Chapter2

EnzymePropertiesofenzymesStructuralfeaturesofenzymesMechanismofenzyme-catalyzedreactionsKineticsofenzyme-catalyzedreactionsInhibitionofenzymesRegulationofenzymesClinicalapplicationsofenzymesNomenclatureContentsSection1

PropertiesofEnzymesA+B→C+D§1.1GeneralConceptsspontaneousreactiononlyifDGisnegative.atequilibriumifDGiszero.spontaneouslyimpossibleifDGispositive.CatalyzedreactionsReactantsneedtopassovertheenergybarrier,

G+.Catalystsreducetheactivationenergyandassistthereactantstopassovertheactivationenergy.FragilestructuresofthelivingsystemsLowkineticenergyofthereactantsLowconcentrationofthereactantsToxicityofcatalystsComplexityofthebiologicalsystemsChemicalreactionsinlivingsystemsarequitedifferentfromthatintheindustrialsituationsbecauseofNeedforspecialcatalysts

Enzymesarecatalyststhathavespecialcharacteristicstofacilitatethebiochemicalreactionsinthebiologicalsystems.Enzyme-catalyzedreactionstakeplaceusuallyunderrelativelymildconditions.

Enzymes§1.2CharacteristicsEnzyme-catalyzedreactionshavethefollowingcharacteristicsincomparisonwiththegeneralcatalyzedreactions:commonfeatures:2“do”and2“don’t”uniquefeatures:3“high”Donotconsumethemselves:nochangesinquantityandqualitybeforeandafterthereactions.Donotchangetheequilibriumpoints:onlyenhancethereactionrates.ApplytothethermodynamicallyallowablereactionsReducetheactivationenergyCommonfeaturesEnzyme-catalyzedreactionshaveveryhighcatalyticefficiency.Enzymeshaveahighdegreeofspecificityfortheirsubstrates.Enzymaticactivitiesarehighly

regulatedinresponsetotheexternalchanges.UniquefeaturesCatalystActivationenergy(cal/M)Nocatalyst18,000Normalcatalyst11,700Hydrogenperoxidase2,000§1.3.aHighefficiencyAcceleratedreactionratesenzymeNon-enzymaticrateconstant(knins-1)enzymaticrateconstant(knins-1)acceleratedreactionrateCarbonicanhydrase10-11068x106Chymotrypsin

4x10-94x10-210-2107Lysozyme3x10-95x10-12x108Triosephosphateisomerase

4x10-64x103109Urease3x10-103x1041014Mandelate

racemase3x10-105x1021.7x1015Alkalinephosphatase10-151021017AbsolutespecificityRelativespecificityStereospecificity

§1.3.bHighspecificityUnlikeconventionalcatalysts,enzymesdemonstratetheabilitytodistinguishdifferentsubstrates.Therearethreetypesofsubstratespecificities.AbsolutespecificityEnzymescanrecognizeonlyonetypeofsubstrateandimplementtheircatalyticfunctions.Enzymescatalyzeoneclassofsubstratesoronekindofchemicalbondinthesametype.RelativespecificityStereospecificityTheenzymecanactononlyoneformofisomersofthesubstrates.LactatedehydrogenasecanrecognizeonlytheL-formbuttheD-formlactate.

Enzyme-catalyzedreactionscanberegulatedinresponsetotheexternalstimuli,satisfyingtheneedsofbiologicalprocesses.Regulationscanbeaccomplishedthroughvaryingtheenzymequantity,adjustingtheenzymaticactivity,orchangingthesubstrateconcentration.

§1.3.cHighregulationSection2

ComponentsofEnzymesAlmostalltheenzymesareproteinshavingwelldefinedstructures.Somefunctionalgroupsarecloseenoughinspacetoformaportioncalledtheactivecenter.Activecenterslooklikeacleftoracrevice.Activecentersarehydrophobic.§2.1ActiveCenterLysozymeResidues(colored)intheactivesitecomefromdifferentpartsofthepolypeptidechain.Bindinggroup:toassociatewiththereactantstoformanenzyme-substratecomplexCatalyticgroup:tocatalyzethereactionsandconvertsubstratesintoproductsTwoessentialgroups

Theactivecenterhastwoessentialgroupsingeneral.ActivecentersSimpleenzymes:consistsofonlyonepeptidechainConjugatedenzymes:

holoenzyme=apoenzyme+cofactor (protein) (non-protein)Cofactors:metalions;smallorganicmolecules§2.2MolecularComponentsMetalionsMetal-activatedenzyme:

ionsnecessarybut

looselybound.Oftenfoundinmetal-activatedenzyme.Metalloenzymes:Ionstightlybound.Particularlyintheactivecenter,transferelectrons,bridgetheenzymeandsubstrates,stabilizeenzymeconformation,neutralizetheanions.SmallsizeandchemicallystablecompoundsTransferringelectrons,protonsandothergroupsVitamin-likeorvitamin-containingmoleculeOrganiccompoundsLooselybindtoapoenzyme.Beabletobeseparatedwithdialysis.AcceptingH+orgroupandleavingtotransferittoothers,orviseversa.CoenzymesProstheticgroupsTightlybindthrougheithercovalentormanynon-covalentinteractions.Remainedboundtotheapoenzymeduringthecourseofreaction.Section3

MechanismofEnzyme-CatalyzedReactionsProximityandorientationarrangementMultielementcatalysisSurfaceeffectTounderstandthemoleculardetailsofthecatalyzedreaction.

Lock-and-keymodelBothEandSarerigidandfixed,sotheymustbecomplementarytoeachotherperfectlyinordertohavearightmatch.Induced-fitmodelThebindinginducesconformationalchangesofbothEandS,forcingthemtogetaperfectmatch.HexokinasecatalyzingglycolysisHexokinase,thefirstenzymeintheglycolysispathway,convertedglucosetoglucose-6-phosphatewithconsumingoneATPmolecule.Twostructuraldomainsareconnectedbyahinge.Uponbindingofaglucosemolecule,domainsclose,shieldingtheactivesiteforwater.

InducedstructuralchangesSection4

KineticsofEnzyme-CatalyzedReactions§4.1ReactionrateThereactionrateisdefinedastheproductformationperunittime.

Theslopeofproductconcentration([P])againstthetimeinagraphicrepresentationiscalledinitialvelocity.Itisofrectangularhyperbolicshape.InitialvelocityReactionvelocitycurveIntermediatestateForminganenzyme-substratecomplex,atransitionstate,isakeystepinthecatalyticreaction.initial intermediate finalK1=rateconstantforESformationK2=rateconstantforESdissociationK3=rateconstantfortheproductreleasedfromtheactivesiteRateconstantsThemathematicalexpressionoftheproductformationwithrespecttotheexperimentalparametersMichaelis-Mentenequationdescribestherelationshipbetweenthereactionrateandsubstrateconcentration[S].§4.2Michaelis-MentenEquation[S]>>[E],changesof[S]isnegligible.K2isnegligiblecomparedwithK1.Steady-state:therateofE-Scomplexformationisequaltotherateofitsdisassociation(backwardE+SandforwardtoE+P)AssumptionsDescribingahyperboliccurve.KmisacharacteristicconstantofE

[S]<<Km時，v∝[S][S]>>Km時，v≈Vmaxthesubstrateconcentrationatwhichenzyme-catalyzedreactionproceedsatone-halfofitsmaximum

velocityKmisindependentof[E].ItisdeterminedbythestructureofE,thesubstrateandenvironmentalconditions(pH,T,ionicstrength,…)SignificanceofKmKmisacharacteristicconstantofE.ThevalueofKmquantifiestheaffinityoftheenzymeandthesubstrateundertheconditionofK3<<K2.ThelargertheKm，thesmallertheaffinity.Kmfor

