Iwamoto A, Omote H, Hanada H, Tomioka N, Itai A, Maeda M, Futai M
Institute of Scientific and Industrial Research, Osaka University, Japan.
J Biol Chem. 1991 Sep 5;266(25):16350-5.
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.
大肠杆菌F1β亚基中的一个序列基序（甘氨酸-甘氨酸-丙氨酸-甘氨酸-缬氨酸-甘氨酸-赖氨酸-苏氨酸，第149 - 156位残基，其中保守残基加下划线）是在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，我们构建了一个携带所有F0F1基因的质粒。该质粒产生了迄今为止报道的最高膜ATP酶活性。将β亚基的第149位甘氨酸替换为丝氨酸可抑制β亚基第174位丝氨酸突变为苯丙氨酸（有缺陷的H⁺ - ATP酶）的影响，但β亚基第150位甘氨酸替换为丝氨酸则没有这种作用。单个突变（β亚基第149位甘氨酸突变为丝氨酸或β亚基第150位甘氨酸突变为丝氨酸）产生了具有改变的二价阳离子依赖性和叠氮化物敏感性的活性酶：β亚基第149位甘氨酸突变为丝氨酸的突变酶对叠氮化物的敏感性降低100倍，基本上没有钙依赖性活性，但在活性氧化磷酸化时具有野生型水平的镁依赖性活性。将β亚基第149位甘氨酸突变为丝氨酸或β亚基第150位甘氨酸突变为丝氨酸与β亚基第174位丝氨酸突变为苯丙氨酸同时引入也降低了钙依赖性活性和叠氮化物敏感性。与我们之前的发现一致（武山，M.，井原，K.，森山，Y.，野见，T.，井田，K.，富冈，N.，板井，A.，前田，M.，和古田，M.（1990）《生物化学杂志》265，21279 - 21284），β亚基第156位苏氨酸突变为丙氨酸或半胱氨酸会损害ATP酶活性，表明第156位的羟基部分对催化活性至关重要。本文讨论了包括二价阳离子结合位点在内的催化位点的可能位置。
