Catalytic sites of mitochondrial ATPase with different properties. Effect of citrate, free ATP and ADP in the presence of dithionite.

The extent of stimulation of the hydrolytic activity of mitochondrial ATPase by the reducing agent dithionite has been found to depend on substrate concentration both for the membrane bound enzyme and for the isolated and purified F1ATPase. The results suggest the existence of three catalytic sites differing in their standard reduction potential. The activating effect of free ATP on the hydrolytic activity of rat liver F1-ATPase has been found to be more pronounced on the reduced form of the enzyme. On the contrary, the inhibitory effect of ADP was higher on the oxidized form of F1-ATPase. Citrate has also been found to be an inhibitor of F1-ATPase; its effect was more pronounced on the reduced form of the enzyme, and exhibited a competitive pattern of inhibition with respect to free ATP. The results obtained have been interpreted in the sense that free ATP and ADP may be modifying the standard reduction potential of the enzyme, and suggest the existence of three independent redox cycles in ATPase governed by the exchange of ADP and Pi for the newly synthesized ATP.