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9/16/16 Enzymesareusedalloveryourbody! Biochemistry 3.1)Enzymes-I Introductiontoenzymestructureand function,andfactorsinvolvingtheir actionsandpathways Prof. Dr. Klaus Heese EnzymesasBiologicalCatalysts Whatisanenzyme? • Almostallenzymesareproteinsthatactas biologicalcatalysts. • Acatalystspeedsupchemicalreactions.Enzymes speedupbiologicalchemicalreactions. • Enzymesarehighlyspecifictoatypeofreaction. • Enzymesmustmaintaintheirspecificshapeinorder tofunction.Anyalterationintheprimary, secondary,tertiary,orquaternaryformsofthe enzymearedetrimental. CO2 +H2O--->H2CO3 • Enzymes areproteins thatincreasetherate ofreactionby loweringtheenergy ofactivation • Theycatalyzenearly DG allthechemical reactionstaking placeinthecellsof thebody • Enzymeshaveunique three-dimensional •Increasedreactionratessometimes106 to1012 shapesthatfitthe increase EnzymesdonotchangeDG,justthereactionrates. shapesofreactants (substrates) (Enzymesalterrates,notequilibria) •Milderreactionconditions •Greatreactionspecificity Enzymescatalyzebystabilizingtransitionstates • FreeenergyGofachemical reactioncanbeplottedovertime • Favorablereactionshavea positivedifference(DG)infree energybetweenthesubstrate andproduct • Thefreeenergyofactivationfor thetransitionstatelimitsthe progressofthereaction • Enzymesactbyreducingthefree energyofthetransitionstate Functionofenzymes Enzymeshavemanyjobs.They: • Breakdownnutrientsintouseablemolecules. • Storeandreleaseenergy(ATP). • Createlargermoleculesfromsmallerones. • Coordinatebiologicalreactionsbetweendifferent systemsinanorganism. 1 9/16/16 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 • AccordingtotheInternationalunionof Biochemistryanenzymenamehastwoparts: - Firstpartisthenameofthesubstratesforthe enzyme. - Secondpartisthetypeofreactioncatalyzedby theenzyme.Thispartendswiththesuffix“ase”. Example:Lactatedehydrogenase ECnumbers • ECnumbersarefourdigits,forexamplea.b.c.d, where“a” istheclass,“b” isthesubclass,“c” is thesub-subclass,and“d” isthesub-sub-subclass. The“b” and“c” digitsdescribethereaction, whilethe“d” digitisusedtodistinguishbetween differentenzymesofthesamefunctionbasedon theactualsubstrateinthereaction. • Example:forAlcohol:NAD +oxidoreductase:EC numberis1.1.1.1 ECnumber Enzymesareclassifiedintosixdifferentgroups accordingtothereactionbeingcatalyzed.The nomenclaturewasdeterminedbytheEnzyme Commissionin1961(withthelatestupdate havingoccurredin1992),henceallenzymes areassignedan“EC” number.The classificationdoesnottakeintoaccount aminoacidsequence(ie,homology),protein structure,orchemicalmechanism. ClassificationofEnzymes TheSixClasses Enzymesareclassifiedaccordingtothetypeofreactiontheycatalyze: • • • • • • EC1.Oxidoreductases EC2.Transferases EC3.Hydrolases EC4.Lyases EC5.Isomerases EC6.Ligases Additionalinformationonthesubclasses,thesub-subclassesandsub-subsubclasses(ie,fullenzymeclassificationandnames)canbefoundatthe referencedweblink. • FromtheWebversion, http://www.chem.qmul.ac.uk/iubmb/enzyme/index.html 2 9/16/16 EC1.Oxidoreductases • EC1.Oxidoreductases:catalyzethetransferof hydrogenoroxygenatomsorelectronsfrom onesubstratetoanother,alsocalledoxidases, dehydrogenases,orreductases.Notethat sincetheseare‘redox’ reactions,anelectron EC2.Transferases • EC2.Transferases – catalyzegrouptransfer reactions,excludingoxidoreductases (which transferhydrogenoroxygenandareEC1). Theseareofthegeneralform: • A-X+B↔BX+A donor/acceptorisalsorequiredtocomplete thereaction. EC3.Hydrolases EC4.Lyases • EC3.Hydrolases– catalyzehydrolytic