ENZYME KINETICS - Enzymes

246 Pages·2004·4.14 MB·English

ENZYME KINETICS
A Modern Approach
ALEJANDRO G. MARANGONI
DepartmentofFoodScience
UniversityofGuelph
AJOHNWILEY&SONS,INC.,PUBLICATION

Thisbookisprintedonacid-freepaper.
Copyright2003byJohnWiley&Sons,Inc.Allrightsreserved.
PublishedbyJohnWiley&Sons,Inc.,Hoboken,NewJersey.
PublishedsimultaneouslyinCanada.
Nopartofthispublicationmaybereproduced,storedinaretrievalsystem,ortransmittedinany
formorbyanymeans,electronic,mechanical,photocopying,recording,scanning,orotherwise,
exceptaspermittedunderSection107or108ofthe1976UnitedStatesCopyrightAct,without
eitherthepriorwrittenpermissionofthePublisher,orauthorizationthroughpaymentofthe
appropriateper-copyfeetotheCopyrightClearanceCenter,Inc.,222RosewoodDrive,Danvers,
MA01923,978-750-8400,fax[phone],oronthewebatwww.copyright.com.Requeststo
thePublisherforpermissionshouldbeaddressedtothePermissionsDepartment,JohnWiley&
Sons,Inc.,111RiverStreet,Hoboken,NJ07030,[phone],fax[phone],e-mail:
[email protected].
LimitofLiability/DisclaimerofWarranty:Whilethepublisherandauthorhaveusedtheirbest
effortsinpreparingthisbook,theymakenorepresentationsorwarrantieswithrespecttothe
accuracyorcompletenessofthecontentsofthisbookandspecificallydisclaimanyimplied
warrantiesofmerchantabilityorfitnessforaparticularpurpose.Nowarrantymaybecreatedor
extendedbysalesrepresentativesorwrittensalesmaterials.Theadviceandstrategiescontained
hereinmaynotbesuitableforyoursituation.Youshouldconsultwithaprofessionalwhere
appropriate.Neitherthepublishernorauthorshallbeliableforanylossofprofitoranyother
commercialdamages,includingbutnotlimitedtospecial,incidental,consequential,orother
damages.
ForgeneralinformationonourotherproductsandservicespleasecontactourCustomerCare
DepartmentwithintheU.S.at877-762-2974,outsidetheU.S.at317-572-3993or
fax[phone].
Wileyalsopublishesitsbooksinavarietyofelectronicformats.Somecontentthatappearsin
print,however,maynotbeavailableinelectronicformat.
Fororderingandcustomerservice,call1-800-CALL-WILEY.
LibraryofCongressCataloging-in-PublicationData:
Marangoni,AlejandroG.,1965-
Enzymekinetics:amodernapproach/AlejandroG.Marangoni.
p.;cm.
ISBN0-471-15985-9(cloth)
1. Enzymekinetics.
[DNLM: 1. Enzymes. 2. Kinetics. 3. Models, Chemical. QU135M311e
2003] I. Title.
QP601.3.M372003
572(cid:1).7—dc21 2002014042
PrintedintheUnitedStatesofAmerica
10987654321
To Dianne,Isaac, andJoshua

CONTENTS
PREFACE xiii
1 TOOLS AND TECHNIQUES OF KINETIC ANALYSIS 1
1.1 Generalities / 1
1.2 Elementary Rate Laws / 2
1.2.1 Rate Equation / 2
1.2.2 Order of a Reaction / 3
1.2.3 Rate Constant / 4
1.2.4 Integrated Rate Equations / 4
1.2.4.1 Zero-Order Integrated Rate
Equation / 4
1.2.4.2 First-Order Integrated Rate
Equation / 5
1.2.4.3 Second-Order Integrated Rate
Equation / 7
1.2.4.4 Third-Order Integrated Rate
Equation / 8
1.2.4.5 Higher-Order Reactions / 9
1.2.4.6 Opposing Reactions / 9
1.2.4.7 Reaction Half-Life / 11
vii
viii CONTENTS
1.2.5 Experimental Determination of Reaction
Order and Rate Constants / 12
1.2.5.1 Differential Method (Initial Rate
Method) / 12
1.2.5.2 Integral Method / 13
1.3 Dependence of Reaction Rates on
Temperature / 14
1.3.1 Theoretical Considerations / 14
1.3.2 Energy of Activation / 18
1.4 Acid–Base Chemical Catalysis / 20
1.5 Theory of Reaction Rates / 23
1.6 Complex Reaction Pathways / 26
1.6.1 Numerical Integration and Regression / 28
1.6.1.1 Numerical Integration / 28
1.6.1.2 Least-Squares Minimization
(Regression Analysis) / 29
1.6.2 Exact Analytical Solution
(Non-Steady-State Approximation) / 39
1.6.3 Exact Analytical Solution (Steady-State
Approximation) / 40
2 HOW DO ENZYMES WORK? 41
3 CHARACTERIZATION OF ENZYME ACTIVITY 44
3.1 Progress Curve and Determination of Reaction
Velocity / 44
3.2 Catalysis Models: Equilibrium and Steady
State / 48
3.2.1 Equilibrium Model / 48
3.2.2 Steady-State Model / 49
3.2.3 Plot of v versus [S] / 50
3.3 General Strategy for Determination of the
Catalytic Constants Km and Vmax / 52
3.4 Practical Example / 53
3.5 Determination of Enzyme Catalytic Parameters
from the Progress Curve / 58
CONTENTS ix
4 REVERSIBLE ENZYME INHIBITION 61
4.1 Competitive Inhibition / 61
4.2 Uncompetitive Inhibition / 62
4.3 Linear Mixed Inhibition / 63
4.4 Noncompetitive Inhibition / 64
4.5 Applications / 65
4.5.1 Inhibition of Fumarase by Succinate / 65
4.5.2 Inhibition of Pancreatic Carboxypeptidase
A by β-Phenylpropionate / 67
4.5.3 Alternative Strategies / 69
5 IRREVERSIBLE ENZYME INHIBITION 70
5.1 Simple Irreversible Inhibition / 72
5.2 Simple Irreversible Inhibition in the Presence of
Substrate / 73
5.3 Time-Dependent Simple Irreversible
Inhibition / 75
5.4 Time-Dependent Simple Irreversible Inhibition in
the Presence of Substrate / 76
5.5 Differentiation Between Time-Dependent and
Time-Independent Inhibition / 78
6 pH DEPENDENCE OF ENZYME-CATALYZED
REACTIONS 79
6.1 The Model / 79
6.2 pH Dependence of the Catalytic Parameters / 82
6.3 New Method of Determining pK Values of
Catalytically Relevant Functional Groups / 84
7 TWO-SUBSTRATEREACTIONS 90
7.1 Random-Sequential Bi Bi Mechanism / 91
7.1.1 Constant [A] / 93
7.1.2 Constant [B] / 93
7.2 Ordered-Sequential Bi Bi Mechanism / 95
7.2.1 Constant [B] / 95
x CONTENTS
7.2.2 Constant [A] / 96
7.2.3 Order of Substrate Binding / 97
7.3 Ping-Pong Bi Bi Mechanism / 98
7.3.1 Constant [B] / 99
7.3.2 Constant [A] / 99
7.4 Differentiation Between Mechanisms / 100
8 MULTISITEAND COOPERATIVE ENZYMES 102
8.1 Sequential Interaction Model / 103
8.1.1 Basic Postulates / 103
8.1.2 Interaction Factors / 105
8.1.3 Microscopic versus Macroscopic
Dissociation Constants / 106
8.1.4 Generalization of the Model / 107
8.2 Concerted Transition or Symmetry Model / 109
8.3 Application / 114
8.4 Reality Check / 115
9 IMMOBILIZED ENZYMES 116
9.1 Batch Reactors / 116
9.2 Plug-Flow Reactors / 118
9.3 Continuous-Stirred Reactors / 119
10 INTERFACIAL ENZYMES 121
10.1 The Model / 122
10.1.1 Interfacial Binding / 122
10.1.2 Interfacial Catalysis / 123
10.2 Determination of Interfacial Area per Unit
Volume / 125
10.3 Determination of Saturation Interfacial Enzyme
Coverage / 127
11 TRANSIENT PHASES OF ENZYMATIC REACTIONS 129
11.1 Rapid Reaction Techniques / 130
11.2 Reaction Mechanisms / 132