enzymes 2: kinetics and inhibition - kimika · enzyme kinecs was described by leonor michaelis and...

ENZYMES2:KINETICSANDINHIBITIONHLeeYuJsuicoJunsay

DepartmentofChemistry

SchoolofScienceandEngineering

AteneodeManilaUniversity1

REVIEWOFKINETICS(GENCHEMII)

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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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 Enzymes(E)associatewiththeirsubstrate(S)toformanEnzyme‐Substratecomplex(ES),thenagerformingtheEScomplex,enzymeworkstoformtheproducts(P).

E+S ES E+Pk1k2

k3

k4


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 ExperimentswereranwithconstantEandincreasingS(similartoyourChem12experiments!)


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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[ ]


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 Usingsteady‐stateassumpCons(concentraConofintermediatesdonotchangewithCme),thefollowingequaConwasderived:

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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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VMAXkcat(ifweknow[E]T)


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v0 =vMAX S[ ]S[ ] + S[ ]

v0 =vMAX2

WhenKM=[S}

KMisconcentraConwhenVmaxishalved


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1v0

=KM

vMAX1S[ ]

+1

vMAX

Lineweaver‐Burkplot

y=mx+b

1/[S]μM‐1 1/V0(min/μM)


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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


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0

10

20

30

40

50

60

70

80

0 200 400 600 800 1000 1200

Ini>alratevs.[S]


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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


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  Canbereversible(noncovalent)orirreversible(covalent)


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  SPECIFICTYPESOFIRREV.INHIB. Group‐specificreagents

  Reactswithexactaminoacids

 SUBSTRATEANALOGS  Analogbindscovalently

 SUICIDEINHIBITION  Inhibitorisprocessed,productinhibitscovalently


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  Bothreversibleandirreversiblecanact COMPETITIVELY NONCOMPETITIVELY UNCOMPETITIVELY

Real‐worldreac>onsaremixedwithhighcharacterofaspecifictype


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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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enzymes 2: kinetics and inhibition - kimika · enzyme kinecs was described by leonor michaelis and...

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