Enzyme Kinetics: Catalysis & Control: A Reference of Theory and Best-Practice Methods PDF
Preview Enzyme Kinetics: Catalysis & Control: A Reference of Theory and Best-Practice Methods
Enzyme Kinetics: Catalysis & Control A Reference of Theory and Best-Practice Methods This page intentionally left blank Enzyme Kinetics: Catalysis & Control A Reference of Theory and Best-Practice Methods Daniel L. Purich Department of Biochemistry and Molecular Biology University of Florida College of Medicine Gainesville, Florida AMSTERDAM(cid:2)BOSTON(cid:2)HEIDELBERG(cid:2)LONDON(cid:2)NEWYORK(cid:2)OXFORD PARIS(cid:2)SANDIEGO(cid:2)SANFRANCISCO(cid:2)SINGAPORE(cid:2)SYDNEY(cid:2)TOKYO AcademicPressisanimprintofElsevier Academic Press is animprint ofElsevier 32 JamestownRoad,London NW1 7BY,UK 30 Corporate Drive,Suite 400, Burlington, MA 01803, USA 525 BStreet, Suite 1900,SanDiego,CA 92101-4495, USA Firstedition 2010 Copyright (cid:2) 2010Elsevier Inc. All rights reserved No part ofthis publicationmay bereproduced, stored ina retrieval system ortransmitted inanyform orby anymeans electronic, mechanical, photocopying, recording or otherwisewithoutthe priorwritten permissionof the publisher. PermissionsmaybesoughtdirectlyfromElsevier’sScience&TechnologyRightsDepartmentinOxford,UK:phone(+44) (0) 1865843830;fax (+44) (0) 1865853333;email: [email protected]. Alternatively, visit the Science and Technology Books website at www.elsevierdirect.com/rights for further information CoverImage:Twoviews of the X-ray crystallographic structure ofthe completeThermusthermophilus 70S ribosome containing boundmessenger RNAand transferRNAs at 5.5-A˚ resolution (from Yusupov, M.M., Yusupova,G. Z., Baucom, A.,Lieberman, K.,Earnest, T. N.,Cate, J. H.,and Noller,H. F. (2001) Science 292, 883–96 with permission). PerhapsNatures’smostcomplicatedmolecularmachine,theribosomeconsistsofRNAandproteinsthatworktogetherto accomplishthe multiple structural,catalytic,and force-generating steps requiredfor the high-fidelitysynthesis and elongationofpolypeptides.Althoughthecenterpieceofthe2009NobelPrizesinChemistry,theribosomeremainsamajor challenge for enzyme scientists and kineticists seekingto unlock itsmanysecrets. Notice No responsibilityis assumed by the publisher for anyinjury and/ordamage topersonsor propertyas amatterof productsliability, negligenceorotherwise, orfrom anyuseoroperation ofanymethods, products,instructions or ideascontained inthe materialherein.Becauseof rapid advances inthe medical sciences, inparticular, independent verification of diagnoses anddrug dosagesshouldbe made British Library Cataloguing-in-PublicationData A catalogue record for this bookis available from the British Library Libraryof CongressCataloging-in-Publication Data A catalog recordfor this book isavailable from the Library ofCongress ISBN:978-0-12-380924-7 For information on all Academic Press publications visitourwebsiteatelsevierdirect.com Typeset byTNQ Books andJournals PvtLtd. www.tnq.co.in Printed and bound inChina 10 11 12 13 1415 10 9 87 6 5 43 2 1 Contents Table of Contents Preface xix toUnderstandtheDetailedMutual ChangesinBothSubstrate andEnzymeDuringCatalysis 34 1.6.2. WeNeedNewApproaches forDeterminingthe Chapter 1. An Introduction to Enzyme ChannelsAllowingEnergy Science 1 FlowDuringEnzyme 1.1. Catalysis 5 Catalysis 37 1.1.1. RootsofCatalysisinthe 1.6.3. WeNeedAdditionalProbes EarliestChemicalSciences 5 ofEnzymeCatalysis 38 1.1.2. SyntheticCatalysts 7 1.6.4. WeNeedtoLearnHow 1.2. BiologicalCatalysis 12 ProteinsFoldandHowto 1.2.1. RootsofEnzymeScience 12 ManipulateProteinStability 38 1.2.2. EnzymeTechnology 13 1.6.5. WeNeedtoDevelopa 1.3. DevelopmentofEnzymeKinetics 15 DeeperUnderstandingof 1.4. TheConceptofaReactionMechanism 19 SubstrateSpecificity 39 1.4.1. Chymotrypsin:ThePrototypical 1.6.6. WeNeedtoDevelopthe BiologicalCatalyst 20 AbilitytoDesignEntirely 1.4.2. Ribozymes 22 NewBiologicalCatalysts 42 1.4.3. Mechanoenzymes 23 1.6.7. WeNeedtoDefinethe 1.5. ExplainingtheEfficiencyofEnzyme EfficientRoutesforObtaining Catalysis 25 HighPotencyEnzymeInhibitors 1.5.1. StabilizationofReaction asDrugsandPesticides 44 TransitionStates 26 1.6.8. WeNeedtoLearnMore 1.5.2. ElectrostaticStabilization AboutInSinguloEnzyme ofTransitionStates 27 Catalysis 45 1.5.3. IntrinsicBindingEnergy 28 1.6.9. WeNeedtoDevelop 1.5.4. ReactingGroup ComprehensiveCatalogs Approximation,Orientation ofEnzymeMechanismsand andOrbitalSteering 28 toUseSuchInformationin 1.5.5. ReactantStateDestabilization 29 FashioningNewMetabolic 1.5.6. Acid/BaseCatalysis 30 Pathways 46 1.5.7. CovalentCatalysis 30 1.6.10. WeNeedtoUnderstand 1.5.8. Transition-StateStabilization HowtoAnalyzetheKinetic byLow-BarrierHydrogen BehaviorofDiscrete Bonds 31 Enzyme-CatalyzedReactions 1.5.9. CatalyticFacilitationby asWellasMetabolic MetalIons 32 PathwaysintheirEnvironment 48 1.5.10. PromotionofCatalysisvia 1.6.11. WeNeedtoDevelop EnzymeConformational TechniquesthatwillFacilitate Flexibility 32 InvestigationofChromosomal 1.5.11. PromotionofCatalysis Remodeling,Epigenetics,and viaForce-Sensingand theGeneticBasisof Force-GatedMechanisms 33 DiseaseandCellSurvival 49 1.6. ProspectsforEnzymeScience 34 1.6.12. WeNeedtoDevelop 1.6.1. WeNeedBetterMethodsfor EffectiveEnzymePreparationsfor AnalyzingEnzymeDynamics UseinDirectEnzymeTherapy 50 v Contents vi Chapter 2. Active Sites and their Chemical 2.4.2. SomeEnzymesExploit SpecializedAmino-Acid Properties 53 ResiduesinCatalysis 78 2.1. EnzymeActiveSites 54 2.5. MetalIonsinEnzymeActiveSites 81 2.1.1. MostEnzymesareProteins, 2.5.1. AGroupofBiologically whichareLinearPolymersof SignificantMetalIonsis a-AminoCarboxylicAcids 55 EssentialforCatalysis 2.1.2. Active-SiteResiduesmaybe bySomeEnzymes 82 ClassifiedwithRespectto 2.5.2. Enzyme-BoundMetalIon theirFunction(s) 57 ComplexesShareStructural 2.1.3. ActiveSitesTypicallyOccupy andChemicalFeatures 84 only2–3percentoftheTotal 