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ENZYMES

THIRDEDITION

RobertA.Copeland

Thisthirdeditionfirstpublished2023 ©2023JohnWiley&Sons,Inc.

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LibraryofCongressCataloging-in-PublicationData Names:Copeland,RobertAllen,author.

Title:Enzymes:apracticalintroductiontostructure,mechanism,anddata analysis/RobertA.Copeland.

Description:Thirdedition.|Hoboken,NJ:Wiley,2023.|Revisededition of:Enzymes/RobertA.Copeland.2nded.c2000.|Includes bibliographicalreferencesandindex.

Identifiers:LCCN2022048916(print)|LCCN2022048917(ebook)|ISBN 9781119793250(cloth)|ISBN9781119793281(adobepdf)|ISBN 9781119793298(epub)

Subjects:LCSH:Enzymes.|Enzymology.

Classification:LCCQP601.C7532023(print)|LCCQP601(ebook)|DDC 572/.7–dc23/eng/20221128

LCrecordavailableathttps://lccn.loc.gov/2022048916

LCebookrecordavailableathttps://lccn.loc.gov/2022048917

CoverDesign:Wiley

CoverImage:CourtesyofRobertA.Copeland

Setin11/13ptTimesLTStdbyStraive,Chennai,India

ToClydeWorthenforteachingmealltheimportant lessons: arigatosensei.

AndtoTheodore(Doc)Jannerforstokingthefire.

