Heterogeneous hydrolysis of substoichiometric ATP by the F1-ATPase from Escherichia coli.

The hydrolysis of 0.3 microM [alpha,gamma-32P]ATP by 1 microM F1-ATPase isolated from the plasma membranes of Escherichia coli has been examined in the presence and absence of inorganic phosphate. The rate of binding of substoichiometric substrate to the ATPase is attenuated by 2 mM phosphate and further attenuated by 50 mM phosphate. Under all conditions examined, only 10-20% of the [alpha,gamma-32P]ATP that bound to the enzyme was hydrolyzed sufficiently slowly to be examined in cold chase experiments with physiological concentrations of non-radioactive ATP. These features differ from those observed with the mitochondrial F1-ATPase. The amount of bound substrate in equilibrium with bound products observed in the slow phase which was subject to promoted hydrolysis by excess ATP was not affected by the presence of phosphate. Comparison of the fluxes of enzyme-bound species detected experimentally in the presence of 2 mM phosphate with those predicted by computer simulation of published rate constants determined for uni-site catalysis (Al-Shawi, M.D., Parsonage, D. and Senior, A.E. (1989) J. Biol. Chem. 264, 15376-15383) showed that hydrolysis of substoichiometric ATP observed experimentally was clearly biphasic. Less than 20% of the substoichiometric ATP added to the enzyme was hydrolyzed according to the published rate constants which were calculated from the slow phase of product release in the presence of 1 mM phosphate. The majority of the substoichiometric ATP added to the enzyme was hydrolyzed with product release that was too rapid to be detected by the methods employed in this study, indicating again that the F1-ATPase from E. coli and bovine heart mitochondria hydrolyze substoichiometric ATP differently.