Ren H, Allison W S
Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, California 92093-0506, USA.
J Biol Chem. 2000 Apr 7;275(14):10057-63. doi: 10.1074/jbc.275.14.10057.
In the crystal structure of bovine mitochondrial F(1)-ATPase (MF(1)) (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628), the side chain oxygen of betaThr(163) interacts directly with Mg(2+) coordinated to 5'-adenylyl beta, gamma-imidodiphosphate or ADP bound to catalytic sites of beta subunits present in closed conformations. In the unliganded beta subunit present in an open conformation, the hydroxyl of betaThr(163) is hydrogen-bonded to the carboxylate of betaGlu(199). Substitution of betaGlu(201) (equivalent to betaGlu(199) in MF(1)) in the alpha(3)beta(3)gamma subcomplex of the F(1)-ATPase from the thermophilic Bacillus PS3 with cysteine or valine increases the propensity to entrap inhibitory MgADP in a catalytic site during hydrolysis of 50 microM ATP. These substitutions lower K(m3) (the Michaelis constant for trisite ATP hydrolysis) relative to that of the wild type by 25- and 10-fold, respectively. Fluorescence quenching of alpha(3)(betaE201C/Y341W)(3)gamma and alpha(3)(betaY341W)(3)gamma mutant subcomplexes showed that MgATP and MgADP bind to the third catalytic site of the double mutant with 8.4- and 4.4-fold higher affinity, respectively, than to the single mutant. These comparisons support the hypothesis that the hydrogen bond observed between the side chains of betaThr(163) and betaGlu(199) in the unliganded catalytic site in the crystal structure of MF(1) stabilizes the open conformation of the catalytic site during ATP hydrolysis.
在牛线粒体F₁ - ATP酶（MF₁）的晶体结构中（亚伯拉罕斯，J.P.，莱斯利，A.G.W.，卢特，R.，以及沃克，J.E.（1994年）《自然》370卷，621 - 628页），β - 苏氨酸（163）的侧链氧原子直接与与结合在处于闭合构象的β亚基催化位点上的5'-腺苷酰β，γ - 亚氨二磷酸或ADP配位的Mg²⁺相互作用。在处于开放构象的未结合配体的β亚基中，β - 苏氨酸（163）的羟基与β - 谷氨酸（199）的羧酸盐形成氢键。将嗜热芽孢杆菌PS3的F₁ - ATP酶的α₃β₃γ亚复合物中的β - 谷氨酸（201）（等同于MF₁中的β - 谷氨酸（199））用半胱氨酸或缬氨酸替代，会增加在50微摩尔ATP水解过程中催化位点截留抑制性MgADP的倾向。相对于野生型，这些替代分别使Kₘ₃（三位点ATP水解的米氏常数）降低了25倍和10倍。α₃（βE201C/Y341W）₃γ和α₃（βY341W）₃γ突变亚复合物的荧光猝灭表明，MgATP和MgADP与双突变体的第三个催化位点结合的亲和力分别比单突变体高8.4倍和4.4倍。这些比较支持了这样一种假说，即在MF₁晶体结构中未结合配体的催化位点上β - 苏氨酸（163）和β - 谷氨酸（199）侧链之间观察到的氢键在ATP水解过程中稳定了催化位点的开放构象。
