title: 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.
abstract: 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 F(1) catalytic sector (subunit stoichiometry, alpha(3)beta(3)gammadeltaepsilon). Lacking is critical information about the chemical events taking place at the catalytic site of each beta-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 Mg(2+), ADP, and orthovanadate (V(i)), a photoreactive phosphate analog with a trigonal bipyramidal geometry resembling that of the gamma-P of ATP in the transition 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 (GGAGVGKT) of a single beta-subunit at alanine 158, implicating this residue as within contact distance of the gamma-P of ATP in the transition state. Here, we report that ADP, although facilitating transition state formation, is not essential. In the presence of Mg(2+) and V(i) alone the catalytic activity of the resultant MgV(i)-F(1) complex is inhibited to nearly the same extent as that observed for the MgADP. V(i)-F(1) complex. Inhibition is not observed with ADP, Mg(2+), or V(i) alone. Significantly, in the presence of ultraviolet light and O(2,) the MgV(i)-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 quantification of the amount of unreacted beta-subunits. These novel findings indicate that Mg(2+) 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 Mg(2+) in transition state formation is proposed here for the first time.
authors.affiliation: Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185, USA.
authors.forename: Y H
authors.initials: YH
authors.lastname: Ko
authors.forename: S
authors.initials: S
authors.lastname: Hong
authors.forename: P L
authors.initials: PL
authors.lastname: Pedersen
mesh_terms: D025181:ATP Synthetase Complexes; D000244:Adenosine Diphosphate / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D008274:Magnesium / Q000737:chemistry*; D008930:Mitochondria, Liver / Q000201:enzymology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry*; D010100:Oxygen / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry; D010710:Phosphates / Q000737:chemistry*; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000737:chemistry*; D006180:Proton-Translocating ATPases / Q000737:chemistry; D051381:Rats; D017207:Rats, Sprague-Dawley; D017421:Sequence Analysis; D014466:Ultraviolet Rays; D014638:Vanadates / Q000737:chemistry
chemical_list: D009097:Multienzyme Complexes; D010446:Peptide Fragments; D010710:Phosphates; D014638:Vanadates; D000244:Adenosine Diphosphate; D025181:ATP Synthetase Complexes; D017856:Phosphotransferases (Phosphate Group Acceptor); D006180:Proton-Translocating ATPases; D008274:Magnesium; D010100:Oxygen