CATALYTIC SITES OF ESCHERICHIA-COLI F1-ATPASE - CHARACTERIZATION OF UNISITE CATALYSIS AT VARIED PH

Using manual rapid-mixing procedures in which small, equal volumes of Escherichia coli F1-ATPase and [gamma-P-32] ATP were combined at final concentrations of 2 and 0.2-mu-M, respectively (i.e., unisite catalysis conditions), it was shown that greater-than-or-equal-to 66% of the P-32 became bound to the enzyme, with the ratio of bound ATP/bound P(i) equal to 0.4 and the rate of dissociation of bound [P-32]P(i) equal to 3.5 x 10(-3) s-1, similar to previously published values. Azide is known to inhibit cooperative but not unisite catalysis in F(1)-ATPase [Noumi, T., Maeda, M., & Futai, M. (1987) FEBS Lett. 213, 381-384]. In the presence of 1 mM sodium azide, 99% of the P-32 became bound to the enzyme, with the ratio of bound ATP/bound P(i) being 0.57. These experiments demonstrated that when conditions are used which minimize cooperative catalysis, most or all of the F1 molecules bind substoichiometric ATP tightly, hydrolyze it with retention of bound ATP and P(i), and release the products slowly. The data justify the validity of previously published rate constants for unisite catalysis. Unisite catalysis in E. coli F1-ATPase was studied at varied pH from 5.5 to 9.5 using buffers devoid of phosphate. Rate constants for ATP binding/release, ATP hydrolysis/resynthesis, P(i) release, and ADP binding/release were measured; the P(i) binding rate constant was inferred from the DELTA-G for ATP hydrolysis. ATP binding was pH-independent; ATP release accelerated at higher pH. The highest K(a)ATP (4.4 x 10(9) M-1) was seen at physiological pH 7.5. ATP hydrolysis and resynthesis were pH-independent, and the equilibrium constant for the cleavage/condensation reaction was around 2 at all pH values, showing catalysis occurred in a sequestered environment. P(i) release was pH-independent, but P(i) binding was drastically slowed at high pH. K(d)P(i), which was around 1 M at pH 5.5-7.5, reached 1.3 X 10(6) M at pH 9.5. ADP release was pH-independent; ADP binding was somewhat pH-sensitive, such that K(d)ADP decreased steadily from 42-mu-M at pH 5.5 to 430 nM at pH 9.5. The data confirm the view that, during normal oxidative phosphorylation, energy input from DELTA-mu(H+) is required for P(i) binding and ATP release. The data also support the idea that two major enzyme conformations are involved in unisite catalysis and that an ionic interaction influences binding of P(i) and release of ATP in one conformation. In contrast to unisite ATP hydrolysis, multisite ATP hydrolysis was pH-dependent in the range pH 5.0-9.5, showing that the rate enhancements deriving from cooperative intersubunit interactions involve ionizable groups on the protein.