J Biol Chem, Vol. 274, Issue 41, 28853-28856, October 8, 1999

COMMUNICATION
Chemical Mechanism of ATP Synthase
MAGNESIUM PLAYS A PIVOTAL ROLE IN FORMATION OF THE TRANSITION STATE WHERE ATP IS SYNTHESIZED FROM ADP AND INORGANIC PHOSPHATE

From the Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185

The chemical mechanism by which ATP synthases catalyze the synthesis of ATP remains unknown despite the recent elucidation of the three-dimensional structures of two forms of the F₁ catalytic sector (subunit stoichiometry, ₃₃). Lacking is critical information about the chemical events taking place at the catalytic site of each -subunit in the transition state. In an earlier report (Ko, Y. H., Bianchet, M. A., Amzel, L. M., and Pedersen, P. L. (1997) J. Biol. Chem. 272, 18875-18881), we provided evidence for transition state formation in the presence of Mg²⁺, ADP, and orthovanadate (V_i), a photoreactive phosphate analog with a trigonal bipyramidal geometry resembling that of the -P of ATP in the transition state of enzymes like myosin. In the presence of ultraviolet light and O_2, the MgADP·V_i-F₁ complex was cleaved within the P-loop (GGAGVGKT) of a single -subunit at alanine 158, implicating this residue as within contact distance of the -P of ATP in the transition state. Here, we report that ADP, although facilitating transition state formation, is not essential. In the presence of Mg²⁺ and V_i alone the catalytic activity of the resultant MgV_i-F₁ complex is inhibited to nearly the same extent as that observed for the MgADP·V_i-F₁ complex. Inhibition is not observed with ADP, Mg²⁺, or V_i alone. Significantly, in the presence of ultraviolet light and O_2, the MgV_i-F₁ complex is cleaved also within the P-loop of a single -subunit at alanine 158 as confirmed by Western blot analyses with two different antibodies, by N-terminal sequence analyses, and by quantification of the amount of unreacted -subunits. These novel findings indicate that Mg²⁺ plays a pivotal role in transition state formation during ATP synthesis catalyzed by ATP synthases, a role that involves both its preferential coordination with P_i and the repositioning of the P-loop to bring the nonpolar alanine 158 into the catalytic pocket. A reaction scheme for ATP synthases depicting a role for Mg²⁺ in transition state formation is proposed here for the first time.