Enzymekinetics

Whystudytherateofenzymecatalyzedreactions?

Studyofreactionratesisanimportanttoolto
investigatethechemicalmechanismofcatalysis

Kineticstudiesprovideinformationonsubstrateand
productaffinitytotheenzyme

Knowledgeofthedynamicpropertiesofenzyme
catalysisisaprerequisiteforthedesignofinhibitors
(drugs)directedagainstacertainenzyme

 Chemicalreactionkinetics

Reactionorderofachemicalprocesscorrespondstothemolecularity
ofthereaction,whichisthenumberofmoleculesthatmustcollide
simultaneouslytogenerateaproduct:

aA+bB+cCproduct(s)

Thevelocityforsuchaprocessisgivenby:

v=k[A]a[B]b[C]c

Wherevisthevelocityofthereactionandktherateconstantofthe
reaction;thereactionorderisthesumoftheexponentialsintherate
equation.

 FirstandSecondorderreactions

Forafirstorderprocess: AP

dP dA
Thereactionvelocity,v,isgivenby: v===k[A]
dt dt

Thereactionvelocityforsuchafirstorderreactionisproportionaltothe
concentrationofA.Suchareactionisalsocalledaunimolecularreaction.

Asecondorderreactioncaninvolvethereactionoftwoidenticalor
differentsubstratemolecules:

A+Aproduct(s)orA+Bproduct(s)

Thevelocityofthereactionisthen:
dP dA
v===k[A] 2 dA dB
orv===k[A][B]
dt dt dt dt

 Thedimensionoftherateconstant

Thedimensionofkdependsonthereaction
order.Thisisduetothefactthatthevelocityofa
reactionismeasuredinmolproductformedper
timeunit(e.g.sec).

Thereforetheunitoftherateconstantofafirst
orderreactionissec1andthatofasecondorder
reactionisM1sec1.

 Determinationofkforafirstorderreaction

Forafirstorderprocess: AP

Asbefore,thereactionvelocity,v,isgivenby:
ln[A]

dA
v==k[A] ln[A]t=0
dt
slope=k
dA =dln[A]=kdt
[A]
Integrationwith[A]t=0to[A]tyields: t
[A]t t

dln[A]=kdt ln[A]t=ln[A]t=0kt
[A]t=0 t=0

[A]t=[A]t=0ekt
Example:radioactivedecay

 Relationshipofhalflifeandrateconstant

Thehalflife(t1/2)isaconstant(dimension:time).Radioactivedecayisusually
expressedinhalfliferatherthanthefirstorderrateconstant:
[A]t=0
Thehalflifeisdefinedasthetimerequiredfor[A] t=0todecreaseto
2

[A]t=0 ln2
=[A]t=0ekt1/2 t1/2=
2 k

 Rateconstantforasecondorderreaction

Considerthefollowingreaction: A+Aproduct(s)
dA
v==k[A] 2
1
dt
[A]t
[A]t t

=k
dA
[A] 2
dt
t=0
[A]t=0
slope=k

1
1 1 [A]t=0 t
=+kt
[A]t [A]t=0
Thistypeofplotcanbeusedto
distinguishbetweenaunimolecular
Thehalflifeforsuchareactionis:
andbimolcularreactioninvolving
A
1
t1/2= 1 Note:halflifedependson[A]t=0
[A]t=0 k

 Theenzymesubstratecomplex

AdrianBrown

BrownandHenriinvestigatedthe
substratedependenceofanenzyme
catalyzedreactionandfoundthatthe
reactionreachedamaximumvelocityat
highsubstrateconcentrations(Vmax)

 BrownandHenrisconclusion:

Theenzymeworksbyformingacomplex(likealockandakey)
withthesusbtrateandactingonitforafiniteperiodoftime.

binding reaction dissociation

E+SESEPE+P
enzyme enzymesubstrate enzymeproduct enzyme
+ complex complex +
substrate product

