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

Young Hee Ko, Sangjin Hong, Peter L. Pedersen

Research output: Contribution to journalArticle

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 F1 catalytic sector (subunit stoichiometry, α3β3γδε). Lacking is critical information about the chemical events taking place at the catalytic site of each β-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 γ-P of ATP in the transition state of enzymes like myosin. In the presence of ultraviolet light and O2, the MgADP·V(i)-F(1) complex was cleaved within the P-loop (GGAGVGKT) of a single β-subunit at alanine 158, implicating this residue as within contact distance of the γ-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)-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, Mg2+, or V(i) alone. Significantly, in the presence of ultraviolet light and O2, the MgV(i)-F(1) complex is cleaved also within the P-loop of a single β-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 β-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.

Original languageEnglish (US)
Pages (from-to)28853-28856
Number of pages4
JournalJournal of Biological Chemistry
Volume274
Issue number41
DOIs
StatePublished - Oct 8 1999

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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