2.5.3. TheChemistryofMetal VolumeofanEnzyme 59 Ion-LigandComplexesis 2.1.4. BindingEnergyOftenIndicates DominatedbytheNatureof theStrengthofEnzyme theirLigancy 85 InteractionswithSubstrates 2.5.4. Field-EffectsInfluencethe andCofactors 60 ColorandMagnetic 2.1.5. TheStructuralOrganization PropertiesofMetalIon ofEnzymescanbeConsidered CoordinationComplexes 87 Hierarchically 61 2.5.5. TheReactionMechanismsof 2.1.6. EnzymesOftenOccurin TransitionMetalComplexes MultipleMolecularForms 63 areDeterminedbytheir 2.2. ForcesAffectingEnzymeStructural Inner-andOuter-Sphere StabilityandInteractions 64 CoordinationBehavior 89 2.2.1. ElectrostaticInteractions 2.5.6. MetalIonsFormComplexes InfluenceEnzymeStructure withEnzymesand/ortheir andInteractions 64 Substrates 93 2.2.2. Ion–DipoleandDipole–Dipole 2.5.7. PropertiesofSelected InteractionsareSpecialized Active-SiteMetalIons 95 ElectrostaticPhenomena 66 2.5.8. ASurveyofMetalIonComplexes 2.2.3. HydrogenBondingMainly withinSelectedEnzymesReveals PlaysaCompensatoryRole KeyFeaturesofBinding-Site inStabilizingProteins 66 Organization 109 2.2.4. HydrophobicInteractions 2.6. ActiveSitesofEnzymesActingon PlayaDominantRolein PolymericSubstrates 112 StabilizingMostProteins 69 2.6.1. ManyEndonucleasesAchievetheir 2.2.5. AlthoughIndividuallyWeak, RemarkableSpecificitybyMeans vanderWaalsInteractions ofSubsiteRecognition 113 aresoNumerousthatthey 2.6.2. ProteasesweretheFirstEnzymes ContributeSignificantly ShowntohaveSubsitesforInteracting toOverallProteinStability 70 withtheirPolymericSubstrates 114 2.2.6. SomeProteinsare 2.6.3. Endo-Glycosidasesalso OccasionallyStabilizedby ExploitSubsitestoAchieveSpecificity 115 p-CationInteractions 70 2.6.4. SubsitesFacilitateSubstrate 2.3. Active-SiteDiversification 70 RecognitionbySignal- 2.3.1. EnzymeDiversificationcan TransducingProteinKinases 115 beExplainedStructurally 71 2.7. BasicOrganicChemistryofEnzyme 2.3.2. CatalyticPromiscuitymay Action 116 ExplaintheEmergenceof 2.7.1. ThereareSixMajorClassesof CatalyticallyDiversified Enzyme-CatalyzedCovalent Enzymes 74 Bond-Making/-Breaking 2.4. AdditionalFunctionalGroupsin Reactions 118 EnzymeActiveSites 77 2.7.2. CarbonhasSeveralReactive 2.4.1. Vitamin-BasedCoenzymesIncrease FormsinEnzymaticMechanisms 119 theChemicalVersatilityofEnzyme 2.7.3. ManyEnzymesUsetheSame ActiveSites 77 GeneralReactionMechanisms Contents vii FirstDiscoveredbyPhysical CoordinatedElectron OrganicChemists 120 TransferReactions 158 2.7.4. NucleophilicSubstitutionisa 2.10.4. Enzyme-CatalyzedElectron WidelyUsedReaction Transfermaybeanalyzed MechanisminEnzymeCatalysis 121 byMarcusTheory 160 2.7.5. Enzyme-CatalyzedElimination 2.10.5. SimpleKineticModelscan ReactionMechanismshaveMany AccountfortheBehaviorof PrecedentsinOrganicChemistry 125 BiologicalElectronTransferReactions 161 2.7.6. EnzymesareHighlyEffectivein 2.10.6. SeveralPrototypicalRedox Forming,Stabilizing,and EnzymesProvideValuable UtilizingCarbanion