Fluorescence titrations of the alpha(3)(betaG(156)C/Y(345)W)(3)gamma, alpha(3)(betaE(199)V/Y(345)W)(3)gamma,
and alpha(3)(betaY(345)W)(3)gamma subcomplexes of TF(1) with nucleotides show that
the betaG(156)C substitution substantially lowers the affinity of catalytic sites
for ATP and ADP with or without Mg(2+), whereas the betaE(199)V substitution increases
the affinity of catalytic sites for nucleotides. Whereas the alpha(3)(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/E(199)V)(3)gamma
hydrolyzes 2 mM ATP at 9% the rate exhibited by the wild-type enzyme. The alpha(3)(betaG(156)C)(3)gamma,
alpha(3)(betaG(156)C/E(199)V)(3)gamma, and alpha(3)(betaG(156)C/E(199)V/Y(345)W)(3)gamma
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,
betaE(199)V, betaY(345)W, and betaE(199)V/Y(345)W subcomplexes hydrolyze substoichiometric
[(3)H]ATP, whereas it is not retained by the betaG(156)C and betaG(156)C/Y(345)W subcomplexes.
Less firmly bound, product [(3)H]ADP is retained when the betaG(156)C/E(199)V and
betaG(156)C/E(199)V/Y(345)W mutants hydrolyze substoichiometric [(3)H]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/E(199)V and betaG(156)C/E(199)V/Y(345)W 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 MgATP 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.