03/08/2016

Chapter6:Enzymes
Weakbondinginteractionsbetweenenzymeandsubstratecatalyzereactions
Enzymesareproteins,allstructuresareessential
Cofactor:additionalchemicalcomponentwithinorganicionssuchasFe2+,

Mg2+,Mn2+,orZn+2
Coenzyme:organicmolecule;transientcarrierofgroups
Prostheticgroup:Coenzymeormetalioncovalentlyboundtoenzyme
Holoenzyme:Enzymewithboundcoenzymeandmetalions
Apoenzyme/apoprotein:proteinpartofholoenzyme

Enzymesareclassifiedbyreactionstheycatalyze
Oxidoreductase:transferofelectrons(H+orOH)
Tranferase:grouptransferreactions
Hydrolase:hydrolysis;transferoffunctionalgroupstowater
Lyases:additionofgroupstodoublebond;formationofdoublebondsby
removalofgroups
Isomerase:transferofgroupstoyieldisomericforms
Ligase:formationofCC,CS,CO,CNbondsbycondensationreactions

Howenzymeswork
Activesite:whereenzymecatalyzedreactionstakeplace
Surfaceislinedwithaminoacidresidueswithsubstituentgroupsthatbind
thesubstrateandcatalyze

Enzymesaffectreactionrates,notequilibria
E+SESEPE+P

ESandEPtransientcomplexes
Coordinatediagramplotsfreeenergyofsystemagainsttheprogressofthe
reaction
Understandardconditions:298K,1atm/101.3kPa,1Mconcentrationof
eachsolute
Horizontalaxisreflectschangeinstructure
BiochemicalstandardfreeenergychangeG
AtpH7
Transitionstate:TopofenergyhillwheremovementtoSorPisequally
probable
Enzymechasestransitionstate,whichlowerstheactivationenergy
ActivationenergyG:differencebetweenenergylevelsofgroundand
transitionstates
Reactionintermediates:Speciesonreactionpathwaythathasafinite
chemicallifetime(ESandEP)
Occupyvalleysinreactioncoordinatediagram
Ratelimitingstep:stepwithhighestactivationenergy;sloweststep

PreciseThermodynamicDefinitions
Equilibriumconstant:Keq=[P]/[S]
G=RTlnKeq
R=8.315J/molK,T=298K
LargenegativevalueforGreflectsfavorablereaction
Equilibriumconstantisdirectlyrelatedtotheoverallstandardfreeenergy
change
Rateconstant=k
Probabilityofreactionundersetofconditions
V=k[S]

CatalyticPowerandSpecificityofEnzymes
Rearrangementofcovalentbondsduringacatalyzedreaction
Catalyticfunctionalgroupsformatransientcovalentbondwithasubstrate
andactivateitforareaction
Covalentinteractionsloweractivationenergyandprovidealowerenergy
reactionpathway
WeaknoncovalentinteractionsduringEScomplexstructurestabilizedby
Hbondsandhydrophobicandionicinteractions
BindingEnergyGB:energyderivedfromenzymesubstrateinteraction
Majorsourceoffreeenergytoloweractivationenergy
Weakinteractionsoptimizedinreactiontransitionstate;activesites
complementarytotransitionstates
Enzymemustbecomplementarytothereactiontransitionstate
Optimalinteractionsbetweensubstrateandenzymeoccuronlyin
transitionstate
Specificity:abilitytodiscriminatebetweensubstrateandcompeting
molecule
Derivedfromformationofweakinteractionsbetweenenzymeand
specificsubstratemolecule
Bindingenergyusedtoovercome:entropy,solvationshellofHbonded
water(stabilizesmolecules),distortionofsubstrates,properalignmentof
functionalgroups
Entropyreduction:largerestrictioninmotionoftwosubstratesthatareto
react
Bindingenergyholdssubstrateinproperorientationtoreact
Constrainingmotionofreactantscanproducerateenhancements
Desolvation:formationofweakbondsbetweensubstrateandenzyme
Weakinteractionsintransitionstatecompensatesdistortionthatsubstratemust
undergo
Inducedfit:enzymeundergoeschangeinconformationinducedbyweak
interactionswithsubstrate
Bringsspecificfunctionalgroupsintoproperpositiontocatalyzereaction
Permitsformationofadditionalweakbondinginteractions

