Energetics of ATP dissociation from the mitochondrial ATPase during oxidative phosphorylation

The dissociation constant (K(dATP)) 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 sub-mitochondrial 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. K(dATP) at 23 °C in the absence of NADH and ADP was 10^-12 M, in the presence of NADH, 3 μM, and in the presence of NADH and 0.1 mM ADP, 60 μM. 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.