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1、THEJOURNALOFBIOLOGICALCHEMISTRYVol.257,No.20,IssueofOctober25.pp.12101-121(16,1982PrintedmU.S.A.MechanismofATPHydrolysisbyBeefHeartMitochondrialATPaseRATEENHANCEMENTSRESULTINGFROMCOOPERATIVEINTERACTIONSBETWEENMULTIPLECATALYTICSITES*(Receivedforpublication,May26,1982)RichardL.Cross$
2、,CharlesGrubmeyerg,andHarveyS.PenefskyFromtheDepartmentofBiochemistry,ThePublicHealthResearchInstituteofthecityofNewYork,hc.,NewYork,NewYork10016Theveryslowturnoverratefor[y3’P]ATPhydroly-catalysis)itwasshown(1)thatsubstratebindsrapidlywithsisatasinglecatalyticsiteonsolublemitochon
3、drialveryhighaffinity(K,=1OI2“I).BindingisfollowedbyrapidATPase(F1)(uni-sitecatalysis)isacceleratedover10‘-developmentofanequilibriumbetweenboundATPandfoldwhenadditionalnonradioactiveATPisaddedtoboundhydrolysisproducts,ADPandPi.Theequilibriumallowbindingatmultiplecatalyticsites.Thi
4、srateen-constantis0.5.TheseobservationssupporttheconceptthathancementincreasesturnovertoV,,withnodetect-enzyme-boundATPisformedfromADPandPiwithalmostablelag,thusdemonstratingthatthehighaffinitycat-zerochangeinfreeenergyandthattheprimaryfunctionofalyticsitepreviouslycharacterized(Gr
5、ubmeyer,C.,energyinputduringoxidativephosphorylationistopromoteCross,R.L.,andPenefsky,H.S.(1982)J.Biol.ChemreleaseofproductATPfromcatalyticsitesonF1’(reviewed257,12092-12100)isanormalcatalyticsite.inRef.2).WhenATPbindingatasecondsiteisratelimitingItisclearfromevidencecurrentlyavail
6、ablethatthemul-forhydrolysisapproximately0.6molofATPpermoloftisubunitATPasecontainsmorethanonecatalyticsite.F1isdetectedatcatalyticsites.ThislevelispredictedChemicalmodificationstudiesandmeasurementofthenum-fromtheequilibriumdistributionofsubstrateandberofnucleotide-bindingsitesind
7、icatethattherearethreeproductsboundatasinglesite.TherateofATPbindingcopiesofthepsubunit(3,4)andthatthissubunitcontainstoasecondcatalyticsite(6X10‘M”s-’)isthesamethecatalyticsite(reviewedinRefs.5and6).Directevidenceastherateofbindingatthefirstsite.AK,,,=30PMandaV,,=300s-’weremeasure
8、dunderconditionsthatforthe