reactions.Includeslipases,esterases, nitrilases,peptidases/proteases.Theseareof thegeneralform: • A-X+H2O↔X-OH+HA • EC4.Lyases – catalyzenon-hydrolytic(coveredin EC3)removaloffunctionalgroupsfrom substrates,oftencreatingadoublebondinthe product;orthereversereaction,i.e. additionof functiongroupsacrossadoublebond. • A-B→A=B+X-Y XY • Includesdecarboxylasesandaldolases inthe removaldirection,andsynthasesintheaddition direction. EC5.Isomerases EC6.Ligases • EC5.Isomerases – catalyzesisomerization reactions,includingracemizations andcistransisomerizations. • EC6.Ligases-- catalyzesthesynthesisof various(mostlyC-X)bonds,coupledwiththe breakdownofenergy-containingsubstrates, usuallyATP 3 9/16/16 Oxidoreductases,Transferases andHydrolases Enzymeactionoverview • Enzymesarelargemoleculesthathaveasmallsection dedicatedtoaspecificreaction.Thissectioniscalled theactive site. • Theactivesitereactswiththedesiredsubstance,called thesubstrate. • Thesubstratemayneedanenvironmentdifferentfrom themostlyneutralenvironmentofthecellinorderto react.Thus,theactivesitecanbemoreacidicorbasic, orprovideopportunitiesfordifferenttypesofbonding tooccur,dependingonwhattypeofsidechainsare presentontheaminoacidsoftheactivesite. Lyases,Isomerases andLigases ActiveSiteofanEnzyme • Theactivesite isaregion withinanenzymethatfits theshapeofsubstrate molecules • Aminoacidside-chains aligntobindthesubstrate throughH-bonding,saltbridges,hydrophobic interactions,etc. • Productsarereleased whenthereactionis complete(theynolonger fitwellintheactivesite) EnzymeSpecificity Enzyme-CatalyzedReactions • Enzymeshavevaryingdegreesofspecificity forsubstrates • Enzymesmayrecognizeandcatalyze: - asinglesubstrate - agroupofsimilarsubstrates - aparticulartypeofbond • Whenasubstrate(S)fitsproperlyinanactivesite,an enzyme-substrate(ES)complex isformed: E +S D ES • WithintheactivesiteoftheES complex,thereaction occurstoconvertsubstratetoproduct(P): ES ® E +P • Theproductsarethenreleased,allowinganother substratemoleculetobindtheenzyme - thiscyclecanberepeatedmillions(orevenmore)times perminute • Theoverallreactionfortheconversionofsubstrateto productcanbewrittenasfollows: E +S D ES ® E +P 4 9/16/16 ExampleofanEnzyme-CatalyzedReaction • Thereactionforthesucrase catalyzedhydrolysisofsucroseto glucoseandfructosecanbewrittenasfollows: E +S D ES ® E +P1 +P2 whereE =sucrase,S =sucrose,P1 =glucoseandP2 =fructose Isoenzymes • Isoenzymes aredifferentformsofanenzymethatcatalyze thesamereactionindifferenttissuesinthebody - theyhaveslightvariationsintheaminoacidsequences ofthesubunitsoftheirquaternarystructure • Forexample,lactatedehydrogenase(LDH),whichconverts lactatetopyruvate,consistsoffiveisoenzymes DiagnosticEnzymes Factorsthataffectenzymeaction • Thelevelsofdiagnosticenzymes inthebloodcanbeused todeterminetheamountofdamageinspecifictissues DenaturingEnzymes (=conformationchange=changeofproteins’3Dstructure) • Whenanenzymeisdenatureditisdamaged. • Denaturingchangestheshape. • Withoutthecorrectshapeenzymeswon’t functionproperly. • HOWareenzymesdenatured? – Temperature – pH Factorsthataffectenzymeaction Enzymesaremostlyaffectedbychangesintemperature andpH. TemperatureandEnzymeActivity • Enzymes aremostactiveatanoptimumtemperature(usually 37°Cinhumans) • Theyshowlittleactivityatlowtemperatures • Activityislostathightemperaturesasdenaturationoccurs • Toohighofatemperaturewilldenaturetheprotein components,renderingtheenzymeuseless. • pHrangesoutsideoftheoptimalrangewillprotonate ordeprotonate thesidechainsoftheaminoacids involvedintheenzyme’sfunctionwhichmaymake themincapableofcatalyzingareaction. 