ENZYMES: Biochemistry, Biotechnology and Clinical Chemistry Second Edition "Talking of education, people have now a-days" (said he) "got a strange opinion that every thing should be taught by lectures. Now, I cannot see that lectures can do so much good as reading the books from which the lectures are taken. I know nothing that can be best taught by lectures, except where experiments are to be shewn. You may teach chymestry by lectures - You might teach making of shoes by lectures!" James Boswell: Life ofS amuel Johnson, 1766 ABOUT THE AUTHORS Trevor Palmer was born in South Yorkshire and graduated from Cambridge University in 1966 with an honours degree in biochemistry, being influenced by (amongst others) Peter Sykes in organic chemistry and Malcolm Dixon in enzymology. He then worked as a clinical biochemist at the Queen Elizabeth Hospital for Children, linked to the Institute of Child Health, University of London, obtaining a PhD for research into inherited disorders. From this emerged the two main interests of his subsequent career, enzymology and evolution, the latter stimulating a further interest in the long-term effects of natural catastrophes. He moved to Nottingham Trent University (then Trent Polytechnic) in 1974, initially as a lecturer in biochemistry, before becoming Head of Department of Life Sciences (1987), Dean of the Faculty of Science and Mathematics (1992), Senior Dean of the University (1998) and Pro Vice-Chancellor for Academic Development (2002), returning to predominantly academic activity as Emeritus Professor in 2006. His books include Understanding Enzymes (1981), Principles of Enzymology for Technological Applications (1993), Controversy - Catastrophism and Evolution (1999) and Perilous Planet Earth (2003). His wife, Jan, teaches psychology and sociology (and is currently a part-time PhD student at Leicester University). Their son, James, is carrying out postdoctoral studies as a Leverhulme Fellow at Nottingham University and their daughter, Caroline, is researching for a PhD at Sheffield University. Philip L. Bonner went to school in Coventry, West Midlands, before graduating from the University of Sussex in 1978 with an honours degree in biochemistry. He then worked as a research assistant at Glaxo plc on Merseyside before leaving to take up a Research Assistant/Demonstrator post at Trent Polytechnic, where he obtained a PhD for research concerning enzymes associated with seed germination. Several postdoctoral appointments followed, at Bristol, Lancaster and Central Lancashire Universities, working on a variety of topics including relaxin, aspartate kinase and phospholipase C, before he was appointed as Senior Lecturer at Nottingham Trent University in 1991. There, he has maintained his research interests in enzymology and analytical biochemistry, working on the role of transglutaminase in plant/animal tissue and methods to isolate and characterise post-translationally-modified MHC peptides. His first single author book, on protein purification, was published in 2007. His wife, Liz, is a manager of an occupational therapist team in Nottingham and their daughter, Francesca, is at junior school. ENZYMES: Biochemistry, Biotechnology and Clinical Chemistry Second Edition Trevor Palmer, BA, PhD, CBiol, FIBiol, FIBMS, FHEA Emeritus Professor in Life Sciences Nottingham Trent University Philip L. Bonner, BSc, PhD Senior Lecturer in Biochemistry Nottingham Trent University WP WOODHEAD PUBLISHING ~ ~ Oxford Cambridge Philadelphia New Delhi For: Caroline, Francesca, James, Jan and Liz Published by Woodhead Publishing Limited, 80 High Street, Sawston, Cambridge CB22 3HJ, UK www.woodheadpublishing.com Woodhead Publishing, 1518 Walnut Street, Suite 1100, Philadelphia, PA 19102-3406, USA Woodhead Publishing India Private Limited, G-2, Vardaan House, 7/28 Ansari Road, Daryaganj, New Delhi – 110002, India www.woodheadpublishingindia.com First edition published by Horwood Publishing Limited, 2001 Second edition published by Horwood Publishing Limited, 2007 Reprinted by Woodhead Publishing Limited, 2011 © T. Palmer and P.L. Bonner, 2007 The authors have asserted their moral rights This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. 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Trademark notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation, without intent to infringe. British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library ISBN 978-1-904275-27-5 Printed by Lightning Source Authors' Preface This book was written, as all textbooks should be, with the requirements of the student firmly in mind. It is intended to provide an informative introduction to enzymology, and to give a balanced, reasonably-detailed, account of all the various theoretical and applied aspects of the subject which are likely to be included in an honours degree course. Furthermore, some of the later chapters may serve as a bridge to more advanced texts for students wishing to proceed further in this area of biochemistry. Although the book is intended mainly for students taking first degree courses which have a substantial biochemistry component, large portions may be of value to students on comparable courses in biological sciences, biomedical sciences or forensic sciences, and even to ones emolled on, in one direction, foundation programmes, or, in the other, MSc or other advanced courses who are approaching the subject of enzymology for the first time (or the first time in many years). No previous knowledge of biochemistry, and little of chemistry, is assumed. Most scientific terms are defined and placed in context when they are first introduced. Enzymology inevitably involves a certain amount of elementary mathematics, and some of the equations which are derived may appear somewhat complicated at first sight; however, once the initial biochemical assumptions have been understood, the derivations usually follow on the basis of simple logic, without involving any difficult mathematical manipulations. Numerical and other problems (with answers) are included, to test and reinforce the student's grasp of certain points. These problems generally use hypothetical data, although the results are often based on findings reported in the biochemical literature. If the size of the book is to be kept reasonable, some things of value have to be left out. The chief aim of this particular book is to help the student understand the concepts involved in enzymology, and the historical context in which they were worked out. It is not a reference book for practising enzymologists, so no comprehensive tables of data or long, finely-detailed accounts are included. Instead, an attempt has been made to give a perspective of each topic, and examples are quoted where appropriate. Credit has been given wherever possible to those responsible for the development of the subject, but many names deserving of mention have been excluded for reasons of space. Authors' Preface xv Individual scientific papers have not, in general, been referred to, but at the end of each chapter is a list of relevant books and articles, to provide context and an up-to date viewpoint, from which references to the original papers may usually be obtained. As with any book at this level, certain topics have been presented in a simplified (possibly even over-simplified) form. However, a considerable effort has been made to avoid giving a distorted account of any topic. It is hoped that this book can provide a foundation for those wishing to pursue more advances studies, and that nothing learned from it will have to be 'unlearned' later. There are good reasons for thinking that this is a realistic hope. For this second edition of Enzymes, we have revised and updated the first edition, reducing coverage of techniques whose use is declining to make room for discussion of topics of greater current and future interest, e.g. expanded bed chromatography, affinity precipitation, immobilized metal affinity chromatography, hydroxyapatite chromatography, hydrophobic charge induction chromatography, lectin affmity chromatography, covalent chromatography, membrane technology, capillary electrophoresis, absorbance fluorescence and lumimetric methods, high-throughput screening methods, 6-His tag and fusion protein technology, mass spectrometry and the use of protein arrays. A completely new section has been added on the use of enzymatic analysis in forensic science, and the final chapter of the first edition has been split into two to allow greater discussion of the rapidly-expanding areas of genomics, bioinformatics and proteomics. Elsewhere, coverage of protein structure, synthesis and function and mechanisms of enzyme activity has been revised to take into account recent developments (e.g. concerning the mechanism of action of lysozyme). Acknowledgements. In the preparation of this new edition, we are grateful for the help of many people, including Lesley Atherton, Mark Crowley, Nick Howard, Elaine James, Caroline Palmer, Jan Palmer and Karen Roberts. However, any errors of fact or interpretation which may have crept into the book are entirely our own responsibility, and we would be grateful if we could be informed about them. Finally, we would like to acknowledge the helpful cooperation of the staff of Horwood Publishing and, in particular, to express our gratitude to, and admiration for, the distinguished scientific publisher, the late Ellis Horwood, without whom this book would never have come into being. Trevor Palmer and Philip Bonner, 2007 1 An Introduction to Enzymes 1.1 WHAT ARE ENZYMES? Enzymes are biological catalysts. They