FOURTH EDITION

BIOCHEMISTRY
CHRISTOPHER K. MATHEWS
Oregon State University

K. E. VAN HOLDE
Oregon State University

DEAN R. APPLING
The University of Texas at Austin

SPENCER J. ANTHONY-CAHILL
Western Washington University

Toronto


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10 9 8 7 6 5 CKV
Library and Archives Canada Cataloguing in Publication
Biochemistry / [edited by] Christopher K. Mathews ... [et al.]. — 4th
U.S. ed.
Includes bibliographical references and index.
ISBN 978-0-13-800464-4
1. Biochemistry. I. Mathews, Christopher K., 1937QH345.B43 2012
572’.3

C2011-902175-7

ISBN 978-0-13-800464-4


ABOUT THE AUTHORS

Christopher K. Mathews is Distinguished Professor Emeritus of Biochemistry
at Oregon State University. He earned his B.A. in chemistry from Reed College

(1958) and Ph.D. in biochemistry from the University of Washington (1962). He
served on the faculties of Yale University and the University of Arizona from 1963
to 1978, when he moved to Oregon State University as chair of the Department of
Biochemistry and Biophysics, a position he held until 2002. His major research
interest is the enzymology and regulation of DNA precursor metabolism and the
intracellular coordination between deoxyribonucleotide synthesis and DNA replication. From 1984 to 1985, Dr. Mathews was an Eleanor Roosevelt International
Cancer Fellow at the Karolinska Institute in Stockholm, and in 1994–1995 he held
the Tage Erlander Guest Professorship at Stockholm University. Dr. Mathews has
published over 175 scientific papers dealing with molecular virology, metabolic
regulation, nucleotide enzymology, and biochemical genetics. He is the author of
Bacteriophage Biochemistry (1971) and coeditor of Bacteriophage T4 (1983) and
Structural and Organizational Aspects of Metabolic Regulation (1990). He was a
coauthor of the three previous editions of Biochemistry. His teaching experience
includes undergraduate, graduate, and medical school biochemistry courses.
He has backpacked and floated the mountains and rivers, respectively, of Oregon
and the Northwest. As an enthusiastic birder he has served as President of the
Audubon Society of Corvallis and is President of the Great Basin Society, which
operates the Malheur Field Station.

K. E. van Holde is Distinguished Professor Emeritus of Biophysics and Biochemistry at Oregon State University. He earned his B.A. (1949) and Ph.D. (1952)
from the University of Wisconsin. Over many years, Dr. van Holde’s major research
interest has been the structure of chromatin; his work resulted in the award of an
American Cancer Society research professorship in 1977. He has been at Oregon
State University since 1967, and was named Distinguished Professor in 1988. He is
a member of the National Academy of Sciences and the American Academy of Arts
and Sciences, and has received Guggenheim, NSF, and EMBO fellowships. He is the
author of over 200 scientific papers and four books in addition to this volume:
Physical Biochemistry (1971, 1985), Chromatin (1988), Principles of Physical
Biochemistry (1998), and Oxygen and the Evolution of Life (2011). He was also coeditor of The Origins of Life and Evolution (1981). His teaching experience includes
undergraduate and graduate chemistry, biochemistry and biophysics, and the


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ABOUT THE AUTHORS

physiology and molecular biology course at the Marine Biological Laboratory at
Woods Hole.

Dean R. Appling is the Lester J. Reed Professor of Biochemistry and Associate
Dean for Research and Facilities for the College of Natural Sciences at the University
of Texas at Austin, where he has taught and done research for the past 26 years. Dean
earned his B.S. in biology from Texas A&M University, and his Ph.D. in biochemistry from Vanderbilt University. The Appling laboratory studies the organization
and regulation of metabolic pathways in eukaryotes, focusing on folate-mediated
one-carbon metabolism. The lab is particularly interested in understanding how
one-carbon metabolism is organized in mitochondria, as these organelles are central players in many human diseases. In addition to coauthoring this book, Dean
has published over 60 scientific papers and book chapters.
As much fun as writing a textbook might be, Dean would rather be outdoors. He
is an avid fisherman and hiker. Recently, Dean and his wife, Maureen, have
become entranced by the birds on the Texas coast. They were introduced to birdwatching by coauthor Chris Mathews and his wife, Kate—an unintended consequence of working on this book!

Spencer J. Anthony-Cahill is a Professor in the Department of Chemistry at
Western Washington University (WWU), Bellingham, WA. Spencer earned his
B.A. in chemistry from Whitman College, and his Ph.D. in bioorganic chemistry
from the University of California, Berkeley. His graduate work, in the lab of Peter
Schultz, focused on the biosynthetic incorporation of unnatural amino acids into
proteins. Spencer was an NIH postdoctoral fellow in the laboratory of Bill
DeGrado (then at DuPont Central Research), where he worked on de novo peptide

design and the prediction of the tertiary structure of the HLH DNA-binding
motif. He then worked for five years as a research scientist in the biotechnology
industry, developing recombinant hemoglobin as a treatment for acute blood loss.
In 1997, Spencer decided to pursue his long-standing interest in teaching and
moved to WWU, where he is today.
Research in the Anthony-Cahill laboratory is directed at the protein engineering of
heme proteins. The primary focus is on circular permutation of human ␤-globin as
a means to develop a single-chain hemoglobin with desirable therapeutic properties. The lab is also pursuing the design of self-assembling protein nanowires.
Outside the classroom and laboratory, Spencer is a great fan of the outdoors—
especially the North Cascades and southeastern Utah, where he has often backpacked, camped, climbed, and mountain biked. Spencer also holds the rank of 3rd
Dan in Aikido, and instructs children and adults at the Kulshan Aikikai Dojo in
Bellingham, WA.


PREFACE

A NEW EDITION
What factors might explain the re-emergence of a well-received biochemistry
textbook (Biochemistry, Third Edition, 2000, by C. K. Mathews, K. E. van Holde,
and K. G. Ahern), some 12 years after publication of the previous edition? In a
rapidly evolving field like biochemistry, textbooks are typically revised every four
or five years to retain their educational value.
Still, biochemistry instructors and students continued to ask when and
whether a fourth edition might appear. While Chris Mathews was interested in
revising and updating the book, his previous coauthors were unable to commit to
a project of this magnitude, and so the search began for a new author team. After
a long and careful selection process, two new coauthors joined Chris Mathews:
Dr. Dean R. Appling, Lester J. Reed Professor of Biochemistry and Associate Dean
for Research and Facilities for the College of Natural Sciences at the University of
Texas at Austin, and Dr. Spencer J. Anthony-Cahill, Professor of Chemistry at

Western Washington University, Bellingham.
Dean Appling is an enzymologist with interests in regulation and organization
of metabolic pathways, with particular emphasis upon folate cofactors and the
metabolism of single-carbon units. Much of his work uses NMR and molecular
genetics to probe metabolic compartmentation and control. Spencer AnthonyCahill’s chief interest is protein folding and design, with current emphasis upon
folding patterns in protein variants that have circularly permuted sequences.
Before assuming his present faculty position, Spencer worked for five years in the
biotechnology industry, an experience that gives him a valuable perspective in
teaching biochemistry. Both Dean and Spencer have used previous editions of
Biochemistry in their own teaching, so they were well aware of the strengths of this
book and areas where fresh attention was needed.
The research interests of the new author team created a natural division of
writing responsibilities. Spencer’s writing was focused upon biomolecular structure and mechanisms, Dean dealt with metabolism and its control, and Chris put
his major effort into genetic biochemistry. However, the project was truly a team
effort. Each chapter draft was scrutinized by all three authors, with revisions made
by each principal draft author before submission to our editors and outside
reviewers. We found our fellow authors to be our strongest critics. And, although
Ken van Holde was not actively involved with this edition, he did review some
drafts and much of his graceful writing remains in this new edition. We are proud
to include him as a coauthor of this new edition.

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PREFACE

EVOLUTION OF THE TEXT
Major Changes

In addition to dealing with the vast amount of new information appearing since
the publication of the third edition in 2000, this new edition introduces three significant changes. First is more emphasis upon biochemical reaction mechanisms
in the enzymes and metabolism chapters. Second is a significant reorganization in
the chapters dealing with intermediary metabolism. The coverage of carbohydrate
metabolism has been unified, so that we now present glycolysis, gluconeogenesis,
glycogen metabolism, and the pentose phosphate pathway in one chapter
(Chapter 13). To accomplish this without excessive expansion of the chapter, we
moved the section on complex carbohydrate metabolism to Chapter 9; instructors
can present this material as part of the metabolism section of the course, if they
prefer. Redox thermodynamics has been moved from Chapter 15 (Biological
Oxidations) to Chapter 3 (Bioenergetics), where it more properly belongs. The
material on interorgan coordination in mammalian metabolism has been split
into two chapters—Chapter 18 (Interorgan and Intracellular Coordination of
Energy Metabolism in Vertebrates) and Chapter 23 (Signal Transduction).
The third major change is the reorganization of genetic biochemistry in the
last major section of the book. As in previous editions, we introduce processes in
biological information transfer early, in Chapter 4, with details presented later. In
addition, we have integrated prokaryotic and eukaryotic informational metabolism, rather than presenting them in separate chapters, as in previous editions.
The four genetic biochemistry chapters in previous editions are now six—
Chapters 24 (Genome Organization), 25 (DNA Replication), 26 (Information
Restructuring), 27 (Transcription and Its Control), 28 (Protein Synthesis and
Processing), and 29 (Control of Gene Expression).

