Evidence for catalytic cooperativity during ATP hydrolysis by beef heart F1-ATPase. Kinetics and binding studies with the photoaffinity label BzATP.
The photoaffinity analog of ATP, 3'-O-(4-benzoyl) benzoyl ATP (BzATP), was used to covalently modify the catalytic sites on the beef heart mitochondrial F1-ATPase. In the absence of actinic illumination, BzATP was a slow substrate for the enzyme (Vmax = 0.19 mumol min-1 mg-1; kcat/Km = 2.2 X 10(6) M-1s-1) and behaved as a classical competitive inhibitor versus ATP (Ki = 0.85 microM). Under photolytic conditions, BzATP inactivated F1 with pseudo first-order kinetics, and the photoinactivation reaction showed rate saturation suggesting specific, reversible binding of BzATP to F1 prior to covalent bond formation. ATP protected against F1 photoinactivation (Kprotect = 0.3 microM) and partially covalently modified F1 yielded the same Km for ATP as unmodified enzyme. These results strongly suggested that BzATP was bound to catalytic sites on the enzyme. In the absence of photolysis, BzATP saturated two binding sites on the F1 (KD = 1.6 microM), and under photolytic conditions, 1 mol of BzATP was shown to be covalently liganded to the beta subunit of the enzyme coincident with 100% loss in ATPase activity. Previous studies with the mitochondrial F1-ATPase have suggested a mechanism involving catalytic cooperativity during ATP hydrolysis. Our demonstration of a molar stoichiometry of 1 for photoinactivation is in accord with this mechanism. It is suggested that either F1 is unable to hydrolyze covalently bound BzATP, or that subsequent to hydrolysis, the BzADP product can not be released from the catalytic site. It is therefore inferred that F1 hydrolytic activity requires cooperativity between multiple, viable catalytic sites and that covalent modification of a single catalytic site is sufficient for complete enzyme inactivation.
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism*; D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D006868:Hydrolysis; D007700:Kinetics; D046911:Macromolecular Substances; D008929:Mitochondria, Heart / Q000201:enzymology*; D010782:Photolysis; D011485:Protein Binding; D006180:Proton-Translocating ATPases / Q000378:metabolism*
D000345:Affinity Labels; D046911:Macromolecular Substances; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D000244:Adenosine Diphosphate; C045291:3'-O-(4-benzoyl)benzoyladenosine diphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases