Zhang Y, Fillingame R H
Department of Biomolecular Chemistry, University of Wisconsin Medical School, Madison 53706.
J Biol Chem. 1994 Feb 18;269(7):5473-9.
Subunit c of the F1F0 type, H(+)-transporting ATP synthase contains an essential Asp that is thought to function in H+ transport. Subunit c folds as a helical hairpin of two transmembrane helices with the essential Asp centered at residue 61 in transmembrane helix-2. Miller et al. (Miller, M. J., Olderburg, M., and Fillingame, R. H. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 4900-4904) have described a functional subunit c variant in which the essential Asp was moved from helix-2 to residue 24 on helix-1 with replacement of Asp61 by Gly. The function of the A24D/D61G subunit c variant is not optimal. In this study, 11 position 61 variants of an A24D subunit c were generated by site-directed mutagenesis in order to test the importance of the position 61 residue. Three functional combinations were found with activities in the order:A24D/D61N > A24D/D61G > or = A24D/D61S. Other substitutions at position 61, including Ala and Cys, did not support function in the A24D protein. Although the A24D/D61N variant showed the highest rates of ATPase-coupled H+ transport, its F0 was inactive in passive H+ transport when F1 was stripped from the membrane. On the other hand, passive H+ transport by A24D/D61G and A24D/D61S stripped membranes approached that of wild type. The defect in function in these two mutants must be ascribed to events related to coupling ATPase and H+ transport. An A24D subunit c (with Asp at both position 24 and 61) was also generated. Its function proved to be pH-dependent. Activity approaching that of wild type was observed at pH 7.0, but function was almost completely lost at pH 7.8. The pH-dependent loss of ATP synthase function led to a slowing of growth on succinate as carbon source on raising the pH from 7.0 to 7.8. In the A24D mutant, with a second Asp at position 61, we postulate that 1 Asp must be protonated before the other can function in H+ transport.
F1F0型H⁺转运ATP合酶的c亚基含有一个必需的天冬氨酸，被认为在H⁺转运中起作用。c亚基折叠成由两个跨膜螺旋组成的螺旋发夹结构，必需的天冬氨酸位于跨膜螺旋-2的第61位残基中心。米勒等人（米勒，M. J.，奥尔德伯格，M.，和菲林加姆，R. H.（1990年）《美国国家科学院院刊》87，4900 - 4904）描述了一种功能性c亚基变体，其中必需的天冬氨酸从螺旋-2移至螺旋-1的第24位残基，天冬氨酸61被甘氨酸取代。A24D/D61G c亚基变体的功能并非最佳。在本研究中，通过定点诱变产生了11种A24D c亚基的61位变体，以测试61位残基的重要性。发现了三种功能组合，活性顺序为：A24D/D61N > A24D/D61G > 或 = A24D/D61S。61位的其他取代，包括丙氨酸和半胱氨酸，在A24D蛋白中不支持功能。尽管A24D/D61N变体显示出最高的ATP酶偶联H⁺转运速率，但其F0在从膜上剥离F1时在被动H⁺转运中无活性。另一方面，A24D/D61G和A24D/D61S剥离膜的被动H⁺转运接近野生型。这两个突变体功能上的缺陷必定归因于与ATP酶和H⁺转运偶联相关的事件。还产生了一个A24D c亚基（第24位和61位均为天冬氨酸）。其功能被证明是pH依赖性的。在pH 7.0时观察到活性接近野生型，但在pH 7.8时功能几乎完全丧失。随着pH从7.0升高到7.8，ATP合酶功能的pH依赖性丧失导致以琥珀酸作为碳源时生长减慢。在A24D突变体中，第61位有第二个天冬氨酸，我们推测一个天冬氨酸必须质子化后另一个才能在H⁺转运中起作用。