PrefacetotheThirdEditionxvii

PrefacetotheSecondEditionxix

PrefacetotheFirstEditionxxi

Acknowledgmentsxxiii

1ABriefHistoryofEnzymology1

KeyLearningPoints / 1

1.1EnzymesinAntiquity / 2

1.2EarlyEnzymology / 3

1.3TheDevelopmentofMechanisticEnzymology / 4

1.4StudiesofEnzymeStructure / 5

1.5EnzymologyToday / 7

1.6Summary / 9 ReferencesandFurtherReading / 9

2ChemicalBondsandReactionsinBiochemistry11

KeyLearningPoints / 11

2.1AtomicandMolecularOrbitals / 12

2.1.1AtomicOrbitals / 12

2.1.2MolecularOrbitals / 15

2.1.3HybridOrbitals / 16

2.1.4ResonanceandAromaticity / 18

2.1.5DifferentElectronicConfigurationsHaveDifferentPotential Energies / 20

2.2ThermodynamicsofChemicalReactions / 22

2.2.1TheTransitionStateofChemicalReactions / 24

2.3Acid–baseChemistry / 27

2.4NoncovalentInteractionsinReversibleBinding / 29

2.4.1ElectrostaticInteractions / 30

2.4.2HydrogenBonding / 30

2.4.3HydrophobicInteractions / 31

2.4.4VanderWaalsForces / 31

2.5RatesofChemicalReactions / 33

2.5.1ReactionOrder / 35

2.5.2ReversibleChemicalReactions / 36

2.5.3MeasurementofInitialVelocity / 37

2.6Summary / 38

ReferencesandFurtherReading / 38

3StructuralComponentsofEnzymes39

KeyLearningPoints / 39

3.1TheAminoAcids / 40

3.1.1PropertiesofAmino-AcidSideChains / 42

3.1.1.1Hydrophobicity / 42

3.1.1.2HydrogenBonding / 42

3.1.1.3Salt-BridgeFormation / 43

3.1.2AminoAcidsasAcidsandBases / 44

3.1.3CationandMetalBinding / 45

3.1.4AnionandPolyanionBinding / 46

3.1.5CovalentBondFormation / 46

3.1.5.1DisulfideBonds / 46

3.1.5.2Phosphorylation / 46

3.1.5.3Glycosylation / 47

3.1.6StericBulk / 47

3.2ThePeptideBond / 48

3.3AminoAcidSequenceorPrimaryStructure / 51

3.4SecondaryStructure / 54

3.4.1TheRight-Handed �� Helix / 55

3.4.2The �� -PleatedSheet / 56

3.4.3 �� Turns / 58

3.4.4OtherSecondaryStructures / 58

3.4.5SupersecondaryStructures / 59

3.5TertiaryStructure / 60

3.5.1Domains / 62

3.6SubunitsandQuaternaryStructure / 64

3.7CofactorsinEnzymes / 67

3.8ConformationalDynamicsandEnzymeFunction / 70

3.9MethodsofProteinStructureDetermination / 75

3.9.1X-rayCrystallography / 76

3.9.2NMRSpectroscopy / 77

3.9.3Cryo-ElectronMicroscopy(Cryo-EM) / 78

3.10Summary / 79

ReferencesandFurtherReading / 80

4Protein–LigandBindingEquilibria83

KeyLearningsPoints / 83

4.1TheEquilibriumDissociationConstant, Kd / 84

4.2TheKineticApproachtoEquilibrium / 86

4.3BindingMeasurementsatEquilibrium / 88

4.3.1DerivationoftheLangmuirIsotherm / 88

4.3.2MultipleBindingSites / 91

4.3.2.1MultipleEquivalentBindingSites / 91

4.3.2.2MultipleNonequivalentBindingSites / 92

4.3.2.3CooperativeInteractionsAmongMultipleBinding Sites / 92

4.3.3CorrectionforNonspecificBinding / 93

4.4GraphicAnalysisofEquilibriumLigand-BindingData / 94

4.4.1DirectPlotsonSemilogScale / 94

4.4.2LinearTransformationsofBindingData:TheWolffPlots / 97

4.5EquilibriumBindingwithLigandDepletion(TightBinding Interactions) / 100

4.6CompetitionAmongLigandsforaCommonBindingSite / 101

4.7ProteinDynamicsinReceptor–LigandBinding / 102

4.8OrthostericandAllostericLigandBindingSites / 104

4.9ExperimentalMethodsforMeasuringLigandBinding / 105

4.9.1MethodsBasedonMassorMobilityDifferences / 105

4.9.1.1EquilibriumDialysis / 105

4.9.1.2MembraneFiltrationMethods / 107

4.9.1.3SizeExclusionChromatography / 109

4.9.1.4MicroscaleThermophoresis / 111

4.9.2SpectroscopicMethods / 113

4.9.2.1FluorescenceSpectroscopy / 113

4.9.2.2SurfacePlasmonResonance / 116

4.9.3Ligand-InducedProteinStabilization / 117

4.9.3.1ThermalDenaturationofProteins / 118

4.9.3.2ChemicalDenaturationofProteins / 120

4.10Summary / 122

ReferencesandFurtherReading / 122

5Steady-StateKineticsofSingle-SubstrateEnzymeReactions125

KeyLearningPoints / 125

5.1TheTimeCourseofEnzymaticReactions / 126

5.2EffectsofSubstrateConcentrationonVelocity / 127

5.3TheRapidEquilibriumModelofEnzymeKinetics / 129

5.4TheSteady-StateModelofEnzymeKinetics / 131

5.5TheSignificanceof kcat and Km / 134

5.5.1 Km / 135

5.5.2 kcat / 135

5.5.3 kcat /Km / 136

5.5.4Diffusion-ControlledReactionsandKineticPerfection / 138

5.6ExperimentalMeasurementof kcat and Km / 139

5.6.1GraphicalDeterminationsfromUntransformedData / 139

5.6.2Lineweaver–BurkPlotsofEnzymeKinetics / 142

5.7OtherLinearTransformationsofEnzymeKineticData / 147

5.7.1Eadie–HofsteePlots / 147

5.7.2Hanes–WolffPlots / 148

5.7.3Eisenthal–Cornish-BowdenDirectPlots / 148

5.8MeasurementsatLowSubstrateConcentrations / 149

5.9DeviationsFromHyperbolicKinetics / 150

5.10Summary / 153 ReferencesandFurtherReading / 153