New Topics
A special challenge in writing a new edition after an interval of so many years was
incorporating the most important of the many spectacular new developments in
molecular life sciences. A partial list of new or significantly revised topics includes:
• Phosphorothioate bonds in DNA (Chapter 4)
• Gene sequence analysis, phylogenetic analysis, proteomic analysis, and
amino acid sequencing by mass spectroscopy (Chapter 5)

• New approaches to classifying protein secondary structure, protein structure prediction, and protein folding energy landscapes (Chapter 6)
• Dynamics of myoglobin, roles of heme proteins in nitric oxide physiology,
and antibody–drug conjugates as anticancer agents (Chapter 7)
• Biological imaging of complex glycoproteins (Chapter 9)
• Lipid rafts (Chapter 10)
• Organic chemical mechanisms of the common biochemical reaction types
(Chapter 12)
• Coordination of energy homeostasis, including mTOR, AMPK, and sirtuins
and protein acetylation (Chapter 18)
• Evolution of metabolic pathways (several chapters); regulation of cholesterol metabolism (Chapter 19)
• Ubiquitin and regulated protein turnover (Chapter 20)
• Methyl group metabolism (Chapter 21)
• Pharmacogenetics (Chapter 22)


PREFACE

• A kinase anchoring proteins (Chapter 23)
• Restriction fragment length polymorphisms, single-nucleotide polymorphisms and genome mapping, chromatin structure, and the centromere
(Chapter 24)
• Double-strand DNA break repair (Chapter 26)
• Structure and function of RNA polymerases (Chapter 27) and of ribosomes
(Chapter 28)
• Apoptosis (Chapter 28)
• The role of Mediator in transcription complexes, DNA methylation and
epigenetics, functional significance of histone modifications, RNA interference, and riboswitches (Chapter 29)

Biochemistry Applications
One feature requested by students and instructors alike is practical applications of
biochemical knowledge—particularly, applications to the health sciences. Unlike

some other textbooks, we prefer to integrate applications with the main text,
instead of setting them apart in boxes. We believe that this makes the text flow
more smoothly.
New applications discussed in this edition include:
• Influenza virus neuraminidase and the action of Tamiflu (Chapter 9)
• Biofuels (Chapter 13)
• Mitochondrial diseases (Chapter 15)
• Artificial photosynthesis (Chapter 16)
• Diabetes, obesity (Chapter 18)
• Calorie restriction and lifespan extension (Chapter 18)
• Methylenetetrahydrofolate reductase variants and disease susceptibility
(Chapter 21)
• Chromosomal translocations and targeted cancer drugs (Chapter 23); mapping disease genes (Chapter 24)
• Patterns of oncogene mutations in cancer (Chapter 23)

Keeping What Works Best
Not everything is new in this edition. We have worked hard to retain and improve
the best-loved features of previous editions, such as an emphasis upon the
physico-chemical concepts upon which biochemical processes and mechanisms
are based, and an emphasis upon the experimental nature of biochemistry. This
latter emphasis is realized with our continued use of the popular Tools of
Biochemistry feature.

TOOLS OF BIOCHEMISTRY
As in past editions, we emphasize the importance of incisive experimental techniques as the engine that drives our increasing understanding of the molecular
nature of life processes. This is accomplished through end-of-chapter essays on
the most important techniques in biochemistry and molecular biology research.
Most of the Tools sections in this edition have been updated or introduced for the
first time. New or significantly modified Tools sections include:
• Introduction to Proteomics; Tandem Mass Spectrometry (Chapter 5)

• Nuclear Magnetic Resonance Spectroscopy (Chapter 6)

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PREFACE

• In Vitro Evolution of Protein Function (Chapter 11)
• Metabolomics (Chapter 12)
• Gene Targeting by Homologous Recombination; Single-Molecule Biochemistry (Chapter 26)
• Microarrays; Chromatin Immunoprecipitation (Chapter 27)
Several Tools sections on manipulating DNA have been combined and moved
earlier in the book, to Chapter 4. The Tools section on radioisotopes in Chapter 12
has been considerably shortened. The material on kinetic isotope methods in analysis of enzyme mechanisms has been strengthened in the Tools section in Chapter 11.

END-OF-CHAPTER PROBLEMS
Wherever possible, we have removed problems that emphasize rote learning and
retained or added problems that require analytical or quantitative thought to be
solved. Several new problems have been added to each chapter. Importantly, we
now include complete solutions to each problem, as well as the answers, at the
back of the book.

ABOUT THE COVER
The cover illustration depicts the structure of the yeast 80S ribosome at 4.15
Ångstrom resolution, based upon X-ray crystallography. This complex RNA- and
protein-containing particle is an enormous molecular machine, which binds the
components of protein synthesis—messenger RNA, transfer RNAs containing activated amino acids, and soluble protein factors that aid in all phases of translation—
initiation, polypeptide chain elongation, and termination.

Tremendous insight into mechanisms of protein synthesis was gained beginning in 2000, when crystal structures for prokaryotic and archaeal ribosomes were
reported. This work was recognized in 2009, with the Nobel Prize in Chemistry to
V. Ramakrishnan, T. A. Steitz, and A. Yonath. Although basic processes in translation are similar in all cells, protein synthesis in eukaryotic cells is much more
complex than in bacteria, particularly with regard to steps in initiation, where
many more soluble protein factors must participate. The eukaryotic ribosome is
correspondingly larger and more complex—about 40% larger than the bacterial
ribosome, with correspondingly more different proteins and larger RNA components. These factors make solving the eukaryotic ribosome structure an even more
formidable problem. This feat was accomplished in several laboratories, beginning in late 2010.
The structure of the yeast ribosome shown here was described by A. BenShem, L. Jenner, G. Yusupova, and M. Yusupov, in Science 330:1203–1209 (2010).
The image on the cover was created by C. Spiegel and S. Anthony-Cahill, working
from atomic coordinates deposited by Ben-Shem and coauthors in the
Brookhaven Protein Database (PBD). Color scheme: 40S particle (PDB ID:
3O30): RNA is in orange; proteins are in slate blue; 60S particle (PDB ID: 3O5H):
RNA is in raspberry red; proteins are in forest green.

SUPPLEMENTS
For Instructors
Instructor resources are password protected and available for download via the
Pearson online catalog at www.pearsonhighered.com. For your convenience,
many of these resources are also available on the Instructor’s Resource CD-ROM
(IRCD) (ISBN 978-0-13-279159-5).


PREFACE

• Test Item File. The fourth edition features a brand new testbank created by
Scott Lefler, Senior Lecturer, Arizona State University, with more than 700
thoughtful questions in editable Word format. The Test Item File can be found on
the IRCD or downloaded from the online catalog.
ã PowerPointđ Presentations. Two sets of PowerPointđ slides are available for

the text. The first consists of all the figures and photos in the textbook in
PowerPoint® format. The second set, created by Bruce Burnham, Associate
Professor of Chemistry, Rider University, consists of PowerPoint® lecture slides
that provide an outline to use in a lecture setting, presenting definitions, key concepts, and figures from the textbook. Both sets of PowerPoint® slides can be found
on the IRCD or downloaded from the online catalog.
• Complete Solutions Manual. As in previous editions, we have created a solutions manual to complement chapter problems in the current edition of our text.
The complete solutions manual, prepared by Sara Codding and Tim Rhoads of
Oregon State University, contains fully worked solutions for those questions that
may benefit from explanations beyond those provided at the back of the book.
Instructors can arrange with the publisher to make this material available to
students (ISBN 978-0-13-292628-7).
• CourseSmart for Instructors. CourseSmart goes beyond traditional expectations, providing instant, online access to the textbooks and course materials you
need at a lower cost for students. And even as students save money, you can save
time and hassle with a digital eTextbook that allows you to search for the most relevant content at the very moment you need it. Whether it’s evaluating textbooks
or creating lecture notes to help students with difficult concepts, CourseSmart can
make life a little easier. See how when you visit www.coursesmart.com/instructors.
• Technology Specialists. Pearson’s Technology Specialists work with faculty
and campus course designers to ensure that Pearson technology products,
assessment tools, and online course materials are tailored to meet your specific
needs. This highly qualified team is dedicated to helping schools take full advantage of a wide range of educational resources by assisting in the integration of a
variety of instructional materials and media formats. Your local Pearson
Education sales representative can provide you with more details on this service
program.

