Introduction to enzyme and coenzyme chemistry third edition

P1: JYS JWST182-fm JWST182-Bugg May 22, 2012 10:0 Printer: Yet to come Trim: 244mm × 168mm Introduction to Enzyme and Coenzyme Chemistry P1: JYS JWST182-fm JWST182-Bugg May 22, 2012 10:0 Printer: Yet to come Trim: 244mm × 168mm Introduction to Enzyme and Coenzyme Chemistry Third Edition T D H BUGG Department of Chemistry, University of Warwick, UK A John Wiley & Sons, Ltd., Publication P1: JYS JWST182-fm JWST182-Bugg May 22, 2012 10:0 Printer: Yet to come Trim: 244mm × 168mm This edition first published 2012 © 2012 John Wiley & Sons, Ltd Registered office John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, United Kingdom For details of our global editorial offices, for customer services and for information about how to apply for permission to reuse the copyright material in this book please see our website at www.wiley.com The right of the author to be identified 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 Wiley also publishes its books in a variety of electronic formats Some content that appears in print may not be available in electronic books Designations used by companies to distinguish their products are often claimed as trademarks All brand names and product names used in this book are trade names, service marks, trademarks or registered trademarks of their respective owners The publisher is not associated with any product or vendor mentioned in this book This publication is designed to provide accurate and authoritative information in regard to the subject matter covered It is sold on 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 should be sought The publisher and the author make no representations or warranties with respect to the accuracy or completeness of the contents of this work and specifically disclaim all warranties, including without limitation any implied warranties of fitness for a particular purpose This work is sold with the understanding that the publisher is not engaged in rendering professional services The advice and strategies contained herein may not be suitable for every situation In view of ongoing research, equipment modifications, changes in governmental regulations, and the constant flow of information relating to the use of experimental reagents, equipment, and devices, the reader is urged to review and evaluate the information provided in the package insert or instructions for each chemical, piece of equipment, reagent, or device for, among other things, any changes in the instructions or indication of usage and for added warnings and precautions The fact that an organization or Website is referred to in this work as a citation and/or a potential source of further information does not mean that the author or the publisher endorses the information the organization or Website may provide or recommendations it may make Further, readers should be aware that Internet Websites listed in this work may have changed or disappeared between when this work was written and when it is read No warranty may be created or extended by any promotional statements for this work Neither the publisher nor the author shall be liable for any damages arising herefrom Library of Congress Cataloging-in-Publication Data Bugg, T D H Introduction to enzyme and coenzyme chemistry / T D H Bugg – 3rd ed p cm Includes bibliographical references and index ISBN 978-1-119-99595-1 (cloth) – ISBN 978-1-119-99594-4 (pbk.) Enzymes QP601.B955 2012 612 0151–dc23 Coenzymes I Title 2012008853 A catalogue record for this book is available from the British Library Cloth ISBN: 9781119995951 Paper ISBN: 9781119995944 Typeset in 10/12pt Times by Aptara Inc., New Delhi, India Instructors can access PowerPoint files of the illustrations presented within this text, for teaching, at: http://booksupport.wiley.com P1: JYS JWST182-fm JWST182-Bugg May 22, 2012 10:0 Printer: Yet to come Trim: 244mm × 168mm Contents Preface ix Representation of Protein Three-Dimensional Structures x From Jack Beans to Designer Genes 1.1 Introduction 1.2 The discovery of enzymes 1.3 The discovery of coenzymes 1.4 The commercial importance of enzymes in biosynthesis and biotechnology 1.5 The importance of enzymes as targets for drug discovery 1 3 All Enzymes Are Proteins 2.1 Introduction 2.2 The structures of the L-␣-amino acids 2.3 The primary structure of polypeptides 2.4 Alignment of amino acid sequences 2.5 Secondary structures found in proteins 2.6 The folded tertiary structure of proteins 2.7 Enzyme structure and function 2.8 Metallo-enzymes 2.9 Membrane-associated enzymes 2.10 Glycoproteins 7 11 12 15 17 20 21 23 Enzymes Are Wonderful Catalysts 3.1 Introduction 3.2 A thermodynamic model of catalysis 3.3 Proximity effects 3.4 The importance of transition state stabilisation 3.5 Acid/base catalysis in enzymatic reactions 3.6 Nucleophilic catalysis in enzymatic reactions 3.7 The use of strain energy in enzyme catalysis 3.8 Desolvation of substrate and active site nucleophiles 3.9 Catalytic perfection 3.10 The involvement of protein dynamics in enzyme catalysis 26 26 28 30 32 36 40 44 45 46 47 Methods for Studying Enzymatic Reactions 4.1 Introduction 4.2 Enzyme purification 4.3 Enzyme kinetics 50 50 50 52 P1: JYS JWST182-fm JWST182-Bugg vi 4.4 4.5 4.6 4.7 May 22, 2012 10:0 Printer: Yet to come Trim: 244mm × 168mm Contents The stereochemical course of an enzymatic reaction The existence of intermediates in enzymatic reactions Analysis of transition states in enzymatic reactions Determination of active site catalytic groups 59 64 68 71 Hydrolytic and Group Transfer Enzymes 5.1 Introduction 5.2 The peptidases CASE STUDY: HIV-1 protease 5.3 Esterases and lipases 5.4 Acyl transfer reactions in biosynthesis (coenzyme A) 5.5 Enzymatic phosphoryl transfer reactions 5.6 Adenosine -triphosphate (ATP) 5.7 Enzymatic glycosyl transfer reactions 5.8 Methyl group transfer: use of S-adenosyl methionine and tetrahydrofolate coenzymes for one-carbon transfers 77 77 79 90 92 93 95 101 102 Enzymatic Redox Chemistry 6.1 Introduction 6.2 Nicotinamide adenine dinucleotide-dependent dehydrogenases 6.3 Flavin-dependent dehydrogenases and oxidases 6.4 Flavin-dependent mono-oxygenases 6.5 CASE STUDY: Glutathione and trypanothione reductases 6.6 Deazaflavins and pterins 6.7 Iron-sulphur clusters 6.8 Metal-dependent mono-oxygenases 6.9 ␣-Ketoglutarate-dependent dioxygenases 6.10 Non-heme iron-dependent dioxygenases 115 115 117 122 128 129 133 135 136 140 141 Enzymatic Carbon–Carbon Bond Formation 7.1 Introduction Carbon–carbon bond formation via carbanion equivalents 7.2 Aldolases CASE STUDY: Fructose 1,6-bisphosphate aldolase 7.3 Claisen enzymes 7.4 Assembly of fatty acids and polyketides 7.5 Carboxylases: Use of biotin 7.6 Ribulose bisphosphate carboxylase/oxygenase (Rubisco) 7.7 Vitamin K-dependent carboxylase 7.8 Thiamine pyrophosphate-dependent enzymes Carbon–carbon bond formation via carbocation intermediates 7.9 Terpene cyclases Carbon–carbon formation through radical intermediates 7.10 Phenolic radical couplings 148 148 149 149 150 153 156 158 161 163 165 168 168 173 173 Enzymatic Addition/Elimination Reactions 8.1 Introduction 8.2 Hydratases and dehydratases 181 181 182 107 P1: JYS JWST182-fm JWST182-Bugg May 22, 2012 10:0 Printer: Yet to come Trim: 244mm × 168mm Contents 8.3 8.4 8.5 Ammonia lyases Elimination of phosphate and pyrophosphate CASE STUDY: 5-Enolpyruvyl shikimate 3-phosphate (EPSP) synthase vii 187 190 191 Enzymatic Transformations of Amino Acids 9.1 Introduction 9.2 Pyridoxal -phosphate-dependent reactions at the ␣-position 9.3 CASE STUDY: Aspartate aminotransferase 9.4 Reactions at the ␤- and ␥ -positions of amino acids 9.5 Serine hydroxymethyltransferase 9.6 N-Pyruvoyl-dependent amino acid decarboxylases 9.7 Imines and enamines in alkaloid biosynthesis 197 197 197 201 204 206 208 208 10 Isomerases 10.1 Introduction 10.2 Cofactor-independent racemases and epimerases 10.3 Keto-enol tautomerases 10.4 Allylic isomerases 10.5 CASE STUDY: Chorismate mutase 213 213 213 216 217 219 11 Radicals in Enzyme Catalysis 11.1 Introduction 11.2 Vitamin B12 -dependent rearrangements 11.3 The involvement of protein radicals in enzyme catalysis 11.4 S-adenosyl-methionine-dependent radical reactions 11.5 Biotin synthase and sulphur insertion reactions 11.6 Radical chemistry in DNA repair enzymes 11.7 Oxidised amino acid cofactors and quinoproteins 225 225 225 229 232 233 234 238 12 Non-Enzymatic Biological Catalysis 12.1 Introduction 12.2 Catalytic RNA 12.3 Catalytic antibodies 12.4 Synthetic enzyme models 242 242 242 246 251 Appendix 1: Cahn-Ingold-Prelog Rule for Stereochemical Nomenclature 258 Appendix 2: Amino Acid Abbreviations 260 Appendix 3: A Simple Demonstration of Enzyme Catalysis 261 Appendix 4: Answers to Problems 263 Index 271 P1: JYS JWST182-Preface JWST182-Bugg May 22, 2012 7:53 Printer: Yet to come Trim: 244mm × 168mm Preface When I was approached about a 3rd edition of the book, I wanted to enhance the interplay of enzyme active site structure with their catalytic mechanisms and also update the book with some more recent literature examples and topics of current research interest in enzymology In the 3rd edition I have redrawn the figures showing protein structures using PyMOL software (see Appendix 3) One advantage of PyMOL is that it is easy to prepare high-resolution images, so hopefully the images in the 3rd edition will be a bit sharper than in the 2nd edition I have used a similar set of examples, but have also added several new examples to supplement the text I have also added some new text: in Chapter I have added some new examples of transition state stabilisation and nucleophilic catalysis to Sections 3.4 and 3.6 and added a new Section 3.8 “Desolvation of substrate and active site nucleophiles”, using the example of S cattleya fluorinase discovered by the group of Professor David O’Hagan I’ve also added some new text and references on the role of protein dynamics in enzyme catalysis, which has been a topic of much discussion and debate in recent years In Chapter I have mentioned the link between hydrogen tunnelling and temperature-independent kinetic isotope effects, also a topic of current interest in enzymology In Chapter I have mentioned the discovery of a covalent intermediate in the lysozyme reaction by the group of Professor Stephen Withers, and expanded the discussion of glycosyltransferases In Chapter 11 I have included a new section 11.6 on “Radical chemistry in DNA repair enzymes”, and included a new figure of the Drosophila melanogaster (6-4) photolyase; I’d like to thank Professor Thomas Carell for helpful discussions and information on this enzyme Finally, in Chapter 12 I have included a figure and discussion about the most ancient catalytic reaction of all: the peptidyl transfer reaction on the ribosome A great deal of new structural data regarding this reaction has emerged in the last 10 years, although the precise catalytic mechanism is still under debate! I’d like to thank colleagues, researchers and students at Warwick and elsewhere for their support and encouragement T D H Bugg University of Warwick January 2012 P1: JYS JWST182-PYMOL JWST182-Bugg May 22, 2012 7:55 Printer: Yet to come Trim: 244mm × 168mm Representation of Protein Three-Dimensional Structures In the 3rd edition I have used the program PyMOL to draw representations of protein threedimensional structures PyMOL was developed by Warren Lyford DeLano and commercialised by DeLano Scientific LLC The software is freely available to educational users from the WWW (http://www.pymol.org), can be downloaded with instructions for its use and is supported by a helpful Wiki page (see below) There are several packages available for representation of protein structures (e.g RASMOL, SwissPDB Viewer) that are freely available and straightforward to use PyMOL allows the user to save all of the information in the current session, to go back and modify later on and to easily render the protein structure images into high-resolution pictures In order to view a protein structure, you must first download the PDB file from the Brookhaven Protein Database, which contains all the data for published X-ray crystal structures and NMR structures of proteins and nucleic acids I have included the PDB filename for each of the pictures that I have drawn in the figure legend I recommend that you download a few of these and try viewing each one on a computer screen; you can turn the structure around and get a really good feel for the three-dimensional structure of the protein You can download the PDB file from http://www.rcsb.org/pdb, or several other web sites Once you have downloaded the PDB file (in PDB text format), then you run the PyMOL program and open the PDB structure file to view the structure You can view the protein backbone in several different ways: as individual atoms (lines or sticks) or protein secondary structure (ribbon or cartoon) In most of the pictures in this edition I have drawn the protein backbone in cartoon format I have then selected certain catalytic amino acid residues and highlighted them in red, and selected any bound substrate analogues or coenzymes and highlighted them in black or red In preparing figures for the book I used only black and red, but on a computer screen you can use a wide range of colours and prepare your own multi-colour pictures! References W.L DeLano, “The PyMOL Molecular Graphics System”, DeLano Scientific, San Carlos, CA (2002) PyMOL home page: http://www.pymol.org PyMOL Wiki page: http://www.pymolwiki.org P1: JYS JWST182-c01 JWST182-Bugg May 21, 2012 10:8 Printer: Yet to come Trim: 244mm × 168mm From Jack Beans to Designer Genes 1.1 Introduction Enzymes are giant macromolecules which catalyse biochemical reactions They are remarkable in many ways Their three-dimensional structures are highly complex, yet they are formed by spontaneous folding of a linear polypeptide chain Their catalytic properties are far more impressive than synthetic catalysts which operate under more extreme conditions Each enzyme catalyses a single chemical reaction on a particular chemical substrate with very high enantioselectivity and enantiospecificity at rates which approach “catalytic perfection” Living cells are capable of carrying out a huge repertoire of enzyme-catalysed