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 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.
Ko
Y H
YH
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185, USA.
Hong
S
S
Pedersen
P L
PL
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
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