Download How Enzymes Work: From Structure to Function

Contents
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Preface
1. Introduction
1.1 General Properties of Enzyme
1.1.1 Enzyme Specificity
1.1.2 Rate Enhancement
1.2 Examples of Enzyme
1.2.1 Neurotransmission and Muscular Action 1.2.2 Gastric Juice and Proton Pump
1.2.3 Genetic Test of Alcohol Sensitivity and
DNA Polymerase
1.2.4 Enzyme Sensor Determination of Glucose
2. Overall Reaction Kinetics
2.1 Road to the Steady State Kinetics
2.1.1 Sucrose Hydrolysis
2.1.2 Henri’s Treatment of the Enzymatic
Reaction
2.1.3 Michaelis–Menten Equation
2.1.4 Briggs and Haldane’s Steady State
Method
2.2 Demonstration of the Enzyme–Substrate
Complex
2.2.1 Peroxidase Reaction
2.2.2 Crystallization of the ES Complex
2.3 Meaning of Steady State
2.3.1 Steady State Model: Tab Model
2.3.2 Application of the Tab Model to the
Enzymatic Reaction
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2.4 Kinetic Parameters
2.4.1 kcat
2.4.2 kcat/Km
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3.1 Enzyme Concentration
3.2 Substrate Concentration
3.2.1 One Substrate Reaction
3.2.2 Two-Substrate Reaction
3.2.2.1 Ordered bi-bi mechanism
3.2.2.2 Random bi-bi mechanism
3.2.2.3 Ping-Pong bi-bi mechanism
3.3 Inhibitor
3.3.1 Reversibility
3.3.2 Derivation of Rate Equations
3.3.2.1 Competitive inhibition
3.3.2.2 Non-competitive Inhibition
3.3.2.3 Uncompetitive Inhibition
3.3.2.4 Mixed-type inhibition
3.3.3 Graphical Method for the Determination
of the Type of Inhibition and Dissociation
Constants
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4.1 Effect of pH
4.1.1 A Basic Model
4.1.2 Graphical Methods to Determine pK Value
4.1.3 Meaning of pK Values
4.2 Thermodynamics in the Enzymatic Reaction
4.2.1 Basics of Thermodynamics
4.2.2 Transition State Theory
4.2.3 Determination of Thermodynamic
Parameters of the Enzymatic Reaction
4.3 Temperature Dependence of the Enzymatic
Reaction
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3. Factors That Affect Enzyme Activity
4. Effect of pH, Temperature, and High Pressure on
Enzymatic Activity
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4.4 Effect of Pressure
4.4.1 Effect of Pressure on the Rate of Reaction
4.4.2 Meaning of the Activation Volume
4.5 The Effect of Temperature and Pressure on
a-Chymotrypsin-Catalyzed Reaction
4.5.1 Ef fect of Temperature
4.5.2 Effect of Pressure
5. Measurement of Individual Rate Constants
5.1 Rapid-Mixing Techniques
5.2 Analysis of the First-Order Reaction
5.2.1 Order of Reaction
5.2.2 Practical Methods to Determine the
First-Order Rate Constant
6. Structure of Protein
6.1 Amino Acids
6.2 Polypeptide and Protein
6.3 Analysis of Primary Structure
6.3.1 Protein Chemical Methods
6.3.2 cDNA Sequencing: Dideoxy Method
6.4 Three-Dimensional Structure
6.4.1 Weak Interactions
6.4.1.1 Electrostatic interaction
6.4.1.2 Hydrogen bond
6.4.1.3 Hydrophobic interaction
6.4.1.4 van der Waals force
6.4.2 Secondary Structures and Their Determination
6.4.2.1 a helix
6.4.2.2 b sheet and b turn
6.4.2.3 Determination of secondary
structures
6.5 Tertiary and Quaternary Structures
6.6 Structural Motif and Loop
6.6.1 Supersecondary Structures: Motifs
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7. Active Site Structure
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7.1 Active Site and Active Center
7.2 Cofactor, Coenzyme, Prosthetic Group
7.2.1 NAD+ (Nicotinamide Adenine Dinucleotide)
and NADP+ (Nicotinamide Adenine
Dinucleotide Phosphate)
7.2.2 Coenzyme A (CoA, CoA-SH)
7.2.3 Flavin Mononucleotide (FMN), Flavin
Adenine Dinucleotide (FAD)
7.2.4 Heme
7.2.5 Pyridoxal Phosphate (PLP)
7.2.6 Folate
7.2.7 Thiamine Pyrophosphate
7.2.8 Biotin
7.2.9 Lipoamide
7.2.10 Protein-Derived Cofactors
7.3 Search of Active Site
7.3.1 Chemical Modification 7.3.1.1 Amino group
7.3.1.2 Carboxyl group
7.3.1.3 Sulfhydryl group
7.3.1.4 Hydroxyl group 7.3.1.5 Guanidino group
7.3.1.6 Imidazole group
7.3.1.7 Indole group
7.3.2 Site-Directed Mutagenesis 7.3.3 Examples of Active Site Studies
7.3.3.1 Chemical modification of l-Phe
oxidase
7.3.3.2 Site-directed mutagenesis of
thermostable l-lactate
dehydrogenase
8. Control of Enzyme Activity
8.1 Regulation by Non-Covalent Interaction
8.2 Regulation by Covalent Modification © 2015 by Taylor & Francis Group, LLC
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8.2.1 Activation of Enzymes by Cleavage of
Polypeptide Chain
8.2.2 Regulation by the Side Chain
Phosphorylation
8.2.2.1 cAMP-dependent protein kinase,
protein kinaseA (PKA) and glycogen
metabolism
8.2.2.2 Regulatory subunit of PKA
8.2.2.3 Catalytic subunit and overall
reaction mechanism of catalysis
8.2.2.4 Phosphoryl transfer reactions
at the active site of the C subunit
9. Preparation of Enzyme
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9.1 Extraction of Enzyme
9.2 Purification of Enzyme
9.2.1 Method to Use the Solubility of Proteins
9.2.1.1 Salting-out
9.2.1.2 Precipitation with organic
solvents
9.2.2 Column Chromatography
9.2.2.1 Ion exchangers
9.2.2.2 Gel filtration
9.2.2.3 Affinity chromatography
9.3 Purity Analysis of Enzyme
9.3.1 Electrophoresis
9.3.2 Sodium Dodecyl Sulfate Polyacrylamide
Gel Electrophoresis
9.3.3 Isoelectric Focusing
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10.1 Introduction
10.2 Preparation of PAO
10.2.1 Preparation of the Cell Extracts
10.2.2 Purification of PAO by Column
Chromatographies
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10. A Case Study: l-Phenylalanine Oxidase (Deaminating
and Decarboxylating)
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10.3 Catalytic Properties of PAO
10.3.1 Stoichiometry of the Reaction Catalyzed
by PAO
10.3.2 Overall Reaction Kinetics
10.3.3 Determination of Kinetic Constants
10.3.4 Hydrogen Quantum Tunneling in the
PAO-Catalyzed Reaction
10.3.4.1 Hydrogen quantum tunneling
(hydrogen tunneling)
10.3.4.2 Hydrogen tunneling in the
PAO-catalyzed reaction
10.4 Structural Properties of PAO
10.4.1 Nucleotide and Its Deduced
Amino Sequences of PAO Gene and
Its Expression
10.4.2 3D Structures of proPAO and PAOpt
10.5 Substrate Specificity and Reaction
Specificity of PAO
Appendix Solutions Index © 2015 by Taylor & Francis Group, LLC
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