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

The chemical mechanism by which the F-1 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'' all 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 gamma-phosphate of ATP. Upon incubating purified rat liver F-1 with MgADP and V-i for 2 h to promote formation of a MgADP.V-i-F-1 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 F-1 (i.e. alpha, beta, gamma, delta, and epsilon), two new electrophoretic species of 17 and 34 kDa, Western blot and N-terminal sequencing analyses identified both bands as arising from the beta subunit with the site of peptide bond cleavage occurring at alanine 158, a conserved residue within F-1-ATPases and the third residue within the nucleotide binding consensus GX(4)GK(T/S) (P-loop). Quantification of the amount of ADP bound within the MgADP.V-i-F-1 complex revealed about 1.0 mol/mol F-1, while quantification of the peptide cleavage products revealed that no more than one beta subunit had been cleaved, Consistent with the cleavage reaction involving oxidation of the methyl group of alanine was the finding that [H-3] from NaB[H-3](4) incorporates into MgADP.V-i-F-1 complex following treatment with ultraviolet light, These novel findings provide information about the transition state involved in the hydrolysis of ATP by a single beta subunit within F-1-ATPases and implicate alanine 158 as residing very near the gamma-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 GX(4)GK(T/S) sequence of many other nucleotide-binding proteins.