F1-ATPase, the C-terminal end of subunit γ is not required for ATP hydrolysis-driven rotation
F1-ATPase, the C-terminal end of subunit γ is not required for ATP hydrolysis-driven rotation
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DOI：
10.1074/jbc.m201998200
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发表时间：
2002-06-28
影响因子：
4.8
通讯作者：
Engelbrecht, S
中科院分区：
文献类型：
--
作者：
Müller, M;Pänke, O;Engelbrecht, S
分离的 F-1-ATP 酶进行的 ATP 水解驱动中心轴（γ 亚基）旋转，该中心轴位于亚基 (alphabeta) (3) 形成的六边形内。 γ 的 C 末端形成一个 α 螺旋，该螺旋正确地适合由 α 和 β 亚基环提供的“疏水轴承”。预计该“轴承”对于旋转功能至关重要。我们通过连续删除 3、6、9、12、15 和 18 个氨基酸残基（大肠杆菌 F-1-ATP 酶）的 C 端来检查该接触区域的重要性。删除了 12 个残基的 F-1 的无负载集合的 ATP 水解活性下降至对照的 24%。缺失 15 或 18 个残基的 EF1 失去活性，可能是因为它无法组装。然而，当荧光肌动蛋白丝加载时，EF1 单分子产生的平均扭矩不受最多 12 个残基删除的影响，其旋转行为也是如此（所有样品在 60 +/- 19% 的观察时间内旋转）。整体的活化能分析显示，从 EF1（全长 gamma）的 54 kJ/mol 适度降低到 EF1(gamma-12) 的 34 kJ/mol。这些观察结果表明，亚基 γ C 末端的完整性在酶的组装过程中提供了结构稳定性和/或路径，但在负载下的旋转作用并不需要它。
ATP hydrolysis by the isolated F-1-ATPase drives the rotation of the central shaft, subunit gamma, which is located within a hexagon formed by subunits (alphabeta)(3). The C-terminal end of gamma forms an alpha-helix which properly fits into the "hydrophobic bearing" provided by loops of subunits alpha and beta. This "bearing" is expected to be essential for the rotary function. We checked the importance of this contact region by successive C-terminal deletions of 3, 6, 9, 12, 15, and 18 amino acid residues (Escherichia coli F-1-ATPase). The ATP hydrolysis activity of a load-free ensemble of F-1 with 12 residues deleted decreased to 24%, of the control. EF1 with deletions of 15 or 18 residues was inactive, probably because it failed to assemble. The average torque generated by a single molecule of EF1 when loaded by a fluorescent actin filament was, however, unaffected by deletions of up to 12 residues, as was their rotational behavior (all samples rotated during 60 +/- 19% of the observation time). Activation energy analysis with the ensemble revealed a moderate decrease from 54 kJ/mol for EF1 (full-length gamma) to 34 kJ/mol for EF1(gamma-12). These observations imply that the intactness of the C terminus of subunit gamma provides structural stability and/or routing during assembly of the enzyme, but that it is not required for the rotary action under load, proper.