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 ␣ 3 (␤T165S) 3 ␥ and wild-type ␣ 3 ␤ 3 ␥ subcomplexes of TF 1 have been compared. Whereas the wild-type complex hydrolyzes 50 M ATP in three kinetic phases, the mutant complex hydrolyzes 50 M ATP with a linear rate. After incubation with a slight excess of ADP in the presence of Mg 2؉ , 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 turnoverdependent 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 Mg 2؉ in the assay medium, whereas the mutant complex is insensitive to increasing concentrations of Mg

Substitution of βGlu201 in the α3β3γ Subcomplex of the F1-ATPase from the Thermophilic Bacillus PS3 Increases the Affinity of Catalytic Sites for Nucleotides

“…The observation that carboxymethylation of ␣ 3 (␤E201C) 3 ␥ increases k cat and K m3 provides additional support for this hypothesis. Also consistent with this hypothesis are earlier reports demonstrating that the ␤T165S substitution in TF 1 increases K m3 and k cat (23,24). Replacement of threonine by serine in this position might decrease the affinity of MgATP bound to closed catalytic sites by interacting less strongly with Mg …”

ATP Hydrolysis and Synthesis of a Rotary Motor V-ATPase from Thermus thermophilus

“…PS3 also entraps an inhibitory Mg-ADP in a catalytic site, resulting in enzyme inhibited for ATP hydrolysis (31). However, this entrapped Mg-ADP in the F 0 F 1 is capable of being released from the catalytic site (32,33). Thus, the F 0 F 1 from PS3 can catalyze both the ATP synthesis/hydrolysis directions.…”

ATP synthesis catalyzed by the mitochondrial F1‐F0 ATP synthase is not a reversal of its ATPase activity

“…Many years ago we have reported that ADP(Mg2+)-deactivated F~-Fo ATPase is capable of ATP synthesis [20] and concluded that the inhibitory ADP binds to a site which is not the same as that where ADP leaves F~ during the steady-state ATP hydrolysis [12,20]. Much effort have been put forward over past 15 years to attribute ADP-specific inhibition to catalytic or non-catalytic ATPase sites but clear-cut consensus does not appear to be reached ( [33][34][35][36] and references cited therein). The results presented in this report strongly support and reinforce our original observation on the catalytic competence of ADP(Mg 2+) inhibited F~-F 0 ATPase in ATP synthesis.…”