6ChemicalMechanismsinEnzymeCatalysis155

KeyLearningPoints / 155

6.1Substrate–ActiveSiteComplementarity / 156

6.2RateEnhancementThroughTransitionStateStabilization / 159

6.3ChemicalMechanismsforTransitionStateStabilization / 162

6.3.1ApproximationofReactants / 163

6.3.2CovalentCatalysis / 166

6.3.2.1NucleophilicCatalysis / 167

6.3.2.2ElectrophilicCatalysis / 168

6.3.3GeneralAcid/BaseCatalysis / 170

6.3.4ConformationalDistortion / 175

6.3.5PreorganizedActiveSiteComplementaritytotheTransition State / 180

6.4TheSerineProteases:AnIllustrativeExample / 182

6.5EnzymaticReactionNomenclature / 187

6.6Summary / 191

ReferencesandFurtherReading / 191

7ExperimentalMeasuresofSteady-StateEnzymeActivity193

KeyLearningPoints / 193

7.1InitialVelocityMeasurements / 194

7.1.1Direct,Indirect,andCoupledAssays / 194

7.1.2AnalysisofProgressCurves:MeasuringTrueSteady-State Velocity / 200

7.1.3ContinuousVersusEndPointAssays / 203

7.1.4Initiating,Mixing,andStoppingReactions / 204

7.1.5TheImportanceofRunningControls / 206

7.2DetectionMethods / 208

7.2.1AssaysBasedonOpticalSpectroscopy / 208

7.2.2AbsorptionMeasurements / 208

7.2.3ChoosinganAnalyticalWavelength / 210

7.2.4OpticalCells / 210

7.2.5ErrorsinAbsorptionSpectroscopy / 212

7.2.6FluorescenceMeasurements / 213

7.2.7InternalFluorescenceQuenchingandEnergyTransfer / 215

7.2.8ErrorsinFluorescenceMeasurements / 217

7.2.9RadioisotopicMeasurements / 220

7.2.10ErrorsinRadioactivityMeasurements / 223

7.2.11OtherDetectionMethods / 223

7.3SeparationMethodsinEnzymeAssays / 224

7.3.1SeparationofProteinsfromLowMolecularWeightSolutes / 224

7.3.2ChromatographicSeparationMethods / 225

7.3.3ElectrophoreticMethodsinEnzymeAssays / 230

7.4FactorsAffectingtheVelocityofEnzymaticReactions / 236

7.4.1EnzymeConcentration / 237

7.4.2pHEffects / 239

7.4.3TemperatureEffects / 245

7.4.4ViscosityEffects / 247

7.4.5IsotopeEffectsinEnzymeKinetics / 249

7.5ReportingEnzymeActivityData / 252

7.6EnzymeStability / 253

7.6.1StabilizingEnzymesDuringStorage / 254

7.6.2EnzymeInactivationDuringActivityAssays / 255

7.7Summary / 258

ReferencesandFurtherReading / 258

8Transient-StateKinetics261

KeyLearningPoints / 261

8.1TimescaleofPre-Steady-StateTurnover / 262

8.2InstrumentationforTransientKineticMeasurements / 264

8.3EstimatingInitialConditionsforTransientKineticMeasurements / 266

8.4ExamplesofSomeCommonTransientKineticReactionMechanisms / 267

8.4.1OneStep,IrreversibleBinding / 267

8.4.2OneStep,ReversibleBinding / 268

8.4.3Consecutive,IrreversibleReaction / 268

8.4.4Consecutive,ReversibleReactionwithaFastFirstStep (Pre-equilibriumReaction) / 269

8.4.5Consecutive,ReversibleReactionwithaFastSecondStep(Enzyme Pre-isomerization) / 271

8.5ExamplesofTransientKineticStudiesfromtheLiterature / 272

8.5.1StudyofSubstrateandInhibitorInteractionswiththeAlzheimer’s Disease β-SiteAmyloidPrecursorProtein-CleavingEnzyme (BACE) / 272

8.5.2StudyoftheMechanismofTime-DependentInhibitionof Staphylococcusaureus PolypeptideDeformylase / 275

8.6Summary / 277

ReferencesandFurtherReading / 278

9EnzymeRegulation279

KeyLearningPoints / 279

9.1ActiveandInactiveConformationalStates / 280

9.2Post-TranslationalModifications / 281

9.2.1ProteolyticProcessing / 282

9.2.2CovalentModificationofAminoAcidSideChains / 288

9.3EnzymeRegulationThroughProtein–ProteinInteractions / 294

9.4Small-MoleculeAllostericLigands / 297

9.4.1HomotropicandHeterotropicAllostery / 298

9.4.2IntramolecularandIntermolecularAllostery / 298

9.5QuantitativeMeasurementsofEnzymeActivationandInhibition / 302

9.5.1ThermodynamicMeasurementofActivator–Enzyme Interactions / 303

9.5.2KineticMeasurementofEnzymeActivationbyPTM / 306

9.6RegulationofProteinKinases / 308

9.6.1KinaseActivationbyPTM / 308

9.6.2KinaseRegulationbyProteinAssociation / 311

9.6.3KinaseActivationbyOligomerization / 312

9.6.4KinaseRegulationbySmall-MoleculeBinding / 313

9.6.5Small-MoleculeMimicryofIntramolecularAllostery / 313

9.7Summary / 314

ReferencesandFurtherReading / 315

10ReversibleInhibitors317

KeyLearningPoints / 317

10.1EquilibriumTreatmentofReversibleInhibition / 319

10.2ThermodynamicModesofReversibleInhibition / 321

10.2.1PureCompetitiveInhibition,ExclusiveBindingtoFreeEnzyme(E): �� =∞ / 321

10.2.2MixedorNoncompetitiveInhibition / 322

10.2.2.1MixedInhibitorsThatBindPreferentiallytotheFree Enzyme(E): ��> 1 / 322

10.2.2.2MixedInhibitorsThatBindEquipotentlytoEandES: �� = 1 / 322

10.2.2.3MixedInhibitorsThatBindPreferentiallytothe Enzyme–SubstrateComplex(ES): ��< 1 / 322