chemical reactions, some of which have little or no precedent in organic chemistry In this book I shall seek to explain from the perspective of organic chemistry what enzymes are, how they work, and how they catalyse many of the major classes of enzymatic reactions 1.2 The discovery of enzymes Historically, biological catalysis has been used by mankind for thousands of years, ever since fermentation was discovered as a process for brewing and bread-making in ancient Egypt It was not until the 19th century A.D however that scientists addressed the question of whether the entity responsible for processes such as fermentation was a living species or a chemical substance In 1897 Eduard Buchner published the observation that cell-free extracts of yeast containing no living cells were able to carry out the fermentation of sugar to alcohol and carbon dioxide He proposed that a species called “zymase” found in yeast cells was responsible for fermentation The biochemical pathway involved in fermentation was subsequently elucidated by Embden and Meyerhof – the first pathway to be discovered The exquisite selectivity of enzyme catalysis was recognised as early as 1894 by Emil Fischer, who demonstrated that the enzyme which hydrolyses sucrose, which he called “invertin”, acts only upon α-D-glucosides, whereas a second enzyme “emulsin” acts only upon β-D-glucosides He deduced that these two enzymes must consist of “asymmetrically built Introduction to Enzyme and Coenzyme Chemistry, Third Edition T D H Bugg © 2012 John Wiley & Sons, Ltd Published 2012 by John Wiley & Sons, Ltd P1: JYS JWST182-c01 JWST182-Bugg May 21, 2012 10:8 Printer: Yet to come Trim: 244mm × 168mm Introduction to Enzyme and Coenzyme Chemistry O H 2N NH2 + H2O Jack bean urease CO2 + NH3 Figure 1.1 Urease molecules”, and that “the enzyme and glucoside must fit each other like a lock and key to be able to exert a chemical influence upon each other” Fischer’s “lock and key” hypothesis remained a powerful metaphor of enzyme action for many years The crystallisation in 1926 of the enzyme urease (see Figure 1.1) from Jack beans by Sumner proved beyond doubt that biological catalysis was carried out by a chemical substance The recognition that biological catalysis is mediated by enzymes heralded the growth of biochemistry as a subject, and the elucidation of the metabolic pathways catalysed by enzymes Each reaction taking place on a biochemical pathway is catalysed by a specific enzyme Without enzyme catalysis the uncatalysed chemical process would be too slow to sustain life Enzymes catalyse reactions involved in all facets of cellular life: metabolism (the production of cellular building blocks and energy from food sources); biosynthesis (how cells make new molecules); detoxification (the breakdown of toxic foreign chemicals); and information storage (the processing of deoxyribonucleic acids) In any given cell there are present several thousand different enzymes, each catalysing its specific reaction How does a cell know when it needs a particular enzyme? The production of enzymes, as we shall see in Chapter 2, is controlled by a cell’s DNA, both in terms of the specific structure of a particular enzyme and the amount which is produced Thus different cells in the same organism have the ability to produce different types of enzymes and to produce them in differing amounts according to the cell’s requirements Since the majority of the biochemical reactions involved in cellular life are common to all organisms, a given enzyme will usually be found in many or all organisms, albeit in different forms and amounts By looking closely at the structures of enzymes from different organisms which catalyse the same reaction, we can in many cases see similarities between them These similarities are due to the evolution and differentiation of species by natural selection So by examining closely the similarities and differences of an enzyme from different species we can trace the course of molecular evolution, as well as learning about the structure and function of the enzyme itself Recent developments in biochemistry, molecular biology and X-ray crystallography now allow a far more detailed insight into how enzymes work at a molecular level We now have the ability to determine the amino acid sequence of enzymes with relative ease, whilst the technology for solving the three-dimensional structure of enzymes is developing apace We also have the ability to analyse their three-dimensional structures using molecular modelling and then to change the enzyme structure rationally using site-directed mutagenesis We are now starting to enter the realms of enzyme engineering, where by rational design we can modify the genes encoding specific enzymes, creating the “designer genes” of the title These modified enzymes could in future perhaps be used to catalyse new types of chemical reactions, or via gene therapy to correct genetic defects in cellular enzymes which would otherwise lead to human diseases P1: JYS JWST182-bapp04 264 JWST182-Bugg May 21, 2012 15:45 Printer: Yet to come Trim: 244mm × 168mm Introduction to Enzyme and Coenzyme Chemistry Chapter Intramolecular base catalysis, with the internal tertiary amine acting as a base to deprotonate an attacking water molecule Tertiary amines are good bases and poor nucleophiles, so nucleophilic catalysis is not feasible Intramolecular acid catalysis (carboxylic acid will be protonated at pH 4) a) Phenoxide ion attacks to form 5-membered ring Effective concentration 7.3 × 104 M Large rate acceleration due to intramolecular nucleophilic attack, 5-membered ring b) Either 1) general base-catalysed attack of water or 2) nucleophilic attack by active site aspartate to give covalent ester intermediate (similar to haloalkane dehalogenase) To examine mechanism (2) could try to detect covalent intermediate (e.g by stopped flow methods) Rate enhancement through transition state stabilisation, bifunctional catalysis, etc In the first catalytic cycle, the oxygen atom introduced into the product comes from the aspartate nucleophile, via hydrolysis of the ester intermediate In subsequent cycles 18 O label becomes incorporated into the active site aspartate and is transferred to product Chapter 787 units/mg ÷ 28 mg/␮mole = 28 ␮moles product/min/␮mole enz So kcat = 0.47 s−1 Use Lineweaver/Burk or Eadie/Hofstee plot vmax = 6.0 nmoles min−1 , Km = 0.71 mM kcat = 2.0 s−1 , kcat /Km = 2,800 M−1 s−1 Retention of stereochemistry Not consistent with an SN 2-type displacement Imine linkage formed between aldehyde group and ε-amino group of an active site lysine residue Enzymatic reaction goes with retention of configuration at phosphorus, whereas non-enzymatic reaction goes with inversion Suggests that non-enzymatic reaction is a single displacement, whereas enzymatic reaction is probably a double displacement reaction proceeding via a phosphoenzyme intermediate Could try to detect phosphoenzyme intermediate using 32 P-labelled substrate In D2 O should see H incorporation into acetaldehyde Chapter Hydrolysis of acyl enzyme intermediate is rate-limiting step in this case Rapid formation of acyl