The α3β3γ Subcomplex of the F1-ATPase from the Thermophilic Bacillus PS3 with the βT165S Substitution Does Not Entrap Inhibitory MgADP in a Catalytic Site during Turnover

Abstract The hydrolytic properties of the mutant α3(βT165S)3γ and wild-type α3β3γ subcomplexes of TF1 have been compared. Whereas the wild-type complex hydrolyzes 50 μM ATP in three kinetic phases, the mutant complex hydrolyzes 50 μM ATP with a linear rate. After incubation with a slight excess of ADP in the presence of Mg2+, the wild-type complex hydrolyzes 2 mM ATP with a long lag. In contrast, prior incubation of the mutant complex under these conditions does not affect the kinetics of ATP hydrolysis. The ATPase activity of the wild-type complex is stimulated 4-fold by 0.1% lauryl dimethylamine oxide, whereas this concentration of lauryl dimethylamine oxide inhibits the mutant complex by 25%. Compared with the wild-type complex, the activity of the mutant complex is much less sensitive to turnover-dependent inhibition by azide. This comparison suggests that the mutant complex does not entrap substantial inhibitory MgADP in a catalytic site during turnover, which is supported by the following observations. ATP hydrolysis catalyzed by the wild-type complex is progressively inhibited by increasing concentrations of Mg2+ in the assay medium, whereas the mutant complex is insensitive to increasing concentrations of Mg2+. A Lineweaver-Burk plot constructed from rates of hydrolysis of 20-2000 μM ATP by the wild-type complex is biphasic, exhibiting apparent Km values of 30 μM and 470 μM with corresponding kcat values of 26 and 77 s−1. In contrast, a Lineweaver-Burk plot for the mutant complex is linear in this range of ATP concentration, displaying a Km of 133 μM and a kcat of 360 s−1.