The βG156C substitution in the F1-ATPase from the thermophilic Bacillus PS3 affects catalytic site cooperativity by destabilizing the closed conformation of the catalytic site

Fluorescence titrations of the alpha(3)(betaG(156)C/(YW)-W-345)(3)gamma, alpha(3)(betaE(199)V/(YW)-W-345)(3)gamma, and alpha(3)(betaY(345)W)(3)gamma subcomplexes of TF1 with nuclectides show that the betaG(156)C substitution substantially lowers the affinity of catalytic sites for ATP and ADP with or without Mg2+, whereas the betaE(199)V substitution increases the affinity of catalytic sites for nucleotides. Whereas the alpha3(betaG(156)C)(3)gamma and alpha(3)(betaE(199)V)(3)gamma subcomplexes hydrolyze 2 mM ATP at 2% and 0.7%, respectively, of the rate exhibited by the wild-type enzyme, the alpha(3)(betaG(156)C/(EV)-V-199)(3)gamma hydrolyzes 2 mM ATP at 9% the rate exhibited by the wild-type enzyme. The alpha(3)(betaG(156)C)(3)gamma, (betaG(156)C/(EV)-V-199)(3)gamma, and alpha(3)(betaG(156)C/E199V)(3)gamma alpha(3)(betaG(156)V/(YW)-W-345)(3)gamma subcomplexes resist entrapment of inhibitory MgADP in a catalytic site during turnover. Product [H-3]ADP remains tightly bound to a single catalytic site when the wild-type, betaE(199)V, betaY(345)W, and betaE(199)V/(YW)-W-345 subcomplexes hydrolyze substoichiometric [3H]ATP, whereas it is not retained by the betaG(156)C and betaG(156)C/(YW)-W-345 subcomplexes. Less firmly bound, product [3H]ADP is retained when the betaG(156)C/(EV)-V-199 and betaG(156)C/(EV)-V-199/(YW)-W-345 mutants hydrolyze substoichiometric [3H]ATP. The Lineweaver-Burk plot obtained with the betaG(156)C mutant is curved downward in a manner indicating that its catalytic sites act independently during ATP hydrolysis. In contrast, the betaG(156)C/(EV)-V-199 and betaG(156)C/(EV)-V-199/(YW)-W-345 mutants hydrolyze ATP with linear Lineweaver-Burk plots, indicating cooperative trisite catalysis. It appears that the betaG(156)C substitution destabilizes the closed conformation of a catalytic site hydrolyzing MYATP in a manner that allows release of products in the absence of catalytic site cooperativity. Insertion of the betaE(199)V substitution into the betaG(156)C mutant restores cooperativity by restricting opening of the catalytic site hydrolyzing MgATP for product release until an open catalytic site binds MgATP.