increase the rate of chemical reactions taking place within living cells without themselves suffering any overall change. The reactants of enzyme-catalysed reactions are termed substrates. Each enzyme is quite specific in character, acting on a particular substrate or substrates to produce a particular product or products. All enzymes are proteins. However, without the presence of a non-protein component called a cofactor, many enzyme proteins lack catalytic activity. When this is the case, the inactive protein component of an enzyme is termed the apoenzyme, and the active enzyme, including cofactor, the holoenzyme. The cofactor may be an organic molecule, when it is known as a coenzyme, or it may be a metal ion. Some enzymes bind cofactors more tightly than others. When a cofactor is bound so tightly that it is difficult to remove without damaging the enzyme, it is sometimes called a prosthetic group. To summarize diagrammatically: < < COE NZYME INACTIVE PROTEIN+ COFACTOR METAL ION ENZYME ACTIVE PROTEIN As we shall see later, both the protein and cofactor components may be directly involved in the catalytic processes taking place. 1.2 A BRIEF HISTORY OF ENZYMES Until the nineteenth century, it was considered that processes such as the souring of milk and the fermentation of sugar to alcohol could only take place through the action of a livitJg organism. In 1833, the active agent breaking down the sugar was partially isolated and given the name diastase (now known as amylase). Sec 1.3] The naming and classification of enzymes 3 A little later, a substance which digested dietary protein was extracted from gastric juice and called pepsin. These and other active preparations were given the general name ferments. Justus von Liebig recognized that these ferments could be non-living materials obtained from living cells, but Louis Pasteur and others still maintained that ferments must contain living material. While this dispute continued, the term ferment was gradually replaced by the name enzyme. This was first proposed by Wilhelm Kfthne in 1878, and comes from the Greek, enzume (i:v?;vµq), meaning 'in yeast'. Appropriately, it was in yeast that a factor was discovered which settled the argument in favour of the inanimate theory of catalysis: brothers Eduard and Hans Buchner showed, in 1897, that sugar fermentation could take place when a yeast cell extract was added even though no living cells were present. In 1926, James Sumner crystallized urease from jack-bean extracts and, in the next few years, many other enzymes were purified and crystallized. Once pure enzymes were available, their structure and properties could be determined, and the findings form the material for most of this book. Today, enzymes still form a major subject for academic research. They are investigated in hospitals as an aid to diagnosis and, because of their specificity of action, are of great value as analytical reagents. Enzymes are still widely used in industry, continuing and extending many processes which have been used since the dawn of history. 1.3 THE NAMING AND CLASSIFICATION OF ENZYMES 1.3.1 Why classify enzymes? There is a long tradition of giving enzymes names ending in '-ase'. The only major exceptions to this are the proteolytic enzymes, i.e. ones involved in the breakdown of proteins, whose names usually end with '-in', e.g. trypsin. The names of enzymes usually indicate the substrate involved. Thus, lactase catalyses the hydrolysis of the disaccharide lactose to its component monosaccharides, glucose and galactose: (1.1) lactose glucose galactose The name lactase is a contraction of the clumsy, but more precise, lactosase. The former is used because it sounds better but it introduces a possible trap for the unwary because it could easily suggest an enzyme acting on the substrate lactate. There is nothing in the name of this enzyme or many others to indicate the type of reaction being catalysed. Fumarase, for example, by analogy with lactase might be supposed to catalyse a hydrolytic reaction, but, in fact, it hydrates fumarate to form malate: -02C.CH=CH.co2 + H20 -02C.CHOH.CH2C02 (1.2) fumarate malate 4 An Introduction to Enzymes [Ch. 1 The names of other enzymes, e.g. transcarboxylase, indicate the nature of the reaction without specifying the substrates (which in the case of transcarboxylase are methylmalonyl-CoA and pyruvate). Some names, such as catalase, indicate neither the substrate nor the reaction ( catalase mediates the decomposition of hydrogen peroxide). Needless to say, whenever a new enzyme has been characterized, great care has usually been taken not to give it exactly the same name as an enzyme catalysing a different reaction. Also, the names of many enzymes make clear the substrate and the nature of the reaction being catalysed. For