南京大学-杨荣武生物化学-英文版-第二篇-EnymeVII

Allostery&Cooperativity:KineticConsequences&StructuralBasisAllostery:WebstersDefinitionMainEntry:al·lo·ste·ricPronunciation:a-lO-'ster-ik,-'stir-Function:adjectiveEtymology:all-+stericDate:1962:of,relatingto,orbeingachangeintheshapeandactivityofaprotein(asanenzyme)thatresultsfromcombinationwithanothersubstanceatapointotherthanthechemicallyactivesite-al·lo·ste·ri·cal·ly/-i-k(&-)lE/adverb-al·lo·ste·ry/'a-lO-ster-E,-stir-/nounAllostery:KeyPointBindingofaligandatasitedifferentfromtheactivesitemodulatestheactivity.Thisbehaviorextendswellbeyondthenormaluseoftheword“allostery”whichisoftenusedtodiscusscooperativeinteractions.Themolecularbasisforallosteryprovidesinsightintomanyregulatorymechanisms.Thatwhichhasbeenlearnedbystudyingallostericallyregulatedenzymes/proteinshasprofoundlyinfluencedourunderstandingofcooperativityandenzymeregulationingeneral.AllosteryvsCooperativityThetermsallosteryandcooperativityhavebeenmuddled.Allosterystrictlyreferstoinfluenceofactivitybyadistantsite.Cooperativityindicatesthattheoccupancyofonesiteinamultisubunitenzymeinfluencesthebindingontheothers.Thisisaformofallostery,butisonlyonemanifestationofageneralphenomena.Unfortunatelyallosteryhadbecomealmostexclusivelyassociatedwiththebehaviorofmulti-subunitenzymes.KineticSignatureofCooperativityinEnzymesMultisubunitenzymesthatexhibitcooperativityshowasigmoidalinitialvelocitycurveincontrasttothehyperboliccurveforindependentsubunits.KineticConsequencesofAllostericEffectorsonCooperativeEnzymesThisisthetraditionalviewoffeed-backinhibitionandregulationin“allosteric”enzymes.ClassicExamplesofAllosteryHemoglobin(notanenzyme)ThiswastheoriginoftheTandRstatesAspartatetranscarbamoylase.ExampleoffeedbackinhibitionThesearegreatexamples,buttherearemanyothers.TypesofRegulationHomotrophicresponses:Thisreferstoallostericmodulationofenzymeactivitybysubstratemolecules.Thisnecessarilymustoccurinmultisubunitenzymes.Heterotrophicresponses:Thisreferstoregulationbynon-substratemoleculesorcombinationsofnon-substrateandsubstratemolecules.Allostericregulationcanbepositiveornegative.ModelsforAllostericRegulationConcerted(conceptuallysimpleandofteneffective)Sequential(probablycorrectbutdifficulttoprove)PostulatesoftheConcertedModelAllostericenzymesarecomposedofidenticalprotomersthatoccupyequivalentpositionsintheenzyme.Eachprotomercontainsabindingsiteforeachspecificligand.Eachprotomercanexistinonlyoneoftwostates.TheR(relaxedorhighsubstrateaffinitystate)orT(tautorlowsubstrateaffinitystate).AllprotomersinenzymemoleculemustbeineithertheRorTstate.TheRandTstatesareinequilibriumwitheachother.Thebindingaffinityofaspecificliganddependsontheconformationoftheenzyme(RorT)andnotontheneighboringsiteoccupancy.Hemoglobin•Theconcertedmechanismforallostericcooperativeenzymesarosefromconsiderationofthebindingcurvesofoxygenbindingtohemoglobin.•Thiswasmodeledasatwostatetransitionfromdeoxy(T)tooxy(R)hemoglobin.Deoxy(TState)Oxy(RState)ConcertedModelAssumes2conformationstates:R&TBindingofsubstrateinducesallsubunitstochangetoRstate.NoT-Rhybrids.Allowsfor+cooperativityonly.+cooperativityTstateRstateSimpleVersionoftheConcertedModel•Thisapproximatemodelimpliesthatthesubstratedoesnotbindtotheinactivestate.Thismustbeagrosssimplificationbutitexplainstheprinciple.Interestingly,itaccountsforalotofenzymaticbehavior(itisthesimplestmodel).Itcannotexplainnegativecooperativity.SequentialModelThebindingofsubstrateswitchesconformationofonlythesubunittowhichitisbound.Conformationalchangeinonesubunitmayortheaffinityofothersubunitshaveforthesubstrate.Allowsfor+or-cooperativity.BoththesequentialandconcertedmodelsaccountforallostericregulationofcooperativeenzymesAllostericactivatorsthataltertheKmofanenzymestabilizethehighaffinitystateoftheactivesite.Allostericactivatorsthatalterthekcatofanenzymestabilizethehighactivitystateoftheactivesite.AllostericinhibitorsthataltertheKmofanenzymestabilizethelowaffinitystateoftheactivesite.Allostericinhibitorsthatalterthekcatofanenzymestabilizethelowactivitystateoftheactivesite.SequentialModelTTTTTTTTTTRRRRRRRRRRSequentialModelforAllostericRegulationofCooperativeEnzymesSequentialModelThistooisagrosssimplificationofreality,butembodiestheconceptthateachbindingeventgeneratesadifferent(group)ofconformationalstates.k1etcaremicroscopicrateconstants.SimplifiedRateEquationsfortheSequentialModelforaHighlyCooperativeenzyme•ThisassumesthattheconcentrationsofES,ES2….andEsn-1areverysmallduetothehighcooperativity.ThisissimilarinformtotheHillequation.vVmax[S]nk'[S]nwherek'k1k2.....knnisreferredtoastheHillconstantandisameasureofthecooperativityoftheenzyme.thegreaterthevalueofn,thehigherthecooperativity.Forthecasewheren=1(nocooperativity)At1/2Vmax[S0.5]givesk'=[S0.5]n.Theseparameterscanbederivedbynon-linearregressionanalysis.Thisisthesimplestkineticmodelforcooperativeenzymes.HillPlot•ThevelocitydataforcooperativeenzymescanbepresentedinalinearformbyuseofEquation:TheslopeofthebestfitlineprovidesanestimatedoftheHillcoefficientn,andtheyinterceptprovidesa