The alpha(3)beta(3)gamma subcomplex of the F-1-ATPase from the thermophilic Bacillus PS3 with the beta T165S substitution does not entrap inhibitory MgADP in a catalytic site during turnover

The hydrolytic properties of the mutant alpha(3)(beta T165S)(3) gamma and wild-type alpha(3) beta(3) gamma subcomplexes of TF1 have been compared. Whereas the wild-type complex hydrolyzes 50 mu M ATP in three kinetic phases, the mutant complex hydrolyzes 50 mu M 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 mu M ATP by the wild-type complex is biphasic, exhibiting apparent K-m values of 30 Cur and 470 mu M with corresponding k(cat) 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 K-m of 133 mu M and a k(cat) of 360 s(-1).