9/16/16

1

3.1)Enzymes-I

Introductiontoenzymestructureandfunction,andfactorsinvolvingtheir

actionsandpathways

Biochemistry

Prof. Dr. Klaus Heese

Enzymesareusedalloveryourbody!

Whatisanenzyme?

• Almostallenzymesareproteinsthatactasbiologicalcatalysts.

• Acatalystspeedsupchemicalreactions.Enzymesspeedupbiologicalchemicalreactions.

• Enzymesarehighlyspecifictoatypeofreaction.

• Enzymesmustmaintaintheirspecificshapeinordertofunction.Anyalterationintheprimary,secondary,tertiary,orquaternaryformsoftheenzymearedetrimental.

EnzymesasBiologicalCatalysts

• Enzymes areproteinsthatincreasetherateofreactionbyloweringtheenergyofactivation

• Theycatalyzenearlyallthechemicalreactionstakingplaceinthecellsofthebody

• Enzymeshaveuniquethree-dimensionalshapesthatfittheshapesofreactants(substrates)

•Increasedreactionratessometimes106 to1012increase

EnzymesdonotchangeDG,justthereactionrates.

(Enzymesalterrates,notequilibria)

•Milderreactionconditions

•Greatreactionspecificity

CO2 +H2O--->H2CO3

DG

Enzymescatalyzebystabilizingtransitionstates

• FreeenergyGofachemicalreactioncanbeplottedovertime

• Favorablereactionshaveapositivedifference(DG)infreeenergybetweenthesubstrateandproduct

• Thefreeenergyofactivationforthetransitionstatelimitstheprogressofthereaction

• Enzymesactbyreducingthefreeenergyofthetransitionstate

Functionofenzymes

Enzymeshavemanyjobs.They:

• Breakdownnutrientsintouseablemolecules.

• Storeandreleaseenergy(ATP).

• Createlargermoleculesfromsmallerones.

• Coordinatebiologicalreactionsbetweendifferentsystemsinanorganism.

9/16/16

2

Naming Enzymes

• Thenameofanenzymeidentifiesthereactingsubstance- usuallyendsin–ase

• Forexample,sucrase catalyzesthehydrolysisofsucrose• Thenamealsodescribesthefunctionoftheenzyme• Forexample,oxidases catalyzeoxidationreactions• Sometimescommonnamesareused,particularlyforthe

digestionenzymessuchaspepsin andtrypsin• Somenamesdescribeboththesubstrateandthefunction• Forexample,alcoholdehydrogenase oxidesethanol

ClassificationofEnzymes• Enzymesareclassifiedaccordingtothetypeofreactionthey

catalyze:

Class Reactionscatalyzed§ Oxidoreductases Oxidation-reduction§ Transferases Transfergroupsofatoms§ Hydrolases Hydrolysis§ Lyases Addatoms/removeatomsto/froma

doublebond§ Isomerases Rearrangeatoms§ Ligases UseATPtocombinemolecules

SystematicName• AccordingtotheInternationalunionofBiochemistryanenzymenamehastwoparts:

- Firstpartisthenameofthesubstratesfortheenzyme.

- Secondpartisthetypeofreactioncatalyzedbytheenzyme.Thispartendswiththesuffix“ase”.

Example:Lactatedehydrogenase

ECnumber

Enzymesareclassifiedintosixdifferentgroupsaccordingtothereactionbeingcatalyzed.ThenomenclaturewasdeterminedbytheEnzymeCommissionin1961(withthelatestupdatehavingoccurredin1992),henceallenzymesareassignedan“EC” number.Theclassificationdoesnottakeintoaccountaminoacidsequence(ie,homology),proteinstructure,orchemicalmechanism.

ECnumbers

• ECnumbersarefourdigits,forexamplea.b.c.d,where“a” istheclass,“b” isthesubclass,“c” isthesub-subclass,and“d” isthesub-sub-subclass.The“b” and“c” digitsdescribethereaction,whilethe“d” digitisusedtodistinguishbetweendifferentenzymesofthesamefunctionbasedontheactualsubstrateinthereaction.

