Post on 24-Sep-2020
ENZYMES2:KINETICSANDINHIBITIONHLeeYuJsuicoJunsay
DepartmentofChemistry
SchoolofScienceandEngineering
AteneodeManilaUniversity1
REVIEWOFKINETICS(GENCHEMII)
2
ChemicalKineCcs
• HowfastwillthereacConproceed?• Itisthestudyofreac>onrates.• REACTIONRATESisthechangeintheconcentraConofareactantorproductwithCme(M/s) AB
!me
RateLaws
• OrRateEqua>onexpressestherateasafuncConofreactantconcentraCons,productconcentraConsandtemperature…
• TheorderofthereacContellsyoubyhowmuchratechangesasyouchangeaparCcularconcentraCon
€
Rate = k A[ ]m B[ ]n
FactorsAffecCngReacConRates
• Concentra>onofreactants
• Temperature
• PhysicalState:SurfaceArea
• Catalysis
ConcentraCon RateofreacCon
Temperature
Surfaceareaofsolidorliquid
Presenceofcatalyst(light,compounds)
RateofreacCon
RateofreacCon
RateofreacCon
CollisionTheory
• AtomsandMoleculesmustcollide.– AffectedbyconcentraCon– Affectedbytemperature
• Theymustcollidewithenoughenergy– Affectedbytemperature
• TheymustcollideintheproperorientaCon
The balanced chemical equation provides information about the beginning and end of reaction. WHAT
The reaction mechanism gives the path of the reaction. HOW
Mechanisms provide a very detailed picture of which bonds are broken and formed during the course of a reaction.
ReacConMechanisms
Mechanismofα‐Chymotrypsin1
1Bugg,T.1997.AnIntroduc!ontoEnzymeandCoenzymeChemistry.
ReacConMechanismsTheoverallprogressofachemicalreacConcanberepresentedatthemolecularlevelbyaseriesofsimpleelementarystepsorelementaryreac-ons.
Anelementarystepisaprocessthatoccursinasingleeventorstep.ThesequenceofelementarystepsthatleadstoproductformaConisthereac-onmechanism.
ThemolecularityisthenumberofmoleculesparCcipaCnginanelementarystep(asinglestep!). unimolecular:onemoleculeintheelementarystep, bimolecular:twomoleculesintheelementarystep,and termolecular:threemoleculesintheelementarystep.(notcommon,staCsCcallyimprobable)
MulC‐stepMechanism Some reactions may take place in a series of
elementary steps: 2NO (g) + O2 (g) 2NO2 (g)
N2O2 is detected during the reaction!
Elementary step: NO + NO N2O2
Elementary step: N2O2 + O2 2NO2 Overall reaction: 2NO + O2 2NO2
+
Elementary steps must add to give the balanced chemical equation. Intermediate : a species which appears in an elementary step which is not a reactant or product.
IdenCfyingIntermediates
Elementary step: NO + NO N2O2
Elementary step: N2O2 + O2 2NO2 Overall reaction: 2NO + O2 2NO2
+
Intermediates are species that appear in a reaction mechanism but not in the overall balanced equation.
An intermediate is always formed in an early elementary step and consumed in a later elementary step.
RateLawsofElementaryStep
The rate law of an elementary step is determined by its molecularity: Unimolecular processes are first order Bimolecular processes are second order Termolecular processes are third order
UnimolecularreacCon A products rate=k[A]
BimolecularreacCon A+Bproducts rate=k[A][B]
BimolecularreacCon A+Aproducts rate=k[A]2
Why?
• Elementarystepsareone‐stepreacCons.
• HoweveryouchangetheconcentraConofonespecieswilldirectlyaffectthe#ofeffecCvecollisions.
HOWDOWEDESCRIBEENZYMEKINETICS?
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EnzymekineCcswasdescribedbyLeonorMichaelisandMaudMenten:
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EnzymekineCcswasdescribedbyLeonorMichaelisandMaudMenten:
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Enzymes(E)associatewiththeirsubstrate(S)toformanEnzyme‐Substratecomplex(ES),thenagerformingtheEScomplex,enzymeworkstoformtheproducts(P).
