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Theenzymeactivesite(features)

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.

Use of Enzyme inhibitors as anti-cancer drugs:

Folate (folic acid)

Transformation of folate to tetrahydrofolate catalyzed by dihydrofolate reductase:

eventually leads to synthesis of thymine nucleotides (DNA metabolism)


Competitive inhibitors of dihydrofolate reductase used in cancer treatment
(resemble folate, bind ~1000x tighter):

SulfaDrugs
ResemblePABAin
structure
Blocksmetabolic
activityofbacteria

Examples of the Physiological (regulatory) Role of Enzyme Inhibitors


Feedback inhibition: The end-product of a biochemical pathway is similar to the
starting product and may (competitively) bind to and inhibit one of the enzymes
in the pathway:

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.

Enzyme inhibitors as anti-bacterial drugs

Penicillin

Most Drugs
and
toxins are
enzyme
inhibitors: