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Bandyopadhyay S, Valder CR, Huynh HG, Ren H, Allison WS: The beta G156C substitution in the F1-ATPase from the thermophilic Bacillus PS3 affects catalytic site cooperativity by destabilizing the closed conformation of the catalytic site. Biochemistry. 2002 Dec 3;41(48):14421-9.
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. |
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