NOVEL INSIGHTS INTO THE CHEMICAL MECHANISM OF ATP SYNTHASE - EVIDENCETHAT 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 hydroly zes and synthesizes ATP remains unknown, For this reason, we have carr ied out studies with orthovanadate (V-i), a phosphate analog which has the potential of ''locking'' all ATPase, in its transition state by f orming 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 r at liver F-1 with MgADP and V-i for 2 h to promote formation of a MgAD P.V-i-F-1 complex, the ATPase activity of the enzyme was markedly inhi bited in a reversible manner, When the resultant complex was formed in the presence of ultraviolet light inhibition could not be reversed, a nd SDS-polyacrylamide gel electrophoresis revealed, in addition to the five known subunit bands characteristic of F-1 (i.e. alpha, beta, gam ma, delta, and epsilon), two new electrophoretic species of 17 and 34 kDa, Western blot and N-terminal sequencing analyses identified both b ands as arising from the beta subunit with the site of peptide bond cl eavage occurring at alanine 158, a conserved residue within F-1-ATPase s and the third residue within the nucleotide binding consensus GX(4)G K(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 quantifica tion of the peptide cleavage products revealed that no more than one b eta subunit had been cleaved, Consistent with the cleavage reaction in volving 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 followi ng treatment with ultraviolet light, These novel findings provide info rmation about the transition state involved in the hydrolysis of ATP b y a single beta subunit within F-1-ATPases and implicate alanine 158 a s 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 th e GX(4)GK(T/S) sequence of many other nucleotide-binding proteins.