BEEF-HEART MITOCHONDRIAL F(1)-ATPASE - INHIBITION BY AZIDOADENYL-5'-YL IMIDODIPHOSPHATES AND COOPERATIVE BINDING OF SUBSTRATE

Two ATP analogs, 2- and 8-azidoadenyl-5'-yl imidodiphosphate, were syn thesized, purified and utilized as inhibitors of soluble beef heart mi tochondrial F1-ATPase under non-photolytical conditions. In the range of 5 muM to 3 mM ATP, the initial rates of ATP hydrolysis in the prese nce and absence of the inhibiting ATP analogs can be adequately descri bed by two pairs of K(m) and V(max) a values (3 muM, 8.5 mumol ATP/min per mg; 255 muM, 42.0 mumol ATP/min per mg). With increasing inhibito r concentrations, the apparent K(m,2) increases as in competitive inhi bition, while V(max,1) decreases as in non-competitive inhibition. The K(i) values derived for both types of inhibition are similar, but str ongly different for 2- and 8-azido-AMP-PNP (4 muM and 460 muM, respect ively). The decrease of the high-affinity V(max) is compensated by an increase in low-affinity catalysis, resulting in a constant sum of max imal velocities. These data can be described by a model where two site s interact with negative cooperativity in binding of substrate.