enzyme kinetics and 1 enzyme kinetics and inhibition pratt cornely ch 7 enzyme kinetics •how...

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  • 2/9/2017

    1

    EnzymeKineticsandInhibition

    Pratt&Cornely Ch 7

    EnzymeKinetics

    Howfastanenzymecatalyzedreactiongoes Whystudyenzymekinetics?

    Helpsusunderstandmechanismofenzyme(howitworks)

    Investigationofmutationsinmetabolicpathways Understandingofregulationofbiochemicalreactions(upordownregulationofcatalyst)

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    SimpleMechanisms

    Chemicalmechanism

    EnzymeCatalyzed

    Howdowemeasurekineticsexperimentally?

    ChemicalKinetics

    Rate:measureproductformedpersecond

    Rateslowsasreactantdisappears

    Measureinitialrate Doasecondexperimentwithmorestartingmaterial,andtheinitialrateisfaster

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    3

    ChemicalKinetics

    Secondaryplot:changeinrateasafunctionofhowmuchsubstrateyoustartedwith

    Linearplotdoesthatmakesense?

    EnzymeKinetics

    Complicatedtwocomponents,treatedseparately

    First,howdoes[enzyme]affectrate(givenlarge[S]?)

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    4

    EnzymeKinetics Next,keepthe[E]constantandlow,andtesthowchangingthe[S]affectsinitialrates

    MichaelisMenton Treatment[Produ

    ct]

    Time

    MichaelisMenton Kinetics

    Rectangularhyperbola Parameters

    Vmax [S]vo =

    Km +[S]

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    GraphicalDeterminationofKineticParameters

    Analyzehyperbola Constructlinearplot Doublereciprical

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    LineweaverBurkAnalysis Howcanyou

    determinekineticparametersVmaxandKm?

    [S]mM[P]at1min(nM)

    1 0.113 0.255 0.3410 0.4530 0.5850 0.61

    0

    0.1

    0.2

    0.3

    0.4

    0.5

    0.6

    0.7

    0 10 20 30 40 50 60

    MMPlot

    y=7.6225x+1.4602R=0.9999

    0

    1

    2

    3

    4

    5

    6

    7

    8

    9

    10

    0 0.2 0.4 0.6 0.8 1 1.2

    LinewaverBurkePlot

    MechanismandAssumptions

    E+S ESE+P Low[E]relativeto[S]

    Steadystate Initialrates

    Nobackrxn Nopdt inhibition

    Derivearateequation

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    InterpretationofShape Low[S]

    Rateverydependenton[S]

    Bindingisratelimiting

    High[S] Rateindependent SaturationofE Chemistryisratelimiting

    MaximumVelocityandtheCatalyticConstant

    Whattwothingscontributetothemaximumvelocitylimit? Amountofenzyme Chemicalabilityofenzyme

    (catalyticconstant) Vmax =[E]kcat Onlykcat tellsusaboutthe

    enzyme Maximum#ofsubstrate

    moleculesperactivesitepersecond

    Turnovernumber

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    Michaelis Constant Km isthe[S]atwhichthe

    reactionreacheshalfitsmaximumvelocity

    Physicalmeaning(assumingequilibriumbinding):Km isthedissociationconstantforES

    Km is[S]atwhichenzymeishalfbound

    Km ismeasureofaffinityofenzymeforS

    LowKm istightbinding

    EnzymeEfficiency Atlow[S],thesecondorderrateconstantiskcat/Km

    Efficientenzymeshavelargekcat/Km Largekcat and/or SmallKm

    Catalyticperfectionat108 or109 M1 S1

    Diffusioncontrol

    Assumelarge[S]andsmall[S]

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    CaseStudy:DiffusionControlledEnzymes

    SuperoxideDismutase:BetterthanDiffusion!

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    CatalyticProficiency

    NonMMKinetics

    Multisubstrate EachsubstratehasitsownKm Random,ordered,pingpong

    Multistepreactions kcat notsimplifiedtok2

    Allostericenzymes cooperativity

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    IrreversibleEnzymeInhibition Affinitylabels

    Testenzymemechanisms

    Serineprotease MechanismbasedInhibitors

    TransitionStateAnalogs

    MechanismBasedInhibitors

    Suicideinhibitors Selectivity Targetingfastgrowingcells

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    12

    DrugByproducts

    Oxidationofxenobiotics byP450enzymes Pharmacology Liverdamagecovalentbindingtocysteine

    TransitionStateAnalog

    Yourbookpresentshighenergyintermediateanalog

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    CaseStudy:Orotidine Decarboxylase

    MechanismofCatalysis

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    ReversibleInhibitionKinetics

    KnowtypesofReversibleInhibition Knoweffectonkineticparameters Understandwhy InterpretMMplots

    CompetitiveInhibition Addedsubstratecanoutcompeteinhibitor

    Drawmechanismwithequilibriumarrows Kmapp:HowdoesaddedIaffectESdissociation?

    Vmax:HowdoesaddinginfiniteSaffectESformation?

    DrawalteredMMandLBplots

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    UncompetitiveInhibition SandIhelpeachotherbind

    Drawmechanismwithequilibriumarrows Kmapp:HowdoesaddedIaffectESdissociation?

    Vmax:HowdoesaddinginfiniteSaffectESformation?

    DrawalteredMMandLBplots

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    MixedInhibition

    Thecontinuumbetweencompetitiveanduncompetitive InhibitormaybindeitherEorES

    Eithermorecompetitiveormoreuncompetitive

    Noncompetitiveismiddleofcontinuum

    Mixedinhibition

    NoncompetitiveInhibition Assumessimplecaseofmixed

    inhibitioninwhichinhibitorbindingequallytoEandES

    Physicalexplanation:inhibitorbindingcauseschangethataffectsreaction,butnotSbinding

    Veryrare(nonexistent) Drawmechanismwith

    equilibriumarrows Kmapp:HowdoesaddedIaffect

    ESdissociation? Vmax:Howdoesaddinginfinite

    SaffectESformation? DrawalteredMMandLBplots

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    FillintheChartInhibition Effect on KM EffectonVmax EffectonVmax/KM

    Competitive DownUncompetitive DownNoncompetitive Down

    Mixed Down

    Problem56[S]M V( noI) V(withI)10 4.63

    nmol/min2.70

    15 5.88 3.4620 6.94 4.7425 9.26 6.0630 10.78 6.4940 12.14 8.0650 14.93 9.71 UseLBplottodetermine

    parameters Whattypeofinhibition? CalculateKi.

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    AllostericRegulation

    Canbeinhibition Negativeeffector Feedbackinhibition PFKregulation

    Mechanism PEPbindinginallostericsitecausesconformationalshiftinneighbor

    AnArg essentialforF6PbindingisreplacedwithGlu

    Tvs.Rstate Cooperative,noeffectonVmax,butonlyapparentKM

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    PositiveEffector

    ADPactswithpositivecooperativity FavorsRstatebybindinginthesameallostericsite,butholdingitopentolockArg intoplace

    DoesADPeffectormakesensephysiologically?

    OtherModesofRegulation

    Transcriptionallevel Compartmentalization Intracellularsignal Covalentmodification