ATP synthase: Conditions under which all catalytic sites of the F₁ moiety are kinetically equivalent in hydrolyzing ATP

Conditions have been reported under which the F₁ moiety of bovine heart ATP synthase catalyzes the hydrolysis of ATP by an apparently cooperative mechanism in which the slow rate of hydrolysis at a single catalytic site (unisite catalysis) is enhanced more than 10⁶-fold when ATP is added in excess to occupy one or both of the other two catalytic sites (multisite catalysis) (Cross, R. L., Grubmeyer, C., and Penefsky, H. S. (1982) J. Biol. Chem. 257, 12101-12105). In the novel studies reported here, and in contrast to the earlier report, we have (a) monitored the kinetics of ATP hydrolysis of F₁ by using nucleotide-depleted preparations and a highly sensitive chemiluminescent assay; (b) followed the reaction immediately upon addition of F₁ to ATP, rather than after prior incubation with ATP; and (c) used a reaction medium with P_i as the only buffer. The following observations were noted. First, regardless of the source of enzyme, bovine or rat, and catalytic conditions (unisite or multisite), the rates of hydrolysis depend on ATP concentration to the first power. Second, the first order rate constant for ATP hydrolysis remains relatively constant under both unisite and multisite conditions declining only slightly at high ATP concentration. Third, the initial rates of ATP hydrolysis exhibit Michaelis-Menten kinetic behavior with a single V_max exceeding 100 μmol of ATP hydrolyzed per min/mg of F₁ (turnover number = 635 s^-1) and a single K_m for ATP of about 57 μM. Finally, the reaction is inhibited markedly by low concentrations of ADP. It is concluded that, under the conditions described here, all catalytic sites that participate in the hydrolysis of ATP within the F₁ moiety of mitochondrial ATP synthase function in a kinetically equivalent manner.