Enzyme kinetics: catalysis & control ; a reference of theory and best-practice methods
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2010
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| 100 | 1 | |a Purich, Daniel L. |e Verfasser |4 aut | |
| 245 | 1 | 0 | |a Enzyme kinetics |b catalysis & control ; a reference of theory and best-practice methods |c Daniel L. Purich |
| 250 | |a 1. ed. | ||
| 264 | 1 | |a Amsterdam [u.a.] |b Elsevier |c 2010 | |
| 300 | |a XXI, 892 S. |b Ill., graph. Darst. | ||
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Datensatz im Suchindex
| _version_ | 1804143084900974593 |
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| adam_text | Titel: Enzyme kinetics
Autor: Purich, Daniel L
Jahr: 2010
Contents
Table of Contents
Preface
Chapter 1. An Introduction to Enzyme
Science
1.1. Catalysis
1.1.1. Roots of Catalysis in the
Earliest Chemical Sciences
1.1.2. Synthetic Catalysts
Biological Catalysis
1.2.1. Roots of Enzyme Science
1.2.2. Enzyme Technology
Development of Enzyme Kinetics
The Concept of a Reaction Mechanism
1.4.1. Chymotrypsin: The Prototypical
Biological Catalyst
1.4.2. Ribozymes
1.4.3. Mechanoenzymes
Explaining the Efficiency of Enzyme
Catalysis
1.2
1.3
1.4
1.5
1.5.1
Stabilization of Reaction
Transition States
Electrostatic Stabil ization
of Transition States
Intrinsic Binding Energy
Reacting Group
Approximation, Orientation
and Orbital Steering
Reactant State Destabil ization
Acid/Base Catalysis
Covalent Catalysis
Transition-State Stabil ization
by Low-Barrier Hydrogen
Bonds
Catalytic Facilitation by
Metal Ions
1.5.10. Promotion of Catalysis via
Enzyme Conformational
Flexibility
1.5.11. Promotion of Catalysis
via Force-Sensing and
Force-Gated Mechanisms
1.6. Prospects for Enzyme Science
1.6.1. We Need Better Methods for
Analyzing Enzyme Dynamics
1.5.2.
1.5.3.
1.5.4.
1.5.5.
1.5.6.
1.5.7.
1.5.8.
1.5.9.
xix to Understand the Detailed Mutual Changes in Both Substrate
and Enzyme During Catalysis 34
1.6.2. We Need New Approaches for Determining the
1 Channels Allowing Energy Flow During Enzyme
5 Catalysis 37
1.6.3. We Need Additional Probes
5 of Enzyme Catalysis 38
7 1.6.4. We Need to Learn How
12 Proteins Fold and How to
12 Manipulate Protein Stability 38
13 1.6.5. We Need to Develop a
15 Deeper Understanding of
19 Substrate Specificity 39
1.6.6. We Need to Develop the
20 Ability to Design Entirely
22 New Biological Catalysts 42
23 1.6.7. We Need to Define the Efficient Routes for Obtaining
25 High Potency Enzyme Inhibitors
as Drugs and Pesticides 44
26 1.6.8. We Need to Learn More About In Singulo Enzyme
27 Catalysis 45
28 1.6.9. We Need to Develop Comprehensive Catalogs of Enzyme Mechanisms and
28 to Use Such Information in
29 Fashioning New Metabolie
30 Pathways 46
30 1.6.10 . We Need to Understand How to Analyze the Kinetic Behavior of Discrete
31 Enzyme-Catalyzed Reactions as Well as Metabolie
32 Pathways in their Environment 48
1.6.11, . We Need to Develop Techniques that will Facilitate
32 Investigation of Chromosomal Remodeling, Epigenetics, and the Genetic Basis of
33 Disease and Cell Survival 49
34 1.6.12. . We Need to Develop Effective Enzyme Preparations for
Use in Direct Enzyme Therapy 50
Contents
Chapter 2. Active Sites and their Chemical
Properties 53
2.1. Enzyme Active Sites 54 2.5.
2.1.1. Most Enzymes are Proteins,
which are Linear Polymers of
a-Amino Carboxylic Acids 55
2.1.2. Active-Site Residues may be
Classified with Respect to
their Function(s) 57
2.1.3. Active Sites Typically Occupy
only 2-3 per cent of the Total
Volume of an Enzyme 59
2.1.4. Binding Energy Often Indicates
the Strength of Enzyme
Interactions with Substrates
and Cofactors 60
2.1.5. The Structural Organization
of Enzymes can be Considered
Hierarchically 61
2.1.6. Enzymes Often Occur in
Multiple Molecular Forms 63
2.2. Forces Affecting Enzyme Structural
Stability and Interactions 64
2.2.1. Electrostatic Interactions
Influence Enzyme Structure
and Interactions 64
2.2.2. Ion-Dipole and Dipole-Dipole
Interactions are Specialized
Electrostatic Phenomena 66
2.2.3. Hydrogen Bonding Mainly
Plays a Compensatory Role
in Stabilizing Proteins 66
2.2.4. Hydrophobie Interactions 2.6.
Play a Dominant Role in
Stabilizing Most Proteins 69
2.2.5. Although Individually Weak,
van der Waals Interactions
are so Numerous that they
Contribute Significantly
to Overall Protein Stability 70
2.2.6. Some Proteins are
Occasionally Stabilized by
u-Cation Interactions 70
2.3. Active-Site Diversifikation 70
2.3.1. Enzyme Diversification can
be Explained Structurally 71
2.3.2. Catalytic Promiscuity may
Explain the Emergence of
Catalytically Diversified
Enzymes 74
2.4. Additional Functional Groups in
Enzyme Active Sites 77
2.4.1. Vitamin-Based Coenzymes Increase
the Chemical Versatility of Enzyme
Active Sites 77
2.7.