SpecificCatalyticGroupsContributetoCatalysis
Properlypositionscatalyticfunctionalgroupsaidincleavageandformationof
bondsby:generalacidbasecatalysis,covalentcatalysis,andmetalioncatalysis
Involvetransientcovalentinteractionwithsubstrateorgrouptransfer
GeneralAcidBaseCatalysis:formationofunstablechargedintermediates
stabilizedbytransferofprotonstoformspeciesthatbreaksdownreadilyto
products
Specificacidbasecatalysis:CatalysisthatusesonlyH+orOHionsin
water
Ifprotonstransferredb/tintermediateandwaterfasterthanintermediate
breaksdowntoreactants,intermediatestabilizedeverytimeitformsandno
effectonrateofreaction
o Noadditionalcatalysisneeded
Generalacidbasecatalysis:protontransfersmediatedbyotherclassesof
molecules
Covalentcatalysis:transientcovalentbondformedbetweenenzymeand
substrate
o Bondcanactivateasubstrateforfurtherreactionspecifictogroup
Metalioncatalysis:Orientsthesubstrateforreactionorstabilizecharged
reactiontransitionstates
o Weakbondinginteractions
o Mediateoxidationreductionreactionsbyreversiblechangesinthe
metalionsoxidationstate

EnzymeKinetics
Initialrate/velocity(V0):
o Low[S],V0increasesalmostlinearlywithanincreasein[S]substrate
V0=(Vmax[S])/Km
o ConcentrationwhereV0ishalfmaximalisKm,theMichaelisconstant
o High[S],V0increasesbysmalleramountsinresponsetoincreasesin
[S]
V0=Vmax
Maximumvelocity(Vmax):increasesinV0areverysmallas[S]increases

o PlateaulikeV0regionoccursduringsaturation
o V0vs.[S]rectangularhyperbola
Slowersecondreactionlimitsrateofoverallreaction
o OverallrateproportionaltoconcentrationofES
Presteadystate:concentrationofESbuildsup;shortperiod
Steadystate:[ES]remainsapproximatelyconstantovertime
o V0generallyreflectssteadystate
MichaelisMentenequation(rateequation):V0=(Vmax[S])/(Km+[S])
o Km=[S]whenV0=.5Vmax
LineweaverBurkEquation:(1/V0)=(Km/Vmax[S])+(1/Vmax)
o Doublereciprocalplot
o SlopeofKm/Vmax
o Interceptof1/Vmaxonthe1/V0(y)axis
o Interceptof1/Kmonthe1/[S](x)axis
Michaelisconstant:Km=(k1+k2)/k1
Dissociationconstant:k2isratelimiting;getridofitintheequation
kcat:limitingrateofanyenzymecatalyzedreactionatsaturation
o Ifreactionhasseveralstepsandoneisclearlyratelimiting,kcatis
equivalenttotherateconstantforthatlimitingstep
o kcat=Vmax/[Et]
[Et]:Freeorunboundenzyme
o V0=(kcat[Et][S])/(km+[S])
o Turnovernumber:numberofsubstratemoleculesconvertedto
productingivenunitoftimeonsingleenzymewhenitissaturated
o Unitsofreciprocaltime
Specificityconstant:Kcat/km
o RateconstantforconversionofE+StoE+P
o Bestwaytocomparecatalyticefficienciesofdifferentenzymes
o Low[S]:V0=(kcat/km)[Et][S]
Secondorderrateconstant:unitsofM1s1
o Largevalueofkcat(rapidturnover)orasmallvalueofKM(high
affinityforsubstrate)makeskcat/KMlarge