• Example:forAlcohol:NAD+oxidoreductase:ECnumberis1.1.1.1

TheSixClasses

• EC1.Oxidoreductases• EC2.Transferases• EC3.Hydrolases• EC4.Lyases• EC5.Isomerases• EC6.Ligases

Additionalinformationonthesubclasses,thesub-subclassesandsub-sub-subclasses(ie,fullenzymeclassificationandnames)canbefoundatthereferencedweblink.• FromtheWebversion,

http://www.chem.qmul.ac.uk/iubmb/enzyme/index.html

ClassificationofEnzymes

Enzymesareclassifiedaccordingtothetypeofreactiontheycatalyze:

9/16/16

3

EC1.Oxidoreductases

• EC1.Oxidoreductases:catalyzethetransferofhydrogenoroxygenatomsorelectronsfromonesubstratetoanother,alsocalledoxidases,dehydrogenases,orreductases.Notethatsincetheseare‘redox’ reactions,anelectrondonor/acceptorisalsorequiredtocompletethereaction.

EC2.Transferases

• EC2.Transferases – catalyzegrouptransferreactions,excludingoxidoreductases (whichtransferhydrogenoroxygenandareEC1).Theseareofthegeneralform:

• A-X+B↔BX+A

EC3.Hydrolases

• EC3.Hydrolases– catalyzehydrolyticreactions.Includeslipases,esterases,nitrilases,peptidases/proteases.Theseareofthegeneralform:

• A-X+H2O↔X-OH+HA

EC4.Lyases• EC4.Lyases – catalyzenon-hydrolytic(coveredinEC3)removaloffunctionalgroupsfromsubstrates,oftencreatingadoublebondintheproduct;orthereversereaction,i.e. additionoffunctiongroupsacrossadoublebond.

• A-B→A=B+X-YXY

• Includesdecarboxylasesandaldolases intheremovaldirection,andsynthasesintheadditiondirection.

EC5.Isomerases

• EC5.Isomerases – catalyzesisomerizationreactions,includingracemizations andcis-transisomerizations.

EC6.Ligases

• EC6.Ligases-- catalyzesthesynthesisofvarious(mostlyC-X)bonds,coupledwiththebreakdownofenergy-containingsubstrates,usuallyATP

9/16/16

4

Oxidoreductases,Transferases andHydrolases Lyases,Isomerases andLigases

Enzymeactionoverview

• Enzymesarelargemoleculesthathaveasmallsectiondedicatedtoaspecificreaction.Thissectioniscalledtheactive site.

• Theactivesitereactswiththedesiredsubstance,calledthesubstrate.

• Thesubstratemayneedanenvironmentdifferentfromthemostlyneutralenvironmentofthecellinordertoreact.Thus,theactivesitecanbemoreacidicorbasic,orprovideopportunitiesfordifferenttypesofbondingtooccur,dependingonwhattypeofsidechainsarepresentontheaminoacidsoftheactivesite.

ActiveSiteofanEnzyme

• Theactivesite isaregionwithinanenzymethatfitstheshapeofsubstratemolecules

• Aminoacidside-chainsaligntobindthesubstratethroughH-bonding,salt-bridges,hydrophobicinteractions,etc.

• Productsarereleasedwhenthereactioniscomplete(theynolongerfitwellintheactivesite)

EnzymeSpecificity• Enzymeshavevaryingdegreesofspecificity forsubstrates• Enzymesmayrecognizeandcatalyze:

- asinglesubstrate- agroupofsimilarsubstrates- aparticulartypeofbond

Enzyme-CatalyzedReactions• Whenasubstrate(S)fitsproperlyinanactivesite,an

enzyme-substrate(ES)complex isformed:E +S D ES

• WithintheactivesiteoftheES complex,thereactionoccurstoconvertsubstratetoproduct(P):ES ® E +P

• Theproductsarethenreleased,allowinganothersubstratemoleculetobindtheenzyme- thiscyclecanberepeatedmillions(orevenmore)timesperminute

• Theoverallreactionfortheconversionofsubstratetoproductcanbewrittenasfollows:E +S D ES ® E +P

9/16/16

5

ExampleofanEnzyme-CatalyzedReaction• Thereactionforthesucrase catalyzedhydrolysisofsucroseto

glucoseandfructosecanbewrittenasfollows:E +S D ES ® E +P1 +P2whereE =sucrase,S =sucrose,P1 =glucoseandP2 =fructose

Isoenzymes• Isoenzymes aredifferentformsofanenzymethatcatalyze

thesamereactionindifferenttissuesinthebody- theyhaveslightvariationsintheaminoacidsequencesofthesubunitsoftheirquaternarystructure

• Forexample,lactatedehydrogenase(LDH),whichconvertslactatetopyruvate,consistsoffiveisoenzymes

DiagnosticEnzymes• Thelevelsofdiagnosticenzymes inthebloodcanbeused

todeterminetheamountofdamageinspecifictissues DenaturingEnzymes

• Whenanenzymeisdenatureditisdamaged.• Denaturingchangestheshape.• Withoutthecorrectshapeenzymeswon’tfunctionproperly.

