Enzymes. Biochemistry, Biotechnology, Clinical Chemistry-Woodhead Publishing

419 Pages·2014·30.53 MB·English

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,
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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

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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.