For Students
• The Chemistry Place for Biochemistry, Fourth Edition. The Chemistry Place
is an online tool that provides students with tutorial aids to help them succeed in
biochemistry. This Website includes animations of key concepts and processes
and self-quizzing created by Scott Napper, Associate Professor, University of
Saskatchewan, to allow students to check their understanding of subject matter.

TheChemistryPlace also contains our Pearson eText. Please visit the site at
www.chemplace.com.
• Pearson eText gives students access to the text whenever and wherever they
have access to the Internet. eText pages look exactly like the printed text, offering
powerful new functionality for students and instructors. Users can create notes,
highlight text in different colors, create bookmarks, zoom, click hyperlinked
words and phrases to view definitions, and view in single-page or two-page view.
Pearson eText allows for quick navigation to key parts of the eText using a table of
contents and provides full-text search. The eText may also offer links to associated
media files, enabling users to access videos, animations, or other activities as they
read the text.

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PREFACE

• CourseSmart for Students. CourseSmart goes beyond traditional expectations, providing instant, online access to the textbooks and course materials you
need at an average savings of 60%. With instant access from any computer and the
ability to search your text, you’ll find the content you need quickly, no matter
where you are. And with online tools like highlighting and note-taking, you can
save time and study efficiently. See all the benefits at www.coursesmart.com/
students.

ACKNOWLEDGMENTS
Although our names appear as authors on the cover of this book, and we expect to
receive most of the credit or criticism resulting therefrom, the book in fact was
created by a large team, with many participants whose contributions rivaled ours.

To begin, this book would never have come into existence but for the enterprise of
Michelle Sartor, now Acting Editor-in-Chief for Humanities and Social Sciences
for Pearson Canada. In her former assignment, she became aware of a continuing
interest in our book, particularly in Canada. Even though a previous attempt at a
fourth edition had aborted, Michelle exercised quiet but effective persistence until
the present author team had been assembled. After Michelle’s reassignment,
Cathleen Sullivan, Executive Editor for Engineering, Science, and Mathematics,
took over, and she has held a steady hand on the tiller through calm seas that occasionally, but only briefly, became rough.
Our day-to-day contact through the writing and development phase was John
Polanszky, Senior Developmental Editor. We appreciated that he gave us much
independence and in general was a calming influence and a source of useful
advice. And we credit him with securing some extremely helpful reviewers, whose
names are listed separately.
When the writing, reviewing, and revisions were completed, we maintained
our valuable contacts with Cathleen and John, but began interacting with a much
larger team of dedicated and skilled professionals, particularly Marissa Lok, inhouse project manager, and Tracy Duff, project manager at PreMediaGlobal. We
exchanged e-mails and phone calls with these ladies nearly every day, and consider
them friends, even though we have yet to meet them in person. Signal contributions were made during this phase by Kelly Birch, copy editor; Stephany Craig,
proofreader; Heather Jackson, permissions researcher; and Greg Miller, Scott
Napper, Mark Jonklaas, Masoud Jelokhani-Niaraki, technical reviewers. Katy
Mehrtens, Publishing Services Director, oversaw the efforts at PreMediaGlobal.
Julia Jevmenova, Marketing Manager at Pearson Canada, impressed us with her
quiet insistence at learning about the substance and content of our book, so that
she could become a truly effective advocate.
We owe a great deal to friends and colleagues in science for advice, updated
information, and graphics. At the risk of neglecting to recognize all who helped
us, the following deserve mention.
Gary Carlton (Arcsine Graphics) produced many of the new figures in
Chapters 6–11. Gary’s attention to detail and quality yielded spectacular results.
Thanks to those researchers who provided new figures for this edition of the text:

Shing Ho (Colorado State University), Figures 4A.6 and 26.24; Jack Benner (New
England Biolabs), Figure 5D.1; Andy Karplus (Oregon State University), Figures 6.12
and 6.13; Scott Delbecq and Rachel Klevit (University of Washington), Figure
6A.10b; Serge Smirnov (Western Washington University), Figure 6A.12; Vlado Gelev
(FBReagents Inc.), Figure 6A.13b; Stephan Grzesiek (University of Basel), Figure
6A.13a; Richard Harris, Figure 6A.14; John Olson (Rice University), Figure 7.6;
Marjorie Longo (University of California, Davis), Figure 10.27; Vamsi Mootha
(MIT), Figure 12B.3; Adrian Keatinge-Clay (University of Texas at Austin), Figure


PREFACE

17.37B; Rowena Matthews (University of Michigan), Figure 21.12; John Tesmer
(University of Michigan), Figure 23.10; Lawrence Loeb (University of Washington),
Figure 23.22; Mike O’Donnell (Rockefeller University), Figures 25.25 and 25.34;
Whitney Yin (University of Texas at Austin), Figure 25.35; David Josephy (University
of Guelph), Figure 26.7; D. G. Vassylyev (University of Alabama at Birmingham),
Figure 27.11; Robin Gutell (University of Texas at Austin), Figure 28.19.
P. Clint Spiegel (Western Washington University) rendered the structure of
the eukaryotic ribosome that appears on the cover.
Special thanks to several colleagues who generously provided feedback on
material in the draft stage, and thereby improved the quality of the final text.
Rachel Klevit (University of Washington), Andrew Baldwin (University of
Toronto), and Serge Smirnov (Western Washington University) reviewed the
material describing optical and NMR spectroscopy in Chapter 6. Tom Brittain
(University of Auckland), John Olson (Rice University), and Antony Mathews
(Pfizer, Inc.) reviewed the material in Chapter 7 describing the structure and
function of globin proteins. Heather Van Epps (Seattle Genetics) provided critical
feedback on the sections of Chapter 10 describing excitable membranes. Jack
Benner (New England Biolabs) reviewed material in Chapter 5 describing proteomics. Andrew Hanson (University of Florida) provided helpful feedback on the

section in Chapter 16 describing photorespiration. John Denu (University of
Wisconsin) shared unpublished data and provided helpful feedback on the section in Chapter 18 describing protein acetylation and sirtuins. Jon Huibregtse
(University of Texas at Austin) provided helpful feedback on the section in
Chapter 20 describing ubiquitin function. Ralph Green (University of California,
Davis) provided helpful feedback on the section in Chapter 20 describing
vitamin B12 and pernicious anemia. JoAnne Stubbe (MIT) provided helpful
feedback on the discussion of ribonucleotide reductase in Chapter 22. John
Tesmer (University of Michigan) provided helpful feedback on the section in
Chapter 23 describing G protein structure and function. Michael Freitag (Oregon
State University) offered valuable information about centromeres, kinetochores,
and epigenetics (Chapters 24 and 29).
As always, our most effective critics were our wives—Kate Mathews, Maureen
Appling, and Yvonne Anthony-Cahill. Barbara van Holde is greatly missed. But
Kate, Maureen, and Yvonne were constant sources of love and support, with occasional pungent advice and criticism. Their patience and enduring support were
the most important elements in seeing this project to a timely and satisfying conclusion.

Christopher K. Mathews
Spencer J. Anthony-Cahill

Dean R. Appling
K. E. van Holde

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PREFACE

REVIEWERS

The following reviewers provided valuable feedback on the manuscript at various stages throughout the writing process.
Nahel Awadallah, Sampson Community College
Stephen L. Bearne, Dalhousie University
Roberto Botelho, Ryerson University
John Brewer, University of Georgia
Robert Brown, Memorial University of Newfoundland
Bruce Burnham, Rider University
Danielle Carrier, University of Ottawa
Lisa Carter, Athabasca University
Amanda Cockshutt, Mount Allison University
Betsey Daub, University of Waterloo
Richard Epand, McMaster University
Eric Gauthier, Laurentian University
Dara Gilbert, University of Waterloo
Masoud Jelokhani, Wilfrid Laurier University
Mark Jonklaas, Baylor University
David Josephy, University of Guelph
Lana Lee, University of Windsor
Elke Lohmeier-Vogel, University of Calgary
Derek McLachlin, University of Western Ontario
Vas Mezl, University of Ottawa
Scott Napper, University of Saskatchewan
Arnim Pause, McGill University
Dorothy Pocock-Goldman, Concordia University
Shauna Reckseidler-Zenteno, Athabasca University
Jim Sandercock, Northern Alberta Institute of Technology
Anthony Siame, Trinity Western University
Anthony Serianni, University of Notre Dame
Ron Smith, Thompson Rivers University
Lakshmaiah Sreerama, St. Cloud State University

