ENZYMES
Enzymes: A Practical Introduction to Structure, Mechanism, and Data Analysis.Robert A. CopelandCopyright 2000 by Wiley-VCH, Inc.ISBNs: 0-471-35929-7 (Hardback); 0-471-22063-9 (Electronic)ENZYMESSECOND EDITION
ENZYMESA Practical Introductionto Structure, Mechanism,and Data AnalysisSECOND EDITIONRobert A. CopelandA JOHN WILEY & SONS, INC., PUBLICATIONNew York / Chichester / Weinheim / Brisbane / Singapore / Toronto
Designations 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 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 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 1976United States Copyright Act, without the prior written permission of the Publisher. Requeststo the Publisher for permission should be addressed to the Permissions Department, JohnWiley & 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 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 sought.ISBN 0-471-22063-9This 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 Worthenfor teaching me all the important lessons:arigato sensei.And to Theodore (Doc) Jannerfor stoking the fire.
CONTENTSPrefacexiAcknowledgmentsxiiiPreface to the First Editionxv1 A Brief History of Enzymology11.1 Enzymes in Antiquity / 21.2 Early Enzymology / 31.3 The Development of Mechanistic Enzymology / 41.4 Studies of Enzyme Structure / 51.5 Enzymology Today / 71.6 Summary / 8References and Further Reading / 102 Chemical Bonds and Reactions in Biochemistry112.1 Atomic and Molecular Orbitals / 112.2 Thermodynamics of Chemical Reactions / 232.3 Acid—Base Chemistry / 292.4 Noncovalent Interactions in Reversible Binding / 322.5 Rates of Chemical Reactions / 352.6 Summary / 41References and Further Reading / 413 Structural Components of Enzymes3.13.23.33.43.542The Amino Acids / 42The Peptide Bond / 53Amino Acid Sequence or Primary Structure / 55Secondary Structure / 57Tertiary Structure / 62vii
viiiCONTENTS3.6 Subunits and Quaternary Structure / 653.7 Cofactors in Enzymes / 683.8 Summary / 71References and Further Reading / 744 Protein‒Ligand Binding Equilibria76The Equilibrium Dissociation Constant, K / 76 The Kinetic Approach to Equilibrium / 78Binding Measurements at Equilibrium / 80Graphic Analysis of Equilibrium Ligand Binding Data / 88Equilibrium Binding with Ligand Depletion (Tight BindingInteractions) / 944.6 Competition Among Ligands for a Common Binding Site / 954.7 Experimental Methods for Measuring Ligand Binding / 964.8 Summary / 107References and Further Reading / 1084.14.24.34.44.55 Kinetics of Single-Substrate Enzyme Reactions1095.1 The Time Course of Enzymatic Reactions / 1095.2 Effects of Substrate Concentration on Velocity / 1115.3 The Rapid Equilibrium Model of Enzyme Kinetics / 1135.4 The Steady State Model of Enzyme Kinetics / 1155.5 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 / 1335.8 Measurements at Low Substrate Concentrations / 1365.9 Deviations from Hyperbolic Kinetics / 1375.10 Transient State Kinetic Measurements / 1415.11 Summary / 145References and Further Reading / 1456 Chemical Mechanisms in Enzyme Catalysis6.16.26.36.46.5146Substrate—Active Site Complementarity / 147Rate Enhancement Through Transition State Stabilization / 151Chemical Mechanisms for Transition State Stabilization / 154The Serine Proteases: An Illustrative Example / 178Enzymatic Reaction Nomenclature / 184
ixCONTENTS6.6 Summary / 186References and Further Reading / 1867 Experimental Measures of Enzyme Activity1887.1 Initial Velocity Measurements / 1887.2 Detection Methods / 2047.3 Separation Methods in Enzyme Assays / 2237.4 Factors Affecting the Velocity of Enzymatic Reactions / 2387.5 Reporting Enzyme Activity Data / 2577.6 Enzyme Stability / 2587.7 Summary / 263References and Further Reading / 2638 Reversible Inhibitors2668.1 Equilibrium Treatment of Reversible Inhibition / 2688.2 Modes of Reversible Inhibition / 2708.3 Graphic Determination of Inhibitor Type / 2738.4 Dose—Response Curves of Enzyme Inhibition / 2828.5 Mutually Exclusive Binding of Two Inhibitors / 2878.6 Structure—Activity Relationships and Inhibitor Design / 2918.6 Summary / 303References and Further Reading / 3039 Tight Binding Inhibitors3059.19.29.39.4Identifying Tight Binding Inhibition / 305Distinguishing Inhibitor Type for Tight Binding Inhibitors / 307Determining K for Tight Binding Inhibitors / 310Use of Tight Binding Inhibitors to Determine Active EnzymeConcentration / 3139.5 Summary / 315References and Further Reading / 31610 Time-Dependent Inhibition10.1 Progress Curves for Slow Binding Inhibitors / 32110.2 Distinguishing Between Slow Binding Schemes / 32510.3 Distinguishing Between Modes of Inhibitor Interaction withEnzyme / 330318