E+S ES E+Pk1k2
k3
k4
EnzymekineCcswasdescribedbyLeonorMichaelisandMaudMenten:
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ExperimentswereranwithconstantEandincreasingS(similartoyourChem12experiments!)
EnzymekineCcswasdescribedbyLeonorMichaelisandMaudMenten:
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Usingsteady‐stateassumpCons(concentraConofintermediatesdonotchangewithCme)…
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1( ) E[ ]T = E[ ] + ES[ ]
(2) dPdt
= v0 = k3 ES[ ]
3( )d ES[ ]dt
= 0 = k1 E[ ] S[ ] − k2 ES[ ] − k3 ES[ ]
EnzymekineCcswasdescribedbyLeonorMichaelisandMaudMenten:
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Usingsteady‐stateassumpCons(concentraConofintermediatesdonotchangewithCme),thefollowingequaConwasderived:
€
v0 =vMAX S[ ]KM + S[ ]
Maximumvelocityofenzymecatalysis
Michaelis‐Mentenconstant
VMAXhappenswhenallenzymesarebeingtransformedtoEScomplexesthatcancreateproducts.
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€
vMAX = k3 E[ ]T
VMAXhappenswhenallenzymesarebeingtransformedtoEScomplexesthatcancreateproducts.
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€
vMAX = k3 E[ ]T
Highk3(some>mescalledkcat),HighvMAX,goodcataly>cac>vity!
KMdescribeshowwelltheenzymeisalachedtothesubstrate(usuallywhenaspecialcaseisadopted,k2>>>k3).
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€
KM =k2 + k3k1
KMdescribeshowwelltheenzymeisalachedtothesubstrate(usuallywhenaspecialcaseisadopted,k2>>>k3).
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€
KM =k2 + k3k1
LowKM,Substrateis>ghtlyboundtoenzyme,MOREchancestoproduceproducts!
Whathappenswhenyouchangesomeparameters?Aslongasthereisenoughsubstrate,higherenzymeconcentra>on,higherrate!
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Whathappenswhenyouchangesomeparameters?Enzymesoperateatspecific/op>mumpH
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Whathappenswhenyouchangesomeparameters?Increasingtemperaturemayincreaseac>vityun>lacertainpoint,aaerwhich,enzymegetsdenatured
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Insummary:
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Enzyme* KM(mM) Kcat(s‐1) kcat/KM
Catalase 0.001 40,000,000 4x1010
Carbonicanhydrase
9 400,000 4.44x105
Chymotrypsin 108 100 0.926
*givenspecificsubstrates
Also,Enzymesoperateunderop>malcondi>ons:pH,temperatureandconcentra>ons
Howdoyougettheseparametersinagraph?
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Howdoyougettheseparametersinagraph?
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VMAXkcat(ifweknow[E]T)
Howdoyougettheseparametersinagraph?
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€
v0 =vMAX S[ ]S[ ] + S[ ]
v0 =vMAX2
WhenKM=[S}
KMisconcentraConwhenVmaxishalved
Howdoyougettheseparametersinagraph?
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Howdoyougettheseparametersinagraph?
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€
1v0
=KM
vMAX1S[ ]
+1
vMAX
Lineweaver‐Burkplot
y=mx+b
1/[S]μM‐1 1/V0(min/μM)
Howdoyougettheseparametersinagraph?
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€
1v0
=KM
vMAX1S[ ]
+1
vMAX
Lineweaver‐Burkplot
[S]μM V0(μM/min)50 10
100 19
150 31
200 38
300 55
400 62
800 68
1000 70
1/[S]μM‐1 1/V0(min/μM)0.0200 0.1000
0.0100 0.0526
0.0067 0.0323
0.0050 0.0263
0.0033 0.0182
0.0025 0.0161
0.0013 0.0147
0.0010 0.0143
Howdoyougettheseparametersinagraph?
