Introduction to Enzyme and Coenzyme Chemistry SECOND EDITION T.D.H. Bugg Bugg/Introduction to Enzyme and Coenzyme Chemistry Final Proof 22.7.2004 3:52pm page i Introduction to Enzyme and Coenzyme Chemistry Bugg/Introduction to Enzyme and Coenzyme Chemistry Final Proof 22.7.2004 3:52pm page ii Bugg/Introduction to Enzyme and Coenzyme Chemistry Final Proof 22.7.2004 3:52pm page iii Introduction to Enzyme and Coenzyme Chemistry Second Edition TIM BUGG Professor of Biological Chemistry, Department of Chemistry, University of Warwick, UK Bugg/Introduction to Enzyme and Coenzyme Chemistry Final Proof 22.7.2004 3:52pm page iv ß 1997, 2004 by Blackwell Publishing Ltd Editorial oYces: Blackwell Publishing Ltd, 9600 Garsington Road, Oxford OX4 2DQ, UK Tel: þ44 (0)1865 776868 Blackwell Publishing Inc., 350 Main Street, Malden, MA 02148-5020, USA Tel: þ1 781 388 8250 Blackwell Publishing Asia Pty Ltd, 550 Swanston Street, Carlton, Victoria 3053, Australia Tel: þ61 (0)3 8359 1011 The right of the Author to be identiWed as the Author of this Work has been asserted in accordance with the Copyright, Designs and Patents Act 1988. 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, mechanical, photocopying, recording or otherwise, except as permitted by the UK Copyright, Designs and Patents Act 1988, without the prior permission of the publisher. First published 1997 by Blackwell Science Second edition published 2004 by Blackwell Publishing Library of Congress Cataloging-in-Publication Data Bugg, Tim. Introduction to enzyme and coenzyme chemistry / Tim Bugg.–2nd ed. p. cm. Includes bibliographical references and index. ISBN 1-4051-1452-5 (pbk. : alk. paper) 1. Enzymes. 2. Coenzymes. I. Title. QP601.B955 2004 5720.7–dc22 2003025117 ISBN 1-4051-1452-5 A catalogue record for this title is available from the British Library Set in 10/13 pt Times by Kolam Information Services Pvt. 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For further information on Blackwell Publishing, visit our website: www.blackwellpublishing.com Bugg/Introduction to Enzyme and Coenzyme Chemistry Final Proof 22.7.2004 3:53pm page v Contents Preface ix Representation of Protein Three-Dimensional Structures x 1 From Jack Beans to Designer Genes 1 1.1 Introduction 1 1.2 The discovery of enzymes 1 1.3 The discovery of coenzymes 3 1.4 The commercial importance of enzymes in biosynthesis and biotechnology 3 1.5 The importance of enzymes as targets for drug discovery 6 2 All Enzymes are Proteins 8 2.1 Introduction 8 2.2 The structures of the l-a-amino acids 8 2.3 The primary structure of polypeptides 10 2.4 Alignment of amino acid sequences 12 2.5 Secondary structures found in proteins 13 2.6 The folded tertiary structure of proteins 16 2.7 Enzyme structure and function 19 2.8 Metallo-enzymes 22 2.9 Membrane-associated enzymes 23 2.10 Glycoproteins 24 3 Enzymes are Wonderful Catalysts 29 3.1 Introduction 29 3.2 A thermodynamic model of catalysis 31 3.3 Proximity eVects 33 3.4 The importance of transition state stabilisation 36 3.5 Acid/base catalysis in enzymatic reactions 37 3.6 Nucleophilic catalysis in enzymatic reactions 41 3.7 The use of strain energy in enzyme catalysis 45 3.8 Catalytic perfection 47 3.9 The involvement of protein dynamics in enzyme catalysis 47 4 Methods for Studying Enzymatic Reactions 51 4.1 Introduction 51 4.2 Enzyme puriWcation 51 4.3 Enzyme kinetics 54 4.4 The stereochemical course of an enzymatic reaction 60 v Bugg/Introduction to Enzyme and Coenzyme Chemistry Final Proof 22.7.2004 3:53pm page vi vi Contents 4.5 The existence of intermediates in enzymatic reactions 68 4.6 Analysis of transition states in enzymatic reactions 71 4.7 Determination of active site catalytic groups 75 5 Enzymatic Hydrolysis and Group Transfer Reactions 81 5.1 Introduction 81 5.2 The peptidases 82 CASE STUDY: HIV-1 protease 96 5.3 Esterases and lipases 98 5.4 Acyl transfer reactions in biosynthesis: use of coenzyme A (CoA) 99 5.5 Enzymatic phosphoryl transfer reactions 102 5.6 Adenosine 50-triphosphate 106 5.7 Enzymatic glycosyl transfer reactions 109 5.8 Methyl group transfer: use of S-adenosyl methionine and tetrahydrofolate coenzymes for one-carbon transfers 112 6 Enzymatic Redox Chemistry 121 6.1 Introduction 121 6.2 Nicotinamide adenine dinucleotide-dependent dehydrogenases 123 6.3 Flavin-dependent dehydrogenases and oxidases 129 6.4 Flavin-dependent mono-oxygenases 134 6.5 CASE STUDY: Glutathione and trypanothione reductases 137 6.6 DeazaXavins and pterins 141 6.7 Iron–sulphur clusters 142 6.8 Metal-dependent mono-oxygenases 143 6.9 a-Ketoglutarate-dependent dioxygenases 147 6.10 Non-haem iron-dependent dioxygenases 148 7 Enzymatic Carbon–Carbon