Table Of ContentMolecular and Cellular Enzymology
Volume I
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·
Jeannine Yon-Kahn Guy Herve´
Molecular and Cellular
Enzymology
Volume I
123
Dr.JeannineYon-Kahn Prof.GuyHerve´
Universite´Paris-SudXI Universite´ParisVI
UMRCNRS CNRSUMR7631
Inst.Biochimie,Biophysique Labo.BiochimiedesSignaux
91405Orsay 96bd.Raspail
Batiment430 75006Paris
France France
[email protected] [email protected]
Originally published in French: Jeannine Yon-Kahn et Guy Hervé, Enzymologie
moléculaire et cellulaire, EDP Sciences, Collection Grenoble Sciences 2005, Tome 1,
ISBN 978-2-86883-816-2
ISBN978-3-642-01227-3 e-ISBN978-3-642-01228-0
DOI10.1007/978-3-642-01228-0
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PREFACE
Enzymology lies at the boundary of several disciplines. It has benefitted from
much progress: in physics, which has lead in particular to the determination of the
three dimensional structures of biological macromolecules and to the study of their
dynamics; in chemistry, with the development of analytical methods and processes
of synthesis; and in biology, with the potential of genetic and cell biological tech-
niques. The coincidence of these methods with those of molecular modelling today
enables us to modify an enzyme in order to make it more stable or to alter its func-
tional properties. These possibilities are of great importance not only for the pre-
cise knowledge of the mechanisms involved in catalytic events and in their regula-
tion, but also to meet the need for applications in biotechnology. Furthermore,
enzymology is of interest to diverse fields in biology and even in medicine where it
contributes, amongst other things, to the development of diagnostics for, and soon
the treatment of, genetic and metabolic diseases. Important developments are ex-
pected due to the entry of biology into the post-genomic era; it is now vital to pro-
cess the information contained within genomes i.e. the structure and function of
proteins coded for by genes. This is why we felt it necessary to bring together in a
single work the ensemble of current knowledge relating to enzymology.
Molecular and Cellular Enzymology addresses not only experienced enzymologists
but also all those, e.g. biologists, medical doctors, industrialists, who are con-
fronted by enzymological problems during their fundamental or applied research.
Equally, it addresses students who will find herein the basics, as well as the most
recent developments in the subject. We hope that this work will be able to stimu-
late in these readers an interest in this field in which an urgent need for training
exists in order to meet both the requirements of research and industrial endeavours.
This book consists of several levels: practical aspects and elementary explanations
are given for the benefit of non-specialists’ understanding; specialists will also find
topics more deeply expounded with the principal bibliographic references cited.
The bibliography, however, is not exhaustive; the choice includes general books
and review articles as well as some specialised articles. In order to facilitate the
task of students, two typographies have been adopted. The main text corresponds to
basic knowledge, whereas text in a smaller font, indented and indicated by (cid:93)(cid:92),
provides more specialised information.
VI MOLECULAR AND CELLULAR ENZYMOLOGY
Part I describes the thermodynamics of enzymatic reactions. At the very begin-
ning, in Chap. 1, there is a succinct reminder of the laws of thermodynamics at
equilibrium, the experimental methods for determining the energetic parameters of
reactions, as well as a study of coupled reactions. Chapter 2 treats protein-ligand
association equilibria and their experimental study. It concerns as much enzymolo-
gists as biologists confronted with the problem of binding molecules to receptors.
The third chapter is devoted to the study of living systems, as open systems far
from equilibrium.
Part II concerns kinetic studies of enzymatic reactions in solution. Chapter 4 is a
reminder of those elements of chemical kinetics indispensable for the understand-
ing of enzyme kinetics. In Chap. 5, all aspects of the kinetics of enzyme reactions
possessing Michaelian behaviour are worked through. The experimental methods
for studying these reactions, along with procedures for data processing and inter-
pretation, are presented in Chap. 6.