10.2.3PureUncompetitiveInhibitors,ExclusiveBindingtothe Enzyme-SubstrateComplex(ES): ��≪ 1 / 323

10.2.4PartialInhibitors / 323

10.3EffectsofInhibitorsonSteady-StateParameters / 324

10.3.1CompetitiveInhibitors / 325

10.3.2NoncompetitiveInhibitors / 329

10.3.3UncompetitiveInhibitors / 330

10.3.4FittingofUntransformedData / 332

10.4Concentration-ResponsePlotsofEnzymeInhibition / 333

10.4.1Concentration-ResponsePlotsforPartialInhibition / 336

10.5EffectsofSubstrateConcentrationonInhibitorConcentration–Response Curves / 337

10.6MutuallyExclusiveBindingofTwoInhibitors / 340

10.7Structure–ActivityRelationshipsandInhibitorDesign / 343

10.7.1SARintheAbsenceofEnzymeStructuralInformation / 343

10.7.2InhibitorDesignBasedonEnzymeStructure / 350

10.8Summary / 353

ReferencesandFurtherReading / 354

11Tight-BindingInhibitors357

KeyLearningPoints / 357

11.1IdentifyingTight-BindingInhibition / 358

11.2DistinguishingInhibitorTypeforTight-BindingInhibitors / 359

11.3Determining Ki forTight-BindingInhibitors / 362

11.4UseofTight-BindingInhibitorstoDetermineActiveEnzymeConcentration / 365

11.5Summary / 368

ReferencesandFurtherReading / 368

12Time-DependentInhibition371

KeyLearningPoints / 371

12.1ProgressCurvesforSlow-BindingInhibitors / 375

12.2DistinguishingBetweenSlow-BindingSchemes / 378

12.2.1SchemeB / 379

12.2.2SchemeC / 379

12.2.3SchemeD / 380

12.3DistinguishingBetweenModesofInhibitorInteractionwithEnzyme / 382

12.4DeterminingReversibility / 384

12.4.1Enzyme-InhibitorResidenceTime / 385

12.5ExamplesofSlow-BindingEnzymeInhibitors / 386

12.5.1SerineProteases / 386

12.5.2ProstaglandinG/HSynthase / 387

12.5.3ChemicalModificationasProbesofEnzymeStructureand Mechanism / 391

12.5.3.1AminoAcidSelectiveChemicalModification / 392

12.5.3.2SubstrateProtectionExperiments / 394

12.5.3.3AffinityLabels / 396

12.6Summary / 398

ReferencesandFurtherReading / 398

13EnzymeReactionswithMultipleSubstrates401

KeyLearningPoints / 401

13.1ReactionNomenclature / 402

13.2Bi–BiReactionMechanisms / 403

13.2.1RandomOrderedBi–BiReactions / 403

13.2.2Compulsory-OrderedBi–BiReactions / 404

13.2.3DoubleDisplacementorPing–PongBi–BiReactions / 406

13.3DistinguishingBetweenRandomandCompulsory-OrderedMechanismsby InhibitionPattern / 407

13.4IsotopeExchangeStudiesforDistinguishingReactionMechanisms / 409

13.5UsingtheKing–AltmanMethodtoDetermineVelocityEquations / 411

13.6Cleland’sNetRateConstantMethodforDetermining Vmax and Vmax /Km / 414

13.7Summary / 416

ReferencesandFurtherReading / 417

14Enzyme–MacromoleculeInteractions419

KeyLearningPoints / 419

14.1MutlitproteinEnzymeComplexes / 420

14.2EnzymeReactionsonMacromolecularSubstrates / 422

14.2.1DifferencesBetweenSmallMoleculeandProteinSubstrateBinding toEnzymes / 422

14.2.2FactorsAffectingProtein–ProteinInteractions / 425

14.2.3SeparationofBindingandCatalyticRecognitionElements / 427

14.2.4NoncompetitiveInhibitionbyActiveSiteBindingMoleculesfor ExositeUtilizingEnzymes / 429