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0
10
20
30
40
50
60
70
80
0 200 400 600 800 1000 1200
Ini>alratevs.[S]
Howdoyougettheseparametersinagraph?
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y=4.6411x+0.0055R²=0.9895
0.0000
0.0200
0.0400
0.0600
0.0800
0.1000
0.1200
0.0000 0.0050 0.0100 0.0150 0.0200 0.0250
1/vvs.1/S
SomeenzymesdonotSTRICTLYobeytheMMtheorem.Wecallthemallostericenzymes…
Moreonthatinthenextchapter!
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ENZYMEINHIBITION
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MoleculeswhichhinderenzymeacCvityarecalledinhibitors.
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Inhibitors:reduceenzymeacCvity ALTERATIONVIA
InfluenceonBINDINGor InfluenceonTURN‐OVERNO.
Mayormaynotresemblesubstrate(transi>onstateanalogs)andmaynotreactorreactveryslowcomparedtosubstrate
CanbeusedasprobesfornatureofacCvesite
MoleculeswhichhinderenzymeacCvityarecalledinhibitors.
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MoleculeswhichhinderenzymeacCvityarecalledinhibitors.
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Canbereversible(noncovalent)orirreversible(covalent)
MoleculeswhichhinderenzymeacCvityarecalledinhibitors.
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SPECIFICTYPESOFIRREV.INHIB. Group‐specificreagents
Reactswithexactaminoacids
SUBSTRATEANALOGS Analogbindscovalently
SUICIDEINHIBITION Inhibitorisprocessed,productinhibitscovalently
MoleculeswhichhinderenzymeacCvityarecalledinhibitors.
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Bothreversibleandirreversiblecanact COMPETITIVELY NONCOMPETITIVELY UNCOMPETITIVELY
Real‐worldreac>onsaremixedwithhighcharacterofaspecifictype
MoleculeswhichhinderenzymeacCvityarecalledinhibitors.
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Gardener‐fling
Husband
Wife
Child
NochildEnzyme
Substrate
Inhibitor
Product
NoProduct
Compe>>veinhibi>onoccurswhenEnzymecanbindtosubstrate(ES)orinhibitor(EI),butnotatthesameCme.InhibitorbindstotheacCvesite.
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The“moral”inhibi>on.OnlythepartnerORthemistress
Compe>>veinhibi>onoccurswhenEnzymecanbindtosubstrate(ES)orinhibitor(EI),butnotatthesameCme.InhibitorbindstotheacCvesite.
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Increasein[S]willincreasethechanceofformingEScomplex,
THUS,inhibi>onmayberelievedwhenthereishigh[S]
Compe>>veinhibi>onincreasesKMbutnotVmax.
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Noncompe>>veinhibi>onoccurswhenInhibitorcanbindtoeitherE(formingEI),ortoES(formingESI).Inhibitorbindstoanotherpartoftheenzyme.
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The“amoralinhibi>on”‐Themistressdoesn’tcareifthepartneristhereornot.Canamachany>me.
Noncompe>>veinhibi>onoccurswhenInhibitorcanbindtoeitherE(formingEI),ortoES(formingESI).Inhibitorbindstoanotherpartoftheenzyme.
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OnceInhibitorbinds,nomoreac>vityisexpected…
Thus,Inhibitorlowers[E]T..
Noncompe>>veinhibi>onlowersVmax,butdoesn’taffectKM.
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Uncompe>>veinhibi>onoccurswhenInhibitorcanbindonlytotheEScomplex(formingESI).Inhibitorbindstoanotherpartoftheenzyme.
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The“immoralinhibi>on”–mistressonlycomesIFpartneristhere.
Uncompe>>veinhibi>onoccurswhenInhibitorcanbindonlytotheEScomplex(formingESI).Inhibitorbindstoanotherpartoftheenzyme.
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Increasingthe[S]willjustallowmoreESItoform.
Uncompe>>veinhibi>onchangesbothVmaxandKM.
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Insummary,
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