Schematicrepresentationofanenzymecatalyzedreaction

 Kineticsofenzymereactions

Recall:Brownobservedthattherateatwhichsucroseisdegradedby
invertaseshowssaturationbehavior,thatisathighsucroseconcentrationsthe
ratebecomesindependentofthesucroseconcentration(zeroorderreaction
withrespecttosucrose).Itwasconcludedthatanenzymecatalyzedreaction
proceedsintwosteps:
k1 k2
[E]+[S] [ES] [P]+[E]
k1
When[S]>>[E]thenalloftheenzymeisinthecomplex[ES]andthe
formationofproductisgivenby:
d[P]
V==k 2[ES]
dt
Theconcentrationof[ES]isacomplexfunctiondependingonthe
individualrateconstantsk1,k1andk2:
d[ES] =k [E][S]k [ES]k [ES]
1 1 2
dt

 Twoapproaches

1. MichaelisMentenkinetic(1913)
(rapidequilibriumassumption)

2. BriggsHaldanekinetic(1925)
(steadystateassumption)

Titlepageof
Michaelis&
Mentensoriginal
paperin
Biochemische
Zeitschriftin
1913

LeonorMichaelis MaudL.Menten
(18751940) (18791960)
 TheMichaelisMentenapproach

k1 k2
[E]+[S] [ES] [P]+[E]
k1
Assumption:k1>>k2i.e.theequilibriumof[E],[S]and[ES]isnot
affectedbyk2:
k1 [E][S] KS=dissociationconstant
KS==
k1 [ES] [ES]=MichaelisMentencomplex

Sinceweassumeequilibriumitfollows:
k1 [ES]
[E][S]k1=[ES]k1solvingfor[E]= (1)
k1 [S]

Inadditionweknowthat:[E]total=[E]+[ES] (2)

Thisrelationshipiscalledtheenzymeconservationequation

 TheMichaelisMentenapproach

k1 [ES]
[E]= (1)
k1 [S]
[E]total=[E]+[ES] (2)
Solvingequation(2)for[E]andsubstituting[E]inequation(1):

k1
[E]total=[ES](1+) (3)
k1[S]
Wealsoknowthatthevelocityofthereactionequals:

v=k2[ES] (4)

Solvingequation(3)and(4)for[ES]andthensubstituting[ES]in
equation(3)with[ES]=v/k2thenyields:

 k2 [E]total k2 [E]total [S]
v==
k1 k1
(1+) [S] +
k1[S] k1

Wedefinek1/k1asKM,theMichaelisMentenconstantandthe
maximalvelocityasvmax=k2[E]total

Thissimplifiestheaboveequationto:

vmax [S] if[S]>>KMthenv=vmax

v= vmax
[S]+KM if[S]=KMthenv=
2

ThereforeKMcanbeviewedasthesubstrateconcentrationwithhalf
maximalvelocity(dimensionM,typicallymMtonM)

 MichaelisMentenplot
Linearplotofsubstrateconcentrationversusvelocity
v yieldsahyperbolicrelationship:

vmax

vmax 1storder zeroorder

2

[S]

KM
 TheBriggsHaldaneapproach

k1 k2
[E]+[S] [ES] [P]+[E]
k1

Assumption:k1~k2i.e.
duringsubstrateturnoverthe
concentrationof[ES]is
constant(steadystate
assumption).Theassumptionis
lessrestrictivethantherapid
equilibriumassumptionby
MichaelisMenten.

d[ES]
=0
dt

 TheBriggsHaldaneapproach

d[ES]
=0 =k1[E][S]k1[ES]k2[ES]
dt

k1[E][S]=k1[ES]+k2[ES]

Wealsoknowthat: [E]total=[E]+[ES],solvingthisequation
For[E]andsubstitutinginthesteadystateequationyields:

k1([E]total[ES])[S]=k1[ES]+k2[ES]

k1[E]total[S]k1[ES][S]=k1[ES]+k2[ES]

k1[E]total[S]=(k1+k2)[ES]+k1[ES][S] :k1

 (k1+k2)
[E]total[S]=[ES]+[ES][S]
k1
Solvingthisequationfor[ES]yields:

[E]total[S] (k1+k2)
[ES]= KM=
(k1+k2) k1
+[S]
k1
[E]total[S] andwithv=k2[ES]
[ES]=
KM+[S]
k2[E]total[S]
v= andwithvmax=k2[E]total
KM+[S]
vmax[S]
Sameequation! v=
KM+[S]

