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 (F-1) was calculated from the ratio of the rate constants for the reverse d issociation step (k(-1)) and the forward binding step (k(+1)). k(-1) f or ATP bound to submitochondrial particles or to submitochondrial part icles washed with KCl so as to activate ATPase activity was accelerate d by about five orders of magnitude during respiratory chain-linked ox idations of NADH, In the presence of NADH and 0.1 mM ADP, k(-1) increa sed more than six orders of magnitude, These energy-dependent dissocia tions of ATP were sensitive to the uncoupler carbonyl cyanide p-triflu oromethyloxyphenylhydrazone. Only small changes in k(+1) were observed in the presence of NADH or NADH and ADP. K-dATP at 23 degrees C in th e absence of NADH and ADP was 10(-12) M, in the presence of NADH, 3 mu M, and in the presence of NADH and 0.1 mM ADP, 60 mu M. Thus, the dis sociation of ATP during the transition from non-energized to energized states was, under these conditions, accompanied by observed free ener gy changes of 8 and 9.7 kcal/mol, respectively.