Catalytic Site Cooperativity of Beef Heart Mitochondrial F, Adenosine Triphosphatase CORRELATIONS OF INITIAL VELOCITY, BOUN6 INTERMEDIATE, AND OXYGEN EXCHANGE MEASUREMENTS WITH AN ALTERNATING THREE-SITE

Previous studies with beef heart mitochondrial F1 ATPase suggest a mechanism in which binding of substrate (ATP) at one catdytic site accelerates the release of products (ADP + Pi) from a separate catalytic site. hitid velocity studies reported here, in harmony with this mechanism, show a biphasic rate response with apparent K,,, values of 250 and 1.7 PM and V,, values of 54 and 2.2 pmol/min/mg. Further, the suggested mechanism leads to an important and previously untested diagnostic prediction for F1 ATPase, namely that high levels of enzyme-bound product wi l be present during turnover at substrate concentrations well below those which give half-maximal velocity. This prediction has been verified by the demonstration that most F1 ATPase molecules retain product ADP at a catalytic site when hydrolyzing ATP at concentrations less than ‘hoo of that for half-maximal velocity. The mechanism also predicts a negative cooperativity of substrate binding, with a slow rate of product formation until all catalytic sites are filIed. The predicted substrate binding pattern is substantiated by the demonstration reported herein that 1 catalytic site/Fl ATPase becomes saturated with only about 1 ,UM ATP present. This singly occupied site shows slow catalytic turnover, and most of the oxygens of Pi formed at 1 p~ ATP have been exchanged with water. Pronounced decrease in this oxygen exchange occurs when the ATP concentration is increased to 10-20 PM, which is still much lower than the concentration of ATP required for halfmaximal velocity. These results cannot be satisfactorily correlated by either independent-site mechanisms or by interacting two-site mechanisms. A simple interacting three-site mechanism is presented that satisfactorily correlates al three sets of results.