5 9/16/16 pHandEnzymeActivity • Enzymes aremostactiveatoptimumpH • Aminoacidswithacidicorbasicside-chainshavethe properchargeswhenthepHisoptimum • ActivityislostatloworhighpHastertiarystructureis disrupted Factorsthataffectenzymeaction Enzymesarealsoaffectedbytheconcentrationofsubstrate, cofactorsandinhibitors,aswellasallostericregulationand feedbackinhibition. OptimumpHforSelectedEnzymes • MostenzymesofthebodyhaveanoptimumpHofabout7.4 • However,incertainorgans,enzymesoperateatlowerand higheroptimumpHvalues EnzymeConcentrationandReactionRate • Therateofreactionincreasesasenzymeconcentration increases(atconstantsubstrateconcentration) • Athigherenzymeconcentrations,moreenzymesare availabletocatalyzethereaction(morereactionsatonce) • Thereisalinearrelationshipbetweenreactionrateand enzymeconcentration(atconstantsubstrateconcentration) • Theconcentrationof substratewilldictatehowmanyenzymes canreact.Toomuchsubstratewillslowtheprocessuntilmore enzymecanbemade. • Theavailabilityofcofactors alsodictateenzymeaction.Toolittle cofactorswillslowenzymeactionuntilmorecofactorsareadded. • Aninfluxofcompetitiveornon-competitiveinhibitorswillnot necessarilyslowtheenzymeprocess,butwillslowtheamountof desiredproduct. SubstrateConcentrationandReactionRate • Therateofreactionincreasesassubstrateconcentration increases(atconstantenzymeconcentration) • Maximumactivity occurswhentheenzymeissaturated (whenallenzymesarebindingsubstrate) • Therelationshipbetweenreactionrateandsubstrate concentrationisexponential,andasymptotes(levelsoff) whentheenzymeissaturated Enzymeactiontheories • LockandKey: Thistheory,postulatedby EmilFischerin1894,proposedthatan enzymeis“structurallycomplementary totheirsubstrates” andthusfittogether perfectlylikealockandkey.Thistheory formedthebasisofmostoftheideasof howenzymeswork,butisnotcompletely correct. 6 9/16/16 Enzymeactiontheories Lock-and-KeyModel • Inthelock-and-keymodel ofenzymeaction: - theactivesitehasarigidshape - onlysubstrateswiththematchingshapecanfit - thesubstrateisakeythatfitsthelockoftheactivesite • Thisisanoldermodel,however,anddoesnotworkforall enzymes Enzymeactiontheories InducedFitModel • Intheinduced-fitmodel ofenzymeaction: - theactivesiteisflexible,notrigid - theshapesoftheenzyme,activesite,andsubstrateadjust tomaximizethefit,whichimprovescatalysis - thereisagreaterrangeofsubstratespecificity • Thismodelismoreconsistentwithawiderrangeofenzymes Enzymecofactors • Acofactor isasubstance,thatisnotaprotein,thatmust bindtotheenzymeinorderfortheenzymetowork. • metalionsascofactors-- Zn2+,Fe2+,Cu2+,others • Acofactorcanbeoforganicorigin.Anorganiccofactoris calledacoenzyme. Enzymeactiontheories • InducedFit: Anenzymethatisperfectlycomplementarytoits substratewouldactuallynotmakeagoodenzymebecausethe reactionhasnoroomtoproceedtothetransitionstateofthe reaction. Togotocompletion,areactionmustgothroughthe transitionstate.Inthelockandkeytheory,thesubstrateorthe enzymecannotchangeconformationstothetransitionstate. Therefore,enzymesmustactuallybecomplementarytothe transitionstate sothereactionmayproceed.Thisideawas researchedbyHaldanein1930,andLinusPaulingin1946.This idealedtheInducedFittheory,postulatedbyDanielKoshland in1958,wheretheenzymeitself canchangeconformationsto facilitatethetransitionstateofthesubstrate.Thischangein conformationoftheenzymeallowsthenecessaryfunctional groupsattheactivesitetomoveclosertothesubstrate, enhancingtheefficiencyofthereaction. InducedFitModel • Enzymescanformtotheshapeofits substrate. Enzymecofactorscont. • Anenzymethatisbondedtoitscofactoriscalleda holoenzyme. • Anenzymethatrequiresacofactor,butisnotbondedto thecofactoriscalledanapoenzyme.Apoenzymes arenot activeuntiltheyarecomplexed withtheappropriate cofactor. • Cofactorsarenotpermanentlybonded.Permanently bondedcofactorsarecalledprostheticgroups. 7 9/16/16 Coenzymes Commoncoenzymes Coenzymes:smallermoleculesthataidinenzymechemistry. Enzymescan: a.Carryoutacid-basereactions b.Transientcovalentbonds c.Charge-chargeinteractions Enzymescannotdo: d.Oxidation- Reduction(=Redox)reactions e.Carbongrouptransfers Manycoenzymesarederivedfromvitamins: • NAD+ (nicotinamide adeninedinucleotide); derived fromniacin(B3 ). • CoenzymeA(CoA);derivedfrompantothenicacid (B5 ). • FAD(flavin adeninedinucleotide);derivedfrom riboflavin(B2 ). Prostheticgroup:permanentlyassociatedwithanenzyme ortransientlyassociated. Holoenzyme:catalyticallyactiveenzymewithcofactor. Apoenzyme: Enzymewithoutitscofactor. Commom Coenzymes Coenzyme Biotin VitaminsareCoenzymeprecursors Reactionmediated Carboxylation Vitamin Coenzyme DeficiencyDisease Biotin Biocytin notobserved Cobalamin (B12) Cobalamin Perniciousanemia Cobalamin (B12) Alkylationtransfers CoenzymeA Acyltransfers Flavin Oxidation-Reduction Lipoic acid Acyltransfers Folicacid tetrahydrofolate Neuraltubedefects Megaloblastic anemia Nicotinamide Oxidation-Reduction Nicotinamide Nicotinamide Pellagra Pyridoxal Phosphate Aminogrouptransfers Pantothenate CoenzymeA Notobserved Tetrahydrofolate One-carbongrouptransfers Thiaminepyrophosphate Aldehydetransfer Thesearewatersolublevitamins.TheFatsolublevitaminsarevitaminsAand D. Humanscannotsynthesizetheseandrelayontheirpresenceinourdiets. Thosewhohaveanunbalanceddietmaynotbereceivingasufficientsupply. Niacin(niacinamide)deficiencyleadstopellagracharacterizedbydiarrhea, dermatitisanddementia.PellagrawasendemicisSouthernUnitedStatesinthe early20thcentury.Niacincanbesynthesizedfromtheessentialaminoacid, tryptophan.Acorndietprevalentatthetimerestrictedtheabsorptionof tryptophancausingadeficiency.Treatmentofcornwithbasecouldreleasethe tryptophan(MexicanIndianstreatedcornwithCa(OH)2 beforemakingtortillas!) Pyridoxine(B6) Pyridoxal phosphate Riboflavin(B2) Thiamine(B1) Notobserved Flavin Thiaminepyrophosphate Notobserved Beriberi Commoncoenzymes Coenzymescanbederivedfromsourcesotherthanvitamins: • ATP(adenosinetriphosphate);derivedfromNADHfrom carbohydratesconsumed.(ATPcanalsoactas neurotransmitter) • CTP(Cytidine triphosphate);derivedfromglutamateand carbamoylphosphate. • PAPS(3'-Phosphoadenosine-5'-phosphosulfate); derivedfrom adenosine5'-phosphosulfate(APS)andsulfateion. 8 9/16/16 Coenzymereactions • Coenzymeshelptotransferafunctionalgrouptoamolecule. Fattyacidsynthesis CoenzymeAisconvertedtoacetyl-CoenzymeA enzymeispyruvatedehydrogenase • Forexample,coenzymeA(CoA)isconvertedtoacetyl-CoAin themitochondriausingpyruvateandNAD+. • Acetyl-CoAcanthenbeusedtotransferanacetyl group (CH3CO)toaidinfattyacidsynthesis. Factorsthataffectenzymeaction • Enzymesthatcanbeactivatedwillbeaffectedbythe amountofactivatororinhibitorattachedtoits allostericsite.Anabundanceofanallostericactivator willconvertmoreenzymestotheactiveformcreating moreproduct. • Enzymesthatarepartofametabolicpathwaymaybe inhibitedbytheveryproducttheycreate.Thisis calledfeedbackinhibition.Theamountofproduct generatedwilldictatethenumberofenzymesusedor activatedinthatspecificprocess. EnzymeActivators • Chemicalsthathelptheenzymework. • Activatorsincreasetheenzymereactionrate. Enzymeactivityandinhibition • The“normal” wayanenzymefunctionsiswhenthespecific substratebindstotheactivesiteandcreatestheproducts. • Asimilarsubstratecanalsobondtotheactivesitecovalently andirreversibly.Thispreventstheenzymefromfunctioning. Irreversibleinhibition. • Asimilarsubstratecanbindtotheactivesite,notpermanently, andpreventsthedesiredsubstratefromenteringtheactivesite. Thischangestheproductsandfunctioningoftheenzyme.Thisis calledcompetitiveinhibition. • Amoleculecanbondtoanotherpartoftheenzymeandcausea changeinconformation.Thischangecausestheactivesiteto changeshapeaswell.Thischangeinshapepreventsthedesired substratefromenteringtheactivesite.Thisiscallednoncompetitiveinhibition. EnzymeInhibitors • Chemicalsthatpreventtheenzymefromworking. • Inhibitorsdecreasetheenzymereactionrate. Active Site Activator X Binding Site Substrate 9 9/16/16 EnzymeInhibitors • Inhibitors(I) aremoleculesthatcausealossof enzymeactivity • Theypreventsubstratesfromfittingintothe activesiteoftheenzyme: E+SD ES® E+P E+I D EI ® no Pformed ExampleofaCompetitiveInhibitor • Malonate isacompetitiveinhibitorofsuccinatedehydrogenase - ithasastructurethatissimilartosuccinate - inhibitioncanbereversedbyaddingsuccinate Reversible Inhibitors (CompetitiveInhibition) • Areversibleinhibitor goes onandoff,allowingthe enzymetoregainactivity whentheinhibitorleaves • Acompetitiveinhibitor is reversibleandhasa structurelikethesubstrate - itcompeteswiththe substratefortheactivesite - itseffectisreversedby increasingsubstrate concentration ReversibleInhibitors(Non-competitiveInhibition) • Anon-competitiveinhibitor hasastructurethatis differentthanthatofthe substrate - itbindstoanallostericsite ratherthantotheactivesite - itdistortstheshapeofthe enzyme,whichaltersthe shapeoftheactivesiteand preventsthebindingofthe substrate • Theeffectcannotbereversed byaddingmoresubstrate, buteventuallyreversedby washingtheinhibitoraway. 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 10 9/16/16 Biochemistry SummaryofEnzymes-I • • • • • • • • • Enzymesaremostlyproteins Theyarehighlyspecifictoareaction Theycatalyzemanyreactionsincludingbreakingdownnutrients,storingand releasingenergy,creatingnewmolecules,andcoordinatingbiological reactions. Enzymesuseanactivesite,butcanbeaffectedbybondingatotherareasof theenzyme. Someenzymesneedspecialmoleculescalledcofactorstocarryouttheir function. Cofactorsthatareorganicinnaturearecalledcoenzymes. Coenzymesareusuallyderivedfromvitamins. Coenzymestransferfunctionalgroupsfortheenzymetheyworkwith. EnzymesareaffectedbychangesinpH,temperature,theamountof substrate,cofactorsandinhibitors,aswellastheamountofallosteric inhibitorsandactivatorsandconcentrationofproductsthatcontrolfeedback inhibition. 3.2)Enzymes-II Introductiontoenzymestructureand function,andfactorsinvolvingtheir actionsandpathways Enzyme- Kinetics Prof. Dr. Klaus Heese TheSixClasses • • • • • • EC1.Oxidoreductases EC2.Transferases EC3.Hydrolases EC4.Lyases EC5.Isomerases EC6.Ligases Additionalinformationonthesubclasses,thesub-subclassesandsub-subsubclasses(ie,fullenzymeclassificationandnames)canbefoundatthe referencedweblink. • FromtheWebversion, http://www.chem.qmul.ac.uk/iubmb/enzyme/index.html Some common types of enzymes Hydrolases Polymerases Nucleases Kinases Proteases Phosphatases Synthases ATP-ases Isomerases Oxidoreductases (dehydrogenases) Enzyme Active Sites 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. Active sites: Usually form a cleft or pocket. Substrates are bound by multiple weak interactions. carboxypeptidase pH,T Lock-Key-model Induced-Fit-Model 11 9/16/16 Properties of Enzymes Reaction Thermodynamics • 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. Rate Acceleration How is Transition State Stabilization Achieved? EA – 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 • The enzyme lowers the activation barrier (EA or DG‡) compared to the uncatalyzed reaction. • DGro is not changed ! Enzymes stabilize the transition state, lowering the activation barrier. transition state EA Enzymes stabilize the transition state, lowering the activation barrier. 