enzyme intermediate, releasing a stoichiometric amount of p-nitrophenol, followed by a slower hydrolysis step Mechanism as for chymotrypsin, via acyl enzyme intermediate Phosphorylation of active site serine by organophosphorus inhibitors gives a tetrahedral adduct resembling the tetrahedral intermediate in the mechanism, which is hydrolysed very slowly Differences in toxicity due to: 1) presence of sulphur on parathion, which de-activates the phosphate ester (in insects this is rapidly oxidised to the phosphate ester, which then kills the insect!); 2) differences in active site structure between human and insect enzymes Antidote binds to choline site through positively-charged pyridinium group Hydroxylamine group is a potent nucleophile which attacks the neighbouring tetrahedral P1: JYS JWST182-bapp04 JWST182-Bugg May 21, 2012 15:45 Printer: Yet to come Trim: 244mm × 168mm Appendix 4: Answers to Problems 265 phosphate ester Thus the rate of hydrolysis of the tetrahedral phosphate adduct is rapidly accelerated Aldehyde is attacked by active site cysteine, generating a thio-hemiacetal intermediate which mimics the tetrahedral intermediate of the normal enzymatic reaction, and is hence bound tightly by the enzyme Acetate kinase gives acyl phosphate intermediate, acetate thiokinase involves acyl adenylate (RCO.AMP) intermediate Glycogen phosphorylase cleaves glucose units successively from end of chain Reaction proceeds with retention of configuration at the anomeric position, so a covalent intermediate is probably formed (cf lysozyme) by attack of an active site carboxylate Displacement by phosphate gives ␣-D-glucose-1-phosphate Phosphoglucose isomerase contains phosphorylated enzyme species which transfers phosphate to C-6 to give 1,6-diphospho-glucose Dephosphorylation at C-1 re-generates phosphoenayme species Glucose-6-phosphatase is straightforward phosphate monoester hydrolysis Defect in glycogen phosphorylase leads to inability to utilise glycogen, so unable to maintain periods of physical exercise Chapter Alcohol dehydrogenase: − 0.16(CH3 CHO) – − 0.32(NAD+ ) = + 0.16V Enoyl reductase: + 0.19(enoyl CoA) – − 0.32(NAD+ ) = + 0.49V Acyl CoA dehydrogenase: + 0.25(cytcox ) – + 0.19(enoyl CoA) = + 0.06V In acyl CoA dehydrogenase the redox potential for the intermediate FAD must be close to + 0.19V if electron transfer is to be thermodynamically favourable This is right at the top end of the redox potential range for flavin Enzyme transfers proR hydrogen of NADPH onto C-3 position of substrate Overall syn addition of hydrogens from NADPH and water Trnasfer of H* onto enzyme-bound NAD+ Resulting C-4 ketone assists the E1cb elimination of C-6 hydroxyl group to give unsaturated ketone intermediate Transfer of H* from cofactor to C-6 gives product Transfer of proS hydrogen of NADPH to FAD Then reverse of acyl CoA dehydrogenase mechanism: transfer of H onto ␤-position giving ␣-radical; electron transfer from flavin semiquinone to give ␣-carbanion; protonation from water at ␣-position Note that the same hydrogen transferred from NADPH to FAD is then transferred to substrate Formation of flavin hydroperoxide intermediate from FADH2 and O2 Attack on flavin hydroperoxide para to phenolic hydroxyl group, followed by elimination of nitrite to give quinone Quinone then reduced to hydroquinone by second equivalent of NADH Mechanism of hydroxylation as for general mechanism, via iron(IV)-oxo species Triple bond of inhibitor is epoxidised to give reactive alkene epoxide intermediate, which re-arranges with 1,2-shift of H* to give a ketene intermediate This is attacked either by water, giving the by-product, or by an active site nucleophile, leading to covalent modification P1: JYS JWST182-bapp04 JWST182-Bugg 266 May 21, 2012 15:45 Trim: 244mm × 168mm Printer: Yet to come Introduction to Enzyme and Coenzyme Chemistry O O O2 , Fe 2+ HN O H* N H α-KG O HN O EnzX O O HN O O N H XEnz N H O H2 O H H* HN O *H H* N H – H H* CO2H HN O O N H Chapter Opening of monosaccharide at C-1 reveals aldehyde substrate Since enzyme requires no cofactors it presumably proceeds through imine linkage at C-2 of pyruvate, followed by deprotonation at C-3 to give enamine intermediate Carbon–carbon bond formation between enamine and aldehyde, followed by hydrolysis of resulting imine linkage Sequential addition of three malonyl CoA units as for fatty acid synthase gives a tetraketide intermediate Formation of carbanion between first and second ketone groups, followed by reaction with thioester terminus, leads to formation of chalcone Formation of carbanion adjacent to thioester terminus, followed by reaction with first ketone group, leads after decarboxylation (␤-keto-acid) to resveratrol Very similar reactions, so similar active sites, but differences in position of carbanion formation and carbon–carbon bond formation Attack of bicarbonate onto phosphate monoester gives enol intermediate and carboxyphosphate Attack of enol at C-3 onto carboxyphosphate gives carboxylated product Reverse of normal biotin mechanism Attack of N-1 of biotin cofactor onto oxaloacetate gives pyruvate and carboxy-biotin intermediate, which carboxylates propionyl CoA to give methylmalonyl CoA Attack of TPP anion onto keto group gives tetrahedral adduct Cleavage of ␣,␤-bond using TPP as electron sink gives enamine intermediate This reacts with aldehyde of second substrate to give another tetrahedral adduct Detachment from cofactor re-generates TPP anion Attack of TPP anion onto ketone gives tetrahedral adduct Decarboxylation gives enamine intermediate as in normal mechanism At this point the enamine intermediate is oxidised by FAD to give acetyl adduct (probably via H transfer followed by single electron transfer) Hydrolysis by attack of water gives acetate product, and re-generates TPP anion FADH2 re-oxidised to FAD by O2 1,3-migration of pyrophosphate to give linalyl PP Attack at C-1 to form 6-membered ring gives tertiary carbonium ion Formation of second ring with concerted attack of pyrophosphate gives product No positional isotopoe exchange in pyrophosphate means that pyrophosphate is bound very tightly (to Mg2+ ) by enzyme, and is not even free to rotate P1: JYS JWST182-bapp04 JWST182-Bugg May 21, 2012 15:45 Trim: 244mm × 168mm Printer: Yet to come Appendix 4: Answers to Problems OPP 267 – OPP OPP OPP Reaction to copalyl PP commenced by protonation of terminal alkene to give tertiary carbonium ion Two ring closures followed by loss of proton to form C C Loss of pyrophosphate followed by formation of 6-membered ring Closure to form final 5membered ring followed by 1,2-alkyl shift and elimination H OPP OPP H H H H + H H H H Aromatic precursor made from tetraketide intermediate which is methylated by SAM: carbanion formation between first & second ketones is followed by attack on terminal thioester carbonyl Formation of phenoxy radical para to methyl group Carbon–carbon bond formation via radical coupling Re-aromatisation of left hand ring followed by attack of phenoxide onto ␣␤-unsaturated ketone of right hand ring Chapter Treat with substrate and NaB3 H4 (or 14 C-substrate + NaBH4 ), degrade labelled enayme by