Mutations in Ser¹⁷⁴ and the glycine-rich sequence (Gly¹⁴⁹, Gly¹⁵⁰, and Thr¹⁵⁶) in the β subunit of Escherichia coli H⁺-ATPase

A sequence motif in the β 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 βGly¹⁴⁹ by Ser suppressed the effect of the βSer¹⁷⁴ → Phe mutation (defective H⁺-ATPase), but βGly¹⁵⁰ → Ser substitution did not have this effect. A single mutation (βGly¹⁴⁹ → Ser or βGly¹⁵⁰ → Ser) gave active enzyme with altered divalent cation dependency and azide sensitivity: the βGly¹⁴⁹ → Ser mutant enzyme had 100-fold lower azide sensitivity and essentially no Ca²⁺-dependent activity, but had the wild-type level of Mg²⁺-dependent activity with active oxidative phosphorylation. Introduction of a βGly¹⁴⁹ → Ser or βGly¹⁵⁰ → Ser mutation with the βSer¹⁷⁴ → Phe mutation also lowered the Ca²⁺-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 βThr¹⁵⁶ → 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.