David Villeneuve, Canadore College
William Willmore, Carleton University
Boris Zhorov, McMaster University


BRIEF CONTENTS

PART 1 The Realm of Biochemistry
CHAPTER 1
CHAPTER 2
CHAPTER 3

1

The Scope of Biochemistry 2
The Matrix of Life: Weak Interactions in an
Aqueous Environment 26
The Energetics of Life 58

PART 2 Molecular Architecture of Living
Matter 89
CHAPTER 4
CHAPTER 5
CHAPTER
CHAPTER
CHAPTER
CHAPTER
CHAPTER

Nucleic Acids 90

Introduction to Proteins: The Primary Level
of Protein Structure 136
6 The Three-Dimensional Structure of
Proteins 177
7 Protein Function and Evolution 234
8 Contractile Proteins and Molecular
Motors 286
9 Carbohydrates: Sugars, Saccharides,
Glycans 309
10 Lipids, Membranes, and Cellular
Transport 359

PART 3 Dynamics of Life: Catalysis and Control
of Biochemical Reactions 409
CHAPTER 11 Enzymes: Biological Catalysts 410
CHAPTER 12 Chemical Logic of Metabolism 475

PART 4 Dynamics of Life: Energy,
Biosynthesis, and Utilization of
Precursors 517
CHAPTER 13 Carbohydrate Metabolism: Glycolysis,
Gluconeogenesis, Glycogen Metabolism, and
the Pentose Phosphate Pathway 518

CHAPTER 14 Citric Acid Cycle and Glyoxylate Cycle 591
CHAPTER 15 Electron Transport, Oxidative Phosphorylation, and Oxygen Metabolism 625
CHAPTER 16 Photosynthesis 672
CHAPTER 17 Lipid Metabolism I: Fatty Acids,
Triacylglycerols, and Lipoproteins 708
CHAPTER 18 Interorgan and Intracellular Coordination of

Energy Metabolism in Vertebrates 753
CHAPTER 19 Lipid Metabolism II: Membrane Lipids,
Steroids, Isoprenoids, and Eicosanoids 775
CHAPTER 20 Metabolism of Nitrogenous Compounds I:
Principles of Biosynthesis, Utilization, and
Turnover 820
CHAPTER 21 Metabolism of Nitrogenous Compounds II:
Amino Acids, Porphyrins, and
Neurotransmitters 862
CHAPTER 22 Nucleotide Metabolism 917
CHAPTER 23 Mechanisms of Signal Transduction 958

PART 5 Information

1001

CHAPTER 24 Genes, Genomes, and Chromosomes 1002
CHAPTER 25 DNA Replication 1036
CHAPTER 26 DNA Restructuring: Repair, Recombination,
Rearrangement, Amplification 1079
CHAPTER 27 Information Readout: Transcription and
Post-transcriptional Processing 1125
CHAPTER 28 Information Decoding: Translation and
Post-translational Protein Processing 1173
CHAPTER 29 Regulation of Gene Expression 1232
Answers to Problems 1276
Glossary 1305
Index 1321

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DETAILED CONTENTS

PART 1
The Realm of Biochemistry
CHAPTER 1
The Scope of Biochemistry

1

2

Biochemistry and the Biological Revolution 2
The Roots of Biochemistry 3
Biochemistry as a Discipline and an Interdisciplinary
Science 7
Biochemistry as a Chemical Science 7
The Chemical Elements of Living Matter
Biological Molecules
8

8

Biochemistry as a Biological Science
12
Distinguishing Characteristics of Living Matter
12
The Unit of Biological Organization: The Cell
14

Biochemistry as a Biological Science: Form and
Function
16
Windows on Cellular Function: The Viruses
17
Biochemistry and the Information Explosion
Summary
19
References
20

18

TOOLS OF BIOCHEMISTRY 1A

Microscopy at Many Levels

TOOLS OF BIOCHEMISTRY 2A

20

Electrophoresis and Isoelectric Focusing

CHAPTER 2
The Matrix of Life: Weak Interactions in an
Aqueous Environment 26
The Nature of Noncovalent Interactions 27
Charge–Charge Interactions
27
Permanent and Induced Dipole Interactions


xiv

Molecular Repulsion at Extremely Close Approach:
The van der Waals Radius
30
Hydrogen Bonds
31
The Role of Water in Biological Processes 33
The Structure and Properties of Water
33
Water as a Solvent
35
Ionic Equilibria
38
Acids and Bases: Proton Donors and Acceptors
38
Ionization of Water and the Ion Product
38
The pH Scale and the Physiological pH Range
40
Weak Acid and Base Equilibria
40
A Closer Look at pKa Values: Factors Affecting Acid
Dissociation
41
Titration of Weak Acids: The Henderson–Hasselbalch
Equation
41
Buffer Solutions

43
Molecules with Multiple Ionizing Groups: Ampholytes,
Polyampholytes, and Polyelectrolytes
45
Interactions Between Macroions in Solution
48
Solubility of Macroions and pH
48
The Influence of Small Ions: Ionic Strength
49
Summary
51
References
52
Problems
52

29

CHAPTER 3
The Energetics of Life

54

58

Energy, Heat, and Work
58
Internal Energy and the State of a System
The First Law of Thermodynamics

60
Enthalpy
62

59


xv

CONTENTS

Entropy and the Second Law of Thermodynamics
63
The Direction of Processes
63
Entropy
63
The Second Law of Thermodynamics
64
Free Energy: The Second Law in Open Systems
65
An Example of the Interplay of Enthalpy and Entropy:
The Transition Between Liquid Water and Ice
65
The Interplay of Enthalpy and Entropy:
A Summary
67
Free Energy and Useful Work
68
Free Energy and Concentration

68
Chemical Potential
69
An Example of How Chemical Potential Is Used: A Close
Look at Diffusion Through a Membrane
70
Free Energy and Chemical Reactions: Chemical
Equilibrium 71
The Free Energy Change and the Equilibrium
Constant 71
Free Energy Calculations: A Biochemical Example
Living Cells Are Not at Equilibrium
73

73

High-Energy Phosphate Compounds: Free Energy Sources
in Biological Systems 75
High-Energy Phosphate Compounds as Energy
Transducers
75
Resonance Stabilization of the Phosphate Products
77
Additional Hydration of the Hydrolysis Products
77
Electrostatic Repulsion Between Charged Products
78
Tautomerization of Product Molecules
78
Water, Protons in Buffered Solutions, and the

“Biochemical Standard State”
79
Phosphate Transfer Potential
80
⌬G ؇؅ for Oxidation/Reduction Reactions
81
Quantitation of Reducing Power: Standard Reduction
Potential
81
Free Energy Changes from Oxidation–Reduction
Reactions
83
Free Energy Changes Under Standard Conditions
84
Calculating Free Energy Changes for Biological Oxidations
Under Nonequilibrium Conditions 84
Summary
85
References
86
Problems
87

PART 2
Molecular Architecture of Living Matter
CHAPTER 4
Nucleic Acids

89


90

The Nature of Nucleic Acids
90
The Two Types of Nucleic Acid: DNA and RNA

90

Properties of the Nucleotides
94
Stability and Formation of the Phosphodiester
Linkage
95
Primary Structure of Nucleic Acids
97
The Nature and Significance of Primary Structure
97
98
DNA as the Genetic Substance: Early Evidence
Secondary and Tertiary Structure of Nucleic Acids
98
The Double Helix
98
Semiconservative Nature of DNA Replication
101
Alternative Nucleic Acid Structures: B and A Helices
103
DNA and RNA Molecules in Vivo
106
The Biological Functions of Nucleic Acids: A Preview

of Molecular Biology 109
Genetic Information Storage: The Genome
109
Replication: DNA to DNA
111
Transcription: DNA to RNA
111
Translation: RNA to Protein
112
Plasticity of Secondary and Tertiary DNA Structure
113
Changes in Tertiary Structure: A Closer Look at
Supercoiling
113
Unconventional Secondary Structures of DNA
115
Stability of Secondary and Tertiary Structure
119
The Helix-to-Random-Coil Transition: Nucleic Acid
Denaturation
119
Superhelical Energy and Changes of DNA
Conformation
121
Summary
122
References
123
Problems
124

TOOLS OF BIOCHEMISTRY 4A

An Introduction to X-Ray Diffraction

125

TOOLS OF BIOCHEMISTRY 4B

Manipulating DNA

129

CHAPTER 5
Introduction to Proteins: The Primary Level
of Protein Structure 136
Amino Acids
136
Structure of the a-Amino Acids
136
Stereochemistry of the a-Amino Acids
138
Properties of Amino Acid Side Chains: Classes of
a-Amino Acids
142
Rare Genetically Encoded Amino Acids
144
Modified Amino Acids
144
Peptides and the Peptide Bond
144

Peptides
144
Polypeptides as Polyampholytes
146
The Structure of the Peptide Bond
148
Stability and Formation of the Peptide Bond
149
Proteins: Polypeptides of Defined Sequence
150


xvi

CONTENTS

Prediction of Secondary and Tertiary Protein
Structure 209
Prediction of Secondary Structure
209
Tertiary Structure Prediction: Computer Simulation
of Folding
210
Quaternary Structure of Proteins
212
Multisubunit Proteins: Homotypic Protein–Protein
Interactions
212
Heterotypic Protein–Protein Interactions
215

Summary
215
References
216
Problems
217

From Gene to Protein
152
The Genetic Code
152
Post-translational Processing of Proteins
153
From Gene Sequence to Protein Function
154
Protein Sequence Homology
156
Summary
158
References
158
Problems
160
TOOLS OF BIOCHEMISTRY 5A

Protein Expression and Purification

161

TOOLS OF BIOCHEMISTRY 5B


Mass, Sequence, and Amino Acid Analyses of
Purified Proteins 166

TOOLS OF BIOCHEMISTRY 6A

Spectroscopic Methods for Studying Macromolecular
Conformation in Solution 219

TOOLS OF BIOCHEMISTRY 5C

How to Synthesize a Polypeptide

172

TOOLS OF BIOCHEMISTRY 6B

Determining Molecular Masses and the Number of
Subunits in a Protein Molecule 228

TOOLS OF BIOCHEMISTRY 5D

A Brief Introduction to Proteomics

175

TOOLS OF BIOCHEMISTRY 6C

CHAPTER 6
The Three-Dimensional Structure

of Proteins 177

Determining the Stability of Proteins

Secondary Structure: Regular Ways to Fold the
Polypeptide Chain 177
Theoretical Descriptions of Regular Polypeptide
Structures
177
Describing the Structures: Helices and Sheets
180
Ramachandran Plots
183
Fibrous Proteins: Structural Materials of Cells and
Tissues 185
The Keratins
186
Fibroin
187
Collagen
188
Elastin
190
Summary
191
Globular Proteins: Tertiary Structure and Functional
Diversity 191
Different Folding for Different Functions
191
Varieties of Globular Protein Structure: Patterns

of Folding
192
Factors Determining Secondary and Tertiary
Structure 195
The Information for Protein Folding
195
The Thermodynamics of Folding
196
The Role of Disulfide Bonds
200
Dynamics of Globular Protein Structure
201
Kinetics of Protein Folding
201
Chaperones
204
Protein Misfolding and Disease
206
Motions Within Globular Protein Molecules

208

CHAPTER 7
Protein Function and Evolution

230

234

Oxygen Transport: The Roles of Hemoglobin and

Myoglobin 235
The Mechanism of Oxygen Binding by Heme Proteins
236
The Oxygen Binding Site
236
Analysis of Oxygen Binding by Myoglobin
238
Oxygen Transport: Hemoglobin
241
Cooperative Binding and Allostery
241
Models for the Allosteric Change in Hemoglobin
245
Changes in Hemoglobin Structure Accompanying
Oxygen Binding
246
A Closer Look at the Allosteric Change in
Hemoglobin
247
Allosteric Effectors of Hemoglobin
251
Response to pH Changes: The Bohr Effect
252
Carbon Dioxide Transport
252
Response to Chloride Ion at the a-Globin N-Terminus
253
2,3-Bisphosphoglycerate
253
Other Functions of the Heme Globins: Reactions with

Nitric Oxide 254
Protein Evolution: Myoglobin and Hemoglobin
as Examples
256
The Structure of Eukaryotic Genes: Exons and
Introns 256
Mechanisms of Protein Mutation
257
Evolution of the Myoglobin–Hemoglobin Family
of Proteins
260


xvii

CONTENTS

Hemoglobin Variants: Evolution in Progress
262
Variants and Their Inheritance
262
Pathological Effects of Variant Hemoglobins
264
Thalassemias: Effects of Misfunctioning Hemoglobin
Genes
265
Immunoglobulins: Variability in Structure Yields Versatility
in Binding 266
The Adaptive Immune Response
267

The Structure of Antibodies
269
Generation of Antibody Diversity
272
T Cells and the Cellular Response
272
The Innate Immune Response
274
AIDS and the Immune Response
275
Antibodies and Immunoconjugates as Potential Cancer
Treatments 275
Summary
276
Appendix: A Brief Look at Multistate and Dynamic Models
of Hemoglobin Allostery
277
References
278
Problems
280
TOOLS OF BIOCHEMISTRY 7A

Immunological Methods

282

CHAPTER 8
Contractile Proteins and Molecular
Motors 286

Muscles and Other Actin–Myosin Contractile Systems
287
Actin and Myosin
287
The Structure of Muscle
289
The Mechanism of Contraction: The Sliding Filament
Model
290
Regulation of Contraction: The Role of Calcium
293
Energetics and Energy Supplies in Muscle
295
Nonmuscle Actin and Myosin
296
Microtubule Systems for Motility
297
Motions of Cilia and Flagella
299
Intracellular Transport
300
Bacterial Motility: Rotating Proteins
304
Summary
307
References
307
Problems
308


CHAPTER 9
Carbohydrates: Sugars, Saccharides,
Glycans 309
Monosaccharides
311
Aldoses and Ketoses
311
Enantiomers
311
Diastereomers
312
Aldose Ring Structures
314

Derivatives of the Monosaccharides
321
Phosphate Esters
321
Alditols
323
Amino Sugars
323
Glycosides
324
Oligosaccharides
324
Oligosaccharide Structures
325
328
Stability and Formation of the Glycosidic Bond

Polysaccharides
329
Storage Polysaccharides
330
Structural Polysaccharides
332
Glycosaminoglycans
334
Bacterial Cell Wall Polysaccharides
336
Glycoproteins
339
N-Linked and O-Linked Glycoproteins
339
Blood Group Antigens
340
Oligosaccharides as Cell Markers
343
Biosynthesis of Glycoconjugates: Amino Sugars
344
Glycoconjugates of Interest
345
O-Linked Oligosaccharides: Blood Group Antigens
346
N-Linked Oligosaccharides: Glycoproteins
347
Microbial Cell Wall Polysaccharides: Peptidoglycan
350
Summary
355

References
356
Problems
357
TOOLS OF BIOCHEMISTRY 9A

Sequencing Oligosaccharides

358

CHAPTER 10
Lipids, Membranes, and Cellular Transport
The Molecular Structure and Behavior of Lipids
Fatty Acids
360
Triacylglycerols: Fats
362
Soaps and Detergents
363
Waxes
364
The Lipid Constituents of Biological Membranes
Glycerophospholipids
364
Sphingolipids and Glycosphingolipids
367
Glycoglycerolipids
368
Cholesterol
369


359
359

364

The Structure and Properties of Membranes and Membrane
Proteins 369
Motion in Membranes
370
372
The Asymmetry of Membranes
Characteristics of Membrane Proteins
373
The Erythrocyte Membrane: An Example of Membrane
Structure
375
Insertion of Proteins into Membranes
378
Evolution of the Fluid Mosaic Model of Membrane
Structure
380
Lipid Curvature and Protein Function
382


xviii

CONTENTS


Transport Across Membranes
382
The Thermodynamics of Transport
383
Nonmediated Transport: Diffusion
385
Facilitated Transport: Accelerated Diffusion
386
Active Transport: Transport Against a Concentration
Gradient
392
Caveolae and Coated Vesicles
396
Excitable Membranes, Action Potentials, and
Neurotransmission 397
The Resting Potential
398
The Action Potential
399
Toxins and Neurotransmission
401
Summary
402
Appendix
402
References
403
Problems
404
TOOLS OF BIOCHEMISTRY 10A


Techniques for the Study of Membranes

406

The Diversity of Enzymatic Function
449
Classification of Protein Enzymes
449
Molecular Engineering of New and Modified
Enzymes
449
Nonprotein Biocatalysts: Catalytic Nucleic Acids
449
The Regulation of Enzyme Activity: Allosteric Enzymes
453
Substrate-Level Control
454
455
Feedback Control
Allosteric Enzymes
456
Aspartate Carbamoyltransferase: An Example of an
Allosteric Enzyme
457
Covalent Modifications Used to Regulate Enzyme
Activity 458
Pancreatic Proteases: Activation by Cleavage
459
A Further Look at Activation by Cleavage: Blood

Clotting
462
Summary
463
References
464
Problems
465
TOOLS OF BIOCHEMISTRY 11A

PART 3
Dynamics of Life: Catalysis and Control
of Biochemical Reactions 409
CHAPTER 11
Enzymes: Biological Catalysts