protease digestion, purify labelled peptide & sequence Overall syn addition Attack of carboxylate onto ␣␤-unsaturated carboxylic acid to give enolate intermediate, which protonates on same face Possibilities are: 1) elimination of water to give enol intermediate, which protonates in ␤-position; 2) 1,2-hydride shift from ␣ to ␤ position (cf Pinacol re-arrangement) In mechanism an ␣-2 H substituent would transferred to ␤ position, in mechanism probably not (unless single acid base responsible for proton transfer) No intramolecular proton transfer observed in practice, so probably mechanism Oxidation of C-3 hydroxyl group by enzyme-bound NAD+ Formation of C-3 ketone assists elimination of methionine by E1cb mechanism, giving ␣␤-unsaturated ketone intermediate Addition of water at C-5 , followed by reduction of C-3 ketone, re-generating enzyme-bound NAD+ Isochorismate synthase could be 1,5-addition of water, but all three reactions can be rationalised by attack of an enzyme active site nucleophile at C-2 and allylic displacment of water, giving a covalent intermediate (see Figure) Attack of water at C-6 gives P1: JYS JWST182-bapp04 268 JWST182-Bugg May 21, 2012 15:45 Trim: 244mm × 168mm Printer: Yet to come Introduction to Enzyme and Coenzyme Chemistry isochorismate Attack of ammonia at C-6 or C-4, followed by elimination of pyruvate, gives anthranilate or p-aminobenzoate respectively – CO2– CO2– EnzX OH2 or NH3 EnzX O OH H+ CO2– O CO2– NH3 Chapter Use of PLP as a four-electron sink Formation of threonine-PLP adduct, followed by deprotonation in ␣-position, gives ketimine intermediate Deprotonation at ␤-position possible using imine as electron sink Either one-base or two-base mechanisms possible for epimerisation process Formation of aldimine adduct followed by ␣-deprotonation gives ␣␤-unsaturated ketimine intermediate This can be attacked by an active site nucleophile at ␥ -position to covalently modify enzyme Formation of aldimine adduct with inhibitor followed by ␣-deprotonation gives a ketimine intermediate which is a tautomeric form of an aromatic amine Rapid aromatisation gives a covalently modified PLP adduct Use of PLP as a four-electron sink Formation of amino acid-PLP adduct followed by ␣-deprotonation Removal of C-3 proS hydrogen using imine as electron sink followed by elimination of phosphate gives ␤␥ -unsaturated intermediate Protonation at ␥ -position, followed by attack of water at ␤-position, and re-protonation at ␣-position Attachment of PLP onto ␣-amino group followed by ␣-deprotonation gives ketiminine intermediate Hydration of ␥ -ketone group is followed by cleavage of ␤␥ -bond, using imine as an electron sink C C cleavage and re-protonation proceeds with overall retention of stereochemistry Chapter 10 One S centre is epimerised by the epimerase enzyme with introduction of an ␣-2 H, but if left to equilibrate both S centres would undergo enzyme-catalysed exchange with H2 O R centre is decarboxylated, with replacement by H, so in principle atoms of H would be found in the L-lysine product Deprotonation at ␥ -position to form dienol intermediate is followed by re-protonation at ␣-position 2-Chlorophenol is processed to give ␦-chloro intermediate Upon deprotonation at the ␥ -position loss of Cl- gives a ␥ ␦-unsaturated lactone This is processed by opening of the lactone, and reduction of the ␣␤-bouble bond by an NADH-dependent reductase Protonation of the dienol at C-5 is followed by attack of water (or an active site nucleophile) at the C-6 ketone Cleavage of the C-5,C-6 bond is then facilitated by the presence of an ␣␤-unsaturated ketone group which can act as an electron sink P1: JYS JWST182-bapp04 JWST182-Bugg May 21, 2012 15:45 Printer: Yet to come Trim: 244mm × 168mm Appendix 4: Answers to Problems 269 Possibilities are: 1) reversible attack of an active site nucleophile at the amide carbonyl, allowing free rotation of the tetrahedral intermediate; 2) a “strain” mechanism in which the enzyme binds the substrate in a strained conformation close to the transition state for rotation of the amide bond Available evidence points to mechanism 2, and it is thought that cyclosporin A acts as a mimic of this strained intermediate [N.B the immunosuppressant activity is due to the complex formed between cyclosporin A and this protein, which is called cyclophilin] Concerted 4-electron pericyclic reaction is a disfavoured process Stepwise reaction possible by transfer of phosphate group to active site group, followed by reaction of enol intermediate with phosphoenzyme species Chapter 11 a) Abstraction of H atom by Ado-CH2 radical at C-2, followed by cyclisation onto C-4 to give a cyclopropyl radical; fragmentation of other C C bond in cyclopropane ring to give product radical b) Formation of radical at C-2, followed by fragmentation of C3-C4 bond to give alkenyl radical, which re-attacks acrylic acid at C-2 to give product radical a) Abstraction of H* to give radical intermediate; cyclisation onto ester carbonyl to give cyclopropyl intermediate; fragmentation to give product radical b) Possibly SAM-dependent radical reaction, or generation by protein radical or heme Fe O Abstraction of hydrogen atom from methyl group of toluene; attack of radical on C C of succinate to give product radical; abstraction of hydrogen atom from glycine ␣-CH2 to give product, and regenerate glycyl radical Generation of tyrosyl radical and Cys radical (1-electron oxidations by Cu centre); radical coupling to form C S bond, followed by re-aromatisation Attack of methanol at quinone C O to form hemiketal intermediate, followed by abstraction of adjacent hydrogen by hemiketal O- and elimination of formaldehyde (using the other C O as electron sink), to give reduced PQQ Chapter 12 In principle the reaction is reversible, which would allow the ribozyme to integrate itself into a piece of RNA (reminiscent of the life cycle of some viruses) A cyclic phosphonate ester would be a good transition state analogue Such an analogue was used to elicit antibodies capable of catalysing this lactonisation reaction with an enantiomeric excess of 94% At neutral pH the tertiary amine will be protonated When exposed to the immune response, this will elicit a complementary antibody containing a negatively-charged carboxylate group in close proximity This group functions as a base for the elimination reaction This “bait and switch” trick has been used in other cases also a) Might expect to mimic cysteine proteases, hydrolysing aromatic ester (and possibly amide) substrates b) Might expect to mimic FAD-containing oxidases, oxidising aromatic amines and aromatic thioethers (to sulphoxides) P1: JYS JWST182-bind JWST182-Bugg May 25, 2012 8:53 Printer: Yet to come Trim: 244mm × 168mm Index acetoacetate decarboxylase 42 acetycholinesterase, catalytic efficiency 47 acetyl coenzyme A (CoA) 63, 94 fatty acid assembly 156–8 N-acetylglucosamine (GlcNAc) 95, 102 N-acetylmuramic acid (MurNAc) 95, 102 acid–base catalysis 36–9 aconitase 184 mechanism 188 activation energy 28 active site of an enzyme 17 determination of catalytic groups 71–3 acyl carrier protein (ACP) 156 acyl CoA dehydrogenase 124, 126 