• HOWareenzymesdenatured?– Temperature– pH

(=conformationchange=changeofproteins’3Dstructure)

Factorsthataffectenzymeaction

Factorsthataffectenzymeaction

EnzymesaremostlyaffectedbychangesintemperatureandpH.

• Toohighofatemperaturewilldenaturetheproteincomponents,renderingtheenzymeuseless.

• pHrangesoutsideoftheoptimalrangewillprotonateordeprotonate thesidechainsoftheaminoacidsinvolvedintheenzyme’sfunctionwhichmaymakethemincapableofcatalyzingareaction.

TemperatureandEnzymeActivity• Enzymes aremostactiveatanoptimumtemperature(usually

37°Cinhumans)• Theyshowlittleactivityatlowtemperatures• Activityislostathightemperaturesasdenaturationoccurs

9/16/16

6

pHandEnzymeActivity• Enzymes aremostactiveatoptimumpH• Aminoacidswithacidicorbasicside-chainshavethe

properchargeswhenthepHisoptimum• ActivityislostatloworhighpHastertiarystructureis

disrupted

OptimumpHforSelectedEnzymes• MostenzymesofthebodyhaveanoptimumpHofabout7.4• However,incertainorgans,enzymesoperateatlowerand

higheroptimumpHvalues

Factorsthataffectenzymeaction

Enzymesarealsoaffectedbytheconcentrationofsubstrate,cofactorsandinhibitors,aswellasallostericregulationandfeedbackinhibition.

• Theconcentrationof substratewilldictatehowmanyenzymescanreact.Toomuchsubstratewillslowtheprocessuntilmoreenzymecanbemade.

• Theavailabilityofcofactors alsodictateenzymeaction.Toolittlecofactorswillslowenzymeactionuntilmorecofactorsareadded.

• Aninfluxofcompetitiveornon-competitiveinhibitorswillnotnecessarilyslowtheenzymeprocess,butwillslowtheamountofdesiredproduct.

EnzymeConcentrationandReactionRate• Therateofreactionincreasesasenzymeconcentration

increases(atconstantsubstrateconcentration)• Athigherenzymeconcentrations,moreenzymesare

availabletocatalyzethereaction(morereactionsatonce)• Thereisalinearrelationshipbetweenreactionrateand

enzymeconcentration(atconstantsubstrateconcentration)

SubstrateConcentrationandReactionRate• Therateofreactionincreasesassubstrateconcentration

increases(atconstantenzymeconcentration)• Maximumactivity occurswhentheenzymeissaturated

(whenallenzymesarebindingsubstrate)• Therelationshipbetweenreactionrateandsubstrate

concentrationisexponential,andasymptotes(levelsoff)whentheenzymeissaturated

Enzymeactiontheories

• LockandKey: Thistheory,postulatedbyEmilFischerin1894,proposedthatanenzymeis“structurallycomplementarytotheirsubstrates” andthusfittogetherperfectlylikealockandkey.Thistheoryformedthebasisofmostoftheideasofhowenzymeswork,butisnotcompletelycorrect.

9/16/16

7

Lock-and-KeyModel• Inthelock-and-keymodel ofenzymeaction:

- theactivesitehasarigidshape- onlysubstrateswiththematchingshapecanfit- thesubstrateisakeythatfitsthelockoftheactivesite

• Thisisanoldermodel,however,anddoesnotworkforallenzymes

Enzymeactiontheories

Enzymeactiontheories• InducedFit: Anenzymethatisperfectlycomplementarytoits

substratewouldactuallynotmakeagoodenzymebecausethereactionhasnoroomtoproceedtothetransitionstateofthereaction. Togotocompletion,areactionmustgothroughthetransitionstate.Inthelockandkeytheory,thesubstrateortheenzymecannotchangeconformationstothetransitionstate.Therefore,enzymesmustactuallybecomplementarytothetransitionstate sothereactionmayproceed.ThisideawasresearchedbyHaldanein1930,andLinusPaulingin1946.ThisidealedtheInducedFittheory,postulatedbyDanielKoshlandin1958,wheretheenzymeitself canchangeconformationstofacilitatethetransitionstateofthesubstrate.Thischangeinconformationoftheenzymeallowsthenecessaryfunctionalgroupsattheactivesitetomoveclosertothesubstrate,enhancingtheefficiencyofthereaction.

