Enzymes E-Book

Robert A. Copeland

• Herausgeber: John Wiley & Sons
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch

Seitenzahl: 1182

Veröffentlichungsjahr: 2023

Beschreibung

ENZYMES A complete and approachable introduction to the study of enzymes, from theory to practice Enzymes catalyze the bulk of important biological processes, both metabolic and biochemical. They are specialized proteins whose function is determined by their structure, understanding which is therefore a key focus of biological, pharmacological, and agrarian research, among many others. A thorough knowledge of enzyme structure, pathways, and mechanisms is a fundamental building block of the life sciences and all others connected to them. Enzymes offers a detailed introduction to this critical subject. It analyzes enzyme proteins at the structural level and details the mechanisms by which they perform their catalyzing functions. The book's in-depth engagement with primary literature and up-to-date research allows it to continuously deploy illustrative examples and connect readers with further research on key subjects. Fully updated after decades as the standard text, this book unlocks a thriving field of biological and biochemical research. Readers of the third edition of Enzymes will also find: * Expanded chapters on steady-state and transient-state enzyme kinetics, structural components of enzymes, and more * New chapters on enzyme regulation, enzyme-macromolecule interactions, enzyme evolution, and enzymes in human health * Key Learning Points at the beginning of each chapter to assist students and instructors Enzymes promises to continue as the standard reference on this subject for practitioners of the life sciences and related fields in both academia and industry.

Leseprobe

Table of Contents

COVER

TITLE PAGE

COPYRIGHT

DEDICATION

PREFACE TO THE THIRD EDITION

PREFACE TO THE SECOND EDITION

PREFACE TO THE FIRST EDITION

ACKNOWLEDGMENTS

1 A BRIEF HISTORY OF ENZYMOLOGY

1.1 ENZYMES IN ANTIQUITY

1.2 EARLY ENZYMOLOGY

1.3 THE DEVELOPMENT OF MECHANISTIC ENZYMOLOGY

1.4 STUDIES OF ENZYME STRUCTURE

1.5 ENZYMOLOGY TODAY

1.6 SUMMARY

REFERENCES AND FURTHER READING

2 CHEMICAL BONDS AND REACTIONS IN BIOCHEMISTRY

2.1 ATOMIC AND MOLECULAR ORBITALS

2.2 THERMODYNAMICS OF CHEMICAL REACTIONS

2.3 ACID–BASE CHEMISTRY

2.4 NONCOVALENT INTERACTIONS IN REVERSIBLE BINDING

2.5 RATES OF CHEMICAL REACTIONS

2.6 SUMMARY

REFERENCES AND FURTHER READING

3 STRUCTURAL COMPONENTS OF ENZYMES

3.1 THE AMINO ACIDS

3.2 THE PEPTIDE BOND

3.3 AMINO ACID SEQUENCE OR PRIMARY STRUCTURE

3.4 SECONDARY STRUCTURE

3.5 TERTIARY STRUCTURE

3.6 SUBUNITS AND QUATERNARY STRUCTURE

3.7 COFACTORS IN ENZYMES

3.8 CONFORMATIONAL DYNAMICS AND ENZYME FUNCTION

3.9 METHODS OF PROTEIN STRUCTURE DETERMINATION

3.10 SUMMARY

REFERENCES AND FURTHER READING

4 PROTEIN–LIGAND BINDING EQUILIBRIA

4.1 THE EQUILIBRIUM DISSOCIATION CONSTANT,

K

4.2 THE KINETIC APPROACH TO EQUILIBRIUM

4.3 BINDING MEASUREMENTS AT EQUILIBRIUM

4.4 GRAPHIC ANALYSIS OF EQUILIBRIUM LIGAND‐BINDING DATA

4.5 EQUILIBRIUM BINDING WITH LIGAND DEPLETION (TIGHT BINDING INTERACTIONS)

4.6 COMPETITION AMONG LIGANDS FOR A COMMON BINDING SITE

4.7 PROTEIN DYNAMICS IN RECEPTOR–LIGAND BINDING

4.8 ORTHOSTERIC AND ALLOSTERIC LIGAND BINDING SITES

4.9 EXPERIMENTAL METHODS FOR MEASURING LIGAND BINDING

4.10 SUMMARY

REFERENCES AND FURTHER READING

NOTE

5 STEADY‐STATE KINETICS OF SINGLE‐SUBSTRATE ENZYME REACTIONS

5.1 THE TIME COURSE OF ENZYMATIC REACTIONS

5.2 EFFECTS OF SUBSTRATE CONCENTRATION ON VELOCITY

5.3 THE RAPID EQUILIBRIUM MODEL OF ENZYME KINETICS

5.4 THE STEADY‐STATE MODEL OF ENZYME KINETICS

5.5 THE SIGNIFICANCE OF AND

K

5.6 EXPERIMENTAL MEASUREMENT OF AND

K

5.7 OTHER LINEAR TRANSFORMATIONS OF ENZYME KINETIC DATA

5.8 MEASUREMENTS AT LOW SUBSTRATE CONCENTRATIONS

5.9 DEVIATIONS FROM HYPERBOLIC KINETICS

5.10 SUMMARY

REFERENCES AND FURTHER READING

6 CHEMICAL MECHANISMS IN ENZYME CATALYSIS

6.1 Substrate–Active Site Complementarity

6.2 RATE ENHANCEMENT THROUGH TRANSITION STATE STABILIZATION

6.3 CHEMICAL MECHANISMS FOR TRANSITION STATE STABILIZATION

6.4 THE SERINE PROTEASES: AN ILLUSTRATIVE EXAMPLE

6.5 ENZYMATIC REACTION NOMENCLATURE

6.6 SUMMARY

REFERENCES AND FURTHER READING

NOTE

7 EXPERIMENTAL MEASURES OF STEADY‐STATE ENZYME ACTIVITY

7.1 INITIAL VELOCITY MEASUREMENTS

7.2 DETECTION METHODS

7.3 SEPARATION METHODS IN ENZYME ASSAYS

7.4 FACTORS AFFECTING THE VELOCITY OF ENZYMATIC REACTIONS

7.5 REPORTING ENZYME ACTIVITY DATA

7.6 ENZYME STABILITY

7.7 SUMMARY

REFERENCES AND FURTHER READING

NOTE

8 TRANSIENT‐STATE KINETICS

8.1 TIMESCALE OF PRE‐STEADY‐STATE TURNOVER

8.2 INSTRUMENTATION FOR TRANSIENT KINETIC MEASUREMENTS

8.3 ESTIMATING INITIAL CONDITIONS FOR TRANSIENT KINETIC MEASUREMENTS

8.4 EXAMPLES OF SOME COMMON TRANSIENT KINETIC REACTION MECHANISMS

8.5 EXAMPLES OF TRANSIENT KINETIC STUDIES FROM THE LITERATURE

8.6 SUMMARY

REFERENCES AND FURTHER READING

9 ENZYME REGULATION

9.1 ACTIVE AND INACTIVE CONFORMATIONAL STATES

9.2 POST‐TRANSLATIONAL MODIFICATIONS

9.3 ENZYME REGULATION THROUGH PROTEIN–PROTEIN INTERACTIONS

9.4 SMALL‐MOLECULE ALLOSTERIC LIGANDS

9.5 QUANTITATIVE MEASUREMENTS OF ENZYME ACTIVATION AND INHIBITION

9.6 REGULATION OF PROTEIN KINASES

9.7 SUMMARY

REFERENCES AND FURTHER READING

10 REVERSIBLE INHIBITORS

10.1 EQUILIBRIUM TREATMENT OF REVERSIBLE INHIBITION

10.2 THERMODYNAMIC MODES OF REVERSIBLE INHIBITION

10.3 EFFECTS OF INHIBITORS ON STEADY‐STATE PARAMETERS

10.4 CONCENTRATION‐RESPONSE PLOTS OF ENZYME INHIBITION

10.5 EFFECTS OF SUBSTRATE CONCENTRATION ON INHIBITOR CONCENTRATION–RESPONSE CURVES

10.6 MUTUALLY EXCLUSIVE BINDING OF TWO INHIBITORS

10.7 STRUCTURE–ACTIVITY RELATIONSHIPS AND INHIBITOR DESIGN

10.8 SUMMARY

REFERENCES AND FURTHER READING

11 TIGHT‐BINDING INHIBITORS

11.1 IDENTIFYING TIGHT‐BINDING INHIBITION

11.2 DISTINGUISHING INHIBITOR TYPE FOR TIGHT‐BINDING INHIBITORS

11.3 DETERMINING

K

i

FOR TIGHT‐BINDING INHIBITORS

11.4 USE OF TIGHT‐BINDING INHIBITORS TO DETERMINE ACTIVE ENZYME CONCENTRATION

11.5 SUMMARY

REFERENCES AND FURTHER READING

12 TIME‐DEPENDENT INHIBITION

12.1 PROGRESS CURVES FOR SLOW‐BINDING INHIBITORS

12.2 DISTINGUISHING BETWEEN SLOW‐BINDING SCHEMES

12.3 DISTINGUISHING BETWEEN MODES OF INHIBITOR INTERACTION WITH ENZYME

12.4 DETERMINING REVERSIBILITY

12.5 EXAMPLES OF SLOW‐BINDING ENZYME INHIBITORS

12.6 SUMMARY

REFERENCES AND FURTHER READING

13 ENZYME REACTIONS WITH MULTIPLE SUBSTRATES

13.1 REACTION NOMENCLATURE

13.2 Bi–Bi REACTION MECHANISMS

13.3 DISTINGUISHING BETWEEN RANDOM AND COMPULSORY‐ORDERED MECHANISMS BY INHIBITION PATTERN

13.4 ISOTOPE EXCHANGE STUDIES FOR DISTINGUISHING REACTION MECHANISMS

13.5 USING THE KING–ALTMAN METHOD TO DETERMINE VELOCITY EQUATIONS

13.6 CLELAND'S NET RATE CONSTANT METHOD FOR DETERMINING

V

MAX

AND

V

MAX

/

K

m

13.7 SUMMARY

REFERENCES AND FURTHER READING

14 ENZYME–MACROMOLECULE INTERACTIONS

14.1 MUTLITPROTEIN ENZYME COMPLEXES

14.2 ENZYME REACTIONS ON MACROMOLECULAR SUBSTRATES

14.3 SUMMARY

REFERENCES AND FURTHER READING

15 COOPERATIVITY IN ENZYME CATALYSIS

15.1 HISTORIC EXAMPLES OF COOPERATIVITY AND ALLOSTERY IN PROTEINS

15.2 MODELS OF ALLOSTERIC BEHAVIOR

15.3 EFFECTS OF COOPERATIVITY ON VELOCITY CURVES

15.4 SIGMOIDAL KINETICS FOR NONALLOSTERIC ENZYMES

15.5 SUMMARY

REFERENCES AND FURTHER READING

16 EVOLUTION OF ENZYMES

16.1 EARLY EARTH CONDITIONS

16.2 NATURAL SELECTION

16.3 GENETIC ALTERATIONS

16.4 ENZYME FAMILIES AND SUPERFAMILIES

16.5 ENZYME PROMISCUITY AS A SPRINGBOARD OF EVOLUTION

16.6 PROTEIN DYNAMICS AND CONFORMATIONAL SELECTION IN EVOLUTION OF NEOFUNCTIONALITY

16.7 ANCESTRAL ENZYME RECONSTRUCTION

16.8 CONTEMPORARY ENZYME EVOLUTION

16.9 SUMMARY

REFERENCES AND FURTHER READING

17 ENZYMES IN HUMAN HEALTH

17.1 ENZYMES AS THERAPEUTIC AGENTS

17.2 ENZYME INHIBITORS AS THERAPEUTIC AGENTS

17.3 ENZYME ESSENTIALITY IN DISEASE

17.4 ENZYME‐MEDIATED TARGET PROTEIN DEGRADATION

17.5 THE ROLE OF ENZYMOLOGY IN DRUG DISCOVERY AND DEVELOPMENT

17.6 SUMMARY

REFERENCES AND FURTHER READING

INDEX

END USER LICENSE AGREEMENT

List of Tables

Chapter 1

Table 1.1 Examples of enzyme inhibitors as potential drugs

Chapter 2

Table 2.1 Electronic configurations of the elements most commonly found in b...

Table 2.2 Values of some useful physicochemical constants.

Table 2.3 Examples of Brønsted–Lowry acids and their conjugate bases.

Table 2.4 Hydrogen bond lengths for H bonds found in proteins.

Table 2.5 Van der Waals radii for atoms in proteins.

Table 2.6 Extent of reaction completion relative at times corresponding to d...

Table 2.7 Reaction order for a few simple chemical reactions.

Chapter 3

Table 3.1 Properties of the natural amino acids

Table 3.2 Typical bond lengths for carbon–oxygen and carbon–nitrogen bonds

Table 3.3 Examples of multisubunit enzymes

Table 3.4 Examples of cofactors found in enzymes

Chapter 4

Table 4.1 Relationship between

K

d

and Δ

G

binding

for receptor–ligand complexe...

Table 4.2 Relationship between ligand concentration relative to

K

d

([L]/

K

d

) ...

Chapter 5

Table 5.1 Relationship between steady‐state kinetic constants and specific s...

Table 5.2 Initial velocity (with random error added) as a function of substr...

Table 5.3 Some popular linear plotting methods for steady‐state enzyme kinet...

Table 5.4 Estimates of the kinetic constants

V

max

and

K

m

from various graphi...

Table 5.5 Setup for an experimental determination of enzyme kinetics using a...