inactivation 126 acyltransferase (AT) 157 reactions 93–5 addition/elimination reactions 181–2 ammonia lyases 187–90 5-enolpyruvyl shikimate-3-phosphate (EPSP) synthase 191–3 hydratases and dehydratases 182–7 phosphate and pyrophosphate elimination 190–1 adenine (A) 10 adenosine -diphosphate (ADP) 101, 158 adenosine -triphosphate (ATP) 66, 101, 160 structure 101 S-adenosyl methionine (SAM) 107–8 biosynthesis 108 methyl group transfer 108 radical reactions 232–3 S-adenosylhomocysteine 108 affinity labelling 72 alanine (Ala) alanine racemase 200 alcohol dehydrogenase 120 active site 120 specificity 122 aldol reaction 149 aldolases 149–50 alkaline phosphatase 97 allylic isomerases 217–8 ␣-helix 13 proteins 15, 16 amide linkages 13 D-amino acid oxidase 124 amino acid transformations 197 aspartate aminotransferase 201–4 imines and enamines in alkaloid biosynthesis 208–9 pyridoxal -phosphate (PLP)-dependent reactions 197–201 N-pyruvoyl-dependent amino acid decarboxylase 208 reactions at ␤- and ␥ -positions 204–6 serine hydroxymethyltransferase 206, 207 amino acids 7–9 abbreviations 260 active site modification 72 codons of DNA 10 sequence alignment 11–12 aminoacyl amino acids 93 ammonia lyases 187–90 antibacterial agents antibodies, catalytic 246–51 anti-bonding orbitals (␲ * ) 123–4 anti- elimination 181, 188 antifungal agents antiparallel ␤-sheet 14, 17 antiviral agents arginine (Arg) active site modification 72 basic sidechain aromatic dihydroxylating dioxygenases 141–2 ascorbic acid (vitamin C) 4, 141 asparagine (Asn) Introduction to Enzyme and Coenzyme Chemistry, Third Edition T D H Bugg © 2012 John Wiley & Sons, Ltd Published 2012 by John Wiley & Sons, Ltd P1: JYS JWST182-bind 272 JWST182-Bugg May 25, 2012 8:53 Printer: Yet to come Trim: 244mm × 168mm Index aspartate (Asp) active site modification 72 hydrophilic sidechain nucleophilic catalysis 41 pKa value 36 aspartate aminotransferase 201–4 mutant 204 aspartyl acid 89 aspartyl proteases 89 assay of enzymes 50–2 asymmetric induction 59 asymmetric organic synthesis AZT bacteriorhodopsin 22 Baeyer–Villiger oxidation 128 beri-beri 165 ␤-sheet 13–14 proteins 15, 16 ␤-turn 15 biosynthesis 148 biotin (vitamin H) 4, 158–60 biotin synthase 233–4 bromelain 79 Cahn–Ingold–Prelog rule 258–9 calciferol (vitamin D) Calvin cycle 162 camphene 169 camphor 168 Cannizzaro reaction 120, 121 carbanion equivalents adolases 149–50 carbanion intermediates 148 carbocation intermediates 148, 168 terpene cyclases 168–73 carbohydrates 102 carbon dioxide 161 carbon–carbon bond formation 148 carbanion equivalents adolases 149 carbocation intermediates 168 terpene cyclases 168–73 carboxylases 158–60 Claisen enzymes 153–6 fatty acid and polyketide assembly 156–8 fructose-1,6-bisphosphate aldolase 150–2 radical intermediates 173 phenolic radical couplings 173–4 ribulose bisphosphate carboxylase/oxygenase (Rubisco) 161–2 thiamine pyrophosphate (TPP)-dependent enzymes 165–8 vitamin K-dependent carboxylase 163–5 carbonium ions 149 carboxylases 158–60 carboxypeptidase 44, 79 active site 87 mechanism 88 case studies aspartate aminotransferase 201–4 chorimate mutase 219–21 5-enolpyruvyl shikimate-3-phosphate (EPSP) synthase 191–3 fructose-1,6-bisphosphate aldolase 150–2 glutathione and trypanothione reductases 129–33 HIV-1 protease 90–1 catalysis, enzymatic efficiencies 55 catalytic antibodies 246–51 catalytic efficiency 55 catalytic RNA 242–6 catechol dioxygenases 141 mechanisms 143 cathepsin 79 cell–cell recognition 23 cephalosporins Chagas’ disease 132 chelating agents 86, 87 chemical competence 68 chiral molecules chiral products 59 nomenclature 59 chirality of amino acids ␤-chloro-D-alanine 200 chloroperoxidases 139 chorimate mutase 219–21 chorismate 182 chorismate synthase 191 chorismic acid 191 chromatography 50 chymotrypsin 79, 80 mechanism 82 Claisen enzymes 153–6 Claisen ester condensation 153–4 clostripain 79 cobalamine (vitamin B12 ) 4, 108 structure 226 vitamin B12 -dependent reactions 225–9 P1: JYS JWST182-bind JWST182-Bugg May 25, 2012 8:53 Printer: Yet to come Trim: 244mm × 168mm Index cobalt-containing metallo-enzymes 20 codons of DNA 10–11 coenzyme A (CoA) 93 coenzymes, discovery of collagen 140 commercial importance of enzymes 3–6 convergent evolution 82 copper-containing metallo-enzymes 20 covalent modification 72 crown ethers 253 cyclodextrins 254 cyclohexanone mono-oxygenase 128 mechanism 130 cyclosporin A cysteine (Cys) active site modification 72 nucleophilic catalysis 41 pKa value 36 polar nucleophilic sidechain cysteine proteases 82–86 cytidine (C) 10, 108, 109 cytochrome b562 15 cytochrome c peroxidase, catalytic efficiency 47 cytochrome P450cam 136, 138 deazaflavins 133–5 dehydratases 182–7 3-dehydroquinate 182 3-dehydroquinate dehydratase 183 3-dehydroshikimate 182 deoxyadenine (dA) 10 deoxycytidine (dC) 10 3-deoxy-D-arabinoheptulosonic acid 7-phosphate (DAHP) 182 deoxyguanine (dG) 10 designer genes deuterium labelling 120–21, 169 deuterium oxide isotope effect 68 Diels-Alder reaction 219 diffusion limit 46 dihydrofolate reductase 48 dihydropyridine ring puckering 122 dihydroxyacetone phosphate (DHAP) 150–2 dimethoate dimethylallyl pyrophosphate (DMAPP) 169, 218 dioextane intermediate 164 dioxygenases ␣-ketoglutarate-dependent 140–1 non-heme iron-dependent 141–4 273 directed evolution 47 DNA codons 10 mutations 11 protein coding transcription 10 DNA repair enzymes 234, 236, 237 domains in proteins 15 L-dopa decarboxylate 201 dopamine 200 drug discovery, importance of enzymes in Eadie-Hofstee plots 56 effective concentration 31 elastase 79, 81 electrophilic attack 148 electrophoresis 52 electrospray mass spectrometry 73 electrostatic interactions of enzymes 18 elimination reaction see addition/elimination reactions emulsin enamine intermediates 150, 154 enamines in alkaloid biosynthesis 208–9 enantioselective acylation 93 enantiospecificity of enyzymes 4–5 endo-cleavage 77, 79 endonucleases 98 enolate intermediates 149 5-enolpyruvyl shikimate-3-phosphate (EPSP) 182, 191 5-enolpyruvyl shikimate-3-phosphate (EPSP) synthase 65, 191–4 enolpyruvyl transferase 194 entropically favoured reactions 30 enzyme catalysis 261–2 enzyme extract preparation 51 enzymes as catalysts 26–8 acid–base catalysis 36–9 active site nucleophiles 45–6 catalytic perfection 46–7 nucleophilic catalysis 40–3 protein dynamics 47 proximity effects 30–2 strain energy 44–5 substrate desolvation 45–6 thermodynamic model 28–9 transition state stabilisation 32–6 P1: JYS JWST182-bind 274 JWST182-Bugg May 25, 2012 8:53 Printer: Yet to come Trim: 244mm × 168mm Index enzymes commercial importance of 3–6 definition discovery 1–2 drug discovery, importance in membrane-associated 21–2 metallo-enzymes 16, 20–21 nature of as proteins purification 50–2 structure and function 17–20 enzyme–substrate intermediate 29 epimerases, cofactor-independent 213–6 erythromycin A 159 erythrose-4-phosphate 182 essential enzymes esterases 27, 92 ethylenediaminetetra-acetic acid (EDTA) 87 exo-cleavage 77, 79 extraction and purification of enzymes 50–2 factor F420 134 factor X 79 farnesyl pyrophosphate 169 fatty acid assembly 156–8 flavin, structure of 134 flavin adenine