InducedFitModel• Intheinduced-fitmodel ofenzymeaction:

- theactivesiteisflexible,notrigid- theshapesoftheenzyme,activesite,andsubstrateadjusttomaximizethefit,whichimprovescatalysis- thereisagreaterrangeofsubstratespecificity

• Thismodelismoreconsistentwithawiderrangeofenzymes

Enzymeactiontheories

InducedFitModel

• Enzymescanformtotheshapeofitssubstrate.

Enzymecofactors

• Acofactor isasubstance,thatisnotaprotein,thatmustbindtotheenzymeinorderfortheenzymetowork.

• metalionsascofactors-- Zn2+,Fe2+,Cu2+,others

• Acofactorcanbeoforganicorigin.Anorganiccofactoriscalledacoenzyme.

• Cofactorsarenotpermanentlybonded.Permanentlybondedcofactorsarecalledprostheticgroups.

Enzymecofactorscont.

• Anenzymethatisbondedtoitscofactoriscalledaholoenzyme.

• Anenzymethatrequiresacofactor,butisnotbondedtothecofactoriscalledanapoenzyme.Apoenzymes arenotactiveuntiltheyarecomplexed withtheappropriatecofactor.

9/16/16

8

Coenzymes

Coenzymes:smallermoleculesthataidinenzymechemistry.Enzymescan:

a.Carryoutacid-basereactionsb.Transientcovalentbondsc.Charge-chargeinteractions

Enzymescannotdo:d.Oxidation- Reduction(=Redox)reactionse.Carbongrouptransfers

Prostheticgroup:permanentlyassociatedwithanenzymeortransientlyassociated.Holoenzyme:catalyticallyactiveenzymewithcofactor.Apoenzyme: Enzymewithoutitscofactor.

Commoncoenzymes

Manycoenzymesarederivedfromvitamins:

• NAD+ (nicotinamide adeninedinucleotide); derivedfromniacin(B3).

• CoenzymeA(CoA);derivedfrompantothenicacid(B5).

• FAD(flavin adeninedinucleotide);derivedfromriboflavin(B2).

Commom Coenzymes

Coenzyme ReactionmediatedBiotin CarboxylationCobalamin (B12) AlkylationtransfersCoenzymeA AcyltransfersFlavin Oxidation-ReductionLipoic acid AcyltransfersNicotinamide Oxidation-ReductionPyridoxal Phosphate AminogrouptransfersTetrahydrofolate One-carbongrouptransfersThiaminepyrophosphate Aldehydetransfer

VitaminsareCoenzymeprecursors

Vitamin Coenzyme DeficiencyDisease

Biotin Biocytin notobserved

Cobalamin (B12) Cobalamin Perniciousanemia

Folicacid tetrahydrofolate NeuraltubedefectsMegaloblastic anemia

Nicotinamide Nicotinamide Pellagra

Pantothenate CoenzymeA Notobserved

Pyridoxine(B6) Pyridoxal phosphate Notobserved

Riboflavin(B2) Flavin Notobserved

Thiamine(B1) Thiaminepyrophosphate Beriberi

Thesearewatersolublevitamins.TheFatsolublevitaminsarevitaminsAandD.

Humanscannotsynthesizetheseandrelayontheirpresenceinourdiets.Thosewhohaveanunbalanceddietmaynotbereceivingasufficientsupply.

Niacin(niacinamide)deficiencyleadstopellagracharacterizedbydiarrhea,dermatitisanddementia.PellagrawasendemicisSouthernUnitedStatesintheearly20thcentury.Niacincanbesynthesizedfromtheessentialaminoacid,tryptophan.Acorndietprevalentatthetimerestrictedtheabsorptionoftryptophancausingadeficiency.Treatmentofcornwithbasecouldreleasethetryptophan(MexicanIndianstreatedcornwithCa(OH)2 beforemakingtortillas!)

Commoncoenzymes

Coenzymescanbederivedfromsourcesotherthanvitamins:

• ATP(adenosinetriphosphate);derivedfromNADHfromcarbohydratesconsumed.(ATPcanalsoactasneurotransmitter)

• CTP(Cytidine triphosphate);derivedfromglutamateandcarbamoylphosphate.

• PAPS(3'-Phosphoadenosine-5'-phosphosulfate); derivedfromadenosine5'-phosphosulfate(APS)andsulfateion.

9/16/16

9

Coenzymereactions

• Coenzymeshelptotransferafunctionalgrouptoamolecule.

• Forexample,coenzymeA(CoA)isconvertedtoacetyl-CoAinthemitochondriausingpyruvateandNAD+.

• Acetyl-CoAcanthenbeusedtotransferanacetyl group(CH3CO)toaidinfattyacidsynthesis.