14.2.5ProcessiveandDistributiveMechanismsofCatalysis / 430

14.2.6EffectofSubstrateConformationonEnzymeKinetics / 434

14.2.7InhibitorBindingtoSubstrates / 434

14.3Summary / 436

ReferencesandFurtherReading / 436

15CooperativityinEnzymeCatalysis439

KeyLearningPoints / 439

15.1HistoricExamplesofCooperativityandAllosteryinProteins / 441

15.2ModelsofAllostericBehavior / 445

15.3EffectsofCooperativityonVelocityCurves / 449

15.4SigmoidalKineticsforNonallostericEnzymes / 452

15.5Summary / 453

ReferencesandFurtherReading / 453

16EvolutionofEnzymes455

KeyLearningPoints / 455

16.1EarlyEarthConditions / 456

16.2NaturalSelection / 456

16.3GeneticAlterations / 459

16.3.1SingleNucleotidePolymorphisms(SNPs) / 459

16.3.2GeneDuplication / 460

16.3.3DeletionsandInsertions / 461

16.3.4TranslocationsandInversions / 461

16.4EnzymeFamiliesandSuperfamilies / 463

16.5EnzymePromiscuityasaSpringboardofEvolution / 467

16.5.1EvolutionofEnzymeSteadyStateParameters / 471

16.6ProteinDynamicsandConformationalSelectioninEvolutionof Neofunctionality / 474

16.7AncestralEnzymeReconstruction / 475

16.7.1MechanismofDrugSelectivityforGleevec / 477

16.7.2OvercomingEpistasistoDefinetheMechanismofSubstrate Specificity / 478

16.7.3RevealingGeneralisttoSpecialistEvolution / 479

16.7.4AncestralSequenceReconstructionasaPracticalTool / 480

16.8ContemporaryEnzymeEvolution / 480

16.9Summary / 483

ReferencesandFurtherReading / 483

17EnzymesinHumanHealth487

KeyLearningPoints / 487

17.1EnzymesasTherapeuticAgents / 487

17.2EnzymeInhibitorsasTherapeuticAgents / 488

17.2.1PropertiesofSmall-MoleculeDrugs / 489

17.2.2EnzymesasDrugTargets / 489

17.3EnzymeEssentialityinDisease / 492

17.3.1ParalogueswithDistinctPhysiologicalRoles / 492

17.3.2DistinctOrthologuesinInfectiousDiseases / 494

17.3.3DiseasesofLifestyle,Environmental,andAging / 497

17.3.4PathogenicAlterationstoEnzymeFunction / 501

17.3.4.1RelatingGeneticAlterationstoDiseaseEssentiality / 502

17.3.4.2EnzymeOverexpression / 505

17.3.4.3ActivatingMutations / 506

17.3.4.4ChromosomalTranslocations / 515

17.3.4.5SyntheticLethality / 518

17.3.5Pro-DrugActivationbyEnzymes / 522

17.4Enzyme-MediatedTargetProteinDegradation / 524

17.5TheRoleofEnzymologyinDrugDiscoveryandDevelopment / 527

17.5.1EnzymeSelectivityAssessment / 529

17.5.2CorrelatingEnzymeInhibitionwithCellularPhenotype / 530

17.5.3HepaticMetabolismofXenobiotics / 533

17.5.4Mutation-BasedDrugResistance / 535

17.6Summary / 537

ReferencesandFurtherReading / 537

PREFACETOTHETHIRDEDITION

“Twentyyearsnow Where’dtheygo?

Twentyyears

Idon’tknow

IsitandIwondersometimes Wherethey’vegone” –LikeaRock,BobSeger

WhenJonathanRose,myeditoratWiley,firstapproachedmeaboutwritingthethirdedition ofthisbook,Iwasabitsurprised.Iaskedhimwhyhethoughtaboutthenewedition.Iwas quiteshockedwhenheremindedmethatithadbeentwentyyearssincethepublicationofthe lastedition.Timetrulydoesfly,andthebiochemicalcommunityhascontinuedtomarchon, makingmanynoveldiscoveriesanddevelopingaplethoraofnewandimprovedtoolsalongthe way.Yet,despitethesegreatadvances,thefundamentalscienceofenzymologyhasretainedits primacyacrosseveryaspectofthelifesciences—frombasictoappliedlifesciences.Indeed, thepreeminenceofenzymologyiswhatfirstmotivatedmetowriteanintroductorybookthat wouldservetheneedsofstudentsandnewcomerstothefield,aswellasserveasakeyreference forlaboratory-basedresearchers.Hence,ithasbeenparticularlyrewardingformetoseethe previouseditionofthistextwidelyusedbythescientificcommunity,asevidencedbyitbeing citedintheprimaryscientificliteraturemorethan1,800times.Itwasclear,however,thatthe previouseditionneededupdating,andthisiswhatultimatelyconvincedmetoacceptJonathan’s recommendationtowritethethirdedition.