dinucleotide (FAD) 123 flavin adenine dinucleotide, reduced (FADH2 ) 123, 129, 142 flavin mononucleotide (FMN) 123 flavin semiquinone 123 flavin-dependent dehydrogenases 122–8 flavin-dependent mono-oxidases 128 flavin-dependent oxidases 122–8 flavodoxin 15, 16 fluorinase enzyme 45 folic acid 4, 108 formaldehyde 108 free energy profiles 29 fructose 105, 106 fructose-1,6-bisphosphate aldolase 60, 150 active site 152 case study 150–2 reaction 150 structure 151 fumarase 186 catalytic efficiency 47 function of enzymes 17–20 galactose oxidase 239 genes 10 mutations 11, 204 geraniol 168 geranyl pyrophosphate 169 glucose 105 glutamate (Glu) active site modification 72 hydrophilic sidechain nucleophilic catalysis 41 pKa value 36 glutamate mutase 229 glutamine (Gln) hydrophilic sidechain glutamine synthetase 66 glutathione (GSH) 129–32 glutathione reductase 129–33 active site 133 mechanism 132 structure 131 glyceraldehyde 3-phosphate (G3P) 150–2 glyceraldehyde 3-phosphate dehydrogenase 40, 41 glycine (Gly) glycolysation 23 glycoproteins 23 glycoside hydrolysis, rate acceleration 27 glycosyl transfer reactions 102–7 glyphosate 6, 193 guanine (G) 10 hairpin ribozyme 245 half cell reactions 115 haloalkane dehalogenase 42 hapten formation 248 helical wheel 25 hemoglobin 15 herbicides 6, 193 histidine (His) active site modification 72 basic sidechain nucleophilic catalysis 41 pKa value 36 histidine ammonia lyase 188 histidine decarboxylase 208 historical perspective commercial importance of enzymes 3–6 discovery of coenzymes discovery of enzymes 1–2 drug discovery, importance of enzymes in P1: JYS JWST182-bind JWST182-Bugg May 25, 2012 8:53 Printer: Yet to come Trim: 244mm × 168mm Index HIV virus 23, 89, 90 HIV-1 protease 90–1 mechanism 91 structure 90 transition state 92 hydratases 182–7 hydrogen bonds 13, 18–19 hydrogen tunnelling 69–70 hydrolases, classes of 78 hydrolysis and group transfer reactions 77–8 rates 26–7, 30–2 stereoselectivity 28 hydrophilic sidechains of amino acids hydrophobic interactions of enzymes 19 hydrophobic sidechains of amino acids 2-hydroxy-3-azido-propionaldehyde 152 p-hydroxybenzoate hydroxylase 129 structure 130 hydroxydecanoyl thioester dehydratase 184 hydroxydecanoyl thioester isomerase 218 hydroxylamine 66 5-hydroxymethylcytidine 108 hypoglycin A 126 imines in alkaloid biosynthesis 208–9 immunoglobulin G (IgG) antibodies 246 structure 247 imprinted polymers 256 influenza virus 23 inhibitors of enymes 6, 57–8 commercially important insecticides intermediates in enzyme reaction 64 chemical interference 66 direct observation 65–6 isotope exchange 66–8 themodynamic model 28–9 transition state analysis 68–70 trapping 66 invertin iron-containing metallo-enzymes 20 iron–sulphur clusters 135–6 isoleucine (Ile) hydrophobic sidechain isomerases 213 allylic isomerases 217–8 chorimate mutase 219–21 275 cofactor-independent racemases and epimerases 213–6 keto-enol tautomerases 216–7 isopentenyl pyrphosphate isomerase (IPP) 218 isopentyl pyrophosphate (IPP) 169 isotope exchange 66–7 isotopes and intermediate analysis 68–70 for stereochemical study 62 Jamaican vomiting sickness 126 kanamycin ␤-keto acids 165 ␤-keto thioesters 157, 158 ketoconazole keto-enol tautomerases 216–7 ␣-ketoglutarate-dependent dioxygenases 140–1 keto-reductase (KR) 158 ketosteroid isomerase 38, 39, 217 and kinetic isotope effect 70–1 catalytic efficiency 47 ketosynthase (KS) 157, 158 kinetic competence 68 kinetic isotope effects 69, 70 kinetics of enzyme reactions 52–9 ␤-lactamase, catalytic efficiency of 47 lactate dehydrogenase 117 leucine (Leu) hydrophobic sidechain leucine aminopeptidase 79, 89 Lewis acids 20–1, 186 lignin 174 limonene 169 linalyl pyrophosphate 169 Lineweaver-Burk plot 56 lipases 27, 92 lipoamide 165 lipoamide cycle 167 lipoic acid synthase 234 local energy maximum 28 local energy minimum 28 lock-and-key hypothesis of enzymes lone pairs of electrons 12 lupinene 208 lysine (Lys) active site modification 72 basic sidechain P1: JYS JWST182-bind 276 JWST182-Bugg May 25, 2012 8:53 Printer: Yet to come Trim: 244mm × 168mm Index lysine (Lys) (Continued) nucleophilic catalysis 41 pKa value 36 lysozyme 17, 102 mechanism 104 structure 105 magnesium-containing metallo-enzymes 20 mannose-containing oligosaccharides 23 membrane proteins 22 membrane-associated enzymes 21–2 mendelate racemase 215 menthol 168 metal-dependent mon-oxygenases 136–9 metallo-enzymes 15, 16, 20–1 metallo-proteases 86–9 methionine (Met) hydrophobic sidechain methionine ␥ -lyase 206 methyl group transfer 107–10 methylaspartase reaction 190 ␤-methylcrotonyl-CoA 160 methylene imidazolone cofactor 188 methylene-tetrahydrofolate 108, 110 methylmalonyl CoA 158 methylmalonyl CoA mutase 227 mevalonate pyrophosphate decarboxylase 191 mevalonic acid 154 Michaelis constant 54 Michaelis-Menten model 53 mirror image structures molybdenum-containing metallo-enzymes 20 monoamine oxidase 124, 126–8 inactivation 126 morphine 3, 174, 208, 209 mutagenesis, site-directed 73 mutations 11, 204 myrcene 169, 171 Nernst equation 117 nicotinamide (niacin) nicotinamide adenine dinucleotide (NAD) 117–22 oxidised form (NAD+ ) 117, 142 reduced form (NADH) 117, 142 nicotinamide adenine dinucleotide, deuterated (NADD) 120–22 nicotinamide adenine dinucleotide, phosphorylated (NADP) 117 oxidised form (NADP+ ) 117 reduced form (NADPH) 117, 129–33 nicotinamide adenine dinucleotide-dependent dehydrogenases 117–22 nicotine 208 NIH shift 134–135 nikkomycin N-linked glycosylation 23 non-enzymatic biological catalysts 242 catalytic antibodies 246–51 catalytic RNA 242–46 synthetic enzyme models 251–55 non-heme iron-dependent dioxygenases 141–4 non-polar (Van der Waals) interactions of enzymes 19 non-superimposable molecular structures 7, 59 N-terminal end of a protein 9, 11 nuclear magnetic resonance (NMR) spectroscopy 17, 47 and protein structure 73 and stereochemical elucidation 62 nucleophilic attack 126, 249 nucleophilic catalysis 40–43 nucleotide bases 10 oligosaccharides 105 O-linked glycosylation 23 orsellinic acid 156 ortho- position on a phenol ring 128 oxidation 115 oxidised amino acid cofactors 238–9 oxonium ions 103 P450 mono-oxygenases 136–39 pantothenic acid papain 79 active site 84 mechanism 85 para- position on a phenol ring 128 parallel ␤-sheet 14, 16 penicillin acylase penicillins pentalenene synthase 171 structure 172 pentalenolactone antibiotics 171 pepsin 79 pepstatin 89 peptidases 79–80 peptide mapping 73 peptidoglycan 95 cross-linking 95 P1: JYS JWST182-bind JWST182-Bugg May 25, 2012 8:53 Printer: Yet to come Trim: 244mm × 168mm Index peroxidases 139 1,10-phenanthroline 87 phenolic hydroxylases 128–9 phenolic radical couplings 173–5 phenyl esters, intramolecular hydrolysis of 30–32 phenyl phthalate 30 phenylalanine (Phe) 8, 181 hydrophobic sidechain phenylalanine ammonia lyase 187–8 mechanisms 189 phenylalanine hydroxylase 135 trans-2-phenylcyclopropylamine 126–27 phenylpyruvate tautomerase 216 phosphate elimination 190–91 phosphatidylcholine 96 phosphodiesterases 99 phosphoenol pyruvate (PEP) 191 phosphoglucose isomerase 217 phosphoramidon 88–9 phosphoryl transfer reactions 95–101 mechanisms 96 stereochemistry 