Chapter 6

Table 6.1 Some examples of enzyme nucleophiles and the covalent intermediate...

Table 6.2 Some examples of electrophilic catalysis in enzymatic reactions

Table 6.3 Steady‐state kinetics of synthetic peptide hydrolysis by pepsin...

Table 6.4 Enzyme classes and subclasses as designated by the recommendations...

Table 6.5 Some examples of enzyme common names and their EC designations

Chapter 7

Table 7.1 Values of

ϕ

, for [B] = 99%, [B]

ss

, useful in designing couple...

Table 7.2 Volumes of stock solutions to prepare experimental and control sam...

Table 7.3 Donor–acceptor pairs for quenching by resonance energy transfer in...

Table 7.4 Properties of radioisotopes that are commonly used in enzyme kinet...

Table 7.5 Results of a hypothetical reaction of dihydroorotate dehydrogenase...

Table 7.6 Some buffers that are useful in enzyme studies

Table 7.7 Examples of amino acid residues with perturbed side chain p

K

, valu...

Table 7.8 Volume of 0.2 M NaOH to be added to 50 mL of 0.2 M Trismaleate sto...

Chapter 8

Table 8.1 Timescale for 95% and 99% reaction completion for different values...

Table 8.2 Timescales of some common transient kinetic methods

Table 8.3 Kinetic and thermodynamic values for PDF inhibition by actinonin...

Chapter 9

Table 9.1 Different forms of hemophilia and the coagulation factor deficienc...

Table 9.2 Examples of PTMs of specific amino acid side chains

Table 9.3 Examples of protein domains that selectively recognize and bind po...

Table 9.4 Some examples of enzyme pairing for writing and erasing specific p...

Table 9.5 Some examples of enzyme activation by protein binding to the catal...

Table 9.6 Some examples of enzymes and corresponding natural protein inhibit...

Table 9.7 Synthetic small‐molecule activators and natural regulators of enzy...

Table 9.8 Impact of TPX2 (1‐43) binding on the steady‐state constants for Au...

Chapter 10

Table 10.1 Effects of different inhibition modalities on steady‐state parame...

Table 10.2 Diagnostic signatures of reversible inhibition modality in double...

Table 10.3 Hypothetical velocity as a function of substrate concentration at...

Table 10.4 Diagnostic patterns of IC

50

changes as a function of [S]/

K

m

for d...

Chapter 12

Table 12.1 Time‐dependent kinetic parameters for carboxylate inhibitors of P...

Table 12.2 Some examples of amino acid–selective chemical modifying agents

Chapter 13

Table 13.1 General nomenclature for enzymatic reactions

Table 13.2 Patterns of dead‐end inhibition observed for the Bi–Bi reaction E...

Table 13.3 Pattern of product inhibition observed for the Bi–Bi reaction E +...

Table 13.4 King‐Altman relationships for a double displacement Bi–Bi reactio...

Chapter 14

Table 14.1 Examples of enzymes that function as multiprotein complexes

Table 14.2 Some examples of enzymes that act on macromolecular substrates

Table 14.3 Some examples of solution conditions that impact protein–protein ...

Table 14.4 Effects of KCl concentration on the steady‐state kinetics and mon...

Table 14.5 Some examples of enzymes that perform multiple rounds of catalysi...

Chapter 16

Table 16.1 The standard DNA codon usage

Table 16.2 Representative enzyme superfamilies

a

Table 16.3 Definitions of terms associated with enzyme promiscuity

Table 16.4 Median values and ranges of steady‐state parameters for contempor...

Chapter 17

Table 17.1 Some physicochemical properties of drug‐like molecules

Table 17.2 Potency of selective inhibitors for human and

H. pylori

DHODase...

Table 17.3 Cellular activity (cell killing) of compound 1 from Copeland et a...

Table 17.4 Some examples of enzyme inhibitors as therapeutic agents for life...

Table 17.5 Examples of oncogenic chromosomal translocations involving protei...

Table 17.6 Enzymatic hydrolysis and solubility of selected prodrugs

Table 17.7 PROTAC programs in clinical development or late‐stage preclinical...

Table 17.8 Some examples of proximal readouts of intracellular enzymatic act...

Table 17.9 The elements of translational chemical biology

List of Illustrations

Chapter 1

Figure 1.1 Photomicrograph of urease crystals (728× magnification), the firs...

Chapter 2

Figure 2.1 Spatial representations of the electron distribution in s and p o...

Figure 2.2 Electron spin represented as rotation of a particle in a magnetic...

Figure 2.3 The Aufbau principle for the order of filling of atomic orbitals,...

Figure 2.4 (A) Schematic representation of two s orbitals on separate hydrog...

Figure 2.5 Combination of two p

z

atomic orbitals by end‐to‐end overlap to fo...

Figure 2.6 Spatial electron distributions of hybrid orbitals: (A) sp hybridi...

Figure 2.7 Hybrid bond formation in ethylene. (A) The bonds are illustrated ...

Figure 2.8 Two common representations tor the benzene molecule. The represen...

Figure 2.9 Enthalpy of hydrogenation observed (black circles) for cyclohexen...

Figure 2.10 Potential energy diagram for the ground and one excited electron...

Figure 2.11 Free energy diagram for the reaction profile of a typical chemic...

Figure 2.12 Exponential decrease in reaction rate (represented by reaction h...

Figure 2.13 Free energy diagram for a chemical reaction that proceeds throug...

Figure 2.14 Hypothetical titration curve for a weak acid illustrating the gr...

Figure 2.15 Potential energy diagram for the van der Waals attraction betwee...

Figure 2.16 Van der Waals radii for the atoms of the amino acid alanine. Fig...

Figure 2.17 Progress curves of product development (circles) and substrate l...

Figure 2.18 Product formation as a function of time for a typical enzyme‐cat...

Chapter 3

Figure 3.1 Side‐chain structures of the 20 natural amino acids. The entire p...

Figure 3.2 Tyrosine participation in hydrogen bonding as (A) a hydrogen dono...

Figure 3.3 Salt bridge formation between a lysine and a glutamic acid residu...

Figure 3.4 The protein coordination sphere of the active‐site zinc of carbon...

Figure 3.5 The structures of the phosphorylated forms of serine, threonine, ...

Figure 3.6 Schematic diagram of a typical peptide bond; numbers are typical ...

Figure 3.7 The cis and trans configurations of the peptide bond.

Figure 3.8 (A) Comparison of the SET domain amino acid sequences of human EZ...

Figure 3.9 The dihedral angles of rotation for an amino acid in a peptide ch...

Figure 3.10 A Ramachandran plot for the amino acid alanine.

Figure 3.11 The right‐handed alpha helix (With kind permission from Dr. Gord...

Figure 3.12 (A) An antiparallel

β

‐pleated sheet composed of two segment...

Figure 3.13 Three common forms of

β

turns.

Figure 3.14 Examples of supersecondary structures: (A) a helical bundle, (B)...

Figure 3.15 Schematic diagram of the secondary structure arrangement of the ...

Figure 3.16 The folding of a polypeptide chain illustrating the hierarchy of...

Figure 3.17 The catalytic triad of the serine protease

α

‐chymotrypsin. ...

Figure 3.18 Cartoon illustration of the domains of a typical transmembrane r...

Figure 3.19 (A) Schematic representation of the multiple domain structure of...

Figure 3.20 Cartoon illustrating the changes in subunit arrangements for a h...

Figure 3.21 Cartoon illustration of the quaternary structure changes that ac...

Figure 3.22 Examples of some common cofactors found in enzymes. The R group ...

Figure 3.23 Structures of the flavin cofactor in its various oxidation state...

Figure 3.24 Covalent adduct formation by flavin cofactors in enzymes. (A) Ox...

Figure 3.25

(

A) Energy landscape for carbon monoxide–bound myoglobin (MbCO), ...

Figure 3.26 (A) Energy landscape for a hypothetical enzyme that can exist in...

Figure 3.27 (A) A microcentrifuge tube as an analogy for the open and closed...

Figure 3.28 Schematic representation of the evolution of conformational micr...

Figure 3.29 Top: illustration of a wave train for the electrical component o...

Figure 3.30 (A) Schematic illustration of the basic set‐up for X‐ray irradia...

Chapter 4

Figure 4.1 van't Hoff plot of the binding of

3

H‐naltrindole to the mouse bra...

Figure 4.2 Time course for approach to equilibrium after mixing of a recepto...

Figure 4.3 Plot of the observed pseudo‐first‐order rate constant (

k

obs

) as a...

Figure 4.4 (A) Langmuir isotherm for formation of the binary RL complex as a...

Figure 4.5 Langmuir isotherm for a receptor with two equivalent ligand bindi...

Figure 4.6 Langmuir isotherm for a receptor with multiple, nonequivalent lig...

Figure 4.7 Idealized data for a ligand‐binding experiment; the data show the...

Figure 4.8 Langmuir isotherm plot on a semilog scale, illustrating the relat...

Figure 4.9 Semilog plot of a Langmuir isotherm for a receptor with multiple,...

Figure 4.10 Double‐reciprocal plot for the data in Figure 4.4. The data in t...

Figure 4.11 (A) Scatchard plot for the ligand‐binding data presented in Figu...

Figure 4.12 Hanes–Wolff plot for the ligand‐binding data from Figure 4.4. In...

Figure 4.13 Binding isotherm for competitive ligand binding.

B

, the fraction...

Figure 4.14 Thermodynamic cycle representing two limiting models of two‐step...

Figure 4.15 Schematic illustration of an equilibrium dialysis apparatus afte...

Figure 4.16 Time course for approach to equilibrium between two chambers of ...

Figure 4.17 (A) Schematic illustration of a 96‐well filter binding apparatus...

Figure 4.18 Binding of 2

′

‐cytidylic acid to the enzyme ribonuclease as...

Figure 4.19 Schematic diagram of a typical setup for MST instrumentation. Se...

Figure 4.20 Schematic illustration of fluorescence polarization, in which a ...

Figure 4.21 Schematic diagram of a typical SPR experimental setup.

Figure 4.22 Idealized SPR sensogram for a ligand binding to and dissociating...

Figure 4.23 Thermal shift analysis of protein denaturation. (A) A typical da...

Figure 4.24 Urea titration of wild‐type HIV‐1 protease in the absence (open ...

Figure 4.25 Correlation plot of Δ

C

M

as a function of

RT

ln(

K

d

) for eight lig...

Chapter 5

Figure 5.1 Reaction progress curves for the loss of substrate [S] and produc...

Figure 5.2 Reaction progress curve for the production of the product during ...

Figure 5.3 (A) Progress curves for a set of enzyme‐catalyzed reactions with ...

Figure 5.4 Development of the steady state for the reaction of cytochrome

c

...

Figure 5.5 (A) Michaelis–Menten plot for the velocity data in Table 5.2. The...

Figure 5.6 Michaelis–Menten plots for restricted data from Table 5.2. (A) Th...

Figure 5.7 Experimental strategy for estimating

K

m

and

V

max

. (A) A limited d...

Figure 5.8 Lineweaver–Burk double‐reciprocal plot for selected data from Tab...

Figure 5.9 Lineweaver–Burk double‐reciprocal plot for the full data set from...

Figure 5.10 Eadie–Hofstee plot of enzyme kinetic data.

Figure 5.11 Hanes–Wolff plot of enzyme kinetic data.

Figure 5.12 Eisenthal–Cornish‐Bowden direct plot of enzyme kinetic data.

Figure 5.13 Michaelis–Menten plot for an enzyme reaction displaying substrat...

Figure 5.14 Effects of multiple enzymes acting on the same substrate. The da...

Figure 5.15 Effects of positive cooperativity on the kinetics of an enzyme‐c...

Figure 5.16 Hill plots for the data from Figure 5.15: log[

v

/(

V

max

−

v

)] is p...

Chapter 6

Figure 6.1 Generic scheme for an enzyme‐catalyzed reaction showing the compo...

Figure 6.2 Schematic illustration of the lock and key model of enzyme–substr...

Figure 6.3 Illustration of three‐point attachment in enzyme–substrate intera...

Figure 6.4 Energy level diagram of an enzyme‐catalyzed reaction and the corr...

Scheme 6.1

Figure 6.5 Illustration of burst phase kinetics. The data represents the pro...

Figure 6.6 Brønsted plots for nucleophilic attack of

p

‐nitrophenyl acetate b...

Figure 6.7 Examples of reactions of amino acids facilitated by electrophilic...

Figure 6.8 Effect of general acid or base concentration on the rate of react...

Figure 6.9 Effect of pH on the rate of reaction for (A) a general base‐catal...

Figure 6.10 Transition state stabilization by a general acid (A) or general ...

Figure 6.11 The rack model of enzyme–substrate interaction and catalysis:

1

,...

Figure 6.12 Schematic illustration of the nonproductive binding model. In th...

Figure 6.13 Schematic illustration of the induced‐strain model: the free enz...

Figure 6.14 The protease subsite nomenclature of Schechter and Berger (1967)...

Figure 6.15 Schematic representation of the general acyl transfer mechanism ...

Figure 6.16 Substrate–active site interactions in the serine proteases. (A) ...

Chapter 7

Figure 7.1 (A) Absorption of ferrocytochrome c as a function of time after a...

Figure 7.2 Typical data for a coupled enzyme reaction illustrating the lag p...

Figure 7.3 Effects of inhibitors on a coupled enzyme reaction: circles, the ...

Figure 7.4 Examples of a progress curve tor an enzyme demonstrating burst ph...

Figure 7.5 A common strategy for initiating an enzymatic reaction in a micro...