Part III covers the formation and structure of enzyme active sites. A discussion
about the origin of enzymatic function is included (Chap. 7), as well as a presenta-
tion of the formation of the functional structure of enzymes (Chap. 8). The diverse
approaches used to determine the topology of enzyme active sites are the subject of
Chap. 9.
Part IV analyses catalytic function, detailing the mechanisms brought into play in
the formation of enzyme-substrate complexes (Chap. 10), the principal catalytic
mechanisms and their involvement in enzyme catalysis (Chap. 11). To illustrate
these as an ensemble, some specific enzymatic systems are studied in Chap. 12,
which represent diverse types of catalytic mechanism. They were chosen based on
structural knowledge of the enzyme molecule and on the extent to which the rela-
tionship between the structure and functional properties has been determined.
Part V explores the regulation of enzymatic activity. It contains an analysis of the
non-covalent regulation of allosteric enzymes, the kinetics of which are treated in
Chap. 13 and their covalent regulation forms the focus in Chap. 14. Multi-functional
enzymes and multi-enzymatic systems are discussed in Chap. 15.
Part VI is devoted to enzymology in a structured environment, whether it be artifi-
cially immobilised enzymes or enzymes linked to well-defined cellular structures.
The theory of metabolic pathway control is also included.
This work thus brings together diverse aspects of enzymology, which only exist in
separate books or articles. For practical reasons we have had no hesitation in recall-
ing certain basic concepts in the different parts, so that the reader may –thanks to a
detailed Index– consult the topic of interest without being obliged to study the pre-
ceding chapters. In a similar vain, the indexing will enable precise points of interest
to be readily looked up.
PREFACE VII
ACKNOWLEDGEMENTS
The authors would like to thank deeply Drs Florence LEDERER, Charis GHELIS and
Michel DESMADRIL for their careful reading of certain parts of this work and for
their constructive suggestions, and in particular Dr Nicolas KELLERSHOHN who
took great care in checking all the equations. These thanks also go to Profs Alain
COZZONE, Bernard BADET, Christian CAMBILLAU and Jacques HAIECH for the docu-
mentation that they provided. The authors thank equally the members of the Reading
Committee, Profs G. LAUQUIN, J.P. MAZAT, B. HOUCHMANDZADEH, M. VAN DER
REST, J. LUNARDI, J. PELMONT and J. RICARD for having carefully read the manu-
script and for communicating their constructive remarks and suggestions.
The authors wish also to thank Philip SIMISTER, Lisa WALTER and Katy WOOD for
their contributions to the translation of the French version of this book and to Julie
RIDARD and Sylvie BORDAGE from Grenoble Sciences for their skilful preparation
and shaping of the two versions.
Jeannine Yon-Kahn
September 2009 Guy Hervé
TABLE OF CONTENTS
VOLUME I
GENERAL INTRODUCTION ............................................................................................................ 1
PART I – THERMODYNAMICS OF ENZYMES REACTIONS
1 – THE LAWS OF THERMODYNAMICS CONCEPT OF CHEMICAL EQUILIBRIUM ........................... 15
1.1. The laws of thermodynamics ...................................................................................... 15
1.1.1. First law.................................................................................................................. 15
1.1.2. Second law ............................................................................................................. 16
1.1.3. Third law ................................................................................................................ 18
1.1.4. Free energy ............................................................................................................. 19
1.2. Concept of equilibrium – Standard free energy .......................................................... 20
1.3. Experimental determination of thermodynamic parameters ....................................... 22
1.3.1. Enthalpy change ..................................................................................................... 22
1.3.2. Free-energy change ................................................................................................ 23
1.3.2.1. Thermochemical analysis ........................................................................................... 24
1.3.2.2. Equilibrium study ...................................................................................................... 24
1.3.2.3. Direct measurement of work supplied by the system ...................................................... 25
1.4. Coupled reactions ....................................................................................................... 27
1.4.1. Definition of energetic coupling ............................................................................ 27
1.4.2. Role of ATP ........................................................................................................... 29
1.4.3. Free energy of hydrolysis of some phosphorylated compounds ............................ 30
1.4.4. Some examples of energetic coupling .................................................................... 32
1.4.4.1. Formation of ATP from the oxidation energy of nutrients .............................................. 32
1.4.4.2. Use of the energy from ATP for chemical work ............................................................. 33
1.4.4.3. Osmotic work ............................................................................................................ 34