Fattyacidsynthesis

CoenzymeAisconvertedtoacetyl-CoenzymeAenzymeispyruvatedehydrogenase

Factorsthataffectenzymeaction

• Enzymesthatcanbeactivatedwillbeaffectedbytheamountofactivatororinhibitorattachedtoitsallostericsite.Anabundanceofanallostericactivatorwillconvertmoreenzymestotheactiveformcreatingmoreproduct.

• Enzymesthatarepartofametabolicpathwaymaybeinhibitedbytheveryproducttheycreate.Thisiscalledfeedbackinhibition.Theamountofproductgeneratedwilldictatethenumberofenzymesusedoractivatedinthatspecificprocess.

Enzymeactivityandinhibition• The“normal”wayanenzymefunctionsiswhenthespecific

substratebindstotheactivesiteandcreatestheproducts.

• Asimilarsubstratecanalsobondtotheactivesitecovalentlyandirreversibly.Thispreventstheenzymefromfunctioning.Irreversibleinhibition.

• Asimilarsubstratecanbindtotheactivesite,notpermanently,andpreventsthedesiredsubstratefromenteringtheactivesite.Thischangestheproductsandfunctioningoftheenzyme.Thisiscalledcompetitiveinhibition.

• Amoleculecanbondtoanotherpartoftheenzymeandcauseachangeinconformation.Thischangecausestheactivesitetochangeshapeaswell.Thischangeinshapepreventsthedesiredsubstratefromenteringtheactivesite.Thisiscallednon-competitiveinhibition.

EnzymeActivators

• Chemicalsthathelptheenzymework.• Activatorsincreasetheenzymereactionrate.

X

Activator

SubstrateBindingSite

ActiveSite

EnzymeInhibitors• Chemicalsthatpreventtheenzymefromworking.• Inhibitorsdecreasetheenzymereactionrate.

9/16/16

10

EnzymeInhibitors

• Inhibitors(I) aremoleculesthatcausealossofenzymeactivity

• Theypreventsubstratesfromfittingintotheactivesiteoftheenzyme:

E+SD ES® E+PE+I D EI ® no Pformed

Reversible Inhibitors (CompetitiveInhibition)

• Areversibleinhibitor goesonandoff,allowingtheenzymetoregainactivitywhentheinhibitorleaves

• Acompetitiveinhibitor isreversibleandhasastructurelikethesubstrate- itcompeteswiththesubstratefortheactivesite- itseffectisreversedbyincreasingsubstrateconcentration

ExampleofaCompetitiveInhibitor• Malonate isacompetitiveinhibitorofsuccinatedehydrogenase

- ithasastructurethatissimilartosuccinate- inhibitioncanbereversedbyaddingsuccinate

ReversibleInhibitors(Non-competitiveInhibition)

• Anon-competitiveinhibitorhasastructurethatisdifferentthanthatofthesubstrate- itbindstoanallostericsiteratherthantotheactivesite- itdistortstheshapeoftheenzyme,whichalterstheshapeoftheactivesiteandpreventsthebindingofthesubstrate

• Theeffectcannotbereversedbyaddingmoresubstrate,buteventuallyreversedbywashingtheinhibitoraway.

IrreversibleInhibitors• Anirreversibleinhibitor destroysenzymeactivity,usuallyby

bondingwithside-chaingroupsintheactivesite

http://upload.wikimedia.org/wikipedia/commons/thumb/6/6f/Major_digestive_enzymes.png/750px-Major_digestive_enzymes.png

Life Sciences-HHMI Outreach. Copyright 2009 President and Fellows of Harvard College

9/16/16

11

SummaryofEnzymes-I• Enzymesaremostlyproteins• Theyarehighlyspecifictoareaction• Theycatalyzemanyreactionsincludingbreakingdownnutrients,storingand

releasingenergy,creatingnewmolecules,andcoordinatingbiologicalreactions.

• Enzymesuseanactivesite,butcanbeaffectedbybondingatotherareasoftheenzyme.

• Someenzymesneedspecialmoleculescalledcofactorstocarryouttheirfunction.

• Cofactorsthatareorganicinnaturearecalledcoenzymes.• Coenzymesareusuallyderivedfromvitamins.• Coenzymestransferfunctionalgroupsfortheenzymetheyworkwith.• EnzymesareaffectedbychangesinpH,temperature,theamountof

substrate,cofactorsandinhibitors,aswellastheamountofallostericinhibitorsandactivatorsandconcentrationofproductsthatcontrolfeedbackinhibition.