Figure 7.6 The importance of running blank controls. (A) Time courses of abs...

Figure 7.7 A typical absorption spectrum of a molecule with an absorption ma...

Figure 7.8 Energy level diagrams for (A) a light‐induced transition from a

π

...

Figure 7.9 Example of the use of difference spectroscopy. (A) Absorption spe...

Figure 7.10 Deviation from Beer's law. Over a small concentration range, the...

Figure 7.11 Resonance energy transfer. In an energy transfer experiment, the...

Figure 7.12 Schematic diagram illustrating the inner filter effect. When a d...

Figure 7.13 Errors in enzyme kinetic measurements due to fluorescence inner ...

Figure 7.14 Radioassay for dihydroorotate dehydrogenase, measuring the incor...

Figure 7.15 Schematic diagram of a TLC‐based enzyme assay. In step 1, a samp...

Figure 7.16 Autoradiograph of a TLC plate demonstrating separation of

14

C‐la...

Figure 7.17 Typical signal from an HPLC chromatograph of a molecule. The sam...

Figure 7.18 Schematic diagram of a protease assay based on SDS‐PAGE separati...

Figure 7.19 Example of activity staining of an enzyme after gel electrophore...

Figure 7.20 Gelatin zymography of a whole cell lysate from Sf9 insect cells ...

Figure 7.21 The relative velocity of an enzymatic reaction, under controlled...

Figure 7.22 The effects of pH on the velocity of a typical enzymatic reactio...

Figure 7.23 (A) Cartoon of the active site structure of α‐chymotrypsin, base...

Figure 7.24 Plot of log(

k

cat

/

K

m

) as a function of pH for a typical enzymatic...

Figure 7.25 Typical profile of the relative activity of an enzyme as a funct...

Figure 7.26 Arrhenius plot of log(

V

max

) of an enzymatic reaction as a functi...

Figure 7.27 Example of a reaction progress curve showing a long lag phase be...

Figure 7.28 Effects of microviscosity on

k

cat

/K

m

for an enzymatic reaction t...

Figure 7.29 Potential energy diagram for an electronic state of a molecule i...

Figure 7.30 Proton inventory plot for reactions involving transfer of one (o...

Figure 7.31 Selwyn's test for enzyme inactivation during an assay. (A) Data ...

Chapter 8

Figure 8.1 Example of burst phase kinetics followed by steady‐state kinetics...

Figure 8.2 Schematic diagram of a typical stopped‐flow instrument for rapid ...

Figure 8.3 Schematic diagram of a typical rapid quench instrument for rapid ...

Figure 8.4 Examples of the form of signal changes, as a function of time, co...

Scheme 8.1

Scheme 8.2

Scheme 8.3

Figure 8.5 Time courses for species A (black circles), B (open circles), and...

Scheme 8.4

Figure 8.6 Replot of

k

obs

as a function of ligand concentration ([L]) for a ...

Scheme 8.5

Figure 8.7 Schematic diagram of the two‐step mechanisms of substrate (top) a...

Figure 8.8 Simulation of the concentrations of species E, EL, and E*L as a f...

Chapter 9

Figure 9.1 Potential energy landscapes for the two conformations of a regula...

Figure 9.2 Schematic diagram of the two blood coagulation pathways that oper...

Figure 9.3 The intrinsic (apoptosome‐mediated) and extrinsic (extracellular ...

Figure 9.4 The three‐enzyme cascade leading to ubquitinylation of a target p...

Figure 9.5 Effect of poly‐Ub chain length (

n

) on the affinity of DHFR‐Ub

n

fo...

Figure 9.6 Chemical structures of serine, phospho‐serine, threonine, phospho...

Figure 9.7 Example of charge‐pairing between arginine 165 (Arg165) and phosp...

Figure 9.8 (A) Schematic representation of the domain structure of human SAR...

Figure 9.9 Schematic illustration of feedback inhibition of a metabolic path...

Figure 9.10 Schematic illustration of the domain interactions in the closed ...

Figure 9.11 Example of enzyme velocity changes as a function of activator co...

Figure 9.12 (A) Structure of the protein kinase PKA showing key structural c...

Figure 9.13 Relative effects of activation segment phosphorylation on the af...

Figure 9.14 Two‐step activation mechanism of PKA. See text for further detai...

Figure 9.15 Ligand binding‐induced activation of RTKs through dimerization, ...

Figure 9.16 Chemical structures of small‐molecule modulators of cAbl kinase ...

Chapter 10

Figure 10.1 Equilibrium scheme for enzyme turnover in the presence and absen...

Figure 10.2 Untransformed (A) and double‐reciprocal (B) plots for the effect...

Figure 10.3 Secondary plot of

K

m

as a function of inhibitor concentration [I...

Figure 10.4 Dixon plot (1/

v

as a function of [I]) for a competitive inhibito...

Figure 10.5 Patterns of lines in the double‐reciprocal plots for mixed (nonc...

Figure 10.6 Secondary plots for the determination of the inhibitor constants...

Figure 10.7 Pattern of lines in the double‐reciprocal plot of an uncompetiti...

Figure 10.8 Direct plots of velocity as a function of substrate concentratio...

Figure 10.9 Concentration–response plot of enzyme fractional activity as a f...

Figure 10.10 Effect of substrate concentration on the IC

50

value of a compet...

Figure 10.11 Concentration–response (A) and Dixon (B) plots for a partial in...

Figure 10.12 Effect of [S]/

K

m

on the IC

50

value of inhibitors of different m...

Figure 10.13 Scheme for simultaneous titration of inhibitor and substrate fo...

Figure 10.14 (A) Yonetani–Theorell plot for two inhibitors I and J that bind...

Figure 10.15 Chemical structures of the substrate (dihydrofolate) and two ty...

Figure 10.16 QSAR correlation plots for the potencies of pteridines (A) and ...

Figure 10.17 (A) Proposed structure of the tetrahedral transition state of t...

Figure 10.18 Interactions of the dihydrofolate reductase active site with th...

Chapter 11

Figure 11.1 (A) Concentration‐response plot of fractional velocity as a func...

Figure 11.2 Double‐reciprocal plot for a tight‐binding competitive inhibitor...

Figure 11.3 (A) The effects of substrate concentration on the IC

50

values of...

Figure 11.4 (A) Determination of “

K

” by the graphical method of Dixon (1972)...

Figure 11.5 Henderson plot for a tight‐binding inhibitor.

Figure 11.6 Plot of fractional velocity as a function of inhibitor concentra...

Figure 11.7 Determination of active enzyme concentration by titration with a...

Figure 11.8 Determination of active enzyme concentration by titration of a f...

Chapter 12

Figure 12.1 Schemes for time‐dependent enzyme inhibition. Scheme A, which de...

Figure 12.2 Examples of progress curves in the presence of varying concentra...

Figure 12.3 Examples of progress curves in the presence of varying concentra...