 MichaelisMentenvs.BriggsHaldane

Althoughbothapproachesyieldthe
samebasicequationforthevelocityof vmax[S]
anenzymecatalysedreaction,the v=
KM+[S]
meaningoftheMichaelisMenten
parameter,KM,differs:

Intherapidequilibriumapproachby k1
MichaelisMentenKMisequivalentto KM=
k1
thetruedissociationconstantKs

Inthesteadystateapproachby (k1+k2)
BriggsHaldanetherateofthe KM=
chemicalstep,k2,ispartoftheKM k1
andhenceitisnotequivalenttothe

dissociationconstant.
 AnalysisofkineticdatatheLineweaverBurkplot
Wehaveseenthatathighsubstrateconcentration,theinitialvelocityofthe
reactionapproachesthemaximalvelocityv maxasymptotically.Inpracticethis
asymptoticvalueisdifficulttodetermineinadirect(hyperbolic)plotofvelocity
vs.substrateconcentration.ThereforeHansLineweaverandDeanBurkhave
linearizedtheMichaelisMentenequationto:

1
v
= ( ) [S]1 + v1
KM

vmax max

Inthisdoublereciprocal
plot1/visplottedvs1/
[S].Theyaxisintercept
yields1/vmaxwhereasthe
xaxisinterceptyields
1/KM.Theslopeofthe
straightlineisequivalent
toKM/vmax.

 Catalyticefficiencyofenzymes

ForanenzymethatobeystheMichaelisMentenkinetics: vmax=k2[E]total
k2isalsocalledkcatorturnovernumberbecauseitreflectsdirectlythe
commitmenttocatalysisandthereforewecanalsowrite:
vmax
vmax=kcat[E]totalorkcat=
[E]total
When[S]<<KMthen[E]~[E]total

k2[E]total[S]
v=
KM+[S]

kcat kcat/KMisa2ndorderrateconstant(M1sec1)
v=[E][S] andassuchreflectstheefficiencyofEto
KM reactwithS

Theseconsiderationalsoallowustodeterminehowfastanenzymecatalyzed
reactioncanproceed:

 Maximalvelocityofanenzymecatalyzedreaction

kcat k2 k1k2
==
KM KM k1+k2

Inthecaseofefficientcatalysisk2>>k1
ThismeansthattheMichaelisMentencomplexdecaysrapidlytothe
product(s)andthebackreactiontofreeenzymeandsubstratearemuch
slower(enzymeiscommittedtocatalysis).Insuchacasetheequation
abovecanberewrittenas:

kcat Sincek1istherateatwhichtheMichaelisMenten
=k1
KM complexforms,thelimitingvalueforthisrateconstantis
therateofencounterofenzymeandsubstrate,i.e.therate
limitedbydiffusion.Thisrateisoftheorder108109M1
sec1.Henceenzymesthatoperateinthisrangehave
achievedmaximalvelocity(catalase:4x10 8M1sec1)

 Determinationofrates

Fromsteadystatemeasurementstwoenzymeparametersareobtained:

1) TheMichaelisMentenParameter(KM)whichmaybe
equivalenttotheenzymesubstratedissociationconstant

2) kcat(turnovernumber)whichmaybeamicroscopicrate
constantoracombinationofseveral

Toobserveindividualratestheapproachtosteadystateneedstobe
observed(presteadystatekinetics);theratesaretypicallyontheorder
of1107sec!

 TheContinuousFlowMethod

Hartridge&Roughton(1923)
Reactantsarecompressedataconstantrategeneratingaconstant
flow
Ataconstantflowratetheageofthesolutionislinearly
proportional tothedistancedowntheflowtube

fromStructureandmechanisminproteinscience,AlanFersht

 TheStoppedFlowMethod
Roughton(1934);improvedbyChance(1940)

Amenableforreactionsthatundergospectralchanges(UVVisabsorbance,
fluorescence,CD)

fromStructureandmechanisminproteinscience,AlanFersht
 TheRapidQuenchFlowTechnique

Requirequenchingofthe
reactionandconcomittant
analysisofthesample
collectedbyanappropriate
analyticalmethod(HPLC,
GCMS,etc.)

fromStructureandmechanisminproteinscience,AlanFersht