Biochemistry

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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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Vice-President, Editorial Director: Gary Bennett Executive Acquisitions Editor: Cathleen Sullivan Marketing Manager: Julia Jevmenova

Senior Developmental Editor: John Polanszky

Project Managers: Marissa Lok and Tracy Duff (PreMediaGlobal) Manufacturing Manager: Susan Johnson

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Proofreader: Stephany Craig Compositor: PreMediaGlobal

Photo and Permissions Researcher: Heather Jackson Art Director: Julia Hall

Cover and Interior Designer: Miriam Blier Cover Image: C. Spiegel and S. Anthony-Cahill

Credits and acknowledgments borrowed from other sources and reproduced, with permission, in this textbook appear on the appropriate page within text.

Copyright © 2013 Pearson Canada Inc. All rights reserved. Manufactured in the United States of America. This publication is protected by copyright and permission should be obtained from the publisher prior to any prohibited reproduction, storage in a retrieval system, or transmission in any form or by any means, electronic, mechanical, photocopying, recording, or likewise. To obtain permission(s) to use material from this work, please submit a written request to Pearson Canada Inc., Permissions Department, 26 Prince Andrew Place, Don Mills, Ontario, M3C 2T8, or fax your request to 416-447-3126, or submit a request to Permissions Requests at www.pearsoncanada.ca.

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., 1937-

QH345.B43 2012 572’.3 C2011-902175-7

ISBN 978-0-13-800464-4

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Christopher K. Mathewsis 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 Holdeis Distinguished Professor Emeritus of Biophysics and Bio- chemistry 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 coed- itor of The Origins of Life and Evolution (1981). His teaching experience includes undergraduate and graduate chemistry, biochemistry and biophysics, and the

ABOUT THE AUTHORS

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ABOUT THE AUTHORS

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

Dean R. Applingis 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 bird- watching by coauthor Chris Mathews and his wife, Kate—an unintended conse- quence of working on this book!

Spencer J. Anthony-Cahillis 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.

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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 Anthony- Cahill’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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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)

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• 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 interfer- ence, 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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• In Vitro Evolution of Protein Function (Chapter 11)

• Metabolomics (Chapter 12)

• Gene Targeting by Homologous Recombination; Single-Molecule Biochem- istry (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 acti- vated 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 begin- ning 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, begin- ning in late 2010.

The structure of the yeast ribosome shown here was described by A. Ben- Shem, 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

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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).

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• 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.

• PowerPoint® Presentations. Two sets of PowerPoint® 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 solu- tions 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 expecta- tions, 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 rel- evant 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 advan- tage 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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• CourseSmart for Students. CourseSmart goes beyond traditional expecta- tions, 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 occa- sionally, 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, in- house 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

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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 occa- sional pungent advice and criticism. Their patience and enduring support were the most important elements in seeing this project to a timely and satisfying con- clusion.

Christopher K. Mathews Dean R. Appling Spencer J. Anthony-Cahill K. E. van Holde

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REVIEWERS

The following reviewers provided valuable feedback on the manuscript at vari- ous 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

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BRIEF CONTENTS

xiii PART 1 The Realm of Biochemistry 1

CHAPTER 1 The Scope of Biochemistry 2

CHAPTER 2 The Matrix of Life: Weak Interactions in an Aqueous Environment 26

CHAPTER 3 The Energetics of Life 58

PART 2 Molecular Architecture of Living Matter 89

CHAPTER 4 Nucleic Acids 90

CHAPTER 5 Introduction to Proteins: The Primary Level of Protein Structure 136

CHAPTER 6 The Three-Dimensional Structure of Proteins 177

CHAPTER 7 Protein Function and Evolution 234 CHAPTER 8 Contractile Proteins and Molecular

Motors 286

CHAPTER 9 Carbohydrates: Sugars, Saccharides, Glycans 309

CHAPTER 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 Phospho-

rylation, 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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PART 1 The Realm of Biochemistry 1

CHAPTER 1 The Scope of Biochemistry 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 8 Biological Molecules 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 18 Summary 19

References 20

TOOLS OF BIOCHEMISTRY 1A Microscopy at Many Levels 20

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 29

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 pK_aValues: 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

TOOLS OF BIOCHEMISTRY 2A

Electrophoresis and Isoelectric Focusing 54

CHAPTER 3 The Energetics of Life 58

Energy, Heat, and Work 58

Internal Energy and the State of a System 59 The First Law of Thermodynamics 60 Enthalpy 62

DETAILED CONTENTS

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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 73 Living Cells Are Not at Equilibrium 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

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 89

CHAPTER 4 Nucleic Acids 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 DNA as the Genetic Substance: Early Evidence 98 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

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 -Amino Acids 136 Stereochemistry of the -Amino Acids 138 Properties of Amino Acid Side Chains: Classes of

-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

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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

Protein Expression and Purification 161 TOOLS OF BIOCHEMISTRY 5B

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

TOOLS OF BIOCHEMISTRY 5C

How to Synthesize a Polypeptide 172 TOOLS OF BIOCHEMISTRY 5D

A Brief Introduction to Proteomics 175

CHAPTER 6 The Three-Dimensional Structure of Proteins 177

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

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

Spectroscopic Methods for Studying Macromolecular Conformation in Solution 219

TOOLS OF BIOCHEMISTRY 6B

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

TOOLS OF BIOCHEMISTRY 6C

Determining the Stability of Proteins 230

CHAPTER 7 Protein Function and Evolution 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 -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

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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

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

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

Stability and Formation of the Glycosidic Bond 328 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

Sequencing Oligosaccharides 358

CHAPTER 10 Lipids, Membranes, and Cellular Transport 359

The Molecular Structure and Behavior of Lipids 359 Fatty Acids 360

Triacylglycerols: Fats 362 Soaps and Detergents 363 Waxes 364

The Lipid Constituents of Biological Membranes 364 Glycerophospholipids 364

Sphingolipids and Glycosphingolipids 367 Glycoglycerolipids 368

Cholesterol 369

The Structure and Properties of Membranes and Membrane Proteins 369

Motion in Membranes 370

The Asymmetry of Membranes 372 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

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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

Techniques for the Study of Membranes 406

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 K_M, k_cat, and k_cat/K_M 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

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 Feedback Control 455 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

How to Measure the Rates of Enzyme-Catalyzed Reactions 468

TOOLS OF BIOCHEMISTRY 11B

Introduction to Protein Engineering of Enzymes 471

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 Eliminations 485

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

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Radioisotopes and the Liquid Scintillation Counter 508 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 Glycolysis 541

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-1,6- Bisphosphatase Substrate Cycle 551

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

Detecting and Analyzing Protein–Protein Interactions 588

CHAPTER 14 Citric Acid Cycle and Glyoxylate Cycle 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

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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 CO₂by an NAD⁺-Linked

Dehydrogenase 607

Step 4: Generation of a Second CO₂by 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

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 Experimental Evidence 646

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

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 of Photosynthesis 699

Regulation of Photosynthesis 700 Photorespiration and the C₄Cycle 701 Evolution of Photosynthesis 703 Summary 705

CHAPTER 17 Lipid Metabolism I: Fatty Acids,

Triacylglycerols, and Lipoproteins 708

Utilization and Transport of Fat and Cholesterol 708

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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 -Oxidation Pathwayb 726

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

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 -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

a Mitochondrial -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 -Oxidation of Fatty Acidsb 732

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-Oxidation of Fatty Acids 733 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

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

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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

B₁₂Coenzymes 853 Summary 858

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

-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

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

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