Figure 12.4 Preincubation time dependence of the fractional velocity of an e...

Figure 12.5 Plot of

k

obs

as a function of inhibitor concentration for a slow...

Figure 12.6 Plot of

k

obs

as a function of inhibitor concentration for a slow...

Figure 12.7 (A) Plot of

k

obs

as a function of inhibitor concentration for a ...

Figure 12.8 (A) Plot of

k

obs

as a function of inhibitor concentration for in...

Figure 12.9 Expected dependence of

k

obs

on substrate concentration for time‐...

Figure 12.10 Examples of slow‐binding α‐aminoboronate peptide inhibitors of ...

Figure 12.11 Examples of carboxylate‐containing NSAIDs that act as slow‐bind...

Figure 12.12 Affinity labeling of PGHS by the bromoacetamido analogue of the...

Figure 12.13 Chemical structures of DuP697 and the generic form of a PGHS2 s...

Figure 12.14 Tsou plot of (

v

i

/

v

0

)

1/

x

as a function of

z

for modification of ...

Figure 12.15 Substrate protection against inactivation by chemical modificat...

Figure 12.16 Examples of photoaffinity labels that can be incorporated into ...

Chapter 13

Figure 13.1 Double‐reciprocal plot for a random ordered bi–bi enzymatic reac...

Figure 13.2 (A) Slope and (B)

y

intercept replots of the data from Figure 13...

Figure 13.3 Double‐reciprocal plot for a double‐displacement (Ping–Pong) bi–...

Figure 13.4 Replots of the data from Figure 13.3 as (A) 1/ versus 1/[B] and...

Figure 13.5 Plots of the equilibrium rate of radioisotope exchange between B...

Chapter 14

Figure 14.1 Plot of binding efficiency as a function of buried surface area ...

Figure 14.2 Plot of normalized values

k

cat

(triangles),

K

m

of ATP (squares),...

Figure 14.3 Cartoon representation of a protein substrate binding to an enzy...

Figure 14.4 Group incorporation into substrate for a distributive (left) and...

Figure 14.5 Time course of substrate (S) depletion (open circles), intermedi...

Figure 14.6 Effect of alternative substrate addition on the time course of p...

Chapter 15

Figure 15.1 Plot of bound molecular oxygen as a function of oxygen concentra...

Figure 15.2 Changes in structure of the active site heme that accompany O

2

b...

Figure 15.3 Cartoons of the interactions of the Trp repressor protein with T...

Figure 15.4 Schematic illustration of the number of possible forms of ligand...

Figure 15.5 Schematic representation of the equilibria involved in substrate...

Figure 15.6 Extension of the Koshland model, from Figure 15.5 to a tetrameri...

Figure 15.7 Schematic representation of the equilibria involved in the bindi...

Figure 15.8 Velocity as a function of substrate concentration plotted in a s...

Chapter 16

Figure 16.1 Evolutionary consequences of gene duplication. In this cartoon, ...

Figure 16.2 Karyotype from a CML patient bearing the Philadelphia chromosome...

Figure 16.3 Schematic illustration of the chromosomal translocation process ...

Figure 16.4 (A) Hand‐drawn ribbon diagram of one monomer from the crystal st...

Figure 16.5 (A and B) Phylogenic analysis, displayed as dendrograms, for the...

Figure 16.6 The magnitude of promiscuous activity (quantified as

k

cat

/

K

M

P

/

k

c

...

Figure 16.7 Schematic illustration of evolution of specialized enzymes (A–D)...

Figure 16.8 Evolution of alternative activity from promiscuous founder enzym...

Figure 16.9 Schematic representation of the experimental process of ancestra...

Figure 16.10 Cartoon illustrating the different mechanism of Gleevec binding...

Figure 16.11 Improvements in catalytic efficiency with number of cycles of l...

Chapter 17

Figure 17.1 Illustration of the concept of surface roughness by analogy to a...

Figure 17.2 Distribution of FDA‐approved anticancer therapeutics by biochemi...

Figure 17.3 Hydrolysis of lactam rings by the enzyme β‐lactamase and example...

Figure 17.4 Structures of ubiquinone and menaquinone cofactors used by human...

Figure 17.5 (A) Comparison of structures of the reaction intermediate specie...

Figure 17.6 Comparison of systemic levels of testosterone and DHT in normal ...

Figure 17.7 Driver amplified genes identified in human cancers by enzymatic ...

Figure 17.8 Schematic illustration of the MAP kinase pathway. Extracellular ...

Figure 17.9 (A) FDA‐approved reversible inhibitors of EGFR kinase activity. ...

Figure 17.10 Analysis of lymphomagenic mutations of EZH2. (A) Steady state v...

Figure 17.11 Chemical structure of tazemetostat (Tazverik®), the first prote...

Figure 17.12 Chemical structures of inhibitors of enzymes involved in oncoge...

Figure 17.13 The concept of synthetic lethality. (A) Genetic synthetic letha...

Figure 17.14 Chemical structures of FDA‐approved PARP inhibitors.

Figure 17.15 (A) Schematic illustration of the PROTAC approach. A heterobifu...

Figure 17.16 Stages of preclinical drug discovery and development and some o...

Figure 17.17 Concentration‐response plots for inhibition of two hypothetical...

Figure 17.18 (A) Plot of hypothetical data showing a correlation between com...

Guide

Cover

Table of Contents

Title Page

Copyright

Dedication

Preface to the Third Edition

Preface to the Second Edition

Preface to the First Edition

Acknowledgments

Begin Reading

Index

End User License Agreement

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ENZYMES

A Practical Introduction to Structure, Mechanism, and Data Analysis

THIRD EDITION

Robert A. Copeland

This third edition first published 2023© 2023 John Wiley & Sons, Inc.

Edition HistoryJohn Wiley & Sons, Inc. (1e, 1996); John Wiley & Sons, Inc. (2e, 2000)

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Library of Congress Cataloging‐in‐Publication DataNames: Copeland, Robert Allen, author.Title: Enzymes : a practical introduction to structure, mechanism, and data analysis / Robert A. Copeland.Description: Third edition. | Hoboken, NJ : Wiley, 2023. | Revised edition of: Enzymes / Robert A. Copeland. 2nd ed. c2000. | Includes bibliographical references and index.Identifiers: LCCN 2022048916 (print) | LCCN 2022048917 (ebook) | ISBN 9781119793250 (cloth) | ISBN 9781119793281 (adobe pdf) | ISBN 9781119793298 (epub)Subjects: LCSH: Enzymes. | Enzymology.Classification: LCC QP601 .C753 2023 (print) | LCC QP601 (ebook) | DDC 572/.7–dc23/eng/20221128LC record available at https://lccn.loc.gov/2022048916LC ebook record available at https://lccn.loc.gov/2022048917

Cover Design: WileyCover Image: Courtesy of Robert A. Copeland

   To Clyde Worthen for teaching me all the important lessons: arigato sensei.