InsightsintoElectronTransfer IntermediatesDuringCatalysis 125 KineticsandMechanisms 162 2.7.7. FreeRadicalsareFormedin 2.10.7. EnzymeElectrodesCombine aSurprisingNumberof theSpecificityofBiological Enzyme-CatalyzedReactions 131 CatalysiswiththeVersatilityof 2.7.8. TheVersatilityofEnzymescan PotentiometryorAmperometry 164 beIllustratedbyConsidering Appendix 168 aSelectedGroupofReaction Mechanisms 134 2.8. DetectingCovalentIntermediatesin Chapter 3. Fundamentals of Chemical EnzymeReactions 137 2.8.1. EnzymesFormaWideRange Kinetics 171 ofEnzyme-SubstrateCovalent 3.1. TimescaleofChemicalProcesses 171 Compounds,andManyare 3.2. TheEmpiricalRateEquation 172 CatalyticallyCompetent 137 3.3. ReactionRate,OrderandMolecularity 174 2.8.2. Side-ReactionsOftenProvide 3.3.1. ReactionRate 174 InvaluableCluesAbout 3.3.2. ReactionOrder 175 MechanismsofEnzyme 3.3.3. Molecularity 176 Catalysis 141 3.3.4. Zero-OrderKinetics 177 2.8.3. SomeEnzyme-Substrate 3.3.5. First-OrderKinetics 177 CovalentCompoundscanbe 3.3.6. Second-OrderKinetics 180 ChemicallyTrapped 143 3.3.7. PseudoFirst-OrderKinetics 180 2.9. BasicsofEnzymeStereochemistry 145 3.4. BasicStrategiesforEvaluatingRateProcesses 181 2.9.1. Definitions 145 3.4.1. Initial-RateMethod 181 2.9.2. TheCahn-Ingold-PrelogSystem 3.4.2. ProgressCurveAnalysis 182 AllowsOnetoAssigntheAbsolute 3.5. CompositeMulti-stage(Multi-step) StereochemicalConfiguration Mechanisms 184 ofChiralCompounds 146 3.5.1. SeriesFirst-OrderKinetics 185 2.9.3. TheProchiralityofMolecules 3.5.2. ReversibleFirst-OrderKinetics 186 mayalsobeSpecified 3.5.3. ReversibleSecond-OrderKinetics 186 Systematically 147 3.5.4. Rapid-Equilibriumand 2.9.4. TheStereochemistryofMethyl Steady-StateTreatments 186 TransferReactionsmaybe 3.5.5. Rate-ControllingSteps 188 AnalyzedUsingEnzymesof 3.5.6. PrinciplesofDetailedBalance 189 KnownStereochemistry 3.5.7. ThermodynamicCyclesfor asReferenceReactions 149 EvaluatingDetailedBalance 190 2.10. ElectronTransferReactions 150 3.5.8. KineticEquivalenceand 2.10.1. TheThermodynamicProperties MechanisticAmbiguity 192 ofOxidation–Reduction 3.6. ThermalEnergy:TheBoltzmann ReactionsareDefinedby DistributionLaw 192 RedoxPotentials 154 3.7. SolutionBehaviorofReacting 2.10.2. TheRedoxBehaviorofComplex Molecules 194 Metalloenzymescanbe 3.7.1. Water:AUniqueSolventfor EvaluatedSpectroscopically BiochemicalProcesses 194 byStoichiometricTitrationTechniques 157 3.7.2. DiffusionLimitationson 2.10.3. RespiratoryChainsare ChemicalProcessesOccurring ComprisedofHighly inWater 196 Contents viii 3.7.3. ElectrostaticEffectson 4.4.2. Beer’sLawisaQuantitative MagnitudeofBimolecular ExpressionLinkingAbsorbance RateConstants 199 toConcentration 240 3.7.4. ReactantDesolvation 200 4.4.3. SomeEnzymeAssaysUse 3.8. Transition-StateTheory 201 AlternativeSubstratesthatare 3.9. ChemicalCatalysis 203 Chromogenic 243 3.9.1. AcceleratingRatewithout 4.5. BasicFluorescenceSpectroscopy 243 AlteringtheEquilibriumPoise 