 123

in lipoproteins, 205, 207f

in amino acid carbon skeleton

citric acid cycle in, 133, 134f

in membranes, 119, 120f, 417-418, 417f

catabolism, 249, 249f

transamination of. See Transamination

Amphipathic molecules, folding and, 6

end products of, 242-243

transporter/carrier systems for, 99

Ampicillin resistance genes, 402, 403f

urea as, 245-247, 246f

glutathione and, 629-630

Amplification, gene, in gene expression

L-glutamate dehydrogenase in,

hormones affecting, 427

regulation, 392-393, 393f

244-245, 244f, 245f

Aminoacyl residues, 18-19

Amylases

transamination of, 243-244, 243f

peptide structure and, 19

diagnostic significance of, 57t

L-Amino acid oxidase, 86-87

Aminoacyl (A/acceptor) site, aminoacyl-

in hydrolysis of starch, 474

in nitrogen metabolism, 244, 244f

tRNA binding to, in protein

β-Amyloid, in Alzheimer disease, 37, 590

Amino acid sequences. See also Protein

synthesis, 368, 368f

Amyloid-associated protein, 590

sequencing

Aminoacyl-tRNA, in protein synthesis, 368

Amyloid precursor proteins, 590

determination of, for glycoproteins, 515t

Aminoacyl-tRNA synthetases, 360, 360f

in Alzheimer disease, 37, 590

primary structure determined by, 18-19

γ-Aminobutyrate, 267, 268f

Amyloidosis, 590-591

repeating, in mucins, 519, 520f

β-Aminoisobutyrate, 300, 301f

Amylopectin, 107, 108f

Amino acids, 2, 14-20, 15-16t. See also

Aminolevulinate (ALA), 270, 273f

Amylopectinosis, 152t

Peptides

in porphyria, 278

Amylose, 107, 108f

646

INDEX

Anabolic pathways/anabolism, 81, 122. See

Anti conformers, 287, 287f

Apo A-IV, 206, 206t

also Endergonic reaction;

Antibiotics

Apo B-48, 206, 206t

Metabolism

amino sugars in, 106

Apo B-100, 206, 206t

Anaerobic glycolysis, 136, 137f, 139

bacterial protein synthesis affected by,

in LDL metabolism, 209, 210f

as muscle ATP source, 574-576, 575f,

371-372

regulation of, 223

575t

folate inhibitors as, 494

Apo C-I, 206, 206t

Analbuminemia, 584

Antibodies, 580, 581. See also

Apo C-II, 206, 206t

Anaphylaxis, slow-reacting substance of,

Immunoglobulins

in lipoprotein lipase activity, 207-208

196

monoclonal, hybridomas in production

Apo C-III, 206, 206t

Anaplerotic reactions, in citric acid cycle,

of, 595-596, 596f

lipoprotein lipase affected by, 208

133

in xenobiotic cell injury, 631, 631f

Apo D, 206, 206t

Anchorin, in cartilage, 551t

Antibody diversity, 591

Apo E, 206, 206t

Andersen’s disease, 152t

DNA/gene rearrangement and, 593-594

receptors for

Androgen response element, 459t

Antichymotrypsin, 583t

in chylomicron remnant uptake,

Androgens

Anticoagulants, coumarin, 604

208-209, 209f

estrogens produced from, 442-445, 444f

Anticodon region, of tRNA, 359, 360, 360f

in LDL metabolism, 209, 210f

receptors for, 471

Antifolate drugs, purine nucleotide

Apoferritin, 586

synthesis of, 440-442, 441f, 443f

synthesis affected by, 293

Apolipoproteins/apoproteins, 205,

Androstenedione

Antigenic determinant (epitope), 33, 591

205-206

estrone produced from, 444f, 445

Antigenicity, xenobiotics altering, cell injury

distribution of, 205-206, 206t

synthesis of, 442, 443f

and, 631, 631f

hemoglobin; oxygenation affecting, 42

Anemias, 47

Antigens, 591

Apomyoglobin, hindered environment for

Fanconi’s, 338

Antihemophilic factor A/globulin (factor

heme iron and, 41, 41f

hemolytic, 136, 143, 609, 619, 620t

VIII), 599f, 600, 600t

Apoproteins. See Apolipoproteins/

glucose-6-phosphate dehydrogenase

deficiency of, 604

apoproteins

deficiency causing, 163,

Antihemophilic factor B (factor IX), 599f,

Apoptosis, 201

169-170, 613, 614f, 619

600, 600t

p53 and, 339

haptoglobin levels in, 584

coumarin drugs affecting, 604

Apo-transketolase, activation of, in thiamin

hyperbilirubinemia/jaundice in, 282,

deficiency of, 604

nutritional status assessment, 489

284, 284t

Antimalarial drugs, folate inhibitors as,

APP. See Amyloid precursor protein

pentose phosphate pathway/

494

Apurinic endonuclease, in base excision-

glutathione peroxidase and, 166,

Antimycin A, respiratory chain affected by,

repair, 337

167f, 169-170

95, 96f

Apyrimidinic endonuclease, in base

primaquine-sensitive, 613

Antioxidants, 91, 119, 611-613, 613t

excision-repair, 337

red cell membrane abnormalities

retinoids and carotenoids as, 119, 482t

Aquaporins, 424-426

causing, 619

vitamin C as, 119

D-Arabinose, 104f, 105t

iron deficiency, 478, 497, 586, 610t

vitamin E as, 91, 119, 486, 487f

Arabinosyl cytosine (cytarabine), 290

megaloblastic

Antiparallel loops, mRNA and tRNA, 360

Arachidonic acid/arachidonate, 113t, 190,

folate deficiency causing, 482t, 492,

Antiparallel β sheet, 32, 33f

190f

494

Antiparallel strands, DNA, 303

eicosanoid formation and, 192, 193f,

vitamin B₁₂ deficiency causing, 482t,

Antiport systems, 426, 426f

194, 194f, 195f

492, 610t

for nucleotide sugars, 517

for essential fatty acid deficiency,

pernicious, 482t, 492

α₁-Antiproteinase (α₁-antitrypsin), 583t,

191-192

recombinant erythropoietin for, 526, 610

589

ARC, 472t, 473

sickle cell. See Sickle cell disease

deficiency of, 589-590, 589f, 590f,

ARE. See Androgen response element

Angiotensin II, 437, 451, 452f

623

Argentaffinoma (carcinoid), serotonin in,

synthesis of, 451-452, 452f

as thrombin inhibitor, 603

266-267

Angiotensin III, 452, 452f

Antiproteinases, 623, 624t

Arginase

Angiotensin-converting enzyme, 451-452,

Antithrombin/antithrombin III, 583t,

in periodic hyperlysinemia, 258

452f

603-604

in urea synthesis, 246f, 247

Angiotensin-converting enzyme inhibitors,

heparin binding to, 547, 603-604

Arginine, 16t, 265, 266f

451-452

α₁-Antitrypsin (α₁-antiproteinase), 583t,

catabolism of, 250, 251f

Angiotensinogen, 451, 452f

589

in urea synthesis, 246f, 247

Anion exchange protein, 615, 615f, 616t

deficiency of, 589-590, 589f, 590f,

Arginosuccinase

Ankyrin, 615f, 616f, 616t, 617

623-624

deficiency of, 248

Anomeric carbon atom, 104

as thrombin inhibitor, 603

in urea synthesis, 246f, 247

Anomers, α and β, 104

APC. See Activated protein C

Arginosuccinate, in urea synthesis, 245,

Anserine, 264, 265, 265f

Apo A-I, 206, 206t, 224

246f, 247

Antennae (oligosaccharide branches), 521

deficiencies of, 228t

Arginosuccinate synthase, 246f, 247

Anterior pituitary gland hormones, blood

Apo A-II, 206t

deficiency of, 247

glucose affected by, 161

lipoprotein lipase affected by, 207-208

Arginosuccinicaciduria, 248

INDEX

647

Aromatase enzyme complex, 442, 444f, 445

Asymmetry

ATP synthase, membrane-located, 96, 97f,

ARS (autonomously replicating sequences),

importin binding and, 501

98f

326, 413

lipid and protein, membrane assembly

Atractyloside, respiratory chain affected by,

Arsenate, oxidation and phosphorylation

and, 511, 512f

affected by, 137, 142

in membranes, 416, 419-420

Attachment proteins, 540, 541f

Arterial wall, intima of, proteoglycans in,

Ataxia-telangiectasia, 338

Autoantibodies, in myasthenia gravis, 431

548

ATCase. See Aspartate transcarbamoylase

Autonomously replicating sequences (ARS),

Arthritis

Atherosclerosis, 205, 607

326, 413

gouty, 299

cholesterol and, 117, 219, 227

Auto-oxidation. See Peroxidation

proteoglycans in, 548

HDL and, 210-211

Autoradiography, definition of, 413

rheumatoid, glycosylation alterations in,

hyperhomocysteinemia and, folic acid

Autotrophic organisms, 82

533

supplements in prevention of,

Avidin, biotin deficiency caused by, 494

Artificial membranes, 421-422

494

Axial ratios, 30

Ascorbate, 167, 168f

LDL receptor deficiency in, 209

Axonemal dyneins, 577

Ascorbic acid (vitamin C), 163, 482t,

lysophosphatidylcholine (lysolecithin)