xCONTENTS10.4 Determining Reversibility / 33210.5 Examples of Slow Binding Enzyme Inhibitors / 33410.6 Summary / 348References and Further Reading / 34911 Enzyme Reactions with Multiple Substrates35011.1 Reaction Nomenclature / 35011.2 Bi Bi Reaction Mechanisms / 35211.3 Distinguishing Between Random and Compulsory OrderedMechanisms by Inhibition Pattern / 35711.4 Isotope Exchange Studies for Distinguishing ReactionMechanisms / 36011.5 Using the King—Altman Method to Determine VelocityEquations / 36211.6 Summary / 364References and Further Reading / 36612 Cooperativity in Enzyme Catalysis36712.1 Historic Examples of Cooperativity and Allostery in Proteins / 36812.2 Models of Allosteric Behavior / 37312.3 Effects of Cooperativity on Velocity Curves / 37912.4 Sigmoidal Kinetics for Nonallosteric Enzymes / 38212.5 Summary / 383References and Further Reading / 384Appendix I. Suppliers of Reagents and Equipment forEnzyme StudiesAppendix II.IndexUseful Computer Software and Web Sitesfor Enzyme Studies385387391
PREFACEIn the four years since the first edition of Enzymes was published, I have beendelighted to learn of the wide acceptance of the book throughout the biochemical 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. 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 inenzymology.In 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. 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 beginner.Hence, 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 enzymology.xi
xiiPREFACEIn developing this second edition I have had fruitful conversations andadvice from a number of colleagues. 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 Pharmaceuticals Company. As always, the love and support of my family has beenmost important in making this work possible.R A. C Wilmington, Delaware
ACKNOWLEDGMENTSIt 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 andfriendship.As 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. 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 pharmaceutical 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 endeavor.Many 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 DeGrado, Steve Betz, Hank George, Jim Wells, and Charles Craik for theircontributions.Finally, and most importantly, I wish to thank my wife, Nancy, and ourchildren, Lindsey and Amanda, for their constant love, support, and encouragement, without which this work could not have been completed.xiii
PREFACETO 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. Enzymes are the catalytic cornerstones of metabolism, and as suchare the focus of intense research within the biomedical community. Indeedenzymes remain the most common targets for therapeutic intervention withinthe pharmaceutical industry. Since ancient times enzymes also have playedcentral roles in many manufacturing processes, such as in the production ofwine, cheese, and breads. During the 1970s and 1980s much of the focus of thebiochemical community shifted to the cloning and expression of proteinsthrough the methods of molecular biology. Recently, some attention has shiftedback to physicochemical characterization of these proteins, and their interactions with other macromolecules and small molecular weight ligands (e.g.,substrates, activators, and inhibitors). Hence, there has been a resurgence ofinterest in the study of enzyme structures, kinetics, and mechanisms of catalysis.The availability of up-to-date, introductory-level textbooks, however, hasnot kept up with the growing demand. I first became aware of this void whileteaching introductory courses at the medical and graduate student level at theUniversity of Chicago. I found that there were a number of excellent advancedtexts that covered different aspects of enzymology with heavy emphasis on thetheoretical basis for much of the science. The more introductory texts that Ifound were often quite dated and did not offer the blend of theoretical andpractical information that I felt was most appropriate for a broad audience ofstudents. I thus developed my own set of lecture notes for these courses,drawing material from a wide range of textbooks and primary literature.In 1993, I left Chicago to focus my research on the utilization of basicenzymology and protein science for the development of therapeutic agents tocombat human diseases. To pursue this goal I joined the scientific staff of theDuPont Merck Pharmaceutical Company. During my first year with thiscompany, a group of associate scientists expressed to me their frustration atbeing unable to find a textbook on enzymology that met their needs forguidance in laboratory protocols and data analysis at an appropriate level andxv
