The Mechanism of ATP Hydrolysis Catalyzed

During ATP hydrolysis, myosin or subfragment 1 catalyzes the exchange of oxygens between water and phosphate, so that on average, each product Pi molecule contains more than one oxygen atom derived from water. Using quenched-flow techniques, the exchange process in both ATP and Pi was studied in the rabbit skeletal muscle subfragment 1 ATPase. The exchange in protein-bound ATP (M**ATP) and protein-bound ADP*Pi (M**.ADP-Pi) was followed as a function of time. The pattern of exchange follows closely a model in which M*.ATP + HzO and M***ADP-Pi are interconverted directly, without any characterizable intermediate between these species. AU oxygen atoms of Pi are equivalent with respect to loss to solvent, and elimination of water to re-form ATP occurs with a rate constant of 16 s-' (at pH 8.0, 1 0.015 M, 20 "c). This is the first direct measurement of the rate constant for the transformation of M* * ADP Pi to M* ATP. Oxygen exchange during steady state ATP hydrolysis in the presence of acto-subfragment 1 also fits well to this model, with actin reducing the time available for M* ATP and M** *ADP= Pi to undergo exchange.