5- or 6-Azacytidine, 290

495-496, 496f

and, 116

8-Azaguanine, 290, 291f

as antioxidant, 119

Atorvastatin, 229

Azathioprine, 290

in collagen synthesis, 38, 496, 535

ATP, 82, 82-85, 287f, 289

5- or 6-Azauridine, 290, 291f

deficiency of, 482t, 496

in active transport, 427-428, 428f

collagen affected in, 38-39, 496,

in coupling, 82, 84

538-539

fatty acid oxidation producing, 182

B blood group substance, 618, 619f

iron absorption and, 478, 496

free energy of hydrolysis of, 82-83, 82t

B (β) cells, pancreatic, insulin produced by,

supplemental, 496

in free energy transfer from exergonic to

160

Asialoglycoprotein receptors

endergonic processes, 82-83, 82f

B cyclins, 333, 334f, 335t

in cotranslational insertion, 506, 506f

hydrolysis of

B gene, Gal transferase encoded by,

in glycoprotein clearance, 517

in muscle contraction, 561-562, 561f

618-619

Asn-GlcNAc linkage

by NSF, 509, 510f

B lymphocytes, 591

in glycoproteins, 521

inorganic pyrophosphate production

in hybridoma production, 595-596, 596f

in glycosaminoglycans, 543

and, 85

B vitamins. See Vitamin B complex

Asparaginase, in amino acid nitrogen

in mitochondrial protein synthesis and

BAC vector. See Bacterial artificial

catabolism, 245, 245f

import, 499

chromosome (BAC) vector

Asparagine, 15t

in muscle/muscle contraction, 556,

Bacteria

in amino acid nitrogen catabolism, 245

561-562, 561f

intestinal, in bilirubin deconjugation, 281

catabolism of, 249, 250f

decrease in availability of, 564

transcription cycle in, 342-343, 342f

synthesis of, 237-238, 238f

sources of, 573-574, 574-576, 575f,

Bacterial artificial chromosome (BAC)

Asparagine synthetase, 238, 238f

575t

vector, 401-402, 402t

Aspartate

in purine synthesis, 293-294, 295f

for cloning in gene isolation, 635t

catabolism of, 249, 250f

respiratory control in maintenance of

in Human Genome project, 634

synthesis of, 237-238, 238f

supply of, 94-95, 97, 97t, 98f,

Bacterial gyrase, 306

in urea synthesis, 246f, 247

134-135

Bacterial promoters, in transcription,

Aspartate 102, in covalent catalysis, 53-54,

structure of, 83f

345-346, 345f

54f

synthesis of

Bacteriophage, definition of, 413

Aspartate aminotransferase (AST/SGOT),

ATP synthase in, 96, 97f, 98f

Bacteriophage lambda (λ), 378-383, 379f,

diagnostic significance of, 57t

in citric acid cycle, 131f, 133, 142,

380f, 381f, 382f

Aspartate transcarbamoylase, 75

143t

BAL. See Dimercaprol

in pyrimidine synthesis, 298f, 299

glucose oxidation yielding, 142, 143t

BAL 31 nuclease, in recombinant DNA

Aspartic acid, 15t

respiratory chain in, 93-95, 98f

technology, 400t

pI of, 17

ATP/ADP cycle, 83, 84f

Balanced chemical equations, 60

Aspartic protease family, in acid-base

ATPase

BamHI, 398, 399t

catalysis, 52, 53f

in active transport, 427-428, 428f

Barbiturates, respiratory chain affected by,

Aspartylglycosaminuria, 532-533, 533t

chaperones exhibiting activity of, 508

95, 96f

Aspirin

copper-binding P-type, mutations in

Basal lamina, laminin as component of,

antiplatelet actions of, 607-608

gene for

540-542

cyclooxygenase affected by, 193

Menkes diseases caused by, 588

Basal metabolic rate, 478

prostaglandins affected by, 190

Wilson disease caused by, 588-589

Base excision-repair of DNA, 336t, 337,

Assembly particles, in absorptive

ATP-binding cassette transporter-1, 210,

337f

pinocytosis, 430

211f

Base pairing in DNA, 7, 303, 304, 305f

AST. See Aspartate aminotransferase

ATP-chaperone complex, 508. See also

matching of for renaturation, 305-306

Asthma, leukotrienes in, 112

Chaperones

recombinant DNA technology and,

Asymmetric substitution, in porphyrins,

ATP-citrate lyase, 134, 135f, 156t, 157

396-397

270, 271f

acetyl-CoA for lipogenesis and, 177

replication/synthesis and, 328-330, 330f

648

INDEX

Base substitution, mutations occurring by,

Bilirubin

Blood cells, 609-625. See also Erythrocytes;

361, 361f, 362

accumulation of (hyperbilirubinemia),

Neutrophils; Platelets

Basement membranes, collagen in, 537

281-284, 284t

Blood clotting. See Coagulation

Bases

conjugated

Blood glucose

conjugate, 10

binding to albumin and, 283

normal, 145

as proton acceptors, 9

reduction of to urobilinogen, 281,

regulation of

strong, 9

282f

clinical aspects of, 161-162, 161f

weak, 9

conjugation of, 280, 280f, 281f

diet/gluconeogenesis/glycogenolysis in,

Bence Jones protein, 595

fecal, in jaundice, 284t

158-161, 159f, 160f

Bends (protein conformation), 32-33,

heme catabolism producing, 278-280,

glucagon in, 160-161

34f

279f

glucokinase in, 159-160, 160f

Beriberi, 482t, 489

liver uptake of, 280-281, 280f, 281f,

glycogen in, 145

Beta-alanine. See β-Alanine

282f

insulin in, 160

Beta anomers, 104

normal values for, 284t

limits of, 158

Beta- (β) carotene, 482, 482t, 483, 483f.

secretion of into bile, 280, 281f

metabolic and hormonal mechanisms