ManyEnzymesCatalyzeReactionswithTwoorMoreSubstrates

 Reactionswith2substratesinvolvetransferofatomorfunctionalgroup
fromonesubstratetoother
Bothsubstratesboundtoenzymeconcurrentlyatsomepointinthecourseof
thereaction,forminganoncovalentternarycomplex
o Substratesbindinarandomsequenceorinaspecificorder
o Intersectinglines
PingPong(doubledisplacement)mechanism
o 1stsubstrateconvertedtoproductanddissociatesbefore2ndsubstrate
binds
o Parallellines
Competitiveinhibitor:competeswithsubstratefortheactivesiteofan
enzyme
o StructurallysimilartosubstrateandformEIcomplex
o V0=(Vmax[S])/(Km+[S])
o =1+[I]/KI
o KI=[E][I]/[EI]
o NoeffectonVmax
o [EI]ishigh,KIislow,ishighcompetition
o Regardlessofconcentrationofcompetitiveinhibitor,highsubstrate
concentrationwillalwaysdisplacetheinhibitor
o =1
KineticTestforDeterminingInhibitionMechanisms
o Doublereciprocalplotwith2setsofrateexperiments,[E]constant
forboth
o Firstset:[S]constantmeasureseffectofincreasing[I]onV0
o Secondset:[I]constantbut[S]varied
o Resultsplottedas1/V0vs.1/[S]
o Slope(Km/Vmax)goesupifIiscompetitive
Uncompetitiveinhibitor:bindsatasitedistinct(allosteric)fromthe
substrateactivesiteandbindsonlytotheEScomplex
o V0=(Vmax[S])/(Km+[S])
o LowersVmaxand
o Vmax/andKm/
Mixedinhibitor:bindsatsitedistinctfromsubstrateactivesite,butbindsto
eithersEorES
o V0=(Vmax[S])/(Km+[S])
o AffectsapparentKmandVmaxVmax/andKm/

 Noncompetitiveinhibition:=
Irreversibleinhibitors:bindcovalentlywithordestroyfunctionalgroupon
enzymeessentialforenzymeactivity,orformstablenoncovalentassociation
Suicideinactivators:unreactiveuntilbindtoactivesiteofspecificenzyme
o Inactivatorconvertedtoveryreactivecompound(mechanismbased
inactivators)thatcombinesirreversiblywithenzyme
Hijacknormalenzymereactiontoinactivateenzyme
pHrangeoverwhichenzymeundergoeschangesinactivityprovideaclueto
typeofaminoacidresidueinvolved
o ChangeinactivitynearpH7reflectstitrationofHisresidue

Chymotrypsin
Serineproteasethathydrolyzespeptidebondsafteranaromaticresidue
o Aromaticaminoacidresidues:Trp,Phe,Tyr
Formstransientcovalentacylenzymeintermediate
Acylationphase:peptidebondcleavedandesterlinkageformedbetween
peptidecarbonylcarbonandenzyme
Deacylationphase:esterlinkagehydrolyzedandnonacylatedenzyme
regenerated
Catalyzeshydrolysisofsmallestersandamides
Structure:primaryconsistsofdisulfidebonds
o Keyactivesiteresidues:Ser195,His57,Asp102
Activesite:HydroxylofSerattackscarbonylofsubstrate
o Negativechargeonoxygenstabilizedbyoxyanionhole(amideN,
includingonefromSer)
o Serinemakestransientlink
Chymotrypsinhydrolysisofesterpnitrophenylacetate(measuredbyrelease
ofcoloredpnitrophenol)hasrapidburstbeforelevelingtoslowerrate
o Rapidacylation,thenslowdeacylationstep
ChangeinkcatreflectsionizationstateofHis57
o DeclineinkcatatlowpHresultsfromprotonationofHis
Changesin1/KmreflectionizationoftheaminogroupofIle16
o GroupformssaltbridgewithAsp194tostabilizeactiveconformation
ofenzyme
o HighpH,grouplosesprotonandsaltbridgeeliminatedand
conformationalchangecloseshydrophobicpocketwherearomatic

aminosidechainofsubstrateinserts
Catalytictraid:Ser195linkedtoHis57andAsp102inHbondingnetwork
o AllowsoxygenofSer195tobenucleophileinacylationphase
Substratebinds:changeconformationcompressHbondbetweenHisand
Asp,resultinginstrongerinteraction(lowbarrierHbond)
o IncreasespKaofHis,allowingHisresiduetoactasbasethatcan
removeprotonfromSerhydroxylgroup
Deprotonationpreventsdevelopmentofunstable+chargeon
SerhydroxylandmakesSersidechainstrongernucleophile
o Ser195attackscarbonylofsubstrate,tetraintermediateformswhere
carbonyloxygenacquiresnegativecharge
Formsinoxyanionhole
ChargestabilizedbyHbondsfromamidegroupsofpeptide
bondsinchymotrypsinbackbone
Chymotrypsin(serineprotease)hasgeneralacidbasecatalysis,covalent
catalysis,&transitionstatestabilization