2.4.2. Some Enzymes Exploit
Specialized Amino-Acid
Residues in Catalysis 78
Metal Ions in Enzyme Active Sites 81
2.5.1. A Group of Biologically
Significant Metal Ions is
Essential for Catalysis
by Some Enzymes 82
2.5.2. Enzyme-Bound Metal Ion
Complexes Share Structural
and Chemical Features 84
2.5.3. The Chemistry of Metal
lon-Ligand Complexes is
Dominated by the Nature of
their Ligancy 85
2.5.4. Field-Effects Influence the
Color and Magnetic
Properties of Metal Ion
Coordination Complexes 87
2.5.5. The Reaction Mechanisms of
Transition Metal Complexes
are Determined by their
Inner- and Outer-Sphere
Coordination Behavior 89
2.5.6. Metal Ions Form Complexes
with Enzymes and/or their
Substrates 93
2.5.7. Properties of Selected
Active-Site Metal Ions 95
2.5.8. A Survey of Metal Ion Complexes
within Selected Enzymes Reveals
Key Features of Binding-Site
Organization 109
Active Sites of Enzymes Acting on
Polymerie Substrates 112
2.6.1. Many Endonucleases Achieve their
Remarkable Specificity by Means
of Subsite Recognition 113
2.6.2. Proteases were the First Enzymes
Shown to have Subsites for Interacting
with their Polymerie Substrates 114
2.6.3. Endo-Glycosidases also
Exploit Subsites to Achieve Specificity 115
2.6.4. Subsites Facilitate Substrate
Recognition by Signal-
Transducing Protein Kinases 115
Basic Organic Chemistry of Enzyme
Action 116
2.7.1. There are Six Major Classes of
Enzyme-Catalyzed Covalent
Bond-Making/-Breaking
Reactions 118
2.7.2. Carbon has Several Reactive
Forms in Enzymatic Mechanisms 119
2.7.3. Many Enzymes Use the Same
General Reaction Mechanisms
Contents
VII
First Discovered by Physical
Organic Chemists
2.7.4. Nucleophilic Substitution is a
Widely Used Reaction
Mechanism in Enzyme Catalysis
2.7.5. Enzyme-Catalyzed Elimination
Reaction Mechanisms have Many
Precedents in Organic Chemistry
2.7.6. Enzymes are Highly Effective in
Forming, Stabilizing, and
Utilizing Carbanion
Intermediates During Catalysis
2.7.7. Free Radicals are Formed in
a Surprising Number of
Enzyme-Catalyzed Reactions
2.7.8. The Versatility of Enzymes can
be lllustrated by Considering
a Selected Group of Reaction
Mechanisms
2.8. Detecting Covalent Intermediates in
Enzyme Reactions
2.8.1. Enzymes Form a Wide Range
of Enzyme-Substrate Covalent
Compounds, and Many are
Catalytically Competent
2.8.2. Side-Reactions Often Provide
Invaluable Clues About
Mechanisms of Enzyme
Catalysis
2.8.3. Some Enzyme-Substrate
Covalent Compounds can be
Chemically Trapped
2.9. Basics of Enzyme Stereochemistry
2.9.1. Definitions
2.9.2. The Cahn-Ingold-Preiog System
Allows One to Assign the Absolute
Stereochemical Configuration
of Chiral Compounds
2.9.3. The Prochirality of Molecules
may also be Specified
Systematically
2.9.4. The Stereochemistry of Methyl
Transfer Reactions may be
Analyzed Using Enzymes of
Known Stereochemistry
as Reference Reactions
2.10. Electron Transfer Reactions
2.10.1. The Thermodynamic Properties
of Oxidation-Reduction
Reactions are Defined by
Redox Potentials
2.10.2. The Redox Behavior of Complex
Metal loenzymes can be
Evaluated Spectroscopically
by Stoichiometric Titration Techniques 157
2.10.3. Respiratory Chains are
Comprised of Highly
120
121
125
125
131
134
137
137
141
143
145
145
146
147
149
150
154
Coordinated Electron
Transfer Reactions 158
2.10.4. Enzyme-Catalyzed Electron
Transfer may be analyzed
by Marcus Theory 160
2.10.5. Simple Kinetic Models can
Account for the Behavior of
Biological Electron Transfer Reactions 161
2.10.6. Several Prototypical Redox
Enzymes Provide Valuable
Insights into Electron Transfer
Kinetics and Mechanisms 162
2.10.7. Enzyme Electrodes Combine
the Specificity of Biological
Catalysis with the Versatility of
Potentiometry or Amperometry 164
Appendix 168
Chapter 3. Fundamentals of Chemical
Kinetics 171
3.1. Timescale of Chemical Processes 171
3.2. The Empirical Rate Equation 172
3.3. Reaction Rate, Order and Molecularity 174
3.3.1. Reaction Rate 174
3.3.2. Reaction Order 175
3.3.3. Molecularity 176
3.3.4. Zero-Order Kinetics 177
3.3.5. First-Order Kinetics 177
3.3.6. Second-Order Kinetics 180
3.3.7. Pseudo First-Order Kinetics 180
3.4. Basic Strategies for Evaluating Rate Processes 181
3.4.1. Initial-Rate Method 181
3.4.2. Progress Curve Analysis 182
3.5. Composite Multi-stage (Multi-step)
Mechanisms 184
3.5.1. Series First-Order Kinetics 185
3.5.2. Reversible First-Order Kinetics 186
3.5.3. Reversible Second-Order Kinetics 186
3.5.4. Rapid-Equilibrium and
Steady-State Treatments 186
3.5.5. Rate-Controlling Steps 188
3.5.6. Principles of Detailed Balance 189
3.5.7. Thermodynamic Cycles for
Evaluating Detailed Balance 190
3.5.8. Kinetic Equivalence and
Mechanistic Ambiguity 192
3.6. Thermal Energy: The Boltzmann
Distribution Law 192
3.7. Solution Behavior of Reacting
Molecules 194
3.7.1. Water: A Unique Solvent for
Biochemical Processes 194
3.7.2. Diffusion Limitations on
Chemical Processes Occurring
in Water 196
VIII
Contents
3.7.3. Electrostatic Effects on
Magnitude of Bimolecular
Rate Constants
3.7.4. Reactant Desolvation
3.8. Transition-State Theory
3.9. Chemical Catalysis
3.9.1. Accelerating Rate without
Altering the Equilibrium Poise
3.9.2. Nucleophilic and Electrophilic
Facilitation
3.9.3. Buffer Catalysis
3.9.4. Autocatalysis
3.10. Reaction Coordinate Diagrams
3.11. Thermodynamic Principles
3.11.1. Chemical Equilibrium
3.11.2. Direction and Extent of
Chemical Reaction
3.11.3. Using AAGto Define Binding
Energetics
3.11.4. Alberty Treatment of
Biochemical Thermodynamics
3.11.5. Some Reacting Systems are
Best Analyzed by Principles
of Non-Equilibrium
Thermodynamics
3.12. Concluding Remarks
199
200
201
203
203
205
206
206
207
210
210
210
211
211
212
214
Chapter 4. Practical Aspects of Measuring
Initial Rates and Reaction
Parameters 215
4.1. Design of Initial-Velocity Enzyme Assays 215
4.1.1. Activity Purity is Sufficient in
Most Initial-Rate Studies 216
4.1.2. Discontinuous and Continuous
Rate Measurements 217
4.1.3. Each Enzyme Rate Assay has Its
Own Special Set of Requirements 220
4.2. Enzyme Purification 232
4.2.1. While Time-Consuming, the Task
of Enzyme Purification is Often
Well Founded 232
4.2.2. Biochemists have Developed a
Powerful Battery of Techniques
for Purifying Enzymes 234
4.3. Coupled (or Auxiliary) Enzyme Assays 235
4.3.1. A Simple Kinetic Treatment
Explains the Lag-Phase in
Coupled Enzyme Assays 238
4.3.2. The Auxiliary Enzyme and Assay
Conditions must be Suited to
the Primary Enzyme Reaction 239
4.4. Basic UVAfisible Absorption
Spectroscopy 240
4.4.1. Absorption Spectra Depend on the
Quantum States of Electron Orbitals 240
4.4.2. Beer s Law is a Quantitative
Expression Linking Absorbance
to Concentration 240
4.4.3. Some Enzyme Assays Use
Alternative Substrates that are
Chromogenic 243
4.5. Basic Fluorescence Spectroscopy 243
4.5.1. Fluorescence Spectra Depend
on Excited-State Relaxation 244
4.5.2. Features of a Research-Grade
Spectrophotofluorimeter 244
4.5.3. The Concentration of Various
Metabolites may be Quantified
Through Fluorescence
Spectrometry 246
4.5.4. Biological Molecules may
Contain Intrinsic or Extrinsic
Fluorescent Reporter Groups 247
4.5.5. Fluorescence Anisotropy is a
Powerful Technique For Quantifying
Binding Interactions 250
4.5.6. Förster (Fluorescence) Resonance
Energy Transfer (FRET) is an
Exquisitely Distance-Sensitive
Probe of Enzymes 252
4.5.7. Continuous Fluorescence Assays
are Now Available for Pi- and
PPi-Producing Reactions 253
4.5.8. Chemiluminescence is a
Photoemissive Process Often
Exploited in Enzyme Rate Assays 254
4.6. Measuring Reaction Rates with Isotopes 255
4.6.1. Stable Isotopes are Versati le
Probes in Enzyme Kinetics 255
4.6.2. Radioisotopes Provide Extremely
Sensitive Assays of Enzyme
Rate Processes 260
Multisubstrate Kinetics and Inhibitor
Kinetics 264
Analysis of Enzyme Rate Data 265
4.8.1. Enzyme Rate Data must be
Appropriately Weighted 266
4.8.2. Quantitative Analysis of Reaction
Progress-Curves can be Used to
Evaluate Rate Parameters 268
4.8.3. Global Analysis Offers Added
Advantages in Statistical Analysis 270
4.9. Working with ATP-Dependent Enzymes 272
4.10. Regenerating Nucleoside
5 -Triphosphate Substrates 275
4.10.1. Protein and Enzyme
Concentration 276
4.10.2. Total Protein Concentration
can be Determined Quantitatively 276
4.10.3. Active Enzyme Concentration can
be Quantified by Several Techniques 276
4.7.
4.8.
Contents
IX
4.11. Equilibrium Constant Determinations 278
4.11.1. Equilibrium Constants can be
Evaluated in a Variety of Ways 279
4.11.2. The Determination of the
Arginine Kinase Reaction
Equilibrium Constant is an
Excellent Example of a
Well-Designed and
Well-Executed Determination 281
4.12. Concluding Remarks 284
Chapter 5. Initial-Rate Kinetics of One-
Substrate Enzyme-Catalyzed
Reactions 287
5.1. Michaelis-Menten Treatment 287
5.1.1. Derivation of the Michaelis-
Menten Equation Reveals how
Key Assumptions Define an
Enzyme s Initial-Rate Behavior 288
5.1.2. Ks, Vm, Vm/Ks, and [S]//Cs are
Rate Parameters Defining an
Enzyme s Initial-Rate Behavior 289
5.1.3. Several Methods for Plotting Initial-
Rate Data are Quite Useful but have
Inherent Limitations 290
5.1.4. The Michaelis-Menten Equation
Predicts a Linear Dependence
of Reaction Rate on the
Concentration of Active Enzyme 292
5.1.5. The Quadratic Formula is
Required When the Enzyme
Concentration Approaches
Substrate Concentration 292
5.1.6. Nonproductive Substrate Binding
Cannot be Detected by the Michaelis-
Menten Treatment 293
5.2. The Briggs-haldane Steady-State
Treatment 293
5.2.1. Derivation of this Rate Equation
Reveals Key Features of
Steady-State Processes 294
5.2.2. Reaction Energetics Determine
the Effect of Increasing Substrate
Concentration on the Conversion
of E + S to E ? S Complex 295
5.2.3. The Corresponding Reverse-Reaction
Rate Equation can now be Written 295
5.2.4. The Haidane Relationship
Constrains the Values of Key
Rate Parameters for Reversible
Enzyme-Catalyzed Reactions 296
5.2.5. The Briggs-Haldane Equation
Requires that an Enzyme
System Satisfies the Steady-
State Assumption 296
5.3. Catalysis Involving Two or More
Intermediates 298
5.3.1. Derivation of the Two-
Intermecliate Gase lllustrates
Why this Treatment is a More
Realistic Representation
of an Enzyme Mechanism 298
5.3.2. A Shortcut can be Taken to
Derive the Steady-State Rate
Equation for the Reverse
Two-Intermediate Reaction
Scheme 298
5.3.3. Multiple Internal Isomerizations are
without Effect on the General
Form ofthe Final Steady-State
Rate Equation 298
5.3.4. The Haidane Relationship
also Constrains the Relative
Magnitudes of Key Rate
Parameters in the Two-
Intermediate Scheme 300
5.4. Additional Comments on Fundamental
Kinetic Parameters 301
5.4.1. The Michaelis Constant has
Several Important Implications,
with Regard to Both Substrate
Affinity and Substrate
Specificity 301
5.4.2. The Turnover Number kcat
Indicates Number of Substrate
Molecules Converted to
Product per Enzyme Active
Site per Second 303
5.4.3. The Specificity Constant
Knax/Km or ^cat/^rn Indicates the
Efficiency of Substrate Capture
by an Enzyme 304
5.4.4. The Commitment to Catalysis
Measures an Enzyme s AbiIity
to Convert the ES Complex
to E ? P, as Compared to
Reconversion of ES to a Prior
Enzyme Form 307
5.4.5. Evolution of Catalytic Proficiency 308
5.4.6. Internal Equilibria and
Energetics of Perfected Enzymes 309
5.5. Reaction Progress Curve Analysis 310
5.6. Ribozyme Kinetics 311
5.7. Proteasome Kinetics 313
5.8. Isomerization Mechanisms 314
5.9. Simultaneous Action of an Enzyme
on Different Substrates 315
5.10. Enantiomeric Enrichment and
Anomeric Specificity 316
5.11. Simultaneous Action of Two Enzymes
on the Same Substrate 318
5.12. Induced-Fit Mechanisms 319
Contents
5.12.1. There are Many Outstanding
Examples of Induced-Fit Binding
Behavior 320
5.12.2. Induced-Fit Energetics may be
Analyzed with Thermodynamic
Cycles 324
5.12.3. Induced-Fit Behavior and
Enzyme Specificity 324
5.12.4. Induced-Fit Behavior
Represents a Considerable
Challenge for Computer-
Based Ligand-Docking 327
5.13. Kinetics of Enzymes Acting on
Polymerie Substrates 327
5.13.1. Processive versus Distributive
Mechanisms 327
5.13.2. Random Scission Kinetics of
Endo-Depolymerases 329
5.13.3. Some Depolymerizing Enzymes
Show Evidence of Substrate-
Assisted Catalysis 330
5.13.4. Microarray and Phage-Display
Profiles of Enzyme Specificity 331
5.13.5. Hidden Nonproductive
Interactions in Steady-State
Treatments of Enzyme Acting
on Polymerie Substrates 332
5.14. Concluding Remarks 333
Chapter 6. Initial-Rate Kinetics of Multi-
Substrate Enzyme-Catalyzed
Reactions 335
6.1. Bisubstrate Kinetic Mechanisms 335
6.1.1. Cleland s Notation Conveniently
Represents Multi-Substrate
Kinetic Mechanisms 336
6.1.2. There are Numerous Examples
of Well-Characterized Bisubstrate
Enzyme Kinetic Mechanisms 338
6.2. Derivation Steady-State Bisubstrate
Rate Equations 341
6.2.1. Fromm s Systematic Method
for Deriving Rate Equations is a
Simple and Reliable Alternative
to the More Confusing King-
Altman Approach 341
6.2.2. The Two-Step Computer-Assisted
Method is a Rapid, Automatic
Way to Obtain Enzyme Rate Laws 343
6.2.3. Cleland s Net Reaction Rate
Method is a Simple, Elegant,
and Reliable Way to Derive Rate
Equations for Unbranched
Kinetic Mechanisms 345
6.2.5.
6.2.6.
6.2.4. Theorell and Chance Defined
a Special Ordered Binary Complex
Mechanism for Two-Substrate
Enzyme Catalyzed Reactions 347
The Steady-State Random
Kinetic Mechanism is Far Too
Complicated for the Unambiguous
Experimental Determination of
Key Rate Parameters 348
The Cha Method Assumes that
Certain Reaction Mechanism
Segments are at Thermodynamic
Equilibrium 349
6.3. Derivation of Rapid Equilibrium Bisubstrate
Rate Equations 349
6.3.1. The Rapid-Equilibrium Assumption
Greatly Facilitates the Derivation
of Rate Laws for Bisubstrate
Random Kinetic Mechanisms 350
6.3.2. The Rapid Equilibrium Treatment
of the Ordered Sequential Kinetic
Mechanism Gives Rise to What
is Probably the Simplest of
Multi-Substrate Rate Laws 350
6.4. Ping Pong Bi Bi Mechanism 352
6.4.1. Ping-Pong Mechanisms have a
Distinctive Steady-State Rate
Equation that Gives Rise to
Parallel-Line Patterns in
1/u-versus-1/[A] and
Mv-versusA/m Plots 352
6.4.2. Ping Pong Enzymes Catalyze
Partial-Exchange Reactions 353
6.4.3. Some Partial Exchange Reactions
can be Mechanistically Ambiguous 354
6.4.4. Burst Kinetics Provide
Information About Rate-
Contributing Steps in Enzyme-
Catalyzed Reaction Mechanisms 356
6.4.5. Certain Hydrolase/Transferase-
Type Enzymes have Distinctive
Kinetic Properties 356
Substrate Inhibition Offers
Insights About Ping-Pong
Reactions 357
Multi-Site Ping Pong Kinetic
Mechanisms Account for the
Transfer of Reactant Moieties
Between Substrate-Binding
Pockets within Topologically Complex
Active Sites 358
6.5. Graphical and Quantitative Analysis
of Bisubstrate Kinetics 359
6.5.1. Re-Plotting Bisubstrate
Experimental Rate Data is a
Useful Way to Derive Key Rate
Parameters 359
6.4.6.
6.4.7.
Contents
6.6.
6.7.
6.8.
6.9.
6.5.2. Haidane Relations can be Used
to Distinguish Kinetic Mechanisms
of Bisubstrate Enzyme-Catalyzed
Reactions
6.5.3. The Dalziel Phi Method is a
Quantitative Approach for
Distinguishing Rival Bisubstrate
Kinetic Mechanisms
6.5.4. Fromm s Point-of-Convergence
Method is Another Method for
Distinguishing Bisubstrate Kinetic
Mechanisms
6.5.5. Crossover-Point Analysis also
Allows Discriminates Bisubstrate
Enzyme Kinetic Mechanisms
6.5.6. Some Multi-Substrate Initial-Rate
Kinetic Data can be Ambiguous
Three-Substrate Enzyme Kinetics
6.6.1. There are Numerous Kinetic Schemes
for Three-Substrate Enzyme Catalyzed
Reactions
6.6.2. There are Several General
Strategies for Reducing the
Complexity of Three-Substrate
Initial-Velocity Experiments
Multisubstrate ISO Mechanisms
Kinetic Properties of Enzymes
Exhibiting Branched Transfer
Pathways
Concluding Comments
Appendix
Chapter 7. Factors Influencing Enzyme
Activity
7.1. Activator Effects on Enzyme Kinetics
7.1.1.
7.1.2.
7.1.3.
7.1.4.
7.1.5.
7.1.6.
7.1.7.
7.1.8.
359
360
362
363
364
366
366
368
370
372
373
374
379
379
381
Definitions
Some Reversible Essential
Activators Bind Before the
Substrate 383
Some Reversible Essential
Activators Bind After the
Substrate 384
Some Enzymes Randomly Bind
Essential Activators and
Substrate 385
Some Essential Activators Bind
to an Otherwise Unreactive Substrate 385
Some Activators Released
During Catalysis Exhibit
Rate-Limiting Rebinding 385
Some Non-Consumed Substrates
Behave as Pseudo-Essential
Activators 386
Enzyme Must Always have
Basal Activity when a
Nonessential Activator is
Absent 387
7.1.9. 3 ,5 -cyclic AMP
Phosphodiesterase
Activation by Ca +-Calmodulin:
AThorough Kinetic Analysis 388
7.1.10. The Method of Continuous
Variation Analysis may be Used
to Determine Activator Binding
Site Number and Affinity 389
7.1.11. Time-Dependent Enzyme
Activation Requires Special
Treatment 390
7.1.12. Some Agents Exhibit Biphasic
Activation and Inhibition
Effects 391
7.2. Metal-Nucleotide Complexes as
Substrates 391
7.2.1. Most ATP-Dependent Enzyme-
Catalyzed Reactions Require
Complexation of ATP4~ with
a Divalent Metal Ion 393
7.2.2. Certain Mechanoenzymes Use
Metal-Free ATP4~ and GTP4 ,
Albeit Slowly 394
7.2.3. Exchange-Inert Metal-Nucleotide
Complexes are Powerful
Mechanistic Probes 395
7.3. pH Effects on Enzyme Kinetics 397
7.3.1. Many Enzymes Exhibit a
Characteristic pH Optimum 397
7.3.2. Enzymes Often Display pH-
Dependent Changes in Activity 398
7.3.3. Several Methods may be
Employed to Estimate Catalytic
p/Ca Values 401
7.3.4. Acetoacetate Decarboxylase
Possesses a Catalytic Lysine
Exhibiting an Atypical
(or Perturbed) pKa Value 404
7.3.5. pKa Values may be Estimated on
the Basis of Protein Structural
Calculations 405
7.3.6. Enzymes Exhibit a Wide Range
of pH-Dependent Behaviors 406
7.3.7. Some Enzymes Undergo pH-
Dependent Changes in Mechanism 407
7.3.8. The pH Kinetics of Bisubstrate
Enzymes can be Complex 408
7.3.9. Bronsted Theory Explains Important
Aspects of Acid/Base Catalysis 409
7.4. Buffer Effects on Enzyme Kinetics 412
7.4.1. Many Factors Influence the
Choice of a Buffer 413
7.4.2. Biochemists Exploit Various
Properties of Selected pH
Buffers 414
XII
Contents
414
415
416
7.4.3. Some Buffers Actively Participate
in Enzyme Catalysis
7.4.4. Some Rate Studies may Require
Buffers of Constant lonic Strength
7.5. lonic Strength Effects on Enzyme
Kinetics
7.5.1. lonic Strength Defines a
Solution s lonic Nature 417
7.5.2. The Debye-Hückel Treatment Explains
How Ions Alter the Thermodynamic
Activity of Solutes 417
7.5.3. Changes in lonic Strength can
Alter the Magnitude of Rate Constants 418
7.5.4. lonic Strength Alters Enzyme
Catalysis Profoundly 419
7.5.5. There are Limits on the
Applicability of lonic Strength 421
7.6. Effect of Organic Solvents on Enzyme
Activity 422
7.7. Temperature Effects on Enzyme
Kinetics 425
7.7.1. Temperature Often Strongly Influences
Enzyme Activity and Stability 425
7.7.2. The Kinetics of Thermal Inactivation
can be Treated Phenomenologically 426
7.7.3. Temperature Alters Both Equilibrium
and Rate Constants 427
7.7.4. Many Nonlinear Arrhenius Plots can
be Explained in Terms of Rate
Compensation 428
7.7.5. The Q10 Parameter is a Semi-
Quantitative Measure of an
Enzyme s Sensitivity to Changes
in Temperature 429
7.7.6. Certain Organisms have their Own
Characteristic Physiologie Temperature 429
7.7.7. Extremophilic Enzymes have
Unusual Structural Stability 430
7.7.8. Some Multi-Subunit Enzymes Exhibit
the Phenomenon of Reversible Cold
Inactivation 433
7.7.9. Cryoenzymology Techniques
Greatly Reduce the Rate
of Enzyme Catalysis 435
7.8. Pressure Effects on Enzyme Kinetics 438
7.9. Effects of Immobilization on Enzyme
Stability and Kinetics 439
7.9.1. Kinetic Behavior of Matrix-
Immobilized Enzymes can be
Substantially Different than the
Behavior of Solution-Phase
Enzymes 442
7.9.2. Enzymes Tethered with Flow Tubes
have Special Kinetic Properties 443
7.9.3. Enzyme Confinement may be
Relevant to Cellular Conditions 443
7.10. Non-Ideality Imposed by Molecular
Crowding 444
7.11. Enzyme Action on Sequestered
Substrates 446
7.12. Interfacial Catalysis 447
7.13. Proofreading Effects on Enzyme
Catalysis 453
7.14. Kinetics of Crystalline Enzymes 457
7.14.1. Accurate Activity Assays of
Crystalline Enzymes can be
Technically Challenging 458
7.14.2. Time-Resolved Laue X-Ray
Crystallography is Quickly
Becoming a Powerful
Mechanistic Tool 459
7.14.3. Direct Measurement of
Reactant Diffusion Rates in
Enzyme Crystals can be
Accomplished by Video
Absorption Spectroscopy 459
7.14.4. Cross-Linking can be an Effective
Tool in Analyzing the Behavior
of Crystalline Enzymes 459
7.15. Probing Enzyme Catalysis Through
Site-Directed Mutagenesis 460
7.15.1. Mutations - Particularly Long-Lived
Naturally Occurring Mutations
- are lntrinsically lnteresting 460
7.15.2. Early Mutagenesis Experiments
Exploited Chemical Modification
to Replace One Naturally
Occurring Amino Acid with Another 461
7.15.3. Alanine Scanning Mutagenesis Often
Provides Useful Clues About Essential
Functional Groups in Enzymes 462
7.15.4. Enzyme Chemists have Adopted
Efficient Strategies for Investigating
Enzyme Catalysis by Site-Directed
Mutagenesis 464
7.15.5. Triose-Phosphate Isomerase:
A Case Study in Directed
Mutagenesis 472
7.15.6. Chemical Rescue is a Method
for Restoring Activity in some
Mutant Enzymes 479
7.15.7. Site-Directed Mutagenesis Suffers
Significant Limitations 480
7.16. Concluding Remarks 483
Chapter 8. Kinetic Behavior of Enzyme
Inhibitors 485
8.1. Scope and Significance of Enzyme
Inhibition
8.1.1. Distinguishing Reversible and
Irreversible Enzyme Inhibitors
485
485
Contents
XIII
8.1.2. Enzyme Inhibitors in Biomedicine
8.1.3. Broader Applications of Enzyme
Inhibitors
8.2. Reversible Enzyme Inhibition
8.2.1. Competitive Inhibition Requires
a Substance to Bind to the Same
Enzyme Form as the Substrate
8.2.2. Noncompetitive Inhibition
Requires an Inhibitor to Bind
to Both E and E-S Forms
8.2.3. Uncompetitive Inhibition Occurs
when an Inhibitor Only Binds to
E-S in One-Substrate Mechanisms
8.2.4. Inhibition can be Linear or
Non-Linear
8.2.5. Cleland Developed Useful Rules
for Analyzing Reversible
Dead-End Inhibition
8.2.6. Some Inhibitors Act
Synergistically
8.3. Substrate Inhibition
8.3.1. Excess Substrate can Give Rise
to Nonlinear Inhibition
8.3.2. Fromm s Alternative Substrate
Inhibition Method Distinguishes
Rival Multi-Substrate Kinetic
Mechanisms
8.3.3. Huang s Constant-Ratio Alternative
Substrate Inhibition Method
Distinguishes Multi-Substrate
Kinetic Mechanisms
8.3.4. Induced Substrate Inhibition is
a Type of abortive Complex
Inhibition
8.4. Product Inhibition
8.4.1. The Alberty/Fromm Strategy Uses
Product Inhibition Patterns to
Distinguish Rival Multi-Substrate
Kinetic Mechanisms
8.4.2. Product Inhibition Equations for
Various Two-Substrate Kinetic
Mechanism Indicate Potentially
Unique Inhibition Patterns
8.4.3. Abortive Complex Formation Alters
Idealized Product Inhibition Patterns
for Two-Substrate Kinetic Mechanisms
8.4.4. A Foster-Neimann Plot Permits
the Analysis of Progress Curves
for Enzyme in the Presence of
Product Inhibition
8.4.5. Product Inhibitors can Provide
Valuable Clues About
Multisubstrate Iso Mechanisms
8.4.6. The Metabolie Significance of
Product Inhibition Merits Greater
Consideration
8.5. Multi-Substrate Geometrie Inhibitors
8.7.
486 8.6.
489
489
489
501
502
504
505
505
506
506
508
510
8.8.