Inthisnewedition,Ihaverefined,augmented,andupdatedeachofthechaptersthatwere presentedinthesecondedition.Beyondthis,Ihaveaddednewchapterstocover,orbetter cover,aspectsofenzymologythathavegainedgreaterimportanceoverthepasttwodecades, andthatwereunder-representedinthepreviousedition.Forexample,Ihavebrokenout steady-state(Chapter7)andtransient-state(Chapter8)enzymekineticsintoseparatechapters, tobettercoverboththeseimportantapproaches.Thechapteronstructuralcomponentsof enzymes(Chapter3)hasbeenaugmentedwithcoverageofmethodsforproteinstructure determination.Likewise,newerexperimentalapproacheshavebeenaddedtothechapterson protein–ligandbindingequilibria(Chapter4),experimentalmeasuresofsteady-stateenzyme activity(Chapter7),andthechaptersonenzymeinhibitors(Chapters10–12).Completely newchaptershavebeenaddedtothisnewedition,tocovertheexpandingareasofenzyme xvii

regulation(Chapter9),enzyme–macromoleculeinteractions(Chapter14),enzymeevolution (Chapter16),andenzymesinhumanhealth(Chapter17).Finally,IhavealsoaddedKey LearningPointsatthebeginningofeachchapter,toaidstudentsandeducatorsineffectively usingthisbookasaclassroomtext.Thisideawassuggestedtomebymyfriendandcolleague, WilliamPitts,forthesecondeditionofmyalliedbook EvaluationofEnzymeInhibitorsin DrugDiscovery (Wiley)andhasbeenwidelyappreciatedbytheacademiccommunity;Ihope thisprovesequallyvaluableforthepresenttext.

Withthesechanges,itismyhopethatthisbookwillcontinuetoservewelltheneedsof thebasicandappliedlifesciencescommunities.Aswithpreviouseditions,thecurrenttext isintendedasanintroductiontoenzymesandausefulreferencesourceforthoseconducting laboratory-basedenzymologyresearch.Ihavetriedtocoverallthetopicswithinthebookin anaccurateandcompletemanner,butalsohavestrivedtouseanapproachablestyle,sothat newcomerstothefieldofenzymologycanbeinitiatedintothiscriticalscienceinawelcoming andunintimidatingfashion.Forthosewishingtodelveintospecificareasofenzymologyin greaterdepth,sufficientreferencesandfurtherreadingsuggestionsareofferedattheendof everychapter.

Aswithallmyscientificwriting,Ihavebenefittedgreatlyfromcommentsandsuggestions frommanycolleaguesandfriends.Beyondthoseacknowledgedintheearliereditions ofthistext,IwishtothankChristopherT.Walsh,JohnKozarich,VernSchramm,David Swinney,JamesWells,ThomasMeek,MargaretPorterScott,ShaneBuker,ChuangLu,Ann Boriack-Sjodin,ScottRibich,KennethDuncan,andalltheemployees,boardmembers,and scientificadvisorsofAccentTherapeutics,Inc.IalsowishtothankDr.AllenSickmierfor hishelpwithsomeoftheillustrationsinthisedition.AspecialthankyoutoProfessorKaren AllenandherlaboratoryatBostonUniversity,forprovidingdetailedcommentsonseveralof thenewchaptersinthisedition.

Finally,andalways,Ithankmyfamily—mywifeNancyandourdaughters,Lindseyand Amanda;theyaremyconstantandnever-failingsourcesoflearning,laughing,andlove.

RobertA.Copeland Lexington,Massachusetts 2022

PREFACETOTHESECOND EDITION

Inthefouryearssincethefirsteditionof Enzymes waspublished,Ihavebeendelightedto learnofthewideacceptanceofthebookthroughoutthebiochemicalcommunity,particularly inthepharmaceuticalcommunity.Duringthistime,anumberofcolleagueshavecontacted metoexpresstheirviewsonthevalueofthetext,andimportantlytomakesuggestionsfor improvementstothecontentandpresentationofsomeconcepts.Ihaveusedthefirsteditionas ateachingsupplementforacourseinwhichIlectureattheUniversityofPennsylvaniaSchoolof Medicine.Frommylectureexperiencesandfromconversationswithstudents,Ihavedeveloped somenewideasforhowtobetterexplainsomeoftheconceptsinthetextandhaveidentified areasthatdeserveexpandedcoverage.Finally,whilethefirsteditionhasbecomepopularwith studentsandindustrialscientists,someofmyacademiccolleagueshavesuggestedaneedfora morein-depthtreatmentofchemicalmechanismsinenzymology.