Bond Formation 156 7.1 Introduction 156 Carbon–carbon bond formation via carbanion equivalents 158 7.2 Aldolases 158 CASE STUDY: Fructose-1,6-bisphosphate aldolase 158 7.3 Claisen enzymes 164 7.4 Assembly of fatty acids and polyketides 166 7.5 Carboxylases: use of biotin 170 7.6 Ribulose bisphosphate carboxylase/oxygenase (Rubisco) 171 7.7 Vitamin K-dependent carboxylase 173 7.8 Thiamine pyrophosphate-dependent enzymes 176 Carbon–carbon bond formation via carbocation intermediates 179 7.9 Terpene cyclases 179 Carbon–carbon bond formation via radical intermediates 183 7.10 Phenolic radical couplings 184 Bugg/Introduction to Enzyme and Coenzyme Chemistry Final Proof 22.7.2004 3:53pm page vii Contents vii 8 Enzymatic Addition/Elimination Reactions 193 8.1 Introduction 193 8.2 Hydratases and dehydratases 194 8.3 Ammonia lyases 199 8.4 Elimination of phosphate and pyrophosphate 202 8.5 CASE STUDY: 5-Enolpyruvyl-shikimate-3-phosphate (EPSP) synthase 204 9 Enzymatic Transformations of Amino Acids 210 9.1 Introduction 210 9.2 Pyridoxal 50-phosphate-dependent reactions at the a-position of amino acids 211 9.3 CASE STUDY: Aspartate aminotransferase 215 9.4 Reactions at b- and g-positions of amino acids 218 9.5 Serine hydroxymethyltransferase 220 9.6 N-Pyruvoyl-dependent amino acid decarboxylases 222 9.7 Imines and enamines in alkaloid biosynthesis 222 10 Isomerases 227 10.1 Introduction 227 10.2 Cofactor-independent racemases and epimerases 227 10.3 Keto–enol tautomerases 230 10.4 Allylic isomerases 231 10.5 CASE STUDY: Chorismate mutase 233 11 Radicals in Enzyme Catalysis 240 11.1 Introduction 240 11.2 Vitamin B12-dependent rearrangements 240 11.3 The involvement of protein radicals in enzyme catalysis 244 11.4 S-adenosyl methionine-dependent radical reactions 246 11.5 Biotin synthase and sulphur insertion reactions 249 11.6 Oxidised amino acid cofactors and quinoproteins 250 12 Non-Enzymatic Biological Catalysis 255 12.1 Introduction 255 12.2 Catalytic RNA 255 12.3 Catalytic antibodies 259 12.4 Synthetic enzyme models 265 Appendices 272 1. Cahn–Ingold–Prelog rule for stereochemical nomenclature 272 2. Amino acid abbreviations 274 3. A simple demonstration of enzyme catalysis 275 4. Answers to problems 277 Index 285 Bugg/Introduction to Enzyme and Coenzyme Chemistry Final Proof 22.7.2004 3:53pm page viii Bugg/Introduction to Enzyme and Coenzyme Chemistry Final Proof 22.7.2004 3:53pm page ix Preface Since the publication of the Wrst edition in 1998, the Weld of chemical biology has, I would say, become more a part of the core research and teaching of Chemistry Departments around the world. As the genes and proteins involved in important biological problems are elucidated, so they become accessible for study at the molecular level by chemists. Enzymology is a core discipline within chemical biology, since enzymes are the biological catalysts that make things happen within cells: they translate gene sequence into biological function. The main feature of the Wrst edition that I wanted to improve was the Wgures. My original intention was to have a case study in each chapter, where I would illustrate the chemical mechanism, with curly arrows, and on the facing page would show the three-dimensional structure of the enzyme active site, and/ or tertiary structure of the enzyme. One of the fascinations of enzymology is the interplay between structure and function, and I wanted to try to convey this to students. In the Wrst edition I was only able to include a set of colour plates in the middle of the book. In the second edition, I have prepared a series of two- colour pictures using Rasmol, to accompany the text. I hope that these pictures convey the desired ideas. I have also updated the Wrst edition with recent observations and references from the literature, and have added a few new topics. I have written a new chapter entitled Radicals in Enzyme Catalysis (Chapter 11), which includes the discovery of protein radicals and the discovery of SAM-dependent radical reactions. I have also mentioned the recent proposals for protein dynamics (Chapter 3) and proton tunnelling (Chapter 4) in enzyme catalysis, in a way that I hope will be accessible to undergraduate students. I would like to thank my colleagues, researchers, and students at Warwick for their encouragement, and hope that the book is useful to chemical biologists everywhere. Tim Bugg University of Warwick ix Bugg/Introduction to Enzyme and Coenzyme Chemistry Final Proof 22.7.2004 3:53pm page x Representation of Protein Three-Dimensional Structures In the second edition I have used the program Rasmol to draw representa- tions of protein three-dimensional structures.