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 complex Entropy cost is paid during binding Rigid reactant complex ® transition state conversion is entropically OK Enzyme Kinetics Kinetics is the study of the rates of reactions • Enzymes endow cells with the remarkable capacity to exert 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 12 9/16/16 Enzyme Kinetics Enzyme Kinetics Reaction Thermodynamics Enzymatic Equations reminder: Consider: [A] + [B] [C] + [D] E+S [C][D] [A][B] Keq = ES EP E+P There are at least three steps……. 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. Enzyme Kinetics The Michaelis-Menten Equation Enzymatic Equations II Kinetics: Vmax and Km E+S ES k1 k-1 EP ES kcat kcat E+P E+P 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] Km = (k-1+ kcat )/k1 Enzyme Kinetics E+S usually kcat <<< k-1, so Km = k-1/k1 = Kd Measures affinity of enzyme for substrate. [S] K m + [S] • Ideal Rate: Enzyme Kinetics Enzyme Kinetics Km = substrate concentration where V = Vmax/2 The Michaelis-Menten equation Effect of Substrate Concentration • when [S]= KM, the equation reduces to • when [S] << KM, the equation reduces to Vmax = the maximum reaction rate. V = Vmax Important Conclusions of Michaels Menten Kinetics • when [S] >> KM, the equation reduces to Reaction rate (V) varies with substrate concentration. v= Vmax [ S ] Km + S • Deviations due to: – Limitation of measurements – Substrate inhibition – Substrate prep contains inhibitors – Enzyme prep contains inhibitors 13 9/16/16 Effect of Substrate Concentration Vmax and Km V= Enzyme Kinetics Enzyme Kinetics Vmax Vmax [S] --------------KM +[S] Vmax dependsontheamountofenzyme. Lineweaver – Burk Double Reciprocal Plots Enzyme Kinetics Enzyme Kinetics Different substrates, Vmax and Km Km is a property of both enzyme and substrate. = ([S]+Km ) Vmax [S] = Vmax [S] ([S]+Km ) = [S] Vmax [S] Km 1 Vmax [S] + + Km Vmax [S] 1 Vmax 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 Enzyme Kinetics 1 v0 1 v0 = • 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 Significance of Km Determination of Kinetic Parameters v0 • 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 • Km is a constant • Small Km means tight binding; high Km means weak binding • Useful to compare Km for different substrates for one enzyme Hexokinase : D-fructose – 1.5 mM D-glucose – 0.15 mM • Useful to compare Km for a common substrate used by several enzymes Hexokinase: D-glucose – 0.15 mM Glucokinase: D-glucose – 20 mM 14 9/16/16 Enzyme Inhibition Enzyme 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 Inhibitors act in a variety of mechanisms • 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 EnzymeActivators • Chemicalsthathelptheenzymework. • Activatorsincreasetheenzymereactionrate. Enzymeactivityandinhibition • The“normal” wayanenzymefunctionsiswhenthespecific substratebindstotheactivesiteandcreatestheproducts. • Asimilarsubstratecanalsobondtotheactivesitecovalently andirreversibly.Thispreventstheenzymefromfunctioning. Irreversibleinhibition. • Asimilarsubstratecanbindtotheactivesite,notpermanently, andpreventsthedesiredsubstratefromenteringtheactivesite. Thischangestheproductsandfunctioningoftheenzyme.Thisis calledcompetitiveinhibition. • Amoleculecanbondtoanotherpartoftheenzymeandcausea