   And to Theodore (Doc) Janner for stoking the fire.

PREFACE TO THE THIRD EDITION

“Twenty years now

Where'd they go?

Twenty years

I don't know

I sit and I wonder sometimes

Where they've gone”

–Like a Rock, Bob Seger

When Jonathan Rose, my editor at Wiley, first approached me about writing the third edition of this book, I was a bit surprised. I asked him why he thought about the new edition. I was quite shocked when he reminded me that it had been twenty years since the publication of the last edition. Time truly does fly, and the biochemical community has continued to march on, making many novel discoveries and developing a plethora of new and improved tools along the way. Yet, despite these great advances, the fundamental science of enzymology has retained its primacy across every aspect of the life sciences—from basic to applied life sciences. Indeed, the preeminence of enzymology is what first motivated me to write an introductory book that would serve the needs of students and newcomers to the field, as well as serve as a key reference for laboratory‐based researchers. Hence, it has been particularly rewarding for me to see the previous edition of this text widely used by the scientific community, as evidenced by it being cited in the primary scientific literature more than 1,800 times. It was clear, however, that the previous edition needed updating, and this is what ultimately convinced me to accept Jonathan's recommendation to write the third edition.

In this new edition, I have refined, augmented, and updated each of the chapters that were presented in the second edition. Beyond this, I have added new chapters to cover, or better cover, aspects of enzymology that have gained greater importance over the past two decades, and that were under‐represented in the previous edition. For example, I have broken out steady‐state (Chapter 7) and transient‐state (Chapter 8) enzyme kinetics into separate chapters, to better cover both these important approaches. The chapter on structural components of enzymes (Chapter 3) has been augmented with coverage of methods for protein structure determination. Likewise, newer experimental approaches have been added to the chapters on protein–ligand binding equilibria (Chapter 4), experimental measures of steady‐state enzyme activity (Chapter 7), and the chapters on enzyme inhibitors (Chapters 10-12). Completely new chapters have been added to this new edition, to cover the expanding areas of enzyme regulation (Chapter 9), enzyme–macromolecule interactions (Chapter 14), enzyme evolution (Chapter 16), and enzymes in human health (Chapter 17). Finally, I have also added Key Learning Points at the beginning of each chapter, to aid students and educators in effectively using this book as a classroom text. This idea was suggested to me by my friend and colleague, William Pitts, for the second edition of my allied book Evaluation of Enzyme Inhibitors in Drug Discovery (Wiley) and has been widely appreciated by the academic community; I hope this proves equally valuable for the present text.

With these changes, it is my hope that this book will continue to serve well the needs of the basic and applied life sciences communities. As with previous editions, the current text is intended as an introduction to enzymes and a useful reference source for those conducting laboratory‐based enzymology research. I have tried to cover all the topics within the book in an accurate and complete manner, but also have strived to use an approachable style, so that newcomers to the field of enzymology can be initiated into this critical science in a welcoming and unintimidating fashion. For those wishing to delve into specific areas of enzymology in greater depth, sufficient references and further reading suggestions are offered at the end of every chapter.

As with all my scientific writing, I have benefitted greatly from comments and suggestions from many colleagues and friends. Beyond those acknowledged in the earlier editions of this text, I wish to thank Christopher T. Walsh, John Kozarich, Vern Schramm, David Swinney, James Wells, Thomas Meek, Margaret Porter Scott, Shane Buker, Chuang Lu, Ann Boriack‐Sjodin, Scott Ribich, Kenneth Duncan, and all the employees, board members, and scientific advisors of Accent Therapeutics, Inc. I also wish to thank Dr. Allen Sickmier for his help with some of the illustrations in this edition. A special thank you to Professor Karen Allen and her laboratory at Boston University, for providing detailed comments on several of the new chapters in this edition.

Finally, and always, I thank my family—my wife Nancy and our daughters, Lindsey and Amanda; they are my constant and never‐failing sources of learning, laughing, and love.

ROBERT A. COPELAND

Lexington, Massachusetts

2022

PREFACE TO THE SECOND EDITION

In the four years since the first edition of Enzymes was published, I have been delighted to learn of the wide acceptance of the book throughout the biochemical community, particularly in the pharmaceutical community. During this time, a number of colleagues have contacted me to express their views on the value of the text, and importantly to make suggestions for improvements to the content and presentation of some concepts. I have used the first edition as a teaching supplement for a course in which I lecture at the University of Pennsylvania School of Medicine. From my lecture experiences and from conversations with students, I have developed some new ideas for how to better explain some of the concepts in the text and have identified areas that deserve expanded coverage. Finally, while the first edition has become popular with students and industrial scientists, some of my academic colleagues have suggested a need for a more in‐depth treatment of chemical mechanisms in enzymology.

In this second edition, I have refined and expanded the coverage of many of the concepts in the text. To help the reader better understand some of the interactions between enzymes and their substrates and inhibitors, a new chapter on protein–ligand binding equilibria has been added (Chapter 4). The chapters on chemical mechanisms in enzyme catalysis (Chapter 6) and on experimental measures of enzyme activity (Chapter 7) have been expanded significantly. The discussions of enzyme inhibitors and multiple substrate reactions (Chapters 8 through 8) have been refined, and, in some cases, alternative treatments have been presented. In all of this, however, I have tried to maintain the introductory nature of the book. There are many excellent advanced texts on catalysis, enzyme mechanisms, and enzyme kinetics, but the level at which these are generally written is often intimidating to the beginner. Hence, as stated in the preface to the first edition, this book is intended to serve as a mechanism for those new to the field of enzymology to develop a reasonable understanding of the science and experimental methods, allowing them to competently begin laboratory studies with enzymes. I have continued to rely on extensive citations to more advanced texts and primary literature as a means for the interested reader to go beyond the treatments offered here and delve more deeply into specific areas of enzymology.

In developing this second edition, I have had fruitful conversations and advice from a number of colleagues. In particular, I wish to thank Andy Stern, Ross Stein, Trevor Penning, Bill Pitts, John Blanchard, Dennis Murphy, and the members of the Chemical Enzymology Department at the DuPont Pharmaceuticals Company. As always, the love and support of my family have been most important in making this work possible.

ROBERT A. COPELAND

Wilmington, Delaware

2000

PREFACE TO THE FIRST EDITION

The latter half of this century has seen an unprecedented expansion in our knowledge and use of enzymes in a broad range of basic research and industrial applications. Enzymes are the catalytic cornerstones of metabolism, and as such is the focus of intense research within the biomedical community. Indeed, enzymes remain the most common targets for therapeutic intervention within the pharmaceutical industry. Since ancient times, enzymes also have played central roles in many manufacturing processes, such as in the production of wine, cheese, and bread. During the 1970s and 1980s, much of the focus of the biochemical community shifted to the cloning and expression of proteins through the methods of molecular biology. Recently, some attention has shifted back to the physicochemical characterization of these proteins and their interactions with other macromolecules and small molecular weight ligands (e.g., substrates, activators, and inhibitors). Hence, there has been a resurgence of interest in the study of enzyme structures, kinetics, and mechanisms of catalysis.