selectedenzymesEnzymeSubstratekm

CatalaseH2O225

HexokinaseATP0.4D-Glucose0.05D-Fructose1.5CarbonicanhydraseHCO3-9

Chemotrypsin

Glycyltyrosinylglycine108N-Benzoyltyrosinamide2.5

GalactosidaseD-Lactose4

Threonine

dehydrataseL-Threonine5ThereactionvelocityofanenzymaticreactionwhenthebindingsitesofEaresaturatedwithsubstrates.Itisproportionalto[E].SignificanceofVmaxVmaxisthereactionratewhentheenzymesaresaturated,andisindependentoftheenzymeconcentration.Thenumberoftheproductsconvertedinaunittimebyoneenzymemoleculewhichissaturated.Turnovernumberk3=Vmax/[E]TodetermineKmandVmaxToidentifythereversiblerepressionLineweaver-BurkplotDouble-reciprocalplotSubstrateconcentrationEnzymeconcentrationTemperaturepHInhibitorsActivators§4.3Factorsaffectingenzyme-catalyzedreaction§4.3.aEffectofsubstrateHasbeendescribedalready.[E]affectstherateofenzyme-catalyzedreactions[S]isheldconstant.When[S]>>[E],V≈[E]§4.3.bEffectofenzyme§4.3.cEffectoftemperatureOptimaltemperature(To)isthecharacteristicTatwhichanenzymehasthemaximalcatalyticpower.35~40

Cforwarmbloodspecies.Reactionratesincreaseby2foldsforevery10

Crise.

HigherTwilldenaturetheenzyme.§4.3.d

EffectofpHOptimalpHisthecharacteristicpHatwhichtheenzymehasthemaximalcatalyticpower.pH7.0issuitableformostenzymes.Particularexamples: pH(pepsin)=1.8 pH(trypsin)=7.8Section5

InhibitionofEnzymeInhibitorsarecertainmoleculesthatcandecreasethecatalyticrateofanenzyme-catalyzedreaction.Inhibitorscanbenormalbodymetabolitesandforeignsubstances(drugsandtoxins).§5.1InhibitorsTheinhibitionprocesscanbeeitherirreversibleorreversible.Theinhibitioncanbecompetitive,non-competitive,orun-competitive.InhibitionprocessesInhibitorsarecovalentlyboundtotheessentialgroupsofenzymes.Inhibitorscannotberemovedwithsimpledialysisorsuper-filtration.Bindingcancauseapartiallossorcompletelossoftheenzymaticactivity.

§5.2IrreversibleinhibitionAcetylcholineaccumulationwillcauseexcitementoftheparasympatheticsystem:omitting,sweating,muscletrembling,pupilcontractionPesticidepoisoningHeavymetalcontainingchemicalsbindtothe–SHgroupstoinactivatetheenzymes.HeavymetalpoisoningInhibitorsareboundtoenzymesnon-covalently.Thereversibleinhibitionischaracterizedbyanequilibriumbetweenfreeenzymesandinhibitor-boundenzymes.§5.3Reversibleinhibition§5.3.aCompetitiveinhibitionCompetitiveinhibitorssharethestructuralsimilaritieswiththatofsubstrates.Competitiveinhibitorscompetefortheactivesiteswiththenormalsubstrates.Inhibitiondependsontheaffinityofenzymesandtheratioof[E]to[S].Lineweaver-BurkplotAs[S]increases,theeffectofinhibitorsisreduced,leadingtonochangeinVmax.Duetothecompetitionforthebindingsites,Kmrises,equivalenttothereductionoftheaffinity.InhibitionfeaturesFH4(tetrahydrofolate)isacoenzymeinthenucleicacidsynthesis,andFH2(dihydrofolate)istheprecursorofFH4.Bacteriacannotabsorbfolicaciddirectlyfromenvironment.Bacteriausep-amino-benzoicacid(PABA),GluanddihydropterintosynthesizeFH2.SulfanilamidederivativessharethestructuralsimilaritywithPABA,blockingtheFH2formationasacompetitiveinhibitor.Example-1:competitiveinhibitorExample-2:competitiveinhibitor§5.3.bNon-competitiveinhibitionInhibitorsbindtoothersitesratherthantheactivesitesonthefreeenzymesortheE-Scomplexes.TheE-Icomplexformationdoesnotaffectthebindingofsubstrates.TheE-I-Scomplexesdonotproceedtoformproducts.Reducingthe[E-S]Vmax↓;unchangedKm.§5.3.cUncompetitiveinhibitionUncompetitiveinhibitorsbindonlytotheenzyme-substratecomplexes.TheE-I-Scomplexesdonotproceedtoformproducts.TheE-I-Scomplexesdonotbackwardtothesubstratesandenzymes.ThisinhibitionhastheeffectsonreducingbothVmaxandKm.Commonlyinthemultiplesubstratereactions.typebindingtargetKmVmaxCompetitiveEonly

