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 α 3 (βG 1 5 6 C/Y 3 4 5 W) 3 γ, α 3 (βE 1 9 9 V/Y 3 4 5 W) 3 γ, and α 3 (βY 3 4 5 W) 3 γ subcomplexes of TF, with nucleotides show that the βG 1 5 6 C substitution substantially lowers the affinity of catalytic sites for ATP and ADP with or without Mg 2 + , whereas the βE 1 9 9 V substitution increases the affinity of catalytic sites for nucleotides. Whereas the α 3 (βG 1 5 6 C) 3 γ and α 3 (βE 1 9 9 V) 3 γ subcomplexes hydrolyze 2 mM ATP at 2% and 0.7%, respectively, of the rate exhibited by the wild-type enzyme, the α 3 (βG 1 5 6 C/E 1 9 9 V) 3 γ hydrolyzes 2 mM ATP at 9% the rate exhibited by the wild-type enzyme. The α 3 -(βG 1 5 6 C) 3 γ α 3 (βG 1 5 6 C/E 1 9 9 V) 3 γ, and α 3 (βG 1 5 6 C/E 1 9 9 V/Y 3 4 5 W) 3 γ subcomplexes resist entrapment of inhibitory MgADP in a catalytic site during turnover. Product [ 3 H]ADP remains tightly bound to a single catalytic site when the wild-type, βE 1 9 9 V, βY 3 4 5 W, and βE 1 9 9 V/Y 3 4 5 W subcomplexes hydrolyze substoichiometric [ 3 H]ATP, whereas it is not retained by the βG 1 5 6 C and βG 1 5 6 C/Y 3 4 5 W subcomplexes. Less firmly bound, product [ 3 H]ADP is retained when the βG 1 5 6 C/E 1 9 9 V and βG 1 5 6 C/E 1 9 9 V/Y 3 4 5 W mutants hydrolyze substoichiometric [ 3 H]ATP. The Lineweaver-Burk plot obtained with the βG 1 5 6 C mutant is curved downward in a manner indicating that its catalytic sites act independently during ATP hydrolysis. In contrast, the βG 1 5 6 C/E 1 9 9 V and βG 1 5 6 C/E 1 9 9 V/Y 3 4 5 W mutants hydrolyze ATP with linear Lineweaver-Burk plots, indicating cooperative trisite catalysis. It appears that the βG 1 5 6 C substitution destabilizes the closed conformation of a catalytic site hydrolyzing MgATP in a manner that allows release of products in the absence of catalytic site cooperativity. Insertion of the βE 1 9 9 V substitution into the βG 1 5 6 C mutant restores cooperativity by restricting opening of the catalytic site hydrolyzing MgATP for product release until an open catalytic site binds MgATP.