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

J Biol Chem. 1999 Oct 8;274(41):28853-6. doi: 10.1074/jbc.274.41.28853.


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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • ATP Synthetase Complexes
  • Adenosine Diphosphate / chemistry*
  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Magnesium / chemistry*
  • Mitochondria, Liver / enzymology
  • Models, Molecular
  • Molecular Sequence Data
  • Multienzyme Complexes / chemistry*
  • Oxygen / chemistry
  • Peptide Fragments / chemistry
  • Phosphates / chemistry*
  • Phosphotransferases (Phosphate Group Acceptor) / chemistry*
  • Proton-Translocating ATPases / chemistry
  • Rats
  • Rats, Sprague-Dawley
  • Sequence Analysis
  • Ultraviolet Rays
  • Vanadates / chemistry


  • Multienzyme Complexes
  • Peptide Fragments
  • Phosphates
  • Vanadates
  • Adenosine Diphosphate
  • ATP Synthetase Complexes
  • Phosphotransferases (Phosphate Group Acceptor)
  • Proton-Translocating ATPases
  • Magnesium
  • Oxygen