410

The Role of Enzymes
410
Chemical Reaction Rates and the Effects of Catalysts:
A Review 411
Reaction Rates, Rate Constants, and Reaction Order
411
Transition States and Reaction Rates
413
Transition State Theory Applied to Catalysis
416
How Enzymes Act as Catalysts: Principles and Examples 418
General Principles: The Induced Fit Model

418
Lysozyme
422
Serine Proteases
425
The Role of Dynamics in Catalysis
429
The Kinetics of Enzymatic Catalysis
431
Reaction Rate for a Simple Enzyme-Catalyzed Reaction:
Michaelis–Menten Kinetics
431
The Significance of KM, kcat, and kcat/KM
433
Analysis of Kinetic Data: Testing the Michaelis–Menten
Equation
435
Multisubstrate Reactions
436
A Closer Look at Some Complex Reactions
437
Single-Molecule Studies of Enzyme Activity
438
Enzyme Inhibition
440
Reversible Inhibition
440
Irreversible Inhibition
444
Cofactors, Vitamins, and Essential Metals

445
Cofactors and What They Do
446
Metal Ions in Enzymes
448

How to Measure the Rates of Enzyme-Catalyzed
Reactions
468
TOOLS OF BIOCHEMISTRY 11B

Introduction to Protein Engineering of Enzymes

CHAPTER 12
Chemical Logic of Metabolism

475

A First Look at Metabolism
475
Freeways on the Metabolic Road Map
477
Central Pathways of Energy Metabolism
477
Distinct Pathways for Biosynthesis and
Degradation
480
Biochemical Reaction Types
481
Nucleophilic Substitutions

482
Nucleophilic Additions
483
Carbonyl Condensations
483
485
Eliminations
Oxidations and Reductions
485
Some Bioenergetic Considerations
486
Oxidation as a Metabolic Energy Source
486
ATP as a Free Energy Currency
489
Major Metabolic Control Mechanisms
498
Control of Enzyme Levels
498
Control of Enzyme Activity
498
Compartmentation
499
Hormonal Regulation
500
Distributive Control of Metabolism
501
Experimental Analysis of Metabolism
502
Goals of the Study of Metabolism

502
Levels of Organization at Which Metabolism Is
Studied
503
Metabolic Probes
505

471


xix

CONTENTS

Summary
References
Problems

506
507
507

TOOLS OF BIOCHEMISTRY 12A

Radioisotopes and the Liquid Scintillation Counter
TOOLS OF BIOCHEMISTRY 12B

Metabolomics

511


PART 4
Dynamics of Life: Energy, Biosynthesis, and
Utilization of Precursors 517
CHAPTER 13
Carbohydrate Metabolism: Glycolysis,
Gluconeogenesis, Glycogen Metabolism,
and the Pentose Phosphate Pathway 518
Glycolysis: An Overview
520
Relation of Glycolysis to Other Pathways
520
Anaerobic and Aerobic Glycolysis
520
The Crucial Early Experiments
522
Strategy of Glycolysis
523
Reactions of Glycolysis
523
Reactions 1–5: The Energy Investment Phase
525
Reactions 6–10: The Energy Generation Phase
529
Metabolic Fates of Pyruvate
535
Lactate Metabolism
535
Isozymes of Lactate Dehydrogenase
536

Ethanol Metabolism
536
Energy and Electron Balance Sheets
539
Gluconeogenesis
540
Physiological Need for Glucose Synthesis
in Animals
541
Enzymatic Relationship of Gluconeogenesis to
541
Glycolysis
Stoichiometry and Energy Balance of
Gluconeogenesis
544
Substrates for Gluconeogenesis
545
Ethanol Consumption and
Gluconeogenesis
548
Roles of Extrahepatic Phosphoenolpyruvate
Carboxykinase
548
Evolution of Carbohydrate Metabolic Pathways
549
Coordinated Regulation of Glycolysis and
Gluconeogenesis
549
The Pasteur Effect
549

Oscillations of Glycolytic Intermediates
550
Reciprocal Regulation of Glycolysis and
Gluconeogenesis
551
Regulation at the Phosphofructokinase/Fructose-1,6Bisphosphatase Substrate Cycle
551

508

Regulation at the Pyruvate Kinase/Pyruvate Carboxylase
+ PEPCK Substrate Cycle
555
Regulation at the Hexokinase/Glucose-6-Phosphatase
Substrate Cycle
556
Entry of Other Sugars into the Glycolytic Pathway
557
Monosaccharide Metabolism
557
Disaccharide Metabolism
560
Polysaccharide Metabolism
561
Hydrolytic and Phosphorolytic Cleavages
561
Starch and Glycogen Digestion
562
Glycogen Metabolism in Muscle and Liver
562

Glycogen Breakdown
562
Glycogen Biosynthesis
563
Biosynthesis of UDP-Glucose
564
The Glycogen Synthase Reaction
564
Formation of Branches
564
Coordinated Regulation of Glycogen Metabolism
565
Structure of Glycogen Phosphorylase
566
Control of Phosphorylase Activity
567
Proteins in the Glycogenolytic Cascade
568
Nonhormonal Control of Glycogenolysis
569
Control of Glycogen Synthase Activity
570
Congenital Defects of Glycogen Metabolism in
Humans
574
Biosynthesis of Other Polysaccharides
575
A Biosynthetic Pathway That Oxidizes Glucose: The Pentose
Phosphate Pathway 575
The Oxidative Phase: Generation of Reducing Power as

NADPH
576
The Nonoxidative Phase: Alternative Fates of Pentose
Phosphates
577
Human Genetic Disorders Involving Pentose Phosphate
Pathway Enzymes
581
Summary
584
References
584
Problems
586
TOOLS OF BIOCHEMISTRY 13A

Detecting and Analyzing Protein–Protein Interactions

CHAPTER 14
Citric Acid Cycle and Glyoxylate Cycle

588

591

Overview of Pyruvate Oxidation and the Citric
Acid Cycle 593
The Three Stages of Respiration
593
Chemical Strategy of the Citric Acid Cycle

594
Discovery of the Citric Acid Cycle
596
Pyruvate Oxidation: A Major Entry Route for Carbon into
the Citric Acid Cycle 597
Coenzymes Involved in Pyruvate Oxidation and the Citric
Acid Cycle 598


xx

CONTENTS

Thiamine Pyrophosphate (TPP)
598
Lipoic Acid (Lipoamide)
598
Flavin Adenine Dinucleotide (FAD)
599
Coenzyme A: Activation of Acyl Groups
601
Action of the Pyruvate Dehydrogenase Complex
602
The Citric Acid Cycle
604
Step 1: Introduction of Two Carbon Atoms as
Acetyl-CoA
605
Step 2: Isomerization of Citrate
605

Step 3: Generation of CO2 by an NAD+-Linked
Dehydrogenase
607
Step 4: Generation of a Second CO2 by an Oxidative
Decarboxylation
608
Step 5: A Substrate-Level Phosphorylation
608
Step 6: A Flavin-Dependent Dehydrogenation
609
Step 7: Hydration of a Carbon–Carbon Double
Bond
610
Step 8: A Dehydrogenation That Regenerates
Oxaloacetate
611
Stoichiometry and Energetics of the Citric Acid Cycle
611
Regulation of Pyruvate Dehydrogenase and the Citric Acid
Cycle 612
Control of Pyruvate Oxidation
612
Control of the Citric Acid Cycle
614
Organization of the Citric Acid Cycle Enzymes
615
Evolution of the Citric Acid Cycle
615
Citric Acid Cycle Malfunction as a Cause of Human
Disease

615
Anaplerotic Sequences: The Need to Replace Cycle
Intermediates 616
Reactions That Replenish Oxaloacetate
616
The Malic Enzyme
618
Reactions Involving Amino Acids
618
Glyoxylate Cycle: An Anabolic Variant of the Citric Acid
Cycle
620
Summary
622
References
623
Problems
624

CHAPTER 15
Electron Transport, Oxidative Phosphorylation,
and Oxygen Metabolism 625
The Mitochondrion: Scene of the Action
626
Oxidations and Energy Generation
628
Free Energy Changes in Biological Oxidations
629
Electron Transport
630

Electron Carriers in the Respiratory Chain
630
Determining the Sequence of Respiratory Electron
Carriers
632
Respiratory Complexes
635
Oxidative Phosphorylation
643
The P/O Ratio: Efficiency of Oxidative
Phosphorylation
643

Oxidative Reactions That Drive ATP Synthesis
644
Mechanism of Oxidative Phosphorylation:
Chemiosmotic Coupling
645
A Closer Look at Chemiosmotic Coupling: The
646
Experimental Evidence
Complex V: The Enzyme System for ATP Synthesis
649
Mitochondrial Transport Systems
657
Shuttling Cytoplasmic Reducing Equivalents into
Mitochondria
659
Energy Yields from Oxidative Metabolism
660

