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 mito chondrial F-1 supported the proposal that ATP is formed in high-affini ty catalytic sites of the enzyme with little of no change in free ener gy and that the major requirement for energy in oxidative phosphorylat ion is for the release of product ATP. The affinity of the membrane-bo und enzyme for ATP during NADH oxidation was calculated from the ratio of the rate constants for the forward binding step (k(+1)) and the re verse dissociation step (k(-1)). k(-1) was accelerated several orders of magnitude by NADH oxidation. In the presence of NADH and ADP an add itional enhancement of k(-1) was observed. These energy-dependent diss ociations of ATP were sensitive to the uncoupler FCCP. k(+1) was affec ted little by NADH oxidation. The dissociation constant (K-dATP) incre ased many orders of magnitude during the transition from nonenergized to energized states.