xviPREFACE TO THE FIRST EDITIONat the same time provide them with some relevant background on the scientificbasis of their experiments. These dedicated individuals asked if I would prepareand present a course on enzymology at this introductory level.Using my lecture notes from Chicago as a foundation, I prepared anextensive set of notes and intended to present a year-long course to a smallgroup of associate scientists in an informal, over-brown-bag-lunch fashion.After the lectures had been announced, however, I was shocked and delightedto find that more than 200 people were registered for this course! The makeupof the student body ranged from individuals with associate degrees in medicaltechnology to chemists and molecular biologists who had doctorates. Thisconvinced me that there was indeed a growing interest and need for a newintroductory enzymology text that would attempt to balance the theoreticaland practical aspects of enzymology in such a way as to fill the needs ofgraduate and medical students, as well as research scientists and technicianswho are actively involved in enzyme studies.The text that follows is based on the lecture notes for the enzymology coursejust described. It attempts to fill the practical needs I have articulated, whilealso giving a reasonable introduction to the theoretical basis for the laboratorymethods and data analyses that are covered. I hope that this text will be of useto a broad range of scientists interested in enzymes. The material coveredshould be of direct use to those actively involved in enzyme research inacademic, industrial, and government laboratories. It also should be useful asa primary text for senior undergraduate or first-year graduate course, inintroductory enzymology. However, in teaching a subject as broad anddynamic as enzymology, I have never found a single text that would cover allof my students’ needs; I doubt that the present text will be an exception. Thus,while I believe this text can serve as a useful foundation, I encourage facultyand students to supplement the material with additional readings from theliterature cited at the end of each chapter, and the primary literature that iscontinuously 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 some of the material in a rather cursoryfashion; it is simply not possible, in a text of this format, to be comprehensivein such an expansive field as enzymology. I hope that the literature citationswill at least pave the way for readers who wish to delve more deeply intoparticular areas. Overall, the intent of this book is to get people started in thelaboratory and in their thinking about enzymes. It provides sufficient experimental and data handling methodologies to permit one to begin to design andperform experiments with enzymes, while at the same time providing atheoretical framework in which to understand the basis of the experimentalwork. Beyond this, if the book functions as a stepping-stone for the reader tomove on to more comprehensive and in-depth treatments of enzymology, it willhave served its purpose.R A. C Wilmington, Delaware
‘‘All the mathematics in the world is no substitute for areasonable amount of common sense.’’W. W. Cleland