Inthissecondedition,Ihaverefinedandexpandedthecoverageofmanyoftheconceptsin thetext.Tohelpthereaderbetterunderstandsomeoftheinteractionsbetweenenzymesand theirsubstratesandinhibitors,anewchapteronprotein–ligandbindingequilibriahasbeen added(Chapter4).Thechaptersonchemicalmechanismsinenzymecatalysis(Chapter6)and onexperimentalmeasuresofenzymeactivity(Chapter7)havebeenexpandedsignificantly. Thediscussionsofenzymeinhibitorsandmultiplesubstratereactions(Chapters8through11) havebeenrefined,and,insomecases,alternativetreatmentshavebeenpresented.Inallofthis, however,Ihavetriedtomaintaintheintroductorynatureofthebook.Therearemanyexcellent advancedtextsoncatalysis,enzymemechanisms,andenzymekinetics,butthelevelatwhich thesearegenerallywrittenisoftenintimidatingtothebeginner.Hence,asstatedinthepreface tothefirstedition,thisbookisintendedtoserveasamechanismforthosenewtothefieldof enzymologytodevelopareasonableunderstandingofthescienceandexperimentalmethods, allowingthemtocompetentlybeginlaboratorystudieswithenzymes.Ihavecontinuedtorely onextensivecitationstomoreadvancedtextsandprimaryliteratureasameansfortheinterested readertogobeyondthetreatmentsofferedhereanddelvemoredeeplyintospecificareasof enzymology.

Indevelopingthissecondedition,Ihavehadfruitfulconversationsandadvicefromanumber ofcolleagues.Inparticular,IwishtothankAndyStern,RossStein,TrevorPenning,BillPitts, JohnBlanchard,DennisMurphy,andthemembersoftheChemicalEnzymologyDepartment

attheDuPontPharmaceuticalsCompany.Asalways,theloveandsupportofmyfamilyhave beenmostimportantinmakingthisworkpossible.

RobertA.Copeland

PREFACETOTHEFIRSTEDITION

Thelatterhalfofthiscenturyhasseenanunprecedentedexpansioninourknowledgeanduseof enzymesinabroadrangeofbasicresearchandindustrialapplications.Enzymesarethecatalytic cornerstonesofmetabolism,andassuchisthefocusofintenseresearchwithinthebiomedicalcommunity.Indeed,enzymesremainthemostcommontargetsfortherapeuticintervention withinthepharmaceuticalindustry.Sinceancienttimes,enzymesalsohaveplayedcentralroles inmanymanufacturingprocesses,suchasintheproductionofwine,cheese,andbread.During the1970sand1980s,muchofthefocusofthebiochemicalcommunityshiftedtothecloning andexpressionofproteinsthroughthemethodsofmolecularbiology.Recently,someattention hasshiftedbacktothephysicochemicalcharacterizationoftheseproteinsandtheirinteractions withothermacromoleculesandsmallmolecularweightligands(e.g.,substrates,activators,and inhibitors).Hence,therehasbeenaresurgenceofinterestinthestudyofenzymestructures, kinetics,andmechanismsofcatalysis.

Theavailabilityofup-to-date,introductoryleveltextbooks,however,hasnotkeptupwith thegrowingdemand.Ifirstbecameawareofthisvoid,whileteachingintroductorycoursesat themedicalandgraduatestudentlevelattheUniversityofChicago.Ifoundthattherewerea numberofexcellentadvancedtextsthatcovereddifferentaspectsofenzymologywithaheavy emphasisonthetheoreticalbasisformuchofthescience.ThemoreintroductorytextsthatI foundwereoftenquitedatedanddidnotoffertheblendoftheoreticalandpracticalinformation thatIfeltwasmostappropriateforabroadaudienceofstudents.Ithusdevelopedmyownset oflecturenotesforthesecourses,drawingmaterialfromawiderangeoftextbooksandprimary literature.

In1993,IleftChicagotofocusmyresearchontheutilizationofbasicenzymologyand proteinscienceforthedevelopmentoftherapeuticagentstocombathumandiseases.Topursue thisgoal,IjoinedthescientificstaffoftheDuPontMerckPharmaceuticalCompany.Duringmy firstyearwiththiscompany,agroupofassociatescientistsexpressedtometheirfrustrationat beingunabletofindatextbookonenzymologythatmettheirneedsforguidanceinlaboratory protocolsanddataanalysisatanappropriatelevelandatthesametimeprovidethemwithsome relevantbackgroundonthescientificbasisoftheirexperiments.Thesededicatedindividuals askedifIwouldprepareandpresentacourseonenzymologyatthisintroductorylevel.

UsingmylecturenotesfromChicagoasafoundation,Ipreparedanextensivesetofnotes andintendedtopresentayear-longcoursetoasmallgroupofassociatescientistsinaninformal, over-brown-bag-lunchfashion.Afterthelectureshadbeenannounced,however,Iwasshocked anddelightedtofindthatmorethan200peoplewereregisteredforthiscourse!Themakeup ofthestudentbodyrangedfromindividualswithassociatedegreesinmedicaltechnologyto xxi

PREFACETOTHEFIRSTEDITION

chemistsandmolecularbiologistswhohaddoctorates.Thisconvincedmethattherewasindeed agrowinginterestandneedforanewintroductoryenzymologytextthatwouldattemptto balancethetheoreticalandpracticalaspectsofenzymologyinsuchawayastofilltheneedsof graduateandmedicalstudents,aswellasresearchscientistsandtechnicianswhoareactively involvedinenzymestudies.

