Energetics of ATP dissociation from the mitochondrial ATPase during oxidative phosphorylation.

The dissociation constant (KdATP) for ATP bound in the high affinity catalytic site of membrane-bound beef heart mitochondrial ATPase (F1) was calculated from the ratio of the rate constants for the reverse dissociation step (k-1) and the forward binding step (k+1). k-1 for ATP bound to submitochondrial particles or to submitochondrial particles washed with KCl so as to activate ATPase activity was accelerated by about five orders of magnitude during respiratory chain-linked oxidations of NADH. In the presence of NADH and 0.1 mM ADP, k-1 increased more than six orders of magnitude. These energy-dependent dissociations of ATP were sensitive to the uncoupler carbonyl cyanide p-trifluoromethyloxyphenylhydrazone. Only small changes in k+1 were observed in the presence of NADH or NADH and ADP. KdATP at 23 degrees C in the absence of NADH and ADP was 10(-12) M, in the presence of NADH, 3 microM, and in the presence of NADH and 0.1 mM ADP, 60 microM. Thus, the dissociation of ATP during the transition from non-energized to energized states was, under these conditions, accompanied by observed free energy changes of 8 and 9.7 kcal/mol, respectively.