511
512
512
513
516
520
521
521
523
8.9.
8.6.2.
8.6.3.
.7.2.
.7.3.
Transition-State Inhibitors 525
8.6.1. The Energetics of Transition-State
Stabilization Explains the
Considerable Inhibitory Potency
of Substances Resembling the
Transition State 525
There are Numerous Examples
of Naturally Occurring and
Synthetically Transition-
State Analogs 527
High-Affinity Binding of Certain
Pro-Transition-State Analogs is
Triggered by Some Enzymes 528
Tight-Binding Reversible Inhibitors 531
8.7.1. Reversible Tight-Binding Inhibitors
Undergo Slow Inhibitor-Induced
Enzyme Conformational
Changes 531
Slow-Binding Inhibitors and
Slow, Tight-Binding Inhibitors
are Time-Dependent 533
Dihydrofolate Reductase Inhibition
by Methotrexate lllustrates Key
Features of Time-Dependent
Reversible Inhibitors 535
8.7.4. ß-Site Amyloid Precursor
Protein-Cleaving Enzyme
Undergoes Time-Dependent
Inhibition by a Statine-Based Peptide 536
Measures of Reversible Inhibitor
Potency 537
8.8.1. Percent Inhibition and Degree
of Inhibition 537
8.8.2. The IC50 Parameter 538
Irreversible Enzyme Inhibition
by Affinity Labeling Agents 539
8.9.1. Baker Advanced the Rational
Design of Active-Site Directed
Irreversible Inhibitors 539
A Simple Rate Equation Explains
Affinity-Labeling Kinetics 540
The Presence of Substrate can
Retard, but Not Block,
Irreversible Inhibition 544
8.9.4. Site-Directed Irreversible Inhibitors
may be Distinguished from Tightly
Bound Reversible Inhibitors 544
8.9.5. pH Often Strongly Influences the
Action of Irreversible Inhibitors 544
8.9.6. Unstable Affinity Reagents
Frequently Undergo Time-
Dependent Deactivation 545
8.9.7. Quiescent Enzyme Inactivators are
Special Irreversible Inhibitors 546
8.9.8. Syncatalytic Affinity-Labeling
Agents React Synchronously
with Catalysis 546
8.9.2.
8.9.3.
Contents
8.10. Photoaffinity Labeling of Enzyme Active
Sites 547
8.10.1. Westheimer First Recognized
the Power and Range of
Photoaffinity Enzyme Reagents 548
8.10.2. Photochemical Reactions
Exhibit Distinctive Properties 548
8.10.3. Even Photoaffinity Reagents Can
Suffer Major Limitations 549
8.11. Mechanism-Based Inhibition 550
8.11.1. Mechanism-Based Inhibitors Proceed
Along Parallel First-Order Paths 552
8.11.2. Mechanism-Based Inhibitors
are Highly Versatile 556
8.11.3. Some Noncovalent Enzyme
Inhibitors Resemble
Mechanism-Based Inhibitors 557
8.12. Designing Highly Effective
Enzyme-Directed Drugs 558
8.12.1. Drug Discovery Focuses on
Druggable Enzyme Targets and
Selection/Evaluation or their Inhibitors 558
8.12.2. Identifying and Perfecting Inhibitory
Potency has Become a Well-
Practiced Art 561
8.12.3. Development of Mechanism-
Based Inhibitors Remains
a Powerful Approach 563
8.12.4. Schramm s Drug Design Strategy
Focuses on Discerning Subtle
Differences in Enzyme Transition
States and Replicating Them
When Designing Inhibitors 563
8.12.5. Pro-Drug Development is another
Viable Approach in Rational Drug
Design 566
8.12.6. Adaptive Inhibition is a New
Approach for Designing Enzyme-
Directed Drugs 567
8.12.7. Lupinski s RuIe-of-Five Index
Predicts the Efficacy of Oral Drugs 568
8.12.8. Fragment-Based Lead Design 569
8.12.9. Distal-Site Drug Potentiation is
Untested Approach for Improving
Efficacy 571
8.12.10. RNA Interference is an Under-
Explored Way to Deplete Target
Enzymes 571
8.12.11. Macromolecules also Offer
Promise as Enzyme Inhibitors 572
8.12.12. Metabolie Control Analysis may
Facilitate the Evaluation of Drug
Action 572
8.13. Concluding Remarks 572
Suggested Reading
Other Authoritative Readings from
Methods in Enzymology
573
574
Chapter 9. Isotopic Probes of Biological
Catalysis 575
9.1. Utility of Isotopes in Defining Enzyme
Stereochemistry 576
9.1.1. Vennesland and Westheimer Established
the Stereochemistry of NADH-
Dependent Hydride Transfer Reactions 576
9.1.2. The Stereochemistry of
Nucleotide-Dependent
Reactions Provides Valuable
Insights into the Chemical
Mechanisms of Phosphoryl and
Nucleotidyl Transfer Reactions 579
9.2. Labeling Substrates for Isotopic
Experiments 585
9.3. Isotope Exchange at and Away
from Equilibrium 586
9.3.1. All Exchange Processes Obey
Simple First-Order Kinetics,
Regardless of the Number or
Nature of Intermediate Steps in
the Overall Chemical Reaction 586
9.3.2. The Basic Experimental Strategy
Focuses on the Atoms (or Groups
of Atoms) Undergoing Exchange 587
9.3.3. Equilibrium Exchange Rate
Equations may be Derived Using
Equilibrium or Steady-State
Approximations 589
9.3.4. Boyer s Strategy for Examining
Equilibrium Isotopic Exchanges
can Define the Order of Substrate
Addition and Product Release 592
9.3.5. Early Measurements Demonstrated
Isotope Exchange, Even with Reversible
Reactions Away from Equilibrium or
with Virtually Irreversible Enzymes 596
9.3.6. A Füller Range of Isotopic
Exchange-Rate Behavior can be
Revealed Using Britton s Flux
Ratio Method 596
9.3.7. Isotopic Rate Measurements Provided the
First Truly Comprehensive Analysis of
Enzyme Transition-State Energetics 599
9.4. Isotope Trapping Method: Enzyme-
Bound Substrate Partitioning Kinetics 603
9.5. Positional Isotope Exchange 606
9.6. Kinetic Isotope Effects 607
Contents
xv
9.6.1. Basic Definitions and Notation
9.6.2. Primary Kinetic Isotope Effects
Reflect Differences in the Respective
Zero-Point Energies of Isotopomers
9.6.3. Some Kinetic Isotope Effects are
Measured by Equilibrium Perturbation
9.6.4. Quantum Mechanical Hydrogen
Tunneling Reveals Important
Information about Reaction Barriers
9.6.5. The Magnitude of Secondary Kinetic
Isotope Effects Distinguishes Sn1- and
S^-type Nucleophilic Mechanisms
9.6.6. Other Reaction Steps may Alter the
Observed Kinetic Isotope Effects
9.6.7. Multiple Isotopically Sensitive Steps
may Influence the Magnitude of the
Observed Kinetic Isotope Effect
9.6.8. Solvent Kinetic Isotope Effects
(SIEs) Occur when Isotopically
Labeled Solvent Molecules are
Used in Rate Measurements
9.6.9. Other Cases lllustrating the Power
of Kinetic Isotope Effect Measurements
9.7. Determining the Rates of Enzyme
Synthesis and Degradation
9.8. Concluding Remarks
Authoritative Readings from the Enzyme
Kinetics and Mechanism volumes of
Methods in Enzymology
Chapter 10. Probing Fast Enzyme
Processes
10.1. Range of Fast Reaction Techniques
