ENZYMES

1 ENZYMESSECOND EDITIONE nzymes: A Practical Introduction to Structure, Mechanism, and Data A. CopelandCopyright 2000 by Wiley-VCH, : 0-471-35929-7 (Hardback); 0-471-22063-9 (Electronic)ENZYMESA Practical Introductionto Structure, Mechanism,and Data AnalysisSECOND EDITIONR obert A. CopelandA JOHN WILEY & SONS, INC., PUBLICATIONNew York / Chichester / Weinheim / Brisbane / Singapore / TorontoDesignations used by companies to distinguish their products are often claimed as trademarks. Inall instances where John Wiley & Sons, Inc., is aware of a claim, the product names appear ininitial capital or . Readers, however, should contact the appropriatecompanies for more complete information regarding trademarks and part of this publication may be reproduced, stored in a retrieval system or transmitted in anyform or by any means, electronic or mechanical, including uploading, downloading, printing,decompiling, recording or otherwise, except as permitted under Sections 107 or 108 of the 1976 UnitedStatesCopyrightAct, the Publisher for permission should be addressed to the Permissions Department, JohnWiley & Sons, Inc.

2 , 605 Third Avenue, New York, NY 10158-0012,(212)850-6011, fax(212)850-6008, E-Mail: PERMREQ publication is designed to provide accurate and authoritative information in regard to thesubject matter covered. It is sold with the understanding that the publisher is not engaged inrendering professional services. If professional advice or other expert assistance is required, theservices of a competent professional person should be 0-471-22063-9 This title is also available in print as ISBN more information about Wiley products, visit our web site at 2000byWiley-VCH, Clyde Worthenfor teaching me all the important lessons:arigato to Theodore (Doc) Jannerfor stoking the to the First Editionxv1 A Brief History of ENZYMES in Antiquity / Early Enzymology / The Development of Mechanistic Enzymology / Studies of Enzyme Structure / Enzymology Today / Summary / 8 References and Further Reading / 102 Chemical Bonds and Reactions in Atomic and Molecular Orbitals / Thermodynamics of Chemical Reactions / Acid Base Chemistry / Noncovalent Interactions in Reversible Binding / Rates of Chemical Reactions / Summary / 41 References and Further Reading / 413 Structural Components of The Amino Acids / The Peptide Bond / Amino Acid Sequence or Primary Structure / Secondary Structure / Tertiary Structure / Subunits and Quaternary Structure / Cofactors in

3 ENZYMES / Summary / 71 References and Further Reading / 744 Protein Ligand Binding The Equilibrium Dissociation Constant,K The Kinetic Approach to Equilibrium / Binding Measurements at Equilibrium / Graphic Analysis of Equilibrium Ligand Binding Data / Equilibrium Binding with Ligand Depletion(Tight BindingInteractions) Competition Among Ligands for a Common Binding Site / Experimental Methods for Measuring Ligand Binding / Summary / 107 References and Further Reading / 1085 Kinetics of Single-Substrate Enzyme Time Course of Enzymatic Reactions / of Substrate Concentration on Velocity / Rapid Equilibrium Model of Enzyme Kinetics / Steady State Model of Enzyme Kinetics / Significance ofk andK / Measurement ofk andK / Linear Transformations of Enzyme Kinetic Data / at Low Substrate Concentrations / from Hyperbolic Kinetics / Transient State Kinetic Measurements / Summary / 145 References and Further Reading / 1456 Chemical Mechanisms in Enzyme Substrate Active Site Complementarity / Rate Enhancement Through Transition State Stabilization / Chemical

4 Mechanisms for Transition State Stabilization / The Serine Proteases: An Illustrative Example / Enzymatic Reaction Nomenclature / Summary / 186 References and Further Reading / 1867 Experimental Measures of Enzyme Initial Velocity Measurements / Detection Methods / Separation Methods in Enzyme Assays / Factors Affecting the Velocity of Enzymatic Reactions / Reporting Enzyme Activity Data / Enzyme Stability / Summary / 263 References and Further Reading / 2638 Reversible Equilibrium Treatment of Reversible Inhibition / Modes of Reversible Inhibition / Graphic Determination of Inhibitor Type / Dose Response Curves of Enzyme Inhibition / Mutually Exclusive Binding of Two Inhibitors / Structure Activity Relationships and Inhibitor Design / Summary / 303 References and Further Reading /

5 3039 Tight Binding Identifying Tight Binding Inhibition / Distinguishing Inhibitor Type for Tight Binding Inhibitors / DeterminingK for Tight Binding Inhibitors / Use of Tight Binding Inhibitors to Determine Active EnzymeConcentration / Summary / 315 References and Further Reading / 31610 Time-Dependent Progress Curves for Slow Binding Inhibitors / Distinguishing Between Slow Binding Schemes / Distinguishing Between Modes of Inhibitor Interaction withEnzyme / Determining Reversibility / Examples of Slow Binding Enzyme Inhibitors / Summary / 348 References and Further Reading / 34911 Enzyme Reactions with Multiple Reaction Nomenclature / Bi Bi Reaction Mechanisms / Distinguishing Between Random and Compulsory OrderedMechanisms by Inhibition Pattern / Isotope Exchange Studies for Distinguishing ReactionMechanisms / Using the King Altman Method to Determine VelocityEquations / Summary / 364 References and Further Reading / 36612 Cooperativity in Enzyme Historic Examples of Cooperativity and Allostery in Proteins / Models of Allosteric Behavior / Effects of Cooperativity on Velocity Curves / Sigmoidal Kinetics for Nonallosteric ENZYMES / Summary / 383 References and Further Reading / 384 Appendix I.

