ROTATION OF SUBUNITS DURING CATALYSIS BY ESCHERICHIA-COLI F1-ATPASE
ROTATION OF SUBUNITS DURING CATALYSIS BY ESCHERICHIA-COLI F1-ATPASE
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DOI：
10.1073/pnas.92.24.10964
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发表时间：
1995-11-21
影响因子：
11.1
通讯作者：
CROSS, RL
中科院分区：
文献类型：
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作者：
DUNCAN, TM;BULYGIN, VV;CROSS, RL
在氧化磷酸化和光磷酸化过程中，F0 F1-ATP酶将质子沿电化学梯度的运动与ATP的合成偶联。一个提出的机制特征仍然是推测性的，该偶联过程需要F0 F1内亚基的旋转。在最近牛F-1的高分辨率结构的指导下[Abrahams，J. P.，莱斯利，A，G，吕特河&步行者，J，E，（1994）Nature（伦敦）370，621-628]，我们已经开发了一种临界测试，用于在来自大肠杆菌的可溶性F-1中中心γ亚基相对于三个催化β亚基的旋转。在牛F-1结构中，γ亚基和三个催化β亚基之一之间的特定接触点包括E的同源物的定位。coli γ-亚基C-87（γ C87）与β-亚基（380）DELSEED（386）序列接近。β D380 C突变使我们能够诱导可溶性大肠杆菌中β和γ C87之间形成特异性二硫键。coli F-1。交联失活的β D380 C-F-1的形成和还原恢复了完全活性。使用交联β D380 C-F-1的解离/重组方法，我们将放射性标记的β亚基掺入F-1的两个非交联β亚基位置。在还原初始非放射性β-γ交联后，仅暴露于催化转换条件下，未标记和放射性标记的β亚基在再氧化后与γ C87具有相似的反应性。结果表明，γ亚基在催化过程中相对于β亚基旋转。
During oxidative and photo-phosphorylation, F0F1-ATP synthases couple the movement of protons down an electrochemical gradient to the synthesis of ATP, One proposed mechanistic feature that has remained speculative is that this coupling process requires the rotation of subunits within F0F1. Guided by a recent, high-resolution structure for bovine F-1 [Abrahams, J. P., Leslie, A, G., Lutter, R. & Walker, J, E, (1994) Nature (London) 370, 621-628], we have developed a critical test for rotation of the central gamma subunit relative to the three catalytic beta subunits in soluble F-1 from Escherichia coli. In the bovine F-1 structure, a specific point of contact between the gamma subunit and one of the three catalytic beta subunits includes positioning of the homolog of E. coli gamma-subunit C-87 (gamma C87) close to the beta-subunit (380)DELSEED(386) sequence. A beta D380C mutation allowed us to induce formation of a specific disulfide bond between beta and gamma C87 in soluble E. coli F-1. Formation of the crosslink inactivated beta D380C-F-1, and reduction restored full activity. Using a dissociation/reassembly approach with cross-linked beta D380C-F-1, we incorporated radiolabeled beta subunits into the two noncrosslinked beta-subunit positions of F-1, After reduction of the initial nonradioactive beta-gamma crosslink, only exposure to conditions for catalytic turnover results in similar reactivities of unlabeled and radiolabeled beta subunits with gamma C87 upon reoxidation. The results demonstrate that gamma subunit rotates relative to the beta subunits during catalysis.