10.2. Flow Techniques
10.2.1. Rapid-Mixing Continuous-Flow
Methods Permit the Detection of
Reaction Intermediates
10.2.2. Chance s Stopped-Flow
Technique Revolutionized the
Investigation of Moderately
Fast Reactions
10.2.3. NAD+ Binding to Alcohol
Dehydrogenase: A Case Study
lllustrating the Utility of the
Stopped-Flow Kinetic Measurements
10.2.4. Rapid-Scan Devices Permit
Spectroscopic Analysis During
Stopped-Flow Experiments
10.2.5. Rapid Mixing/Quenching Devices
Permit Kinetic Analysis of a Wide
Range of Chemical Reactions
10.2.6. Freeze-Quench Approaches Rely on
a Sudden Drop in Temperature to
Arrest Otherwise Highly Dynamic
Processes
608
609
612
10.2.7. Ribonucleotide Reductase
Catalysis is Gainfully Examined
by Rapid Freeze-Quench Techniques
10.2.8. Burst-Phase Kinetics also
Reveal Key Features of Enzyme Action
613
616
619
10.3. Relaxation Kinetics
10.3.1. Chemical Relaxation
Encompasses a Robust Range
of Techniques
10.3.2. While Conceptually
Straightforward, Relaxation Rate
Analysis Offers Powerful Insight
into Complicated Chemical Processes
10.3.3. The Temperature-jump Method
is Probably the Most Versatile
Chemical Relaxation Technique
622 10.4. Stopped-flow and Temperature-Jump
Techniques Provide Powerful Insights
into Enzyme Catalysis
10.4.1. Ribonuclease
10.4.2. Aminotransferase Catalysis:
A Case Study in Temperature-
Jump Kinetics
10.4.3. Dihydrofolate Reductase:
Another Outstanding Example
10.5. Other Relaxation Techniques
10.5.1. Pressure-Jump Methods Increase
the Versatility of Relaxation
Studies
10.5.2. Concentration Analysis (CCA)
10.6. Other Rapid Reaction Methods
10.6.1. Flash Photolysis
10.6.2. Pulsed Radiolysis
10.7. Data Analysis
10.8. Concluding Remarks
623
627
631
634
635
637
638
641
641
642
645
647
649
653
Chapter 11. Regulatory Behavior of
Enzymes
11.1. Overview of Enzyme Regulation
11.2. General Strategies for Measuring
Ligand Binding
11.3. The Hill Equation
11.4. The Scatchard Equation
11.4.1. A Modified Scatchard Equation
Accounts for Steric Hindrance
Amongst Sites
11.4.2. The Scatchard Analysis may be
Extended to Deal with Two
Classes of Binding Interactions -
One Strong and One Weak
11.5. Wyman s Linked Function Analysis
11.6. The Monod-Wyman-Changeux Model
11.6.1. Several Key Properties of
Allosteric Systems Suggested
the Symmetry-Conserving
MWC Model
654
655
656
658
658
666
669
669
670
671
672
672
673
674
675
677
678
680
685
685
688
691
693
693
694
694
695
695
XVI
Contents
11.6.2. MWC Ligand Saturation Functions
are Simple Polynomials Accounting
for Ligand Binding to One or Two
Conformationally Distinct States
of the Enzyme
11.6.3. The Saturation Function may be
Generalized to Explain Ligand
Binding to an Oligomer with n
Symmetry-Conserved Sites
11.6.4. The MWC Model Also Accounts
for the Effects of Positive and
Negative Allosteric Modifiers
11.7. The Koshland-Nemethy-Filmer Model
11.7.1. The KNF Model is Rooted in
Adair s Treatment of Polyvalent
Ligand Binding Interactions
11.7.2. The KNF Model Incorporates
Elements of Pauling s Site
Interaction Model
11.7.3. The KNF Model Accounts for
Both Positive and Negative
Cooperativity
11.7.4. White not an Enzyme,
Hemoglobin Provided Many
Clues About Allostery
11.7.5. Negative Cooperativity
Distinguishes KNF Models
from MWC Models
11.7.6. Fraction-of-the-Sites Behavior:
The Case of Escherichia
coli Alkaline Phosphatase
11.8. Other Cooperativity Models
11.8.1. Hybrid Cooperativity Models
11.8.2. The Duke, Le Novere and Bray
Conformational Spread Model
11.8.3. V-Type Allosteric Systems
11.9. Oligomerization-Dependent Changes
in Enzyme Activity
11.9.1. Enzyme Self-Association
can Alter Catalytic Activity
11.9.2. Some Substrates Alter Enzyme
Oligomerization and Catalytic
Activity
11.10. Hysteresis
11.11. Enzyme Amplification Cascades
11.12. Substrate Channeling
11.12.1. Several Criteria Define
Substrate Channeling
11.12.2. Tryptophan Synthase is an
Outstanding Example of
Substrate Channeling
11.12.3. The Once-Confusing Story
of NAD+ Transfer Between
Dehydrogenases lllustrates
the Need for Careful Studies
on Substrate Channeling
11.12.4. Substrate Hydration may
also Affect Channeling
Measurements
11.13. Metabolie Control Analysis
696 11.14. Concluding Comments
698
Chapter 12. Single-Molecule Enzyme
Kinetics
722
723
726
729
12.1. General Comments on Single-Molecule
699 Enzyme Kinetics 729
699 12.2. Demonstration of Single-Molecule
Reaction Rates 730
12.3. Kinetic Treatment of Single-Molecule
700 Enzyme Behavior 733
12.4. Video Microscopy 737
12.4.1. Kinesin Takes One 8-nm Step
700 12.4.2. per ATP Molecule Hydrolyzed Dark-Field Microscopy Affords Direct Observation of 737
701 Microtubule Assembly/ Disassembly Dynamics 738
12.5. Optica I Tweezers 739
702 12.5.1. Optical Tweezers Facilitated Single-Molecule Studies on RNA Polymerase 740
703 12.5.2. Optical Trapping Facilitates Single-Molecule Studies of RecA Polymerization on
705 Double-Stranded DNA 742
707 12.5.3. Actin-Based Listeria Motility
707 Exhibits Monomer-Sized Stepping 742
708 12.6. Atomic Force Microscopy 744
709 12.7. Near-Field Optical Microscopy 745
12.8. Fluorescence Microscopy 746
709 12.8.1. Epifluorescence Permits Uniform Sample Illumination 746
709 12.8.2. Fluorescence Microscopy Permits Direct Observation of Rotatory Catalysis 748
711 12.8.3. Total Internal Reflection
712 Fluorescence Microscopy
713 (TIRFM) Exploits Evanescent
718 12.8.4. Wave Phenomena Single Dihydrofolate Reductase 749
720 12.8.5. Molecules Blink During Catalysis Single-Molecule Fluorescence Facilitates Observation 749
721 12.8.6. of Dextran Binding to Bacterial Glucosyltransferase Single-Molecule Fluorescence also Provides a Way to Analyze the Conformational Dynamics 750
722 of Staphylococcal Nuclease Catalysis 751
Contents
XVII
12.9. Fluorescence Correlation Spectroscopy (FCS) 751
12.9.1. FCS Detects Emitted Light
Fluctuations within Extremely
Small Volumes 752 13.5
12.9.2. One- and Two-Photon FCS
Provides a Highly Versati le
Enzyme Probe 753
12.9.3. Basic Kinetic Theory 754
12.9.4. Proteolyse Cleavage may be
Fruitfully Investigated by FCS 755
12.9.5. Endonucleolytic Cleavage has
also been Examined by FCS 757
12.10. Zero-Mode Waveguides for
Single-Molecule Analysis 757
12.11. Prospects 758
13.6.
