Bandyopadhyay Sanjay, Valder Carolina R, Huynh Hue G, Ren Huimiao, Allison William S
Department of Chemistry & Biochemistry, University of California at San Diego, La Jolla, California 92093-0601, USA.
Biochemistry. 2002 Dec 3;41(48):14421-9. doi: 10.1021/bi026243g.
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.
用核苷酸对TF₁的α(3)(βG(156)C/Y(345)W)(3)γ、α(3)(βE(199)V/Y(345)W)(3)γ和α(3)(βY(345)W)(3)γ亚复合物进行荧光滴定表明，βG(156)C取代显著降低了催化位点对有或无Mg²⁺时ATP和ADP的亲和力，而βE(199)V取代增加了催化位点对核苷酸的亲和力。野生型酶水解2 mM ATP的速率分别为100%，而α(3)(βG(156)C)(3)γ和α(3)(βE(199)V)(3)γ亚复合物水解2 mM ATP的速率分别为2%和0.7%，α(3)(βG(156)C/E(199)V)(3)γ水解2 mM ATP的速率为野生型酶的9%。α(3)(βG(156)C)(3)γ、α(3)(βG(156)C/E(199)V)(3)γ和α(3)(βG(156)C/E(199)V/Y(345)W)(3)γ亚复合物在周转过程中能抵抗催化位点中抑制性MgADP的截留。当野生型、βE(199)V、βY(345)W和βE(199)V/Y(345)W亚复合物水解亚化学计量的[(3)H]ATP时，产物[(3)H]ADP紧密结合在单个催化位点上，而βG(156)C和βG(156)C/Y(345)W亚复合物则不保留产物。当βG(156)C/E(199)V和βG(156)C/E(199)V/Y(345)W突变体水解亚化学计量的[(3)H]ATP时，结合不太牢固的产物[(3)H]ADP被保留。用βG(156)C突变体得到的Lineweaver - Burk图向下弯曲，表明其催化位点在ATP水解过程中独立起作用。相反βG(156)C/E(199)V和βG(156)C/E(199)V/Y(345)W突变体水解ATP时，Lineweaver - Burk图呈线性，表明是协同三位点催化。似乎βG(156)C取代以一种允许在没有催化位点协同性的情况下释放产物的方式破坏了水解MgATP催化位点的封闭构象。将βE(199)V取代插入βG(156)C突变体中，通过限制水解MgATP的催化位点打开以释放产物，直到开放的催化位点结合MgATP，从而恢复协同性。