3.2)Enzymes-II

Introductiontoenzymestructureandfunction,andfactorsinvolvingtheir

actionsandpathwaysEnzyme- Kinetics

Biochemistry

Prof. Dr. Klaus Heese

TheSixClasses

• EC1.Oxidoreductases• EC2.Transferases• EC3.Hydrolases• EC4.Lyases• EC5.Isomerases• EC6.Ligases

Additionalinformationonthesubclasses,thesub-subclassesandsub-sub-subclasses(ie,fullenzymeclassificationandnames)canbefoundatthereferencedweblink.• FromtheWebversion,

http://www.chem.qmul.ac.uk/iubmb/enzyme/index.html

Some common types of enzymes

HydrolasesNucleasesProteasesSynthasesIsomerases

PolymerasesKinasesPhosphatasesATP-asesOxidoreductases (dehydrogenases)

Enzyme Active Sites

Active sites:The region that binds substrate.Only a small fraction of the enzyme.Formed from AAs in different parts of the sequence.

carboxypeptidase

Enzyme Active Sites

Active sites:Usually form a cleft or pocket.

Substrates are bound by multiple weak interactions.pH,T

Lock-Key-modelInduced-Fit-Model

9/16/16

12

Properties of Enzymes

• Enzymes are highly efficient and specific catalysts.

• Enzymes alter rates, not equilibria.

• Enzymes stabilize transition states.

• Reaction rates depend on concentrations of enzymes, substrates, and on the efficiency of the enzyme.

Reaction Thermodynamics

Enzymes stabilize the transition state, lowering the activation barrier.

Rate Acceleration

• The enzyme lowers the activation barrier (EA orDG‡) compared to the uncatalyzedreaction.

• DGro is not changed !

EA

EA

Enzymes stabilize the transition state, lowering the activation barrier.

How is Transition State Stabilization Achieved?

– acid-base catalysis: give and take protons– covalent catalysis: change reaction paths– metal ion catalysis: use redox cofactors, pKa shifters– electrostatic catalysis: preferential interactions with

transition state

How to Lower DG‡?Enzymes organizes reactive groups into

proximity (---> induced-fit-model)

• Uncatalyzed bimolecular reactions: two free reactants ® single restricted transition state conversion is entropically unfavorable

• Uncatalyzed unimolecular reactions: flexible reactant ® rigid transition state conversion is entropically unfavorable for flexible reactants

• Catalyzed reactions:Enzyme uses the binding energy of substrates to organize the reactants to a fairly rigid ES complexEntropy cost is paid during bindingRigid reactant complex ® transition state conversion is entropically OK

Enzyme Kinetics

• Enzymes endow cells with the remarkable capacity toexert kinetic control over thermodynamic potentiality

• Enzymes are the agents of metabolic function• What we want to be able to determine:

– Maximum velocity– Substrate affinity– Inhibitor affinity

• What it can tell us:– Flow through metabolic pathways– Utilization of substrates

• What can we do with the information:– Control and manipulate metabolic events

Kinetics is the study of the rates of reactions

9/16/16

13

Reaction Thermodynamicsreminder: Consider:

[A] + [B] [C] + [D]

Keq =

Keq depends only on the nature of the products and the reactants.Reaction will proceed spontaneously only when the change in free energy (DG) is negative.

[C][D][A][B]

Enzyme Kinetics

Enzymatic Equations

E + S ES EP E + P

There are at least three steps…….

Enzyme Kinetics

Enzymatic Equations II

E + S ES E + Pk1

k-1

kcat

E + S ES EP E + P

usually kcat <<< k-1, so Km = k-1/k1 = Kd

Km = (k-1+ kcat )/k1

atsteadystate:forward=reversereaction:d[ES]/dt =-d[ES]/dt <--->k1 [E][S]=k-1+kcat [ES]

=[E][S]=(k-1+kcat )/k1 [ES]--->with[E]total=[E]t and[S]0 =[S]attimepoint0:[E]=[E]t –[ES]--->([E]t –[ES])[S]=Km [ES]--->[ES]=[S][E]/Km +[S]--->istheproductformationcontrollingthevelocityofthereaction:--->V0 =kcat [ES]=kcat {[S][E]/Km +[S]};if[S]highsothatenzymefullysatisfied:Vmax =kcat [E]t--->V0 =Vmax [S]/Km +[S]

Enzyme KineticsKinetics: Vmax and Km

Reaction rate (V) varies with substrate concentration.

Vmax = the maximum reaction rate.

Km = substrate concentration where V = Vmax/2

Measures affinity of enzyme for substrate.