=NoncompetitiveEorES=

UncompetitiveESonly

SummaryofinhibitionActivatorsarethecompoundswhichbindtoanenzymeoranenzyme-substratecomplextoenhancetheenzymaticactivitywithoutbeingmodifiedbytheenzymes.ActivatorMetalionsessentialactivators:noenzymaticactivitywithoutit Mg2+ofhexokinasenon-essentialactivators:enhancingthecatalyticpower.ActivatorsEnzymaticactivityisameasureofthecapabilityofanenzymeofcatalyzingachemicalreaction.Itdirectlyaffectsthereactionrate.Internationalunit(IU):theamountofenzymerequiredtoconvert1μmolofsubstratetoproductperminuteunderadesignatedcondition.EnzymaticactivityDeterminationoftheenzymaticactivityrequirespropertreatmentofenzymes,excessamountofsubstrate,optimalTandpH,…Onekatal

istheamountofenzymethatconverts1molofsubstratepersecond.IU=16.67×10-9katInadditiontoenzymes,otherchemicalspeciesoftenparticipateinthecatalysis.

Cofactor:chemicalspeciesrequiredbyinactiveapoenzymes(proteinonly)toconvertthemselvestoactiveholoenzymes.§2.2MolecularComponentsCofactorsActivatorions:looselyandreversiblybound,oftenparticipateinthebindingofsubstrates.Metalionsofmetalloenzymes:tightlybound,andfrequentlyparticipatedirectlyincatalyticreactions.EssentialionsTransferelectronLinkageofSandE；KeepconformationofE-ScomplexNeutralizeanionFunctionofmetalionsActasgroup-transferreagentstosupplyactivesiteswithreactivegroupsnotpresentonthesidechainsofaminoacidsCosubstrates:Prostheticgroups:CoenzymesThesubstratesinnature.Theirstructuresarealteredforsubsequentreactions.Shuttlemobilemetabolicgroupsamongdifferentenzyme-catalyzedreactions.CosubstratesSupplytheactivesiteswithreactivegroupsnotpresentonthesidechainsofAAresidues.Canbeeithercovalentlyattachedtoitsapoenzymesorthroughmanynon-covalentinteractions.Remainedboundtotheenzymeduringthecourseofthereaction.ProstheticgroupsMetabolitecoenzymes:theyaresynthesizedfromthecommonmetabolites.severalNTP,ATP(mostabundant),UDP-glucoseVitamin-derivedcoenzymes:theyarederivativesofvitamins,andcanonlybeobtainedfromnutrients.NADandNADP+,FADandFMN,lipidvitamins,…CoenzymesUntilrecently,alltheenzymesareknowntobeproteins.Ribonucleicacidsalsodemonstratethecatalyticability.Ribozymeshavetheabilitytoself-cleave.Theyarehighlyconservative,anindicationofthebiologicalevolutionandtheprimaryenzyme.§2.3RibozymeFamilyofserineprotease§5.3.aCompetitiveinhibition§5.3.bNon-competitiveinhibition§5.3.cUncompetitiveinhibitionSection6

RegulationofEnzymeManybiologicalprocessestakeplaceataspecifictime;ataspecificlocationandataspecificspeed.Thecatalyticcapacityistheproductoftheenzymeconcentrationandtheirintrinsiccatalyticefficiency.Thekeystepofthisprocessistoregulateeithertheenzymaticactivityortheenzymequantity.MaintenanceofanorderedstateinatimelyfashionandwithoutwastingresourcesConservationofenergytoconsumejustenoughnutrientsRapidadjustmentinresponsetoenvironmentalchanges

