Beef heart mitochondrial F1-ATPase: inhibition by azidoadenyl-5'-yl imidodiphosphates and cooperative binding of substrate.

Two ATP analogs, 2- and 8-azidoadenyl-5'-yl imidodiphosphate, were synthesized, purified and utilized as inhibitors of soluble beef heart mitochondrial F1-ATPase under non-photolytical conditions. In the range of 5 microM to 3 mM ATP, the initial rates of ATP hydrolysis in the presence and absence of the inhibiting ATP analogs can be adequately described by two pairs of Km and Vmax values (3 microM, 8.5 mumol ATP/min per mg; 255 microM, 42.0 mumol ATP/min per mg). With increasing inhibitor concentrations, the apparent Km,2 increases as in competitive inhibition, while Vmax,1 decreases as in non-competitive inhibition. The Ki values derived for both types of inhibition are similar, but strongly different for 2- and 8-azido-AMP-PNP (4 microM and 460 microM, respectively). The decrease of the high-affinity Vmax is compensated by an increase in low-affinity catalysis, resulting in a constant sum of maximal velocities. These data can be described by a model where two sites interact with negative cooperativity in binding of substrate.