V = Vmax

[S] Km + [S]

The Michaelis-Menten equation

The Michaelis-Menten Equation

kcat

Enz

yme

Kin

etic

s

Important Conclusions of Michaels -Menten Kinetics

• when [S]= KM, the equation reduces to

• when [S] >> KM, the equation reduces to

• when [S] << KM, the equation reduces to

Enz

yme

Kin

etic

s

Effect of Substrate Concentration

• Ideal Rate:

• Deviations due to:

– Limitation of measurements

– Substrate inhibition

– Substrate prep contains inhibitors

– Enzyme prep contains inhibitors

SKSVv

m +=

][max

Enz

yme

Kin

etic

s

9/16/16

14

Vmax

Enz

yme

Kin

etic

s

Effect of Substrate Concentration

V=Vmax [S]KM +[S]---------------

Vmax and Km

Vmax dependsontheamountofenzyme.

Enz

yme

Kin

etic

s

Different substrates, Vmax and Km

Km is a property of both enzyme and substrate.

Enz

yme

Kin

etic

s

Lineweaver – Burk Double Reciprocal Plots• It is difficult to determine Vmax experimentally• The equation for a hyperbola can be transformed into

the equation for a straight line by taking the reciprocal of each side

• The formula for a straight line is y = mx + b

• A plot of 1/V versus 1/[S] will give a straight line with slope of KM/Vmax and y intercept of 1/Vmax

• Such a plot is known as a Lineweaver-Burk double reciprocal plot

Enz

yme

Kin

etic

s

Determination of Kinetic Parameters

v0 = Vmax[S]([S]+Km)

1

v0= ([S]+Km)

Vmax[S] = [S]Vmax[S] +

KmVmax[S]

1

v0= Km

Vmax

1[S] +

1Vmax

Linearized double-reciprocal plot is good for analysis of two-substrate data or inhibition.Lineweaver – Burk Double Reciprocal Plots

V and [S] can be determined

experimentally

Significance of Km

• Km is a constant• Small Km means tight binding; high Km means weak

binding• Useful to compare Km for different substrates for one

enzymeHexokinase : D-fructose – 1.5 mM

D-glucose – 0.15 mM• Useful to compare Km for a common substrate used

by several enzymesHexokinase: D-glucose – 0.15 mMGlucokinase: D-glucose – 20 mM

Enz

yme

Kin

etic

s

9/16/16

15

Enzyme InhibitionEnzyme inhibitors are important for a variety of reasons

1) they can be used to gain information about the shape on the enzyme active site and the amino acid residues in the active site.

2) they can be used to gain information about the chemical mechanism.

3) they can be used to gain information about the regulation or control of a metabolic pathway.

4) they can be very important in drug design.

Enzyme inhibition

Tool to study enzymatic reactions.Important in host/pathogen interactions.Important in drug design.

Irreversible (suicide) inhibition (eg - nerve gas).Reversible inhibition:

competitive (eg. - transition state analogues).non-competitive.uncompetitive.

Type of inhibition can be determined experimentally.

Inhibition Patterns

• An inhibitor may bind at the same site as one of the substrates – these inhibitors structurally resemble the substrate

• An inhibitor may bind at an alternate site affecting catalytic activity without affecting substrate binding

• Many inhibitors do both• Most common types

– Competitive– Mixed or Non-competitive– Uncompetitive

Inhibitors act in a variety of mechanismsEnzymeactivityandinhibition

• The“normal”wayanenzymefunctionsiswhenthespecificsubstratebindstotheactivesiteandcreatestheproducts.

• Asimilarsubstratecanalsobondtotheactivesitecovalentlyandirreversibly.Thispreventstheenzymefromfunctioning.Irreversibleinhibition.

• Asimilarsubstratecanbindtotheactivesite,notpermanently,andpreventsthedesiredsubstratefromenteringtheactivesite.Thischangestheproductsandfunctioningoftheenzyme.Thisiscalledcompetitiveinhibition.

• Amoleculecanbondtoanotherpartoftheenzymeandcauseachangeinconformation.Thischangecausestheactivesitetochangeshapeaswell.Thischangeinshapepreventsthedesiredsubstratefromenteringtheactivesite.Thisiscallednon-competitiveinhibition.

EnzymeActivators

• Chemicalsthathelptheenzymework.• Activatorsincreasetheenzymereactionrate.

X

Activator

SubstrateBindingSite

ActiveSite

EnzymeInhibitors• Chemicalsthatpreventtheenzymefromworking.• Inhibitorsdecreasetheenzymereactionrate.