Thetextthatfollowsisbasedonthelecturenotesfortheenzymologycoursejustdescribed.It attemptstofillthepracticalneedsIhavearticulated,whilealsogivingareasonableintroduction tothetheoreticalbasisforthelaboratorymethodsanddataanalysesthatarecovered.Ihopethat thistextwillbeofusetoabroadrangeofscientistsinterestedinenzymes.Thematerialcovered shouldbeofdirectusetothoseactivelyinvolvedinenzymeresearchinacademic,industrial, andgovernmentlaboratories.Italsoshouldbeusefulasaprimarytextforseniorundergraduate orfirst-yeargraduatecourses,inintroductoryenzymology.However,inteachingasubjectas broadanddynamicasenzymology,Ihaveneverfoundasingletextthatwouldcoverallofmy students’needs;Idoubtthatthepresenttextwillbeanexception.Thus,whileIbelievethistext canserveasausefulfoundation,Iencouragefacultyandstudentstosupplementthematerial withadditionalreadingsfromtheliteraturecitedattheendofeachchapter,andtheprimary literaturethatiscontinuouslyexpandingourviewofenzymesandcatalysis.

Inattemptingtoprovideabalancedintroductiontoenzymesinasingle,readablevolume,I havehadtopresentsomeofthematerialinarathercursoryfashion;itissimplynotpossible,in atextofthisformat,tobecomprehensiveinsuchanexpansivefieldasenzymology.Ihopethat theliteraturecitationswillatleastpavethewayforreaderswhowishtodelvemoredeeplyinto particularareas.Overall,theintentofthisbookistogetpeople started inthelaboratoryandin theirthinkingaboutenzymes.Itprovidessufficientexperimentalanddatahandlingmethodologiestopermitonetobegintodesignandperformexperimentswithenzymes,whileatthesame timeprovidingatheoreticalframeworkinwhichtounderstandthebasisoftheexperimental work.Beyondthis,ifthebookfunctionsasastepping-stoneforthereadertomoveontomore comprehensiveandin-depthtreatmentsofenzymology,itwillhaveserveditspurpose.

RobertA.Copeland

Wilmington,Delaware 1995

ACKNOWLEDGMENTS

Itismygreatpleasuretothankthemanyfriendsandcoworkerswhohavehelpedmeinthe preparationofthiswork.Manyoftheoriginallecturenotesfromwhichthistexthasdeveloped weregeneratedwhileIwasteachingacourseonbiochemistryforfirst-yearmedicalstudentsat theUniversityofChicago,alongwiththelateHowardS.Tager.Howardcontributedgreatlyto mydevelopmentasateacherandwriter.Hisuntimelydeathwasagreatlosstomanyofusin thebiomedicalcommunity;Idearlymisshisguidanceandfriendship.

AsdescribedinthePreface,thenotesonwhichthistextisbasedweresignificantlyexpanded andreorganizedtodevelopacourseofenzymologyforemployeesandstudentsattheDuPont MerckPharmaceuticalCompany.Iamgratefulformanydiscussionswithstudentsduringthis course,whichhelpedtorefinethefinalpresentation.IespeciallythankDianaBlessingtonfor theoriginalsuggestionofacourseofthisnature.Thatagraduate-levelcourseofthistypecould bepresentedwithinthestructureofafor-profitpharmaceuticalcompanyspeaksvolumesforthe insightandprogressivenessofthemanagementofDuPontMerck.IparticularlythankJames M.Trzaskos,RobertC.Newton,RonaldL.Magolda,andPieterB.Timmermansfornotonly toleratingbutembracingthisendeavor.

Manycolleaguesandcoworkerscontributedsuggestionsandartworkforthistext.Ithank JuneDavis,PetraMarchand,DianeLombardo,RobertLombardo,JohnGiannaras,Jean Williams,RandiDowling,DrewVanDyk,RobBruckner,BillPitts,CarlDecicco,Pieter Stouten,JimMeek,BillDe-Grado,SteveBetz,HankGeorge,JimWells,andCharlesCraik fortheircontributions.

Finally,andmostimportantly,Iwishtothankmywife,Nancy,andourchildren,Lindseyand Amanda,fortheirconstantlove,support,andencouragement,withoutwhichthisworkcould nothavebeencompleted.