The presence of phosphate at a catalytic site suppresses the formation of the MgADP-inhibited form of F-1-ATPase

F-1-ATPase is inactivated by entrapment of MgADP in catalytic sites and reactivated by MgATP or P-i. Here, using a mutant alpha(3)beta(3)gamma complex of thermophilic F-1-ATPase (alphaW463F/betaY341W) and monitoring nucleotide binding by fluorescence quenching of an introduced tryptophan, we found that P-i interfered with the binding of MgATP to F-1-ATPase, but binding of MgADP was interfered with to a lesser extent. Hydrolysis of MgATP by F-1-ATPase during the experiments did not obscure the interpretation because another mutant, which was able to bind nucleotide but not hydrolyse ATP (alphaW463F/betaE190Q/betaY341W), also gave the same results. The half-maximal concentrations of P-i that suppressed the MgADP-inhibited form and interfered with MgATP binding were both approximate to 20 mM. It is likely that the presence of P-i at a catalytic site shifts the equilibrium from the MgADP-inhibited form to the enzyme-MgADP-P-i complex, an active intermediate in the catalytic cycle.