Mechanism of ATP synthesis by mitochondrial ATP synthase from beef heart.

Previous studies of the rate constants for the elementary steps of ATP hydrolysis by the soluble and membrane-bound forms of beef heart mitochondrial F1 supported the proposal that ATP is formed in high-affinity catalytic sites of the enzyme with little or no change in free energy and that the major requirement for energy in oxidative phosphorylation is for the release of product ATP. The affinity of the membrane-bound enzyme for ATP during NADH oxidation was calculated from the ratio of the rate constants for the forward binding step (k+1) and the reverse dissociation step (k-1). k-1 was accelerated several orders of magnitude by NADH oxidation. In the presence of NADH and ADP an additional enhancement of k-1 was observed. These energy-dependent dissociations of ATP were sensitive to the uncoupler FCCP. k+1 was affected little by NADH oxidation. The dissociation constant (KdATP) increased many orders of magnitude during the transition from nonenergized to energized states.