Catalytic Activity of the α3β3γ Complex of F1-ATPase without Noncatalytic Nucleotide Binding Site

Abstract A mutant α3β3γ complex of F1-ATPase from thermophilic Bacillus PS3 was generated in which noncatalytic nucleotide binding sites lost their ability to bind nucleotides. It hydrolyzed ATP at an initial rate with cooperative kinetics (Km(1), 4 μM; Km(2), 135 μM) similar to the wild-type complex. However, the initial rate decayed rapidly to an inactivated form. Since the inactivated mutant complex contained 1.5 mol of ADP/mol of complex, this inactivation seemed to be caused by entrapping inhibitory MgADP in a catalytic site. Indeed, the mutant complex was nearly completely inactivated by a 10 min prior incubation with equimolar MgADP. Analysis of the progress of inactivation after initiation of ATP hydrolysis as a function of ATP concentration indicated that the inactivation was optimal at ATP concentrations in the range of Km(1). In the presence of ATP, the wild-type complex dissociated the inhibitory [3H]ADP preloaded onto a catalytic site whereas the mutant complex did not. Lauryl dimethylamineoxide promoted release of preloaded inhibitory [3H]ADP in an ATP-dependent manner and partly restored the activity of the inactivated mutant complex. Addition of ATP promoted single-site hydrolysis of 2′,3′-O-(2,4,6-trinitrophenyl)-ATP preloaded at a single catalytic site of the mutant complex. These results indicate that intact noncatalytic sites are essential for continuous catalytic turnover of the F1-ATPase but are not essential for catalytic cooperativity of F1-ATPase observed at ATP concentrations below ~300 μM.