203 4.5.1. FluorescenceSpectraDepend 3.9.2. NucleophilicandElectrophilic onExcited-StateRelaxation 244 Facilitation 205 4.5.2. FeaturesofaResearch-Grade 3.9.3. BufferCatalysis 206 Spectrophotofluorimeter 244 3.9.4. Autocatalysis 206 4.5.3. TheConcentrationofVarious 3.10. ReactionCoordinateDiagrams 207 MetabolitesmaybeQuantified 3.11. ThermodynamicPrinciples 210 ThroughFluorescence 3.11.1. ChemicalEquilibrium 210 Spectrometry 246 3.11.2. DirectionandExtentof 4.5.4. BiologicalMoleculesmay ChemicalReaction 210 ContainIntrinsicorExtrinsic 3.11.3. UsingDDGtoDefineBinding FluorescentReporterGroups 247 Energetics 211 4.5.5. FluorescenceAnisotropyisa 3.11.4. AlbertyTreatmentof PowerfulTechniqueForQuantifying BiochemicalThermodynamics 211 BindingInteractions 250 3.11.5. SomeReactingSystemsare 4.5.6. Fo¨rster(Fluorescence)Resonance BestAnalyzedbyPrinciples EnergyTransfer(FRET)isan ofNon-Equilibrium ExquisitelyDistance-Sensitive Thermodynamics 212 ProbeofEnzymes 252 3.12. ConcludingRemarks 214 4.5.7. ContinuousFluorescenceAssays areNowAvailableforPi-and PPi-ProducingReactions 253 Chapter 4. Practical Aspects of Measuring 4.5.8. Chemiluminescenceisa Initial Rates and Reaction PhotoemissiveProcessOften ExploitedinEnzymeRateAssays 254 Parameters 215 4.6. MeasuringReactionRateswithIsotopes 255 4.1. DesignofInitial-VelocityEnzymeAssays 215 4.6.1. StableIsotopesareVersatile 4.1.1. ‘‘ActivityPurity’’isSufficientin ProbesinEnzymeKinetics 255 MostInitial-RateStudies 216 4.6.2. RadioisotopesProvideExtremely 4.1.2. DiscontinuousandContinuous SensitiveAssaysofEnzyme RateMeasurements 217 RateProcesses 260 4.1.3. EachEnzymeRateAssayhasIts 4.7. MultisubstrateKineticsandInhibitor OwnSpecialSetofRequirements 220 Kinetics 264 4.2. EnzymePurification 232 4.8. AnalysisofEnzymeRateData 265 4.2.1. WhileTime-Consuming,theTask 4.8.1. EnzymeRateDatamustbe ofEnzymePurificationisOften AppropriatelyWeighted 266 WellFounded 232 4.8.2. QuantitativeAnalysisofReaction 4.2.2. BiochemistshaveDevelopeda Progress-CurvescanbeUsedto PowerfulBatteryofTechniques EvaluateRateParameters 268 forPurifyingEnzymes 234 4.8.3. GlobalAnalysisOffersAdded 4.3. Coupled(orAuxiliary)EnzymeAssays 235 AdvantagesinStatisticalAnalysis 270 4.3.1. ASimpleKineticTreatment 4.9. WorkingwithATP-DependentEnzymes 272 ExplainstheLag-Phasein 4.10. RegeneratingNucleoside CoupledEnzymeAssays 238 59-TriphosphateSubstrates 275 4.3.2. TheAuxiliaryEnzymeandAssay 4.10.1. ProteinandEnzyme ConditionsmustbeSuitedto Concentration 276 thePrimaryEnzymeReaction 239 4.10.2. TotalProteinConcentration 4.4. BasicUV/VisibleAbsorption canbeDeterminedQuantitatively 276 Spectroscopy 240 4.10.3. ActiveEnzymeConcentrationcan 4.4.1. AbsorptionSpectraDependonthe beQuantifiedbySeveralTechniques 276 QuantumStatesofElectronOrbitals 240 Contents ix 4.11. EquilibriumConstantDeterminations 278 5.3. CatalysisInvolvingTwoorMore 4.11.1. EquilibriumConstantscanbe Intermediates 298 EvaluatedinaVarietyofWays 