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Enzymes: A Practical Introduction to Structure, Mechanism, and Data Analysis Robert A Copeland Copyright 2000 by Wiley-VCH, Inc ISBNs: 0-471-35929-7 (Hardback); 0-471-22063-9 (Electronic) ENZYMES SECOND EDITION ENZYMES A Practical Introduction to Structure, Mechanism, and Data Analysis SECOND EDITION Robert A Copeland A JOHN WILEY & SONS, INC., PUBLICATION New York / Chichester / Weinheim / Brisbane / Singapore / Toronto Designations used by companies to distinguish their products are often claimed as trademarks In all instances where John Wiley & Sons, Inc., is aware of a claim, the product names appear in initial capital or Readers, however, should contact the appropriate companies for more complete information regarding trademarks and registration Copyright " 2000 by Wiley-VCH, Inc All rights reserved No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form 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 United States Copyright Act, without the prior written permission of the Publisher Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 605 Third Avenue, New York, NY 10158-0012, (212) 850-6011, fax (212) 850-6008, E-Mail: PERMREQ!WILEY.COM This publication is designed to provide accurate and authoritative information in regard to the subject matter covered It is sold with the understanding that the publisher is not engaged in rendering professional services If professional advice or other expert assistance is required, the services of a competent professional person should be sought ISBN 0-471-22063-9 This title is also available in print as ISBN 0-471-35929-7 For more information about Wiley products, visit our web site at www.Wiley.com To Clyde Worthen for teaching me all the important lessons: arigato sensei And to Theodore (Doc) Janner for stoking the fire CONTENTS Preface xi Acknowledgments xiii Preface to the First Edition xv A Brief History of Enzymology 1.1 Enzymes in Antiquity / 1.2 Early Enzymology / 1.3 The Development of Mechanistic Enzymology / 1.4 Studies of Enzyme Structure / 1.5 Enzymology Today / 1.6 Summary / References and Further Reading / 10 Chemical Bonds and Reactions in Biochemistry 11 2.1 Atomic and Molecular Orbitals / 11 2.2 Thermodynamics of Chemical Reactions / 23 2.3 Acid—Base Chemistry / 29 2.4 Noncovalent Interactions in Reversible Binding / 32 2.5 Rates of Chemical Reactions / 35 2.6 Summary / 41 References and Further Reading / 41 Structural Components of Enzymes 3.1 3.2 3.3 3.4 3.5 42 The Amino Acids / 42 The Peptide Bond / 53 Amino Acid Sequence or Primary Structure / 55 Secondary Structure / 57 Tertiary Structure / 62 vii viii CONTENTS 3.6 Subunits and Quaternary Structure / 65 3.7 Cofactors in Enzymes / 68 3.8 Summary / 71 References and Further Reading / 74 Protein‒Ligand Binding Equilibria 76 The Equilibrium Dissociation Constant, K / 76 ! The Kinetic Approach to Equilibrium / 78 Binding Measurements at Equilibrium / 80 Graphic Analysis of Equilibrium Ligand Binding Data / 88 Equilibrium Binding with Ligand Depletion (Tight Binding Interactions) / 94 4.6 Competition Among Ligands for a Common Binding Site / 95 4.7 Experimental Methods for Measuring Ligand Binding / 96 4.8 Summary / 107 References and Further Reading / 108 4.1 4.2 4.3 4.4 4.5 Kinetics of Single-Substrate Enzyme Reactions 109 5.1 The Time Course of Enzymatic Reactions / 109 5.2 Effects of Substrate Concentration on Velocity / 111 5.3 The Rapid Equilibrium Model of Enzyme Kinetics / 113 5.4 The Steady State Model of Enzyme Kinetics / 115 5.5 The Significance of k and K / 120 "#$ % 5.6 Experimental Measurement of k and K / 124 "#$ % 5.7 Other Linear Transformations of Enzyme Kinetic Data / 133 5.8 Measurements at Low Substrate Concentrations / 136 5.9 Deviations from Hyperbolic Kinetics / 137 5.10 Transient State Kinetic Measurements / 141 5.11 Summary / 145 References and Further Reading / 145 Chemical Mechanisms in Enzyme Catalysis 6.1 6.2 6.3 6.4 6.5 146 Substrate—Active Site Complementarity / 147 Rate Enhancement Through Transition State Stabilization / 151 Chemical Mechanisms for Transition State Stabilization / 154 The Serine Proteases: An Illustrative Example / 178 Enzymatic Reaction Nomenclature / 184 ix CONTENTS 6.6 Summary / 186 References and Further Reading / 186 Experimental Measures of Enzyme Activity 188 7.1 Initial Velocity Measurements / 188 7.2 Detection Methods / 204 7.3 Separation Methods in Enzyme Assays / 223 7.4 Factors Affecting the Velocity of Enzymatic Reactions / 238 7.5 Reporting Enzyme Activity Data / 257 7.6 Enzyme Stability / 258 7.7 Summary / 263 References and Further Reading / 263 Reversible Inhibitors 266 8.1 Equilibrium Treatment of Reversible Inhibition / 268 8.2 Modes of Reversible Inhibition / 270 8.3 Graphic Determination of Inhibitor Type / 273 8.4 Dose—Response Curves of Enzyme Inhibition / 282 8.5 Mutually Exclusive Binding of Two Inhibitors / 287 8.6 Structure—Activity Relationships and Inhibitor Design / 291 8.6 Summary / 303 References and Further Reading / 303 Tight