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

The chemical mechanism by which ATP synthases catalyze the synthesis of ATP remains unknown despite the recent elucidation of the three-dimensional st ructures of two forms of the F-1 catalytic sector (subunit stoichiometry, a lpha(3)beta(3)gamma delta epsilon). Lacking is critical information about t he chemical events taking place at the catalytic site of each P-subunit in the transition state. In an earlier report (Ko, Y. H., Planchet, M. A., Amz el, 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+, A DP, and orthovanadate (V-i), a photoreactive phosphate analog with a trigon al bipyramidal geometry resembling that of the gamma-P of ATP in the transi tion state of enzymes like myosin, In the presence of ultraviolet light and O-2, the MgADP.V-i-F-1 complex was cleaved within the P-loop (GG (A) under bar GVGKT) of a single P-subunit at alanine 158, implicating this residue as within contact distance of the gamma-P of ATP in the transition state. H ere, we report that ADP, although facilitating transition state formation, is not essential. In the presence of Mg2+ and V-i alone the catalytic activ ity of the resultant MgVi-F-1 complex is inhibited to nearly the same exten t as that observed for the MgADP.V-i-F-1 complex. Inhibition is not observe d with ADP, Mg2+, or V-i alone. Significantly, in the presence of ultraviol et light and O-2, the MgVi-F-1 complex is cleaved also within the P-loop of a single beta-subunit at alanine 158 as confirmed by Western blot analyses with two different antibodies, by N-terminal sequence analyses, and by qua ntification of the amount of unreacted beta-subunits. These novel findings indicate that Mg2+ plays a pivotal role in transition state formation durin g ATP synthesis catalyzed by ATP synthases, a role that involves both its p referential coordination with P-i and the repositioning of the P-loop to br ing the nonpolar alanine 158 into the catalytic pocket. A reaction scheme f or ATP synthases depicting a role for Mg2+ in transition state formation is proposed here for the first time.