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

A. Iwamoto, H. Omote, H. Hanada, N. Tomioka, A. Itai, M. Maeda, M. Futai

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48 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)16350-16355
Number of pages6
JournalJournal of Biological Chemistry
Volume266
Issue number25
Publication statusPublished - Nov 1 1991
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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