INDEXAbsorption spectroscopy, 189, 205errors in, 210Acid-base catalysis, 155, 164pH effects on, 166Acid-base chemistry, 29of amino acid side chains, 45, 48Activation energy, 27, 152Active site complementarity, 147Active site preorganization, 155, 176Active site structure, 147Active site titration, 197, 313Active site triad of serine proteases, 63,178Activity staining, in gel electrophoresis,234Acyl-enzyme intermediates, 158, 162, 179Affinity labeling, 346AIDS, 9, 67Allosteric constant, 377Allosteric effectors, 368Allostery, 367Alpha carbon, of amino acids, 42Alpha helix, 58Alpha-aminoboronate peptides, asinhibitors of serine proteases, 335Alpha-amylase, 3Amino acid sequence, 7, 55Amino acids, 42physicochemical properties of, 43side chain structures of, 44Amino terminus, 55Ancient references to enzymes, 2Anion and polyanion binding inproteins, 50Antibodies, 178, 233Apoenzyme, 69Approximation of reactants, 155Aromaticity, 20Arrhenius equation, 28, 249Arrhenius plots, 250Aryl azides, 346Aspartate carbamoyltransferase, 373Aspirin, as an inhibitor of prostaglandinsynthase, 335Atomic orbitals, 11ATPases, 52Aufbau principle, 14Autoradiography, 219, 227Beer’s law, 206Benzophenones, 346Beta pleated sheet, 60Beta turns, 61Bi bi reactions, 352Bohr model of atoms, 12Bond lengths, of peptide components, 53Bonding and antibonding orbitals, 15Briggs and Haldane steady stateapproach, 115Bromoacetamido-affinity labels, asinhibitors of prostaglandinsynthase, 336Brönsted-Lowry acids and bases, 29, 48Brönsted equations, 167391
392INDEXBrönsted plots, 160, 169Buffering capacity, 31Buffers used in enzyme assays, 242Burst phase kinetics, 159, 196Carbonic anhydrase, 49Carboxyl terminus, 56Carboxypeptidase, 179Carrier proteins, 260Catalytic antibodies, 178Cation and metal binding in proteins, 49Chemical bonds, 11Chemical mechanisms of catalysis, 146Chemical modification, 341Cheng and Prusoff equations, 285Chromatography, 102, 224Chymotrypsin, 63, 179Cis-prolyl bonds in enzymes, 55Cis-trans peptide bonds, 54Coenzymes, see CofactorsCofactors, 68effects on velocity, 240Comformational distortion, 170Competitive binding, 95Competitive inhibitors, 273, 358Compulsory ordered reactions, 354Computer software for enzyme studies,387Concerted transition model ofcooperativity, 373Conjugate bases, 29Consumer products, use of enzymes in, 1Continuous assays, 199Controls, importance of in experimentalmeasurements, 202Coomassie brilliant blue, 231Cooperativity, 86, 139, 367effects on velocity curves, 139, 379in inhibitor binding, 381models of, 373Cooperativity index, 380Coulombic attractive forces, 32Coupled reactions, 25, 190Covalent catalysis, 158Covalent modification, 50, 341CPM (Counts per minute), 219Curie (Ci), 219Cytochrome c, 189Cytochrome oxidase, 25,
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 .
Restriction enzymes and DNA ligases represent the cutting and joining functions in DNA manipulation. All other enzymes involved in genetic engineering fall under the broad category of enzymes known as DNA modifying enzymes. These enzymes are involved in the degradation, synthesis and alteration of the nucle
metabolism? We can group enzymes together according to the types of reactions they catalyse. –Hydrolase enzymes control hydrolysis reactions. –Oxidoreductase enzymes control redox reactions. –Transferase enzymes move whole chemical groups (eg. methyl, amino, carboxyl). –Isomerase enzymes change the shape of a molecule (creating an .
Enzymes in Organic Chemistry! March 25th, 2013! Enzymes Enzymes are biological catalysts. They increase the rate at which equilibrium is reached, but they do not affect the equilibrium. Enzymes differ from ordinary chemical catalyst
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Organisms use protein catalysts called enzymes to accelerate many metabolic reactions to useful rates. Enzymes are mainly made of proteins, although many enzymes also incorporate non-protein cofactors, such as vitamins or metals. Enzymes lower the Ea of a reaction because substrates bind to a specific
20 Production of industrial enzymes and some applications in fermented foods 622 A. Godfrey 20.1 Introduction 622 20.2 Brief history of industrial enzymes 622 20.3 Modem approach to enzyme production 625 20.4 Enzymes from animal materials 626 20.5 Enzymes from plant materials 629 20.6 Principal steps in extraction of animal and plant tissues 630
Enzymes are compounds that facilitate chemical reactions. These compounds are mostly proteins found in living organisms and are very important for cells to live and function. For example, the food that you eat is broken down into smaller pieces by different enzymes known as the digestive enzymes. Some
Chapter 6: Energy Flow in Cells Enzymes Biological Catalysts Unique Properties of Enzymes (compared to other catalysts): 1) Enzymes are specific (High Specificity) 2) Enzyme activity is regulated: A) Regulate synthesis of enzyme B) Regulate active state of enzyme Enzymes synthesized