64 phosphorylation by ATP 102 phosphotyrosine peptides 98 photosynthesis 115, 161–2 phylloquinone (vitamin K) vitamin K-dependent carboxylase 163–4 ␲-cation interaction 171 ␣-pinene 169 ␤-pinene 169 pinene synthase 169 PKa values and catalysis 36 polyketide assembly 156, 158 polyketide synthase 158 polypeptides see also proteins hydrolysis 79 polysaccharides 102, 105 hydrolysis 79 positional isotope exchange 67 Prelog’s rule 59 preorganisation 32 primary metabolites 148 primary structure of proteins 9–11 prochiral selectivity 60–64 proline (Pro) prolyl hydroxylase 140, 141 propanediol dehydrase 225 proteases, commercially available 80 277 protein domains 15 protein folding 12–15 protein radicals 229–31 proteinase K 79 proteins amino acid sequence alignment 11–12 enzymes as glycoproteins 23–4 polypeptide nature of 7–9 three-dimensional structure primary structure 9–11 secondary structure 12–15 tertiary structure 15–17 quaternary structure 15 protocatechuate 4,5-dioxygenase 143 proton abstraction 184 proximity effects 30–2 pterins 133 purification and extraction of enzymes 50–2 pyridoxal (vitamin B6 ) pyridoxal -phosphate (PLP)-dependent reactions amino acid ␣-position 197–201 amino acid ␤- and ␥ -positions 192–5 pyridoxamine -phosphate (PMP) 202 pyrophosphate elimination 190–91 pyrroloquinolone (PQQ) 238, 239 pyruvate decarboxylase 166 pyruvate formate lysase 230–32 pyruvic acid 166 N-pyruvoyl-dependent amino acid decarboxylase 208 quaternary structure of proteins 15 quinine quinoproteins 238–9 racemases, cofactor-independent 213–16 racemic mixtures, resolution using enzymes radical intermediates 150, 171 phenolic radical couplings 173–5 radicals 225 S-adenosyl methionine (SAM) 232–4 biotin synthase and sulphur insertion reactions 233–34 DNA repair enzymes 234, 235–238 oxidised amino acid cofactors and quinoproteins 238–9 protein radicals 229–32 vitamin B12 -dependent reactions 225–26 rate acceleration 28 P1: JYS JWST182-bind 278 JWST182-Bugg May 25, 2012 8:53 Printer: Yet to come Trim: 244mm × 168mm Index rates of reaction 26 diffusion limit 46 reaction mechanisms 50 active site catalytic group determination 71–3 intermediates 64 chemical interference 66–4 direct observation 65–6 isotope exchange 66–7 tapping 66 transition state analysis 68–9 kinetics 52–58 stereochemical course 59–60 chiral product generation 59 prochiral selectivity 59–60 redox chemistry 115–16 deazaflavins and pterins 133–4 flavin-dependent dehydrogenases and oxidases 122–23 flavin-dependent mono-oxidases 127–9 glutathione and trypanothione reductases 129–30 iron–sulphur clusters 135–6 ␣-ketoglutarate-dependent dioxygenases 140–1 metal-dependent mono-oxygenases 128–29 nicotinamide adenine dinucleotide-dependent dehydrogenases 117–18 non-heme iron-dependent dioxygenases 140–1 redox enzymes 118 redox potential 115–6 redox reagents, metal ions as 21 reduction 115 renin 79 retinol (vitamin A) reversible inhibition 56–7 riboflavin (vitamin B2 ) 3, 122, 133, 257 ribonuclease A mechanism 100 structure 99 ribonucleotide reductase 229–30 ribosomes 12, 86 ribozyme 242–46 ribulose bisphosphate carboxylase/oxygenase (Rubisco) 161–2 RNA catalytic 242–46 messenger (mRNA) 10, 242 ribosomal (rRNA) 242 transfer (tRNA) 242 salutaridine synthase 174 SAM (S-adenosyl methionine) 107 biosynthesis 107 methyl group transfer 108 radical reactions 232–4 scurvy 141 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) 52, 53 secondary deuterium isotope effect 70 secondary metabolites 148 secondary structure of proteins 12–15 selectivity definition 28 prochiral 59–60 self-splicing reaction 242–3 semi-synthetic penicillins sequence motifs 12 serine (Ser) nucleophilic catalysis 40 polar nucleophilic sidechain serine esterase 93 serine hydroxymethyltransferase 206, 207 serine proteases 81 inhibitors 81 possible evolution 86 shikimate 182 shikimate pathway 182, 183 shikimate-3-phosphate 191 sialic acid 107 sialyltransferase 107 signal transmission across biological membranes 22 site-directed mutagenesis 150 snake venom 98 sodium cyanoborohydride trapping 88 specific activity 52 specificity, definition 28 stabilisation of intermediates 148 statine 89 steady state approximation 53 stearic acid 156 stereochemical course of enzyme reactions 59–60 chiral product generation 59 phosphate release 64 prochiral selectivity 60–64 stereochemical nomenclature 258–9 stereoselectivity in hydrolysis 27 stop codons 10 stopped flow apparatus 58 P1: JYS JWST182-bind JWST182-Bugg May 25, 2012 8:53 Printer: Yet to come Trim: 244mm × 168mm Index strain energy and catalysis 44–5 streptomycin structures of enzymes 17–19 strychnine 208, 209 substrate selectivity of enzymes 17 subtilisin 79, 81 succinate dehydrogenase 124 sucrose 105, 106 sucrose phosphorylase 106 suicide inhibitors 184 sulochrin oxidase 173 sulphur insertion reactions 233–34 superoxide 124 superoxide dismutase 15, 16 catalytic efficiency 46 syn- elimination 181 synthetic enzyme models 251–52 taxol terpene cyclases 168–70 terpenes 168 tertiary structure of proteins 15–17 tetrahydrofolate 108 thalidomide thermodynamic model of catalysis 28–9 thermolysin 40, 44, 79 active site 87 inhibitors 88 mechanism 88 thiamine (vitamin B1 ) 3, 165 thiamine pyrophosphate (TPP)-dependent enzymes 165–6 thiazolium ring 165 thioesterase (TE) 158 three-dimensional structure of proteins secondary structure 12–15 tertiary structure 15–17 quaternary structure 15 threonine (Thr) nucleophilic catalysis 40 polar nucleophilic sidechain threonine dehydratase 204–5 thrombin 80 thymidine (dT) T-lymphocyte cells 91 tocopherol (vitamin E) transferases 77 classes 78 279 transition states 28 analysis 68–9 in enzymatic reactions 68–9 stabilisation 32–4 transketolase reaction 168, 179 transpeptidases 77 trapping 66 2,4,5-trihydroxyphenylalanine quinone (TPQ) 239 triose phosphate isomerase, catalytic efficiency of 47 triplet codons 86 tritium labelling 227 trypanothione reductase (TR) 132–4 trypsin 80–1 acitive site 84 tryptophan (Trp) 8, 181 active site modification 72 hydrophobic sidechain tryptophylquinone (TTQ) 239 tunnelling, hydrogen 69 turnover number 52 tyrosine (Tyr) 8, 170 active site modification 72 nucleophilic catalysis 41 pKa value 37 universal genetic code 10 urease uridine (U) 10 uridine diphosphate (UDP) 106–7 UV assay 51–2 valine (Val) hydrophobic sidechain Van der Waals (non-polar) interactions of enzymes 19 viruses 23 vitamin K-dependent carboxylase 163–5 vitamins, discovery of water elimination 182 X-ray crystallography 15, 73 zinc finger DNA-binding 20 zinc-containing metallo-enzymes 20, 21 zymase ... Coenzyme (TPP) Coenzyme (FAD, FMN) Coenzyme (NAD) Coenzyme (PLP) Coenzyme Coenzyme, anti-oxidant Calcium homeostasis Anti-oxidant Coenzyme Coenzyme, anti-oxidant Coenzyme (tetrahydrofolate) Coenzyme. .. a second enzyme “emulsin” acts only upon β-D-glucosides He deduced that these two enzymes must consist of “asymmetrically built Introduction to Enzyme and Coenzyme Chemistry, Third Edition T... Printer: Yet to come Trim: 244mm × 168mm Introduction to Enzyme and Coenzyme Chemistry O H 2N NH2 + H2O Jack bean urease CO2 + NH3 Figure 1.1 Urease molecules”, and that “the enzyme and glucoside