Reasonsforregulation

Controllinganenzymethatcatalyzestherate-limitingreactionwillregulatetheentiremetabolicpathway,makingthebiosystemcontrolmoreefficient.Ratelimitingreactionisthereactionwhoseratesetbyanenzymewilldictatethewholepathway,namely,theslowestoneorthe“bottleneck”step.ZymogenactivationAllostericregulationCovalentmodification§6.1RegulationofEActivity

Certainproteinsaresynthesizedandsecretedasaninactiveprecursorofanenzyme,calledzymogen.Selectiveproteolysisoftheseprecursorsleadstoconformationalchanges,andactivatestheseenzymes.Itistheconformationalchangesthateitherforman

activesiteoftheenzymeorexposetheactivesitetothesubstrates.§6.1.aZymogenactivationHormones:proinsulinDigestiveproteins:trypsinogen,…Funtionalproteins:factorsofbloodclottingandclotdissolutionConnectivetissueproteins:procollagenWidevarietiesActivationofchymotrypsin

AcascadereactioningeneralToprotectthezymogensfrombeingdigestedToexertfunctioninappropriatetimeandlocationStoreandtransportenzymesFeaturesofzymogen

activationAllostericenzymesarethosewhoseactivitycanbeadjustedbyreversible,non-covalentbindingofaspecificmodulatortotheregulatorysites,specificsitesonthesurfaceofenzymes.Allostericenzymesarenormallycomposedofmultiplesubunitswhichcanbeeitheridenticalordifferent.§6.1.bAllostericregulation

Themultiplesubunitsare catalyticsubunits regulatorysubunitsKineticplotofvversus[S]issigmoidalshape.Demonstratingeitherpositiveornegativecooperativeeffect.

Therearetwoconformationalforms,TandR,whichareinequilibrium.ModulatorsandsubstratescanbindtotheRformonly;theinhibitorscanbindtotheTform.PropertiesofallostericenzymesAllostericcurve

Activationofproteinkinase

C:catalyticportionsR:regulatoryportionsAvarietyofchemicalgroupsonenzymescouldbemodifiedinareversibleandcovalentmanner.Suchmodificationcanleadtothechangesoftheenzymaticactivity.§6.1.cCovalentmodification

phosphorylation-dephosphorylation

adenylation-deadenylationmethylation-demethylationuridylation-deuridylationribosylation-deribosylationacetylation-deacetylationCommonmodifications

PhosphorylationTwoactiveforms(highandlow)CovalentmodificationEnergyneededAmplificationcascadeSomeenzymescanbecontrolledbyallostericandcovalentmodification.FeaturesofcovalentmodificationConstitutiveenzymes(house-keeping):enzymeswhoseconcentrationessentiallyremainsconstantovertimeAdaptiveenzymes:enzymeswhosequantityfluctuateasbodyneedsandwell-regulated.Regulationofenzymequantityisaccomplishedthroughthecontrolofthegenesexpression.§6.2RegulationofEQuantityInducer:substratesorstructurallyrelatedcompoundsthatcaninitiatetheenzymesynthesisRepressor:compoundsthatcancurtail

thesynthesisofenzymesinananabolicpathwayinresponsetotheexcessofanmetaboliteBothareciselements,trans-actingregulatoryproteins,andspecificDNAsequenceslocatedupstreamofgenesControllingthesynthesisEnzymesareimmortal,andhaveawiderangeoflifetime.LDH45-6days,amylase3-5hours.Theydegradeoncenotneededthroughproteolyticdegradation.Thedegradationspeedcanbeinfluencedbythepresenceofligandssuchassubstrates,coenzymes,andmetalions,nutrientsandhormones.ControllingthedegradationLysosomicpathway:

UndertheacidicconditioninlysosomesNoATPrequiredIndiscriminativedigestionDigestingtheinvadingorlonglifetimeproteins