279 5.3.1. DerivationoftheTwo- 4.11.2. TheDeterminationofthe IntermediateCaseIllustrates ArginineKinaseReaction WhythisTreatmentisaMore EquilibriumConstantisan RealisticRepresentation ExcellentExampleofa ofanEnzymeMechanism 298 Well-Designedand 5.3.2. AShortcutcanbeTakento Well-ExecutedDetermination 281 DerivetheSteady-StateRate 4.12. ConcludingRemarks 284 EquationfortheReverse Two-IntermediateReaction Scheme 298 5.3.3. MultipleInternalIsomerizationsare Chapter 5. Initial-Rate Kinetics of One- withoutEffectontheGeneral Substrate Enzyme-Catalyzed FormoftheFinalSteady-State Reactions 287 RateEquation 298 5.3.4. TheHaldaneRelationship 5.1. Michaelis-MentenTreatment 287 alsoConstrainstheRelative 5.1.1. DerivationoftheMichaelis- MagnitudesofKeyRate MentenEquationRevealshow ParametersintheTwo- KeyAssumptionsDefinean IntermediateScheme 300 Enzyme’sInitial-RateBehavior 288 5.1.2. KS,Vm,Vm/KS,and[S]/KSare 5.4. AdditionalCommentsonFundamental RateParametersDefiningan KineticParameters 301 Enzyme’sInitial-RateBehavior 289 5.4.1. TheMichaelisConstanthas SeveralImportantImplications, 5.1.3. SeveralMethodsforPlottingInitial- withRegardtoBoth‘‘Substrate RateDataareQuiteUsefulbuthave Affinity’’andSubstrate InherentLimitations 290 Specificity 301 5.1.4. TheMichaelis-MentenEquation PredictsaLinearDependence 5.4.2. TheTurnoverNumberkcat IndicatesNumberofSubstrate ofReactionRateonthe MoleculesConvertedto ConcentrationofActiveEnzyme 292 ProductperEnzymeActive 5.1.5. TheQuadraticFormulais SiteperSecond 303 RequiredWhentheEnzyme ConcentrationApproaches 5.4.3. The‘‘SpecificityConstant’’ SubstrateConcentration 292 Vmax/Kmorkcat/KmIndicatesthe EfficiencyofSubstrateCapture 5.1.6. NonproductiveSubstrateBinding byanEnzyme 304 CannotbeDetectedbytheMichaelis- MentenTreatment 293 5.4.4. TheCommitmenttoCatalysis MeasuresanEnzyme’sAbility 5.2. TheBriggs-haldaneSteady-State toConverttheE$SComplex Treatment 293 toE$P,asComparedto 5.2.1. DerivationofthisRateEquation ReconversionofE$StoaPrior RevealsKeyFeaturesof EnzymeForm 307 Steady-StateProcesses 294 5.4.5. EvolutionofCatalyticProficiency 308 5.2.2. ReactionEnergeticsDetermine 5.4.6. InternalEquilibriaand theEffectofIncreasingSubstrate EnergeticsofPerfectedEnzymes 309 ConcentrationontheConversion ofEþStoE$SComplex 295 5.5. ReactionProgressCurveAnalysis 310 5.2.3. TheCorrespondingReverse-Reaction 5.6. RibozymeKinetics 311 RateEquationcannowbeWritten 295 5.7. ProteasomeKinetics 313 5.2.4. TheHaldaneRelationship 5.8. IsomerizationMechanisms 314 ConstrainstheValuesofKey 5.9. SimultaneousActionofanEnzyme RateParametersforReversible onDifferentSubstrates 315 Enzyme-CatalyzedReactions 296 5.10. EnantiomericEnrichmentand 5.2.5. TheBriggs-HaldaneEquation AnomericSpecificity 316 RequiresthatanEnzyme 5.11. SimultaneousActionofTwoEnzymes SystemSatisfiestheSteady- ontheSameSubstrate 318 StateAssumption 296 5.12. Induced-FitMechanisms 319
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