Binding Inhibitors 305 9.1 9.2 9.3 9.4 Identifying Tight Binding Inhibition / 305 Distinguishing Inhibitor Type for Tight Binding Inhibitors / 307 Determining K for Tight Binding Inhibitors / 310 & Use of Tight Binding Inhibitors to Determine Active Enzyme Concentration / 313 9.5 Summary / 315 References and Further Reading / 316 10 Time-Dependent Inhibition 10.1 Progress Curves for Slow Binding Inhibitors / 321 10.2 Distinguishing Between Slow Binding Schemes / 325 10.3 Distinguishing Between Modes of Inhibitor Interaction with Enzyme / 330 318 x CONTENTS 10.4 Determining Reversibility / 332 10.5 Examples of Slow Binding Enzyme Inhibitors / 334 10.6 Summary / 348 References and Further Reading / 349 11 Enzyme Reactions with Multiple Substrates 350 11.1 Reaction Nomenclature / 350 11.2 Bi Bi Reaction Mechanisms / 352 11.3 Distinguishing Between Random and Compulsory Ordered Mechanisms by Inhibition Pattern / 357 11.4 Isotope Exchange Studies for Distinguishing Reaction Mechanisms / 360 11.5 Using the King—Altman Method to Determine Velocity Equations / 362 11.6 Summary / 364 References and Further Reading / 366 12 Cooperativity in Enzyme Catalysis 367 12.1 Historic Examples of Cooperativity and Allostery in Proteins / 368 12.2 Models of Allosteric Behavior / 373 12.3 Effects of Cooperativity on Velocity Curves / 379 12.4 Sigmoidal Kinetics for Nonallosteric Enzymes / 382 12.5 Summary / 383 References and Further Reading / 384 Appendix I Suppliers of Reagents and Equipment for Enzyme Studies Appendix II Index Useful Computer Software and Web Sites for Enzyme Studies 385 387 391 PREFACE In the four years since the first edition of Enzymes was published, I have been delighted to learn of the wide acceptance of the book throughout the biochemical community, and particularly in the pharmaceutical community During this time a number of colleagues have contacted me to express their views on the value of the text, and importantly to make suggestions for improvements to the content and presentation of some concepts I have used the first edition as a teaching supplement for a course in which I lecture at the University of Pennsylvania School of Medicine From my lecture experiences and from conversations with students, I have developed some new ideas for how to better explain some of the concepts in the text and have identified areas that deserve expanded coverage Finally, while the first edition has become popular with students and industrial scientists, some of my academic colleagues have suggested a need for a more in-depth treatment of chemical mechanisms in enzymology In this second edition I have refined and expanded the coverage of many of the concepts in the text To help the reader better understand some of the interactions between enzymes and their substrates and inhibitors, a new chapter on protein—ligand binding equilibria has been added (Chapter 4) The chapters on chemical mechanisms in enzyme catalysis (Chapter 6) and on experimental measures of enzyme activity (Chapter 7) have been expanded significantly The discussions of enzyme inhibitors and multiple substrate reactions (Chapters through 11) have been refined, and in some cases alternative treatments have been presented In all of this, however, I have tried to maintain the introductory nature of the book There are many excellent advanced texts on catalysis, enzyme mechanisms, and enzyme kinetics, but the level at which these are generally written is often intimidating to the beginner Hence, as stated in the preface to the first edition, this book is intended to serve as a mechanism for those new to the field of enzymology to develop a reasonable understanding of the science and experimental methods, allowing them to competently begin laboratory studies with enzymes I have continued to rely on extensive citations to more advanced texts and primary literature as a means for the interested reader to go beyond the treatments offered here and delve more deeply into specific areas of enzymology xi xii PREFACE In developing this second edition I have had fruitful conversations and advice from a number of colleagues In particular, I wish to thank Andy Stern, Ross Stein, Trevor Penning, Bill Pitts, John Blanchard, Dennis Murphy, and the members of the Chemical Enzymology Department at the DuPont Pharmaceuticals Company As always, the love and support of my family has been most important in making this work possible R A C Wilmington, Delaware ... seen an unprecedented expansion in our knowledge and use of enzymes in a broad range of basic research and industrial applications Enzymes are the catalytic cornerstones of metabolism, and as... within the biomedical community Indeed enzymes remain the most common targets for therapeutic intervention within the pharmaceutical industry Since ancient times enzymes also have played central roles... primary literature that is continuously expanding our view of enzymes and catalysis In attempting to provide a balanced introduction to enzymes in a single, readable volume I have had to present