13.7.
13.8.
13.9.
Chapter 13. Mechanoenzymes: Catalysis,
Force Generation and
Kinetics 761
13.1. Brief Overview of Energase-Type Reactions 761
13.2. The Driving Force for Affinity-
Modulated Molecular Motors 766
13.3. Qualitative Features of Force-Induced
Noncovalent Bond Rupture 770
13.3.1. Bond Energetics may be Described
as Potential Energy Functions 770
13.3.2. Kramers Developed an Insightful
Bond Rupture Model 770
13.3.3. Noncovalent Bonding
Interactions are Inherent to
Mechanoenzyme Action 771
13.3.4. Noncovalent Bonds may be
Classified as Ideal, Slip,
and Catch Bonds 773
13.3.5. Green Fluorescent Protein
Unfolding/Refolding is
Force-Dependent 775
13.4. Keller-Bustamante Treatment of
Molecular Motor Behavior 776
13.4.1. Motor Molecu le Motions are
Analyzed in Terms of State
Space and the Potential of
Mean Force 777
13.4.2. Molecular Motors Operate
Stochastically 778
13.4.3. Motors Walk on the Potential
Energy Surface During Chemical
and Positional Transitions 778
Calcium Ion Pump: Chemical
Specificity versus Vectorial
Specificity 779
Actomyosin Mechanism 782
GTP-Regulatory Proteins 782
AAA* Mechanoenzymes 783
13.8.1. The AAA ATPases Possess
Common Structural Elements 784
13.8.2. DNA Processivity Clamp
Loader: An AAA4 Mechanoenzyme 785
Gradient-Driven Mechanoenzymatic
Processes 788
13.10. ATP Synthase: Boyer s Binding
Change Mechanism 790
13.11. Role of ATP in Protein Folding 792
13.11.1. GroEL/GroES is a Model
Foldase System 792
13.11.2. GroEL/GroES Mediates an
Annealing/Folding Cycle 792
13.12. Actoclampin Molecular Motors 793
13.12.1. Hill-Type Mechanisms for
Force Generation by
Polymerizing Free-ended
Filaments 794
13.12.2. The Actoclampin Hypothesis:
Concerning the Existence
and Action of Cytoskeletal
Filament End-Tracking
Motors 794
13.12.3. Processive Single-Filament
End-Tracking by ActA-VASP
Complex 795
13.12.4. Properties of Actoclampin Motors 798
13.13. Concluding Remarks and Prospects 802
References 807
Appendix 845
Glossary 847
Index 865
|
| any_adam_object | 1 |
| author | Purich, Daniel L. |
| author_facet | Purich, Daniel L. |
| author_role | aut |
| author_sort | Purich, Daniel L. |
| author_variant | d l p dl dlp |
| building | Verbundindex |
| bvnumber | BV036515391 |
| classification_rvk | VK 8700 WC 4350 WD 5050 |
| classification_tum | CHE 827f |
| ctrlnum | (OCoLC)815878854 (DE-599)BVBBV036515391 |
| discipline | Chemie / Pharmazie Biologie Chemie |
| edition | 1. ed. |
| format | Book |
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| id | DE-604.BV036515391 |
| illustrated | Illustrated |
| indexdate | 2024-07-09T22:42:03Z |
| institution | BVB |
| isbn | 9780123809247 |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-020437518 |
| oclc_num | 815878854 |
| open_access_boolean | |
| owner | DE-11 DE-19 DE-BY-UBM DE-M49 DE-BY-TUM |
| owner_facet | DE-11 DE-19 DE-BY-UBM DE-M49 DE-BY-TUM |
| physical | XXI, 892 S. Ill., graph. Darst. |
| publishDate | 2010 |
| publishDateSearch | 2010 |
| publishDateSort | 2010 |
| publisher | Elsevier |
| record_format | marc |
| spelling | Purich, Daniel L. Verfasser aut Enzyme kinetics catalysis & control ; a reference of theory and best-practice methods Daniel L. Purich 1. ed. Amsterdam [u.a.] Elsevier 2010 XXI, 892 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Enzymkatalyse (DE-588)4152480-9 gnd rswk-swf Enzymkinetik (DE-588)4133166-7 gnd rswk-swf Enzymkinetik (DE-588)4133166-7 s DE-604 Enzymkatalyse (DE-588)4152480-9 s HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=020437518&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Purich, Daniel L. Enzyme kinetics catalysis & control ; a reference of theory and best-practice methods Enzymkatalyse (DE-588)4152480-9 gnd Enzymkinetik (DE-588)4133166-7 gnd |
| subject_GND | (DE-588)4152480-9 (DE-588)4133166-7 |
| title | Enzyme kinetics catalysis & control ; a reference of theory and best-practice methods |
| title_auth | Enzyme kinetics catalysis & control ; a reference of theory and best-practice methods |
| title_exact_search | Enzyme kinetics catalysis & control ; a reference of theory and best-practice methods |
| title_full | Enzyme kinetics catalysis & control ; a reference of theory and best-practice methods Daniel L. Purich |
| title_fullStr | Enzyme kinetics catalysis & control ; a reference of theory and best-practice methods Daniel L. Purich |
| title_full_unstemmed | Enzyme kinetics catalysis & control ; a reference of theory and best-practice methods Daniel L. Purich |
| title_short | Enzyme kinetics |
| title_sort | enzyme kinetics catalysis control a reference of theory and best practice methods |
| title_sub | catalysis & control ; a reference of theory and best-practice methods |
| topic | Enzymkatalyse (DE-588)4152480-9 gnd Enzymkinetik (DE-588)4133166-7 gnd |
| topic_facet | Enzymkatalyse Enzymkinetik |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=020437518&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT purichdaniell enzymekineticscatalysiscontrolareferenceoftheoryandbestpracticemethods |