Novel insights into the chemical mechanism of ATP synthase. Evidence that in the transition state the γ-phosphate of ATP is near the conserved alanine within the P-loop of the β-subunit

The chemical mechanism by which the F₁ moiety of ATP synthase hydrolyzes and synthesizes ATP remains unknown. For this reason, we have carried out studies with orthovanadate (V(i)), a phosphate analog which has the potential of 'locking' an ATPase, in its transition state by forming a MgADP·V(i) complex, and also the potential, in a photochemical reaction resulting in peptide bond cleavage, of identifying an amino acid very near the γ-phosphate of ATP. Upon incubating purified rat liver F₁ with MgADp and V(i) for 2 h to promote formation of a MgADP·V(i)-F₁ complex, the ATPase activity of the enzyme was markedly inhibited in a reversible manner. When the resultant complex was formed in the presence of ultraviolet light inhibition could not be reversed, and SDS-polyacrylamide gel electrophoresis revealed, in addition to the five known subunit bands characteristic of F₁ (i.e. α, β, γ, δ, and ε), two new electrophoretie species of 17 and 34 kDa. Western blot and N-terminal sequencing analyses identified both bands as arising from the β subunit with the site of peptide bond cleavage occurring at alanine 158, a conserved residue within F₁-ATPases and the third residue within the nucleotide binding consensus GX₄GK(T/S) (P-loop). Quantification of the amount of ADP bound within the MgADP·V(i)-F₁ complex revealed about 1.0 mol/mol F₁, while quantification of the peptide cleavage products revealed that no more than one β subunit had been cleaved. Consistent with the cleavage reaction involving oxidation of the methyl group of alanine was the finding that [³H] from NaB[³H]₄ incorporates into MgADP·V(i)-F₁ complex following treatment with ultraviolet light. These novel findings provide information about the transition state involved in the hydrolysis of ATP by a single β subunit within F₁-ATPases and implicate alanine 158 as residing very near the γ-phosphate of ATP during catalysis. When considered with earlier studies on myosin and adenylate kinase, these studies also implicate a special role for the third residue within the GX₄GK(T/S) sequence of many other nucleotide-binding proteins.