1.4.4.4. Mechanical work ....................................................................................................... 34
Bibliography ........................................................................................................................ 35
2 – PROTEIN-LIGAND ASSOCIATION EQUILIBRIA ........................................................................ 37
2.1. Proteins possessing a single ligand-binding site ........................................................ 37
2.2. Proteins possessing several equivalent and independent sites ................................... 38
2.3. Proteins possessing n independent and non-equivalent sites ..................................... 41
X MOLECULAR AND CELLULAR ENZYMOLOGY
2.4. Proteins possessing n equivalent but dependent sites ................................................. 42
2.4.1. Equivalent sites presenting an electrostatic dependence ........................................ 42
2.4.2. Equivalent sites presenting steric or conformational interactions .......................... 44
2.4.2.1. Phenomenological aspect ........................................................................................... 44
2.4.2.2. Interaction energy between sites ................................................................................. 46
2.4.2.3. Empirical equations .................................................................................................. 47
2.5. Linked functions .......................................................................................................... 48
2.6. Methods to study ligand binding ................................................................................. 51
2.6.1. Equilibrium dialysis ............................................................................................... 51
2.6.2. Dynamic dialysis .................................................................................................... 52
2.6.3. Measuring protein-ligand interactions in a biphasic water-polymer system .......... 55
2.6.4. Size-exclusion chromatography ............................................................................. 55
2.6.5. Ultrafiltration ......................................................................................................... 56
2.6.6. Ultracentrifugation ................................................................................................. 56
2.6.7. Direct spectrophotometric methods........................................................................ 57
2.6.8. Direct titration of a number of active sites ............................................................. 57
2.6.9. Interpretation of experimental data ........................................................................ 58
Bibliography ........................................................................................................................ 61
3 – LIVING BEINGS, OPEN SYSTEMS ........................................................................................... 63
3.1. Living beings are open systems, far from equilibrium ................................................ 63
3.1.1. Conservation of mass in open systems ................................................................... 64
3.1.2. Energy conservation in open systems: expression of the first law ......................... 65
3.1.3. Entropy production in open systems: second law .................................................. 66
3.1.4. Entropy production due to chemical reactions: chemical affinity .......................... 68
3.1.5. Entropy production and rate of irreversible phenomena ........................................ 70
3.1.5.1. Irreversible phenomena in the vicinity of the equilibrium .............................................. 70
3.1.5.2. Irreversible phenomena far from equilibrium ............................................................... 74
3.2. Exchange of matter and energy with the environment ................................................ 79
Bibliography ........................................................................................................................ 82
PART II – KINETICS OF ENZYMES REACTIONS IN SOLUTION
4 – CHEMICAL KINETICS ............................................................................................................ 85
4.1. Order of chemical reactions ....................................................................................... 85
4.1.1. Fundamental law of chemical kinetics ................................................................... 85
4.1.2. Determining the order of a reaction ....................................................................... 86
4.1.3. First-order reactions ............................................................................................... 88
4.1.4. Reversible first-order reactions .............................................................................. 90
4.1.5. Simultaneous first-order reactions.......................................................................... 92
4.1.6. Second-order reactions ........................................................................................... 93
4.1.6.1. First case: a ≠ b ...................................................................................................... 94
0 0
4.1.6.2. Second case: a = b .................................................................................................. 94
0 0
4.1.7. Reversible second-order reactions.......................................................................... 94
4.1.8. Dimerisation equilibrium ....................................................................................... 95
