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Thecatalyticsiteisrelativelysmallcompared
withtherestoftheenzyme.Whyaremany
enzymessobigthen?
Thecatalyticsiteisathreedimensionalentity
Substratesareboundtoenzymesbymultiple
weak,noncovalentinteractions(electrostatic
bonds,hydrogenbonds,vanderWaalsforces,
hydrophobicinteractions)
Ribonuclease
Catalyticsitesformcleftsor
crevices
Substratemoleculesboundwithincleft
Water(unlessinvolvedincatalysis)isnormally
excluded
Overallnonpolarcharacterofcleftcanenhance
bindingofsubstrate
Cleftmayalsocontainpolarresidueswhichmaytake
oncatalyticpropertieswithinthisnonpolar
microenvironment(exceptiontotheruleregarding
hydrophobiccorepresentinmanyglobularproteins)
ActivesiteofcytochromeP450
Activesiteinvolvesaminoacidsfar
apartintheprimarysequenceofa
protein(example:lysozyme)
Thespecificityofbindingdepends
onthepreciselydefinedarrangement
ofatomsinanactivesite
EmilFischer(over
100yearsago):came
upwiththelockand
keyhypothesisto
describeenzyme
substrateinteractions
Inducedfitmodel:amore
refinedmodelthattakes
intoaccounttheenzyme
assumesacomplimentary
shapetothatofitssubstrate
onlyaftersubstratebindsto
theenzyme.
Moredynamicscenario
comparedtothelockand
keyhypothesis
MichaelisMentenmodelof
enzymekinetics(Vmax&Km)
Keyelementintheirmodelistheexistence
oftheEScomplex
Rateofcatalysis(V)increaseswith
increasing[S],whereVisdefinedasthe
numberofmolesofproductformedper
second
Whenenzymeconcentrationsareconstant,
Vislinearlyproportionalto[S]WHEN[S]
ISSMALL.
Athigh[S](whenSisinvastexcessofthe
[enzyme]),Visnearlyindependentof[S]
TheMichaelisMentenequation
Km&Vmax
Km=theMichaelisconstant
Definedasthe[substrate]atwhichthe
reactionrateishalfofitsmaximalvalue
Usedtodefinerelativeaffinityofan
enzymeforitssubstrate
ThehighertheKmvalue,thelowerthe
affinityandviceversa
Vmax:describesthemaximalrateofproduct
formationwhen[S]ishigh(i.e.,invastexcessof
enzyme).
Undersuchconditionsalloftheexistingpool
ofenzymeactivesitesarefull
FromVmaxanenzymesturnovernumbercanbe
determined(expressedasthenumberofsubstrate
moleculesconvertedintoproductperunittime)
Doublereciprocal(lineweaver
Burk)plot
UsedtocalculateKm&
Vmax
Alsousedtocharacterize
mechanismsofenzyme
inhibitionbyspecific
compounds
Dataexpressedas1/V
versus1/[S]:givesa
straightline
CalculatingKmandVmax
Allostericenzymesdonotconform
toMichaelisMentenkinetics
YieldasigmoidalcurveonaVversusS
plot(nothyperbolicasseenunder
MichaelisMentenconditions)
Sigmoidalcurveindicatescooperative
binding(bindingofonemoleculeofS
affectsaffinityandbindingof
additionalSmolecules)
Regulatorymoleculescanalter
activityofallostericenzymes
Enzymeinhibition
ForenzymesthatobeyMichaelisMenten
laws,compoundsthatreversiblyinhibit
enzymeactivitycanbekineticallyclassified
Considertwogeneraltypes:
Competitiveinhibitors
Noncompetitiveinhibitors
Competitivevs.
noncompetitive
inhibitors
Competitiveinhibitors
Yinterceptthesameregardlessofwhether
inhibitorispresentorabsent,BUTtheslope
differsbetweenthetwolines
Competitiveinhibitors
DonotalterVmax
IncreaseKm
Competitiveinhibitioncanbeovercomeby
increasingsubstrateconcentration
Blocksubstratebindingtotheactivesiteof
anenzyme
Examplesofcompetitiveinhibitors
Alcohol(alcoholdehydrogenase)
UpCA(RNase)
DHFRinhibitors(DNAmetabolicinhibitor
oftumors)
Sulfadrugs(antibacterialdrugs)
Physiologicalexamples:feedback
inhibition,pancreatictrypsininhibitor
Enzymeinhibition&automobile
antifreeze
Ethyleneglycol(EG)isaconstituentof
antifreeze
EGnottoxicbutisconvertedtooxalicacid
whichformcrystalsinthekidneysleading
toextensivetissuedamageandrenalfailure
FirststepofconversionofEGtooxalicacid
isitsoxidationtoanaldehydebyalcohol
dehydrogenase
Thisreactioninhibitedbyethanolwhich
competeswithEGforbindingtothe
alcoholdehydrogenase
InhibitionofRNaseby
UpCA
An example of a
typical competitive
inhibitor:
UpCA has a very
similar structure
to the genuine
substrate, but is
chemically unable
to undergo reaction.
SulfaDrugs
ResemblePABAin
structure
Blocksmetabolic
activityofbacteria
Anotherexampleofregulatorycompetitive
inhibition:Inhibition
byPancreaticTrypsinInhibitor
Noncompetitiveinhibitors
PlotsconvergeontheXaxisinthe
presenceorabsenceofinhibitor
Noncompetitiveinhibitors
DonotalterKm
DecreaseVmax
Noncompetitiveinhibitioncannotbe
overcomebyaddingexcesssubstrate
Bindtoasiteoutsideofcatalyticsiteof
enzymeandactbydecreasingtheturnover
numberofanenzyme
Innoncompetitiveinhibitionwhy
isVmaxdecreasedwhileKm
remainsunchanged?
Theinhibitorlowersthe
concentrationoffunctionalenzyme
Theremaininguninhibitedenzymebehaveslike
amoredilutesolutionofthatenzyme(assumes
[inhibitor]islimiting)
Inotherwords,thesubstratecanstillbindto
enzymealoneorenzymecomplexedwiththe
inhibitor.Butonlyfreeenzymewillcatalyzethe
reaction.
Sincethepooloffreeenzymeislowerinpresence
ofinhibitor,Vmaxwillalsobelower
IrreversibleEnzymeInhibitors
Inhibitorbecomescovalentlylinkedtothe
enzyme
Attachmentoftenoccursattheactivesite
Examples:5fluorouracil,DIPF(nervegas),
penicillin
SuicideInhibitors
Irreversibleenzymeinhibitors
Participateintheenzymaticreactionlikethe
substrate
Atsomepointinthereactiontheygetstuck
andbecomepermanentlylinkedtotheenzyme.
Example:5Fluorouracil,asuicideinhibitor
whichtargetsthymidylatesynthaseandisused
incancertreatement.
TScannotcatalyze
reaction
5Fluorouracil
Adeadlyapplicationofirreversibleenzyme
inhibition
DIPF(NerveGas)
DIPFbecomespermanently
linkedtotheactivesiteserine
ofserineproteases
Thetoxiceffectcomesfrom
inactivationof
acetylcholinesterase
Thenormalfunctionofthis
serineproteaseistodigestthe
neuromusculartransmitter
acetylcholine
Whenacetylcholinesteraseis
inactivatedacetylcholine
persists.Thisleadstomuscle
paralysisanddeath.
Penicillin
Most Drugs
and
toxins are
enzyme
inhibitors: