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:
Use of Enzyme inhibitors as anti-cancer drugs:
SulfaDrugs
ResemblePABAin
structure
Blocksmetabolic
activityofbacteria
Examples of the Physiological (regulatory) Role of Enzyme Inhibitors
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.
5Fluorouracil
TScannotcatalyze
reaction
Adeadlyapplicationofirreversibleenzyme
inhibition
DIPF(NerveGas)
DIPFbecomespermanently
linkedtotheactivesiteserine
ofserineproteases
Thetoxiceffectcomesfrom
inactivationof
acetylcholinesterase
Thenormalfunctionofthis
serineproteaseistodigestthe
neuromusculartransmitter
acetylcholine
Whenacetylcholinesteraseis
inactivatedacetylcholine
persists.Thisleadstomuscle
paralysisanddeath.
Enzyme inhibitors as anti-bacterial drugs
Penicillin
Most Drugs
and
toxins are
enzyme
inhibitors: