The apparent absolute requirement of adenosine diphosphate for the inorganic phosphate--water exchange of oxidative phosphorylation.

With suitable submitochondrial particles from the heart, the rate of the inorganic phosphate e HOH exchange during substrate oxidation in the absence of ADP is less than l/1000 of the rate in the presence of ADP. The concentration dependence of the exchange on ADP shows simple saturation behavior with an apparent K, of 0.32 mM. For net oxidative phosphorylation under similar conditions, but with a hexokinase-glucose trap’present, the apparent K,,, for ADP is 0.58 mr& The ADP analogue, adenosine methylene diphosphonate, is not detectably phosphorylated, does not stimulate the Pi + HOH exchange in the absence of ADP, and does not inhibit the exchange in the presence of ADP. The submitochondrial particles show the presence of an oligomycin and a 2,4-dinitrophenol-sensitive ADP $ ATP exchange. These results are consistent with the interpretation that the Pi z=+. HOH exchange results from dynamic reversal of ATP formation at the catalytic site, and that in the phosphorylation reaction the Crst covalent compound formed from Pi or ADP is ATP.