6 Suppliers of Reagents and Equipment forEnzyme Studies385 Appendix II. Useful Computer Software and Web Sitesfor Enzyme Studies387 Index391xCONTENTSPREFACEIn the four years since the first edition ofEnzymeswas published, I have beendelighted to learn of the wide acceptance of the book throughout the biochemi-cal community, and particularly in the pharmaceutical community. During thistime a number of colleagues have contacted me to express their views on thevalue of the text, and importantly to make suggestions for improvements to thecontent and presentation of some concepts. I have used the first edition as ateaching supplement for a course in which I lecture at the University ofPennsylvania School of Medicine. From my lecture experiences and fromconversations with students, I have developed some new ideas for how to betterexplain some of the concepts in the text and have identified areas that deserveexpanded coverage.

7 Finally, while the first edition has become popular withstudents and industrial scientists, some of my academic colleagues havesuggested a need for a more in-depth treatment of chemical mechanisms this second edition I have refined and expanded the coverage of many ofthe concepts in the text. To help the reader better understand some of theinteractions between ENZYMES and their substrates and inhibitors, a newchapter on protein ligand binding equilibria has been added(Chapter 4). Thechapters on chemical mechanisms in enzyme catalysis(Chapter 6)and onexperimental measures of enzyme activity(Chapter 7)have been expandedsignificantly. The discussions of enzyme inhibitors and multiple substratereactions(Chapters 8 through 11)have been refined, and in some casesalternative treatments have been presented.

8 In all of this, however, I have triedto maintain the introductory nature of the book. There are many excellentadvanced texts on catalysis, enzyme mechanisms, and enzyme kinetics, but thelevel at which these are generally written is often intimidating to the , as stated in the preface to the first edition, this book is intended to serveas a mechanism for those new to the field of enzymology to develop areasonable understanding of the science and experimental methods, allowingthem to competently begin laboratory studies with ENZYMES . I have continuedto rely on extensive citations to more advanced texts and primary literature asa means for the interested reader to go beyond the treatments offered here anddelve more deeply into specific areas of developing this second edition I have had fruitful conversations andadvice from a number of colleagues.

9 In particular, I wish to thank Andy Stern,Ross Stein, Trevor Penning, Bill Pitts, John Blanchard, Dennis Murphy, andthe members of the Chemical Enzymology Department at the DuPont Phar-maceuticals Company. As always, the love and support of my family has beenmost important in making this work A. C Wilmington, DelawarexiiPREFACEACKNOWLEDGMENTSIt is a great pleasure for me to thank the many friends and coworkers whohave helped me in the preparation of this work. Many of the original lecturenotes from which this text has developed were generated while I was teachinga course on biochemistry for first-year medical students at the University ofChicago, along with the late Howard S. Tager. Howard contributed greatly tomy development as a teacher and writer. His untimely death was a great lossto many of us in the biomedical community; I dearly miss his guidance described in the Preface, the notes on which this text is based weresignificantly expanded and reorganized to develop a course of enzymology foremployees and students at the DuPont Merck Pharmaceutical Company.

10 I amgrateful for the many discussions with students during this course, whichhelped to refine the final presentation. I especially thank Diana Blessington forthe original suggestion of a course of this nature. That a graduate-level courseof this type could be presented within the structure of a for-profit pharmaceuti-cal company speaks volumes for the insight and progressiveness of themanagement of DuPont Merck. I particularly thank James M. Trzaskos,Robert C. Newton, Ronald L. Magolda, and Pieter B. Timmermans for notonly tolerating, but embracing this colleagues and coworkers contributed suggestions and artwork forthis text. I thank June Davis, Petra Marchand, Diane Lombardo, RobertLombardo, John Giannaras, Jean Williams, Randi Dowling, Drew Van Dyk,Rob Bruckner, Bill Pitts, Carl Decicco, Pieter Stouten, Jim Meek, Bill De-Grado, Steve Betz, Hank George, Jim Wells, and Charles Craik for , and most importantly, I wish to thank my wife, Nancy, and ourchildren, Lindsey and Amanda, for their constant love, support, and encour-agement, without which this work could not have been THE FIRST EDITIONThe latter half of this century has seen an unprecedented expansion in ourknowledge and use of ENZYMES in a broad range of basic research and industrialapplications.