9/16/16

16

EnzymeInhibitors

• Inhibitors(I) aremoleculesthatcausealossofenzymeactivity

• Theypreventsubstratesfromfittingintotheactivesiteoftheenzyme:

E+SD ES® E+PE+I D EI ® no Pformed

ReversibleInhibitors(CompetitiveInhibition)

• Areversibleinhibitor goesonandoff,allowingtheenzymetoregainactivitywhentheinhibitorleaves

• Acompetitiveinhibitor isreversibleandhasastructurelikethesubstrate- itcompeteswiththesubstratefortheactivesite- itseffectisreversedbyincreasingsubstrateconcentration

ExampleofaCompetitiveInhibitor• Malonate isacompetitiveinhibitorofsuccinatedehydrogenase

- ithasastructurethatissimilartosuccinate- inhibitioncanbereversedbyaddingsuccinate

• A competitive inhibitor reduces the amount of free enzyme available for substrate binding thus increasing the Km for the substrate

• The effect of a competitive inhibitor can be overcome with high concentrations of the substrate

Competitive Inhibition

• Unimolecular Reaction

• Bimolecular Reaction

Competitive Inhibition Competitive Inhibition

Change of Lineweaver – Burk graph by enzyme inhibition

9/16/16

17

ReversibleInhibitors(Non-competitiveInhibition)• Anon-competitiveinhibitor

hasastructurethatisdifferentthanthatofthesubstrate- itbindstoanallostericsiteratherthantotheactivesite- itdistortstheshapeoftheenzyme,whichalterstheshapeoftheactivesiteandpreventsthebindingofthesubstrate

• Theeffectcannotbereversedbyaddingmoresubstrate,buteventuallyreversedbywashingtheinhibitoraway.

Mixed or Non-Competitive Inhibition• The inhibitor can bind to both free enzyme and the ES

complex• The affinity of the inhibitor to the two complexes might be

different– If binding of inhibitor changes the affinity for the substrate, Km will be changed and called mixed inhibition

– If only Vmax affected called Non-competitive inhibitor

Non-Competitive

Change of Lineweaver – Burk graph by enzyme inhibition

Mixed Inhibition

• The result will be decrease in Vmax and either an increase or decrease in Km

• The effect of an non-competitive inhibitor can only be partially overcome by high concentrations of the substrate

Mixed Inhibition Uncompetitive Inhibition• An uncompetitive

inhibitor binds to the enzyme substrate complex but not to free enzyme

• The result is a decrease in Vmaxand Km

• The effect of an uncompetitive inhibitor can not be overcome by high concentrations of the substrate

9/16/16

18

Uncompetitive Inhibition Uncompetitive

IrreversibleInhibitors• Anirreversibleinhibitor destroysenzymeactivity,usuallyby

bondingwithside-chaingroupsintheactivesite

Reactionoftheirreversibleinhibitordiisopropylfluorophosphate (DFP)withaserineprotease;DFPisairreversiblecholinesteraseinhibitor:

Diisopropyl fluorophosphate isaverypotentneurotoxin.ItsLD50inratsis1.3mg/kg.Itcombineswiththeaminoacidserineattheactivesiteoftheenzymeacetylcholinesterase, anenzymethatdeactivatestheneurotransmitteracetylcholine.Neurotransmittersareneededtocontinuethepassageofnerveimpulsesfromoneneurontoanother(ortothe(striated)muscle)acrossthesynapse.Oncetheimpulsehasbeentransmitted,acetylcholinesterase functionstodeactivatetheacetylcholinealmostimmediatelybybreakingitdown.Iftheenzymeisinhibited,acetylcholineaccumulatesandnerveimpulsescannotbestopped,causingprolongedmusclecontraction.Paralysisoccursanddeathmayresultsincetherespiratorymusclesareaffected.

SummaryofEnzymes-II• Enzymesaremostlyproteins• Theyarehighlyspecifictoareaction• Theycatalyzemanyreactionsincludingbreakingdownnutrients,storingand

releasingenergy,creatingnewmolecules,andcoordinatingbiologicalreactions.

• Enzymesuseanactivesite,butcanbeaffectedbybondingatotherareasoftheenzyme.

• Someenzymesneedspecialmoleculescalledcofactorstocarryouttheirfunction.

• Cofactorsthatareorganicinnaturearecalledcoenzymes.• Coenzymesareusuallyderivedfromvitamins.• Coenzymestransferfunctionalgroupsfortheenzymetheyworkwith.• EnzymesareaffectedbychangesinpH,temperature,theamountof

substrate,cofactorsandvarioustypesofinhibitors.