Mechanism of Inhibition by C-terminal α-Helices of the ε Subunit of Escherichia coli FoF1-ATP Synthase
Mechanism of Inhibition by C-terminal α-Helices of the ε Subunit of Escherichia coli FoF1-ATP Synthase
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DOI：
10.1074/jbc.m109.003798
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发表时间：
2009-06-26
影响因子：
4.8
通讯作者：
Noji, Hiroyuki
中科院分区：
文献类型：
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作者：
Iino, Ryota;Hasegawa, Rie;Noji, Hiroyuki
已知细菌FoF 1-ATP合酶（FoF 1）的α-亚基，一种旋转马达蛋白，抑制该酶的ATP水解反应。抑制作用由α-螺旋的C-末端构象调节，并且“延伸”而不是“发夹折叠”状态负责抑制。虽然ATP水解的抑制C-末端结构域的ATP已被广泛研究，对ATP合成的影响还没有完全了解。在这项研究中，我们产生了一个大肠杆菌FoF 1（EFoF 1）突变体，其中的β亚基缺乏的C-末端结构域（FoF 1 Δ C），和ATP合成驱动的酸碱转换（Δ pH值）和K+-缬氨霉素扩散电位（Δ Psi）进行了详细比较与野生型酶（FoF 1 Δ WT）。FoF 1 Δ WT的转换数（k（cat））比FoF 1 Δ C低几倍，而FoF 1 Δ WT的米氏常数（K-m）较高。FoF 1 Δ WT和FoF 1 Δ Delta C的活性对Delta pH和Delta Psi的各种组合的依赖性是相似的，这表明ATP合成中的限速步骤没有被Δ WT的C-末端结构域改变。与FoF 1 Δ C的相应值相比，溶解的FoF 1 Δ WT也显示出较低的k（cat）和较高的K-m值用于ATP水解。这些结果表明，C-末端结构域的EFoF 1的γ亚基减慢多个基本步骤的ATP合成/水解反应，通过限制旋转的γ亚基。
The epsilon subunit of bacterial FoF1-ATP synthase (FoF1), a rotary motor protein, is known to inhibit the ATP hydrolysis reaction of this enzyme. The inhibitory effect is modulated by the conformation of the C-terminal alpha-helices of epsilon, and the "extended" but not "hairpin-folded" state is responsible for inhibition. Although the inhibition of ATP hydrolysis by the C-terminal domain of epsilon has been extensively studied, the effect on ATP synthesis is not fully understood. In this study, we generated an Escherichia coli FoF1 (EFoF1) mutant in which the epsilon subunit lacked the C-terminal domain (FoF1 epsilon Delta C), and ATP synthesis driven by acid-base transition (Delta pH) and the K+-valinomycin diffusion potential (Delta Psi) was compared in detail with that of the wild-type enzyme (FoF1 epsilon WT). The turnover numbers (k(cat)) of FoF1 epsilon WT were severalfold lower than those of FoF1 epsilon Delta C.FoF1 epsilon WT showed higher Michaelis constants (K-m). The dependence of the activities of FoF1 epsilon WT and FoF1 epsilon Delta C on various combinations of Delta pH and Delta Psi was similar, suggesting that the rate-limiting step in ATP synthesis was unaltered by the C-terminal domain of epsilon. Solubilized FoF1 epsilon WT also showed lower k(cat) and higher K-m values for ATP hydrolysis than the corresponding values of FoF1 epsilon Delta C. These results suggest that the C-terminal domain of the epsilon subunit of EFoF1 slows multiple elementary steps in both the ATP synthesis/hydrolysis reactions by restricting the rotation of the gamma subunit.