Non-lysosomicpathway:DigesttheproteinsofshortlifetimeLabelingbyubiquitinfollowedbyhydrolysisATPneededDegradationpathwayEnzymes/pathwaysincellularorganelles

organelleEnzyme/metabolicpathwayCytoplasmAminotransferases,peptidases,glycolysis,hexose

monophosphateshunt,fattyacidssynthesis,purineandpyrimidinecatabolismMitochondriaFattyacidoxidation,aminoacidoxidation,Krebscycle,ureasynthesis,electrontransportchainandoxidativephosphorylationNucleusBiosynthesisofDNAandRNAEndoplasmicreticulumProteinbiosynthesis,triacylglycerolandphospholipidssynthesis,steroidsynthesisandreduction,cytochromeP450,esteraseLysosomesLysozyme,phosphatases,phospholipases,proteases,lipases,nucleasesGolgiapparatusGlucose6-phosphatase,5’-nucleotidase,glucosyl-andgalactosyl-transferasePeroxisomesCalatase,urate

oxidase,D-aminoacidoxidase,longchainfattyacidoxidaseSection7

ClinicalApplicationsPlasmaspecificorplasmafunctionalenzymes:Normallypresentintheplasmaandhavespecificfunctions.Highactivitiesinplasmathaninthetissues.Synthesizedinliverandenterthecirculation.Impairmentinliverfunctionorgeneticdisorderleadstoafallintheactivities.§7.1FundamentalConceptsNon-plasmaspecificorplasmanon-functionalenzymes:eithertotallyabsentoratalowconcentrationinplasmaInthenormalturnoverofcells,intracellularenzymesarereleasedintobloodstream.AnorgandamagedbydiseasesmayelevatethoseenzymesAgroupofenzymesthatcatalyzethesamereactionbutdifferfromeachotherintheirstructure,substrateaffinity,Vmax,andregulatoryproperties.Duetogenedifferentiation:thedifferentgeneproductsordifferentpeptidesofthesamegenePresentindifferenttissuesofthesamesystem,orsubcellularcomponentsofthesamecell§7.2Isoenzyme

Synthesizedfromdifferentgenes(malate

dehydrogenaseincytosolversusinmitochondria)Oligomericformsofmorethanonetypeofsubunits(lactatedehydrogenase)Differentcarbohydratecontent(alkalinephosphatase)Reasonsforisoenzyme

5isoenzymes,LDH1–LDH5TetramerMsubunits(Mformuscle),basicHsubunits(Hforheart),acidicDifferentcatalyticactivitiesUsedasthemarkerfordiseasediagnosisLactatedehydrogenase(LDH)LDH1(H4)inheartmuscleconvertslactatetopyruvate,andthentoacetylCoA.LDH5(M4)inskeletalmuscleconvertspyruvatetolactate.3isoenzymes,BB,BM,MMDimericform:M(muscle)orB(brain)CPK2isundetectable(<2%)inserumforhealthyindividuals,andelevatedto20%inthefirst6-18hrsaftermyocardialinfarction.Usedasaearliestreliableindicatorofmyocardialinfarction.Creatine

phosphokinaseUsefulness:EnzymeassaysprovideimportantinformationconcerningthepresenceandseverityofdiseasesProvideameansofmonitoringthepatient’sresponseapproaches:MeasuringtheenzymaticactivitiesdirectlyUsedasagentstomonitorthepresenceofsubstrates§7.3DiagnosticApplicationsEnzymaticactivitychangesElectrophoresisofLDH

Serumenzymes(elevated)DiseasesAmylaseAcutepancreatitisSerumglutamatepyruvate

transaminase(SGPT)Liverdiseases(hepatitis)Serumglutamateoxaloacetate

transaminase(SGOT)Heartattack(myocardialinfarction)AlkalinephosphataseRickets,obstructivejaundiceAcidphosphataseCancerofprostateglandLactatedehydrogenase(LDH)Heartattack,liverdiseasesγ-glutamyl

transpeptidase(GGT)Alcoho