The Mitochondrial Genome and Disease
661
Mitochondria and Evolution
662
Oxygen as a Substrate for Other Metabolic Reactions
663
Oxidases and Oxygenases
663
Cytochrome P450
664
Reactive Oxygen Species, Antioxidant Defenses, and
Human Disease
665
Summary
668
References
668
Problems
670

CHAPTER 16
Photosynthesis

672

The Basic Processes of Photosynthesis
673
The Chloroplast
675
The Light Reactions

677
Absorption of Light: The Light-Harvesting System
677
Photochemistry in Plants and Algae: Two Photosystems
in Series
680
An Alternative Light Reaction Mechanism: Cyclic
Electron Flow
691
Reaction Center Complexes in Photosynthetic
Bacteria
692
Artificial Photosynthesis
694
The Dark Reactions: The Calvin Cycle
695
Stage I: Carbon Dioxide Fixation and Sugar
Production
695
Stage II: Regeneration of the Acceptor
698
A Summary of the Light and Dark Reactions in Two-System
Photosynthesis 699
The Overall Reaction and the Efficiency
699
of Photosynthesis
Regulation of Photosynthesis
700
Photorespiration and the C4 Cycle
701

Evolution of Photosynthesis
703
Summary
705
References
706
Problems
707

CHAPTER 17
Lipid Metabolism I: Fatty Acids,
Triacylglycerols, and Lipoproteins

708

Utilization and Transport of Fat and Cholesterol

708


xxi

CONTENTS

Fats as Energy Reserves
710
Fat Digestion and Absorption
710
Transport of Fat to Tissues: Lipoproteins
712

Cholesterol Transport and Utilization in Animals
716
Mobilization of Stored Fat
721
Fatty Acid Oxidation
723
Early Experiments
723
Fatty Acid Activation and Transport into
Mitochondria
724
The b-Oxidation Pathway
726
Mitochondrial b-Oxidation Involves Multiple
Isozymes 728
Energy Yield from Fatty Acid Oxidation 729
Oxidation of Unsaturated Fatty Acids
730
Oxidation of Fatty Acids with Odd-Numbered
Carbon Chains
731
Control of Fatty Acid Oxidation
732
Peroxisomal b-Oxidation of Fatty Acids
732
733
a-Oxidation of Fatty Acids
Ketogenesis
733
Fatty Acid Biosynthesis

736
Relationship of Fatty Acid Synthesis to Carbohydrate
Metabolism
736
Early Studies of Fatty Acid Synthesis
736
Biosynthesis of Palmitate from Acetyl-CoA
737
Elongation of Fatty Acid Chains
744
Fatty Acid Desaturation
744
Control of Fatty Acid Synthesis
745
Variant Fatty Acid Synthesis Pathways That Lead to
Antibiotics
747
Biosynthesis of Triacylglycerols
748
Biochemical Insights into Obesity
750
Summary
750
References
751
Problems
752

CHAPTER 18
Interorgan and Intracellular Coordination of

Energy Metabolism in Vertebrates 753
Interdependence of the Major Organs in Vertebrate Fuel
Metabolism 753
Fuel Inputs and Outputs
754
Metabolic Division of Labor Among the Major
Organs
754
Hormonal Regulation of Fuel Metabolism
757
Actions of the Major Hormones
758
Coordination of Energy Homeostasis
761
Responses to Metabolic Stress: Starvation, Diabetes
768
Starvation
768
Diabetes
770
Summary
773
References
773
Problems
774

CHAPTER 19
Lipid Metabolism II: Membrane Lipids,
Steroids, Isoprenoids, and Eicosanoids


775

Metabolism of Glycerophospholipids
775
Biosynthesis of Glycerophospholipids in
Bacteria
776
Glycerophospholipid Metabolism in
Eukaryotes
781
Metabolism of Sphingolipids
790
Steroid Metabolism
794
Some Structural Considerations
795
Biosynthesis of Cholesterol
795
Bile Acids
803
Steroid Hormones
804
Other Isoprenoid Compounds
808
Lipid-Soluble Vitamins
808
Other Terpenes
811
Eicosanoids: Prostaglandins, Thromboxanes, and

Leukotrienes 811
Some Historical Aspects
812
Structure
813
Biosynthesis and Catabolism
813
Biological Actions
815
Summary
817
References
817
Problems
819

CHAPTER 20
Metabolism of Nitrogenous Compounds I:
Principles of Biosynthesis, Utilization, and
Turnover 820
Utilization of Inorganic Nitrogen: The Nitrogen
Cycle
822
Biological Nitrogen Fixation
823
Nitrate Utilization
825
Utilization of Ammonia: Biogenesis of Organic
Nitrogen 826
Glutamate Dehydrogenase: Reductive Amination

of a-Ketoglutarate
827
Glutamine Synthetase: Generation of Biologically Active
Amide Nitrogen
827
Asparagine Synthetase: A Similar Amidation
Reaction
831
Carbamoyl Phosphate Synthetase: Generation of
an Intermediate for Arginine and Pyrimidine
Synthesis
831
The Nitrogen Economy: Aspects of Amino Acid Synthesis
and Degradation 832
Metabolic Consequences of the Absence of Nitrogen
Storage Compounds
832
Biosynthetic Capacities of Organisms
832
Transamination
833


xxii

CONTENTS

Protein Turnover
834
Quantitative Features of Protein Turnover

834
Biological Importance of Protein Turnover
835
Intracellular Proteases and Sites of Turnover
836
Chemical Signals for Turnover
837
Amino Acid Degradation and Metabolism of Nitrogenous
End Products 840
Common Features of Amino Acid Degradation
Pathways
840
Detoxification and Excretion of Ammonia
840
The Krebs–Henseleit Urea Cycle
841
Transport of Ammonia to the Liver
844
Coenzymes Involved in Nitrogen Metabolism
845
Pyridoxal Phosphate
845
Tetrahydrofolate Coenzymes and One-Carbon
Metabolism
848
B12 Coenzymes
853
Summary
858
References

859
Problems
860

CHAPTER 21
Metabolism of Nitrogenous Compounds II: Amino
Acids, Porphyrins, and Neurotransmitters 862
Pathways of Amino Acid Degradation
862
Pyruvate Family of Glucogenic Amino Acids
863
Oxaloacetate Family of Glucogenic Amino Acids
865
a-Ketoglutarate Family of Glucogenic Amino Acids
865
Succinyl-CoA Family of Glucogenic Amino Acids
866
Acetoacetate/Acetyl-CoA Family of Ketogenic Amino
Acids
870
Amino Acids as Biosynthetic Precursors
874
S-Adenosylmethionine and Biological Methylation
874
S-Adenosylmethionine and Polyamines
880
Other Precursor Functions of Glutamate
882
Nitric Oxide and Creatine Phosphate
883

Tyrosine Utilization in Animals
885
Aromatic Amino Acid Utilization in Plants
887
Porphyrin and Heme Metabolism
889
Biosynthesis of Tetrapyrroles: The Succinate–Glycine
Pathway
889
Degradation of Heme in Animals
893
Amino Acids and Their Metabolites as Neurotransmitters
and Biological Regulators 895
Biosynthesis of Serotonin and Catecholamines
895
Amino Acid Biosynthesis
897
Synthesis of Glutamate, Aspartate, Alanine, Glutamine,
and Asparagine
898
Synthesis of Methionine, Threonine, and Lysine from
Aspartate
898
Metabolism of Sulfur-Containing Amino Acids
901
Synthesis of Proline, Ornithine, and Arginine from
Glutamate
903

Hydroxyproline and Collagen

905
Synthesis of Serine and Glycine from
3-Phosphoglycerate
906
Synthesis of Valine, Leucine, and Isoleucine from
Pyruvate
907
Synthesis of the Aromatic Amino Acids from Glycolytic
Intermediates: The Shikimic Acid Pathway
908
Synthesis of Histidine from Glycolytic
Intermediates
911
Summary
913
References
913
Problems
915

CHAPTER 22
Nucleotide Metabolism

917

Outlines of Pathways in Nucleotide Metabolism
917
Biosynthetic Routes: De Novo and Salvage Pathways
917
Nucleic Acid Degradation and the Importance of

Nucleotide Salvage
918
PRPP: A Central Metabolite in De Novo and Salvage
Pathways
920
De Novo Biosynthesis of Purine Nucleotides
920
Early Studies on De Novo Purine Synthesis
920
Purine Synthesis from PRPP to Inosinic Acid
921
Synthesis of ATP and GTP from Inosinic Acid
924
Regulation of De Novo Purine Biosynthesis
925
Utilization of Adenine Nucleotides in Coenzyme
Biosynthesis
925
Purine Degradation and Clinical Disorders of Purine
Metabolism 926
Formation of Uric Acid
926
Excessive Accumulation of Uric Acid: Gout
927
Salvage of Purines and Lesch–Nyhan Syndrome
929
Unexpected Consequences of Defective Purine
Catabolism: Immunodeficiency
929
Pyrimidine Nucleotide Metabolism

