On the rate of F1-ATPase turnover during ATP hydrolysis by the single catalytic site. Evidence that hydrolysis with a slow rate of product release does not occur at the alternating active site.

Under conditions of molar excess of enzyme, isolated F1-ATPase from beef heart mitochondria catalyses ATP hydrolysis biphasically. The rate constants for product release are approximately 10(-1) and 10(-4)-10(-3) s-1, respectively. The slow phase of ATP hydrolysis is insensitive to EDTA. [gamma-32P]ATP splitting in the slow phase cannot be chased from the enzyme during several catalytic turnovers. It follows from these results that the slow single-site hydrolysis of ATP (kcat approximately 10(-4) s-1), initially observed by Grubmeyer et al. [(1982) J. Biol. Chem. 257, 12092-12100], is not carried out by the normal catalytic site. In contrast, the phase of rapid ATP hydrolysis (kcat approximately 10(-1) s-1) is completely prevented by EDTA and is believed to be the normal function of the normal catalytic site of F1-ATPase.