THE ALPHA(3)BETA(3)GAMMA SUBCOMPLEX OF THE F1-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 com pared. Whereas the wild-type complex hydrolyzes 50 mu M ATP in three k inetic 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 c ontrast, prior incubation of the mutant complex under these conditions does not affect the kinetics of ATP hydrolysis. The ATPase activity o f the wild-type complex is stimulated 4-fold by 0.1% lauryl dimethylam ine oxide, whereas this concentration of lauryl dimethylamine oxide in hibits 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 muta nt complex does not-entrap substantial inhibitory MgADP in a catalytic site during turnover, which is supported by the following observation s. ATP hydrolysis catalyzed by the wild-type complex is progressively inhibited by increasing concentrations of Mg2+ in the assay medium, wh ereas the mutant complex is insensitive to increasing concentrations o f Mg2+. A Lineweaver-Burk plot constructed from rates of hydrolysis of 20-2000 mu M ATP by the wild-type complex is biphasic, exhibiting app arent K-m values of 30 Cur and 470 mu M with corresponding k(cat) valu es of 26 and 77 s(-1). In contrast; a Lineweaver-Burk plot for the mut ant 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).