changeinconformation.Thischangecausestheactivesiteto changeshapeaswell.Thischangeinshapepreventsthedesired substratefromenteringtheactivesite.Thisiscallednoncompetitiveinhibition. EnzymeInhibitors • Chemicalsthatpreventtheenzymefromworking. • Inhibitorsdecreasetheenzymereactionrate. Active Site Activator X Binding Site Substrate 15 9/16/16 EnzymeInhibitors • Inhibitors(I) aremoleculesthatcausealossof enzymeactivity • Theypreventsubstratesfromfittingintothe activesiteoftheenzyme: E+SD ES® E+P E+I D EI ® no Pformed ExampleofaCompetitiveInhibitor • Malonate isacompetitiveinhibitorofsuccinatedehydrogenase - ithasastructurethatissimilartosuccinate - inhibitioncanbereversedbyaddingsuccinate ReversibleInhibitors(CompetitiveInhibition) • Areversibleinhibitor goes onandoff,allowingthe enzymetoregainactivity whentheinhibitorleaves • Acompetitiveinhibitor is reversibleandhasa structurelikethesubstrate - itcompeteswiththe substratefortheactivesite - itseffectisreversedby increasingsubstrate concentration Competitive Inhibition • 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 Competitive Inhibition • Unimolecular Reaction • Bimolecular Reaction Change of Lineweaver – Burk graph by enzyme inhibition 16 9/16/16 ReversibleInhibitors(Non-competitiveInhibition) • Anon-competitiveinhibitor hasastructurethatisdifferent thanthatofthesubstrate - itbindstoanallostericsite ratherthantotheactivesite - itdistortstheshapeofthe enzyme,whichaltersthe shapeoftheactivesiteand preventsthebindingofthe substrate • Theeffectcannotbereversed byaddingmoresubstrate,but eventuallyreversedbywashing theinhibitoraway. 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 Mixed Inhibition 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 Uncompetitive Inhibition • An uncompetitive inhibitor binds to the enzyme substrate complex but not to free enzyme • The result is a decrease in Vmax and Km • The effect of an uncompetitive inhibitor can not be overcome by high concentrations of the substrate 17 9/16/16 Uncompetitive Inhibition IrreversibleInhibitors • Anirreversibleinhibitor destroysenzymeactivity,usuallyby bondingwithside-chaingroupsintheactivesite Uncompetitive Reactionoftheirreversibleinhibitor diisopropylfluorophosphate (DFP)witha serineprotease; DFPisairreversiblecholinesteraseinhibitor: Diisopropyl fluorophosphate isaverypotent neurotoxin.ItsLD50inratsis1.3mg/kg.It combineswiththeaminoacidserineatthe activesiteoftheenzyme acetylcholinesterase, anenzymethat deactivatestheneurotransmitter acetylcholine.Neurotransmittersareneeded tocontinuethepassageofnerveimpulses fromoneneurontoanother(ortothe (striated)muscle)acrossthesynapse.Once theimpulsehasbeentransmitted, acetylcholinesterase functionstodeactivate theacetylcholinealmostimmediatelyby breakingitdown.Iftheenzymeisinhibited, acetylcholineaccumulatesandnerve impulsescannotbestopped,causing prolongedmusclecontraction.Paralysis occursanddeathmayresultsincethe respiratorymusclesareaffected. SummaryofEnzymes-II • • • • • • • • • Enzymesaremostlyproteins Theyarehighlyspecifictoareaction Theycatalyzemanyreactionsincludingbreakingdownnutrients,storingand releasingenergy,creatingnewmolecules,andcoordinatingbiological reactions. Enzymesuseanactivesite,butcanbeaffectedbybondingatotherareasof theenzyme. Someenzymesneedspecialmoleculescalledcofactorstocarryouttheir function. Cofactorsthatareorganicinnaturearecalledcoenzymes. Coenzymesareusuallyderivedfromvitamins. Coenzymestransferfunctionalgroupsfortheenzymetheyworkwith. EnzymesareaffectedbychangesinpH,temperature,theamountof substrate,cofactorsandvarioustypesofinhibitors. 18