McMillan Duncan G G, Watanabe Rikiya, Ueno Hiroshi, Cook Gregory M, Noji Hiroyuki
From the Department of Applied Chemistry, Graduate School of Engineering, The University of Tokyo, Tokyo 113-8656, Japan and
From the Department of Applied Chemistry, Graduate School of Engineering, The University of Tokyo, Tokyo 113-8656, Japan and.
J Biol Chem. 2016 Nov 11;291(46):23965-23977. doi: 10.1074/jbc.M116.743633. Epub 2016 Sep 13.
FF ATP synthases are bidirectional molecular motors that translocate protons across the cell membrane by either synthesizing or hydrolyzing ATP. Alkaliphile ATP synthases are highly adapted, performing oxidative phosphorylation at high pH against an inverted pH gradient (acid/alkaline). Unlike mesophilic ATP synthases, alkaliphilic enzymes have tightly regulated ATP hydrolysis activity, which can be relieved in the presence of lauryldimethylamine oxide. Here, we characterized the rotary dynamics of the Caldalkalibacillus thermarum TA2.A1 F ATPase (TA2F) with two forms of single molecule analysis, a magnetic bead duplex and a gold nanoparticle. TA2F rotated in a counterclockwise direction in both systems, adhering to Michaelis-Menten kinetics with a maximum rotation rate (V) of 112.4 revolutions/s. TA2F displayed 120° unitary steps coupled with ATP hydrolysis. Torque measurements revealed the highest torque (52.4 piconewtons) derived from an F molecule using fluctuation theorem. The implications of high torque in terms of extreme environment adaptation are discussed.
FF型ATP合酶是双向分子马达，可通过合成或水解ATP使质子跨细胞膜转运。嗜碱菌ATP合酶具有高度适应性，能在高pH值下逆着反向pH梯度（酸/碱）进行氧化磷酸化。与嗜温ATP合酶不同，嗜碱酶的ATP水解活性受到严格调控，在存在月桂基二甲基氧化胺的情况下这种调控可被解除。在此，我们通过两种单分子分析形式，即磁珠双链体和金纳米颗粒，对嗜热栖热放线菌TA2.A1 F型ATP酶（TA2F）的旋转动力学进行了表征。TA2F在两个系统中均沿逆时针方向旋转，遵循米氏动力学，最大旋转速率（V）为112.4转/秒。TA2F在ATP水解时呈现120°的单位步幅。扭矩测量显示，利用涨落定理从一个F分子获得的最高扭矩为52.4皮牛。文中讨论了高扭矩在极端环境适应性方面的意义。