example, there is little ambiguity about the reaction catalysed by malate dehydrogenase. This enzyme mediates the removal of hydrogen from malate to produce oxaloacetate: -0 C.C.CH .C0.2 + NADH + H+ (1.3) 2 2 II 0 oxaloacetate However, malate dehydrogenase, like many other enzymes, has been known by more than one name. So, because of the lack of consistency in the nomenclature, it became apparent as the list of known enzymes rapidly grew that there was a need for a systematic way of naming and classifying enzymes. A commission was appointed by the International Union of Biochemistry (later re-named the International Union of Biochemistry and Molecular Biology, IUBMB), and its report, published in 1964, forms the basis of the currently accepted system. Revised editions of the report were published in 1972, 1978, 1984 and 1992. An electronic version is now maintained by the IUBMB on an accessible web-site, and this is updated on a regular basis. 1.3.2 The Enzyme Commission's system of classification The Enzyme Commission divided enzymes into six main classes, on the basis of the total reaction catalysed. Each enzyme was assigned a code number, consisting of four elements, separated by dots. The first digit shows to which of the main classes the enzyme belongs, as follows: First digit Enzyme class Type of reaction catalysed Oxidoreductases Oxidation/Reduction reactions 2 Transferases Transfer of an atom or group between two molecules (excluding reactions in other classes) 3 Hydro lases Hydrolysis reactions 4 Lyases Removal of a group from substrate (not by hydrolysis) 5 Isomerases Isomerization reactions 6 Ligases The synthetic joining of two molecules, coupled with the breakdown of the pyrophosphate bond in a nucleoside triphosphate Sec. 1.3] The naming and classification of enzymes 5 The second and third digits in the code further describe the kind of reaction being catalysed. There is no general rule, because the meanings of these digits are defmed separately for each of the main classes. Some examples are given later in this chapter. Note that, for convenience, and in line with normal practice, some structures are written in a slightly simplified form in the lists provided. So, for example, in the case of the acyl group, which is transferred in reactions catalysed by E.C. 2.3 enzymes, it should be understood that the structure written -COR represents: -C-R II 0 Enzymes catalysing very similar but non-identical reactions, e.g. the hydrolysis of different carboxylic acid esters, will have the same first three digits in their code. The fourth digit distinguishes between them by defining the actual substrate, e.g. the actual carboxylic acid ester being hydrolysed. However, it should be noted that isoenzymes, that is to say, different enzymes catalysing identical reactions, will have the same four figure classification. There are, for example, five different isoenzymes of lactate dehydrogenase within the human body and these will have an identical code. The classification, therefore, provides only the basis for a unique identification of an enzyme. The particular isoenzyme and its source still have to be specified. It should also be noted that all reactions catalysed by enzymes are reversible to some degree and the classification which would be given to the enzyme for the catalysis of the forward reaction would not be the same as that for the reverse reaction. The classification used is that of the most important direction from the biochemical point of view, or according to some convention defined by the Commission. For example, for oxidation/reduction involving the interconversion of NADH and NAD+ (see section 11.5.2) the classification is usually based on the direction where NAD+ is the electron acceptor rather than that where NADH is the electron donor. Some problems are given at the end of this chapter to help the student become familiar with this system of classification. 1.3.3 The Enzyme Commission's recommendations on nomenclature The Commission assigned to each enzyme a systematic name in addition to its existing trivial name. This systematic name includes the name of the substrate or substrates in full and a word ending in '-ase' indicating the nature of the process catalysed. This word is either one of the six main classes of enzymes or a subdivision of one of them. When a reaction involves two types of overall change, e.g. oxidation and decarboxylation, the second function is indicated in brackets, e.g. oxidoreductase (decarboxylating). Examples are given below. The systematic name and the Enzyme Commission (E.C.) classification number unambiguously describe the reaction catalysed by an enzyme and should always be included in a report of an investigation of an enzyme, together with the source of enzyme, e.g. rat liver mitochondria.