The availability of up‐to‐date, introductory level textbooks, however, has not kept up with the growing demand. I first became aware of this void, while teaching introductory courses at the medical and graduate student level at the University of Chicago. I found that there were a number of excellent advanced texts that covered different aspects of enzymology with a heavy emphasis on the theoretical basis for much of the science. The more introductory texts that I found were often quite dated and did not offer the blend of theoretical and practical information that I felt was most appropriate for a broad audience of students. I thus developed my own set of lecture notes for these courses, drawing material from a wide range of textbooks and primary literature.

In 1993, I left Chicago to focus my research on the utilization of basic enzymology and protein science for the development of therapeutic agents to combat human diseases. To pursue this goal, I joined the scientific staff of the DuPont Merck Pharmaceutical Company. During my first year with this company, a group of associate scientists expressed to me their frustration at being unable to find a textbook on enzymology that met their needs for guidance in laboratory protocols and data analysis at an appropriate level and at the same time provide them with some relevant background on the scientific basis of their experiments. These dedicated individuals asked if I would prepare and present a course on enzymology at this introductory level.

Using my lecture notes from Chicago as a foundation, I prepared an extensive set of notes and intended to present a year‐long course to a small group of associate scientists in an informal, over‐brown‐bag‐lunch fashion. After the lectures had been announced, however, I was shocked and delighted to find that more than 200 people were registered for this course! The makeup of the student body ranged from individuals with associate degrees in medical technology to chemists and molecular biologists who had doctorates. This convinced me that there was indeed a growing interest and need for a new introductory enzymology text that would attempt to balance the theoretical and practical aspects of enzymology in such a way as to fill the needs of graduate and medical students, as well as research scientists and technicians who are actively involved in enzyme studies.

The text that follows is based on the lecture notes for the enzymology course just described. It attempts to fill the practical needs I have articulated, while also giving a reasonable introduction to the theoretical basis for the laboratory methods and data analyses that are covered. I hope that this text will be of use to a broad range of scientists interested in enzymes. The material covered should be of direct use to those actively involved in enzyme research in academic, industrial, and government laboratories. It also should be useful as a primary text for senior undergraduate or first‐year graduate courses, in introductory enzymology. However, in teaching a subject as broad and dynamic as enzymology, I have never found a single text that would cover all of my students' needs; I doubt that the present text will be an exception. Thus, while I believe this text can serve as a useful foundation, I encourage faculty and students to supplement the material with additional readings from the literature cited at the end of each chapter, and the primary literature that is continuously expanding our view of enzymes and catalysis.

In attempting to provide a balanced introduction to enzymes in a single, readable volume, I have had to present some of the material in a rather cursory fashion; it is simply not possible, in a text of this format, to be comprehensive in such an expansive field as enzymology. I hope that the literature citations will at least pave the way for readers who wish to delve more deeply into particular areas. Overall, the intent of this book is to get people started in the laboratory and in their thinking about enzymes. It provides sufficient experimental and data handling methodologies to permit one to begin to design and perform experiments with enzymes, while at the same time providing a theoretical framework in which to understand the basis of the experimental work. Beyond this, if the book functions as a stepping‐stone for the reader to move on to more comprehensive and in‐depth treatments of enzymology, it will have served its purpose.

ROBERT A. COPELAND

Wilmington, Delaware

1995

ACKNOWLEDGMENTS

It is my great pleasure to thank the many friends and coworkers who have helped me in the preparation of this work. Many of the original lecture notes from which this text has developed were generated while I was teaching a course on biochemistry for first‐year medical students at the University of Chicago, along with the late Howard S. Tager. Howard contributed greatly to my development as a teacher and writer. His untimely death was a great loss to many of us in the biomedical community; I dearly miss his guidance and friendship.

As described in the Preface, the notes on which this text is based were significantly expanded and reorganized to develop a course of enzymology for employees and students at the DuPont Merck Pharmaceutical Company. I am grateful for many discussions with students during this course, which helped to refine the final presentation. I especially thank Diana Blessington for the original suggestion of a course of this nature. That a graduate‐level course of this type could be presented within the structure of a for‐profit pharmaceutical company speaks volumes for the insight and progressiveness of the management of DuPont Merck. I particularly thank James M. Trzaskos, Robert C. Newton, Ronald L. Magolda, and Pieter B. Timmermans for not only tolerating but embracing this endeavor.

Many colleagues and coworkers contributed suggestions and artwork for this text. I thank June Davis, Petra Marchand, Diane Lombardo, Robert Lombardo, John Giannaras, Jean Williams, Randi Dowling, Drew Van Dyk, Rob Bruckner, Bill Pitts, Carl Decicco, Pieter Stouten, Jim Meek, Bill De‐Grado, Steve Betz, Hank George, Jim Wells, and Charles Craik for their contributions.

Finally, and most importantly, I wish to thank my wife, Nancy, and our children, Lindsey and Amanda, for their constant love, support, and encouragement, without which this work could not have been completed.

“All the mathematics in the world is no substitute for a reasonable amount of common sense.”

W. Wallace (Mo) Cleland

1A BRIEF HISTORY OF ENZYMOLOGY

KEY LEARNING POINTS

Enzyme reactions have been used since antiquity in the production of bread, cheeses, and alcoholic beverages. Early references to the action of enzymes can be found in Homer's Iliad and the Codex of Hammurabi.

Systematic studies of enzyme activity began in the eighteenth century in studies of food digestion from gastric juices taken from the stomachs of birds and mammals (including humans).

In the late nineteenth and early twentieth centuries, mathematical models for enzyme activity began to be derived and published.

From these early studies, the modern science of enzymology has developed. Today, enzymology is studied as basic science but is also widely studied in a variety of commercial applications, including medicine, agriculture, food and beverage making, cleaning products, and much more.

Life depends on a well‐orchestrated series of chemical reactions. Many of these reactions, however, proceed too slowly on their own to sustain life. Hence, nature has designed catalysts, which we now refer to as enzymes, to greatly accelerate the rates of these chemical reactions. The catalytic power of enzymes facilitates life processes in essentially all life forms from viruses to man. Many enzymes retain their catalytic potential after extraction from the living organism, and it did not take long for mankind to recognize and exploit the catalytic power of enzymes for commercial purposes. In fact, the earliest known references to enzymes are from ancient texts dealing with the manufacture of cheeses, bread, and alcoholic beverages, and for the tenderizing of meats. Today enzymes continue to play key roles in many food and beverage manufacturing processes and are ingredients in numerous consumer products, such as laundry detergents (which dissolve protein‐based stains with the help of proteolytic enzymes). Enzymes are also of fundamental interest in th