Mutations in Ser174 and the glycine-rich sequence (Gly149, Gly150, and Thr156) in the beta subunit of Escherichia coli H(+)-ATPase.
Mutations in Ser174 and the glycine-rich sequence (Gly149, Gly150, and Thr156) in the beta subunit of Escherichia coli H(+)-ATPase.
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大肠杆菌 H()-ATP 酶 β 亚基中 Ser174 和富含甘氨酸的序列（Gly149、Gly150 和 Thr156）中的突变。
DOI：
10.1016/s0021-9258(18)55304-7
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发表时间：
1991
期刊：
影响因子：
--
通讯作者：
M. Futai
中科院分区：
文献类型：
--
作者：
A. Iwamoto;H. Omote;H. Hanada;N. Tomioka;Akiko Itai;Masatomo Maeda;M. Futai
大肠杆菌 F1 β 亚基中的序列基序（Gly-Gly-Ala-Gly-Val-Gly-Lys-Thr，残基 149-156，其中保守残基用下划线表示）是许多核苷酸结合蛋白中发现的富含甘氨酸的序列之一。在本研究中，我们构建了携带所有 F0F1 基因的质粒。该质粒具有迄今为止报道的最高的膜 ATP 酶活性。用Ser取代βGly149可以抑制βSer174——Phe突变（H(+)-ATP酶缺陷）的作用，但βGly150——Ser取代没有这种作用。单一突变（β Gly149---Ser或β Gly150---Ser）使活性酶具有改变的二价阳离子依赖性和叠氮化物敏感性：β Gly149---Ser突变酶的叠氮化物敏感性降低100倍，并且基本上没有Ca（2+）依赖性活性，但具有野生型水平的Mg（2+）依赖性活性和活性氧化磷酸化。引入β Gly149——Ser或β Gly150——Ser突变与β Ser174——Phe突变也降低了Ca(2+)依赖性活性和叠氮化物敏感性。与我们之前的发现一致（Takeyama, M., Ihara, K., Moriyama, Y., Noumi, T., Ida, K., Tomioka, N., Itai, A., Maeda, M., and Futai, M. (1990) J. Biol. Chem. 265, 21279-21284），β Thr156—Ala 或 Cys 突变受损 ATP 酶活性，表明 156 位的羟基部分对于催化活性至关重要。讨论了包括二价阳离子结合位点的催化位点的可能位置。
A sequence motif in the beta subunit of Escherichia coli F1 (Gly-Gly-Ala-Gly-Val-Gly-Lys-Thr, residue 149-156, where conserved residues are underlined) is one of the glycine-rich sequences found in many nucleotide binding proteins. In this study, we constructed a plasmid carrying all the F0F1 genes. This plasmid gave the highest membrane ATPase activity so far reported. Substitution of beta Gly149 by Ser suppressed the effect of the beta Ser174—-Phe mutation (defective H(+)-ATPase), but beta Gly150—-Ser substitution did not have this effect. A single mutation (beta Gly149—-Ser or beta Gly150—-Ser) gave active enzyme with altered divalent cation dependency and azide sensitivity: the beta Gly149—-Ser mutant enzyme had 100-fold lower azide sensitivity and essentially no Ca(2+)-dependent activity, but had the wild-type level of Mg(2+)-dependent activity with active oxidative phosphorylation. Introduction of a beta Gly149—-Ser or beta Gly150—-Ser mutation with the beta Ser174—-Phe mutation also lowered the Ca(2+)-dependent activity and azide sensitivity. Consistent with our previous findings (Takeyama, M., Ihara, K., Moriyama, Y., Noumi, T., Ida, K., Tomioka, N., Itai, A., Maeda, M., and Futai, M. (1990) J. Biol. Chem. 265, 21279-21284), a beta Thr156—-Ala or Cys mutation impaired ATPase activity, suggesting that the hydroxyl moiety at position 156 is essential for the catalytic activity. The possible location of the catalytic site including divalent cation binding site(s) is discussed.