931
De Novo Biosynthesis of the Pyrimidine Ring
931
Control of Pyrimidine Biosynthesis in Bacteria
933
Multifunctional Enzymes in Eukaryotic Pyrimidine
Synthesis
933
Salvage Synthesis and Pyrimidine Catabolism
934
Glutamine-Dependent Amidotransferases
934
Deoxyribonucleotide Biosynthesis and Metabolism
935
Reduction of Ribonucleotides to Deoxyribonucleotides
936
Biosynthesis of Thymine Deoxyribonucleotides
942
Deoxyuridine Nucleotide Metabolism
943
Salvage Routes to Deoxyribonucleotide Synthesis
944
Thymidylate Synthase: A Target Enzyme for
Chemotherapy
945
Flavin-Dependent Thymidylate Synthase: A Novel Route
to dTMP 949


xxiii


CONTENTS

Virus-Directed Alterations of Nucleotide Metabolism

950

Biological and Medical Importance of Other Nucleotide
Analogs 951
Nucleotide Analogs as Chemotherapeutic Agents
951
Nucleotide Analogs and Mutagenesis
953
Nucleotide-Metabolizing Enzymes as Selectable
Genetic Markers
954
Summary
955
References
955
Problems
956

CHAPTER 23
Mechanisms of Signal Transduction
An Overview of Hormone Action

958

960


Hierarchical Nature of Hormonal Control

962

Synthesis of Peptide Hormone Precursors

963

Signal Transduction: Receptors
963
Experimental Study of Receptors
964
Agonists and Antagonists
965
Classes of Catecholamine Receptors
965
Receptors and Adenylate Cyclase as Distinct Components
of Signal Transduction Systems
965
Transducers: G Proteins
968
Actions of G Proteins
968
Structure of G Proteins
969
Consequences of Blocking GTPase
969
G Proteins in the Visual Process
970

A Closer Look at G Protein Subunits
970
Modulating the Hormonal Stimulus
971
Effectors: Adenylate Cyclase

972

Second-Messenger Systems
972
Cyclic AMP
972
Cyclic GMP and Nitric Oxide
973
Calcium Ion
974
Phosphoinositides
974
Receptor Tyrosine Kinases

995

PART 5
Information

1001

CHAPTER 24
Genes, Genomes, and Chromosomes


1002

Prokaryotic and Eukaryotic Genomes
1003
Size of the Genome
1003
Repetitive Sequences
1003
Gene Families
1007
Restriction and Modification
1008
Biology of Restriction and Modification
1009
Properties of Restriction and Modification
Enzymes
1011
Determining Genome Nucleotide Sequences
1014
Mapping Large Genomes
1014
Physical Organization of Genes: The Nucleus,
Chromosomes, and Chromatin
1019
Chromosomes
1019
Chromatin
1022
The Nucleosome
1023

Higher-Order Chromatin Structure in the Nucleus
The Cell Cycle
1026
Phases of Mitosis
1027
The Centromere and Kinetochore
1027
Control of the Cell Cycle
1029
Summary
1030
References
1031
Problems
1032
Polymerase Chain Reaction

CHAPTER 25
DNA Replication
980

Signal Transduction, Oncogenes, and Cancer
982
Viral and Cellular Oncogenes
983
Oncogenes in Human Tumors
985
Oncogenes and the Central Growth Factor Activation
Pathway
987

The Cancer Genome Mutational Landscape
989
Neurotransmission
990
The Cholinergic Synapse
990
Fast and Slow Synaptic Transmission
992
Functions of Specific Neurotransmitters
993
Drugs That Act in the Synaptic Cleft
994
Peptide Neurotransmitters and Neurohormones
994
Signaling in Bacteria and Plants

997
998
1000

1025

TOOLS OF BIOCHEMISTRY 24A

977

Steroid and Thyroid Hormones: Intracellular Receptors

Summary
References

Problems

1034

1036

Early Insights into DNA Replication
1036
DNA Polymerases: Enzymes Catalyzing Polynucleotide
Chain Elongation
1039
Structure and Activities of DNA Polymerase I
1039
A Brief Review of Microbial Genetics
1041
Multiple DNA Polymerases
1043
Structure and Mechanism of DNA Polymerases
1045
Other Proteins at the Replication Fork
1046
Discontinuous DNA Synthesis
1047
RNA Primers
1047
The DNA Polymerase III Holoenzyme
1049
Single-Strand DNA-Binding Proteins: Maintaining
Optimal Template Conformation
1051

Helicases: Unwinding DNA Ahead of the Fork
1053


xxiv

CONTENTS

Topoisomerases: Relieving Torsional Stress
1054
A Model of the Replisome
1059
Proteins in Eukaryotic DNA Replication
1060
DNA Polymerases
1060
Other Eukaryotic Replication Proteins
1061
Replication of Chromatin
1062
Initiation of DNA Replication
1063
Requirements for Initiation of Replication
1064
Initiation of E. coli DNA Replication at oric
1064
Initiation of Eukaryotic Replication
1066
Mitochondrial DNA Replication
1066

Replication of Linear Genomes
1067
Linear Virus Genome Replication
1067
Telomerase
1068
Fidelity of DNA Replication
1068
3¿ Exonucleolytic Proofreading
1069
Polymerase Insertion Specificity
1069
RNA Viruses: The Replication of RNA
Genomes
1072
RNA-Dependent RNA Replicases
1072
Replication of Retroviral Genomes
1072
Summary
1074
References
1075
Problems
1076
TOOLS OF BIOCHEMISTRY 25A

Two-Dimensional Gel Electrophoretic Analysis
of DNA Topoisomers
1078


CHAPTER 26
DNA Restructuring: Repair, Recombination,
Rearrangement, Amplification 1079
DNA Repair
1080
Types and Consequences of DNA Damage
1080
Direct Repair of Damaged DNA Bases: Photoreactivation
and Alkyltransferases
1083
Nucleotide Excision Repair: Excinucleases
1085
Base Excision Repair: DNA N-Glycosylases
1087
1090
Mismatch Repair
Daughter-Strand Gap Repair
1092
Translesion Synthesis
1093
Double-Strand Break Repair
1096
Recombination
1097
Classification of Recombination Processes
1097
Homologous Recombination
1099
Site-Specific Recombination

1105
Gene Rearrangements
1107
Immunoglobulin Synthesis: Generating Antibody
Diversity
1107
Transposable Genetic Elements
1110
Retroviruses
1114
Gene Amplification
1115
Summary
1117
References
1117
Problems
1119

TOOLS OF BIOCHEMISTRY 26A

Gene Targeting by Homologous Recombination

1120

TOOLS OF BIOCHEMISTRY 26B

Single-Molecule Biochemistry

1122


CHAPTER 27
Information Readout: Transcription
and Post-transcriptional Processing

1125

DNA as the Template for RNA Synthesis
1126
The Predicted Existence of Messenger RNA
1126
T2 Bacteriophage and the Demonstration of
Messenger RNA
1127
RNA Dynamics in Uninfected Cells
1128
Enzymology of RNA Synthesis: RNA Polymerase
1129
Biological Role of RNA Polymerase
1130
Structure of RNA Polymerase
1131
Mechanism of Transcription
1133
Initiation of Transcription: Interactions with
Promoters
1133
Initiation and Elongation: Incorporation of
Ribonucleotides
1135

Punctuation of Transcription: Promoter
Recognition
1138
Punctuation of Transcription: Termination
1140
Transcription and Its Control in Eukaryotic Cells
1143
RNA Polymerase I: Transcription of the Major
Ribosomal RNA Genes
1143
RNA Polymerase III: Transcription of Small RNA
Genes
1144
RNA Polymerase II: Transcription of Structural
Genes
1148
Chromatin Structure and Transcription
1152
Termination of Transcription
1155
Post-transcriptional Processing
1155
Bacterial mRNA Turnover
1155
Post-transcriptional Processing in the Synthesis
of Bacterial rRNAs and tRNAs
1156
Processing of Eukaryotic Messenger RNA
1158
Alternative Splicing

1161
RNA Editing
1162
Summary
1163
References
1164
Problems
1165
TOOLS OF BIOCHEMISTRY 27A

Footprinting: Identifying Protein-Binding Sites
on DNA 1166
TOOLS OF BIOCHEMISTRY 27B

Mapping Transcriptional Start Points
TOOLS OF BIOCHEMISTRY 27C

DNA Microarrays

1170

TOOLS OF BIOCHEMISTRY 27D

Chromatin Immunoprecipitation

1171

1168