Effects of mutations of conserved Lys-155 and Thr-156 residues in the phosphate-binding glycine-rich sequence of the F1-ATPase β subunit of Escherichia coli

Hiroshi Omote, Masatomo Maeda, Masamitsu Futai

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Abstract

βLys-155 in the glycine-rich sequence of the β subunit of Escherichia coli F1-ATPase has been shown to be near the γ-phosphate moiety of ATP by affinity labeling (Ida, K., Noumi, T., Maeda, M., Fukui, T., and Futai, M. (1991) J. Biol. Chem. 266, 5424-5429). For examination of the roles of βLyS-155 and βThr-156, mutants (βLys-155 → Ala, Ser, or Thr; βThr-156 → Ala, Cys, Asp, or Ser; βLyS-155/βThr-156 → βThr-155/βLys-156; and βThr-156/βVal-157 → βAla-156/βThr-157) were constructed, and their properties were studied extensively. The βSer-156 mutant was active in ATP synthesis and had ∼1.5-fold higher membrane ATPase activity than the wild type. Other mutants were defective in ATP synthesis, had 1 in their membranes as the wild type. Purified mutant enzymes (βAla-155, βSer-155, βAla-156, and βCys-156) showed low rates of multisite (1 values of the mutant enzymes for unisite catalysis were lower than that of the wild type: not detectable with the βAla-156 and βCys-156 enzymes and 102-fold lower with the βAla-155 and βSer-155 enzymes. The βThr-156 → Ala or Cys enzyme showed an altered response to Mg2+, suggesting that βThr-156 may be closely related to Mg2+ binding. These results suggest that βLys-155 and βThr-156 are essential for catalysis and are possibly located in the catalytic site, although βThr-156 could be replaced by a serine residue.

Original languageEnglish
Pages (from-to)20571-20576
Number of pages6
JournalJournal of Biological Chemistry
Volume267
Issue number29
Publication statusPublished - Oct 15 1992
Externally publishedYes

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Proton-Translocating ATPases
Glycine
Escherichia coli
Phosphates
Mutation
Enzymes
Adenosine Triphosphate
Catalysis
Membranes
Viperidae
Labeling
Serine
Adenosine Triphosphatases
Catalytic Domain

ASJC Scopus subject areas

  • Biochemistry

Cite this

Effects of mutations of conserved Lys-155 and Thr-156 residues in the phosphate-binding glycine-rich sequence of the F1-ATPase β subunit of Escherichia coli. / Omote, Hiroshi; Maeda, Masatomo; Futai, Masamitsu.

In: Journal of Biological Chemistry, Vol. 267, No. 29, 15.10.1992, p. 20571-20576.

Research output: Contribution to journalArticle

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abstract = "βLys-155 in the glycine-rich sequence of the β subunit of Escherichia coli F1-ATPase has been shown to be near the γ-phosphate moiety of ATP by affinity labeling (Ida, K., Noumi, T., Maeda, M., Fukui, T., and Futai, M. (1991) J. Biol. Chem. 266, 5424-5429). For examination of the roles of βLyS-155 and βThr-156, mutants (βLys-155 → Ala, Ser, or Thr; βThr-156 → Ala, Cys, Asp, or Ser; βLyS-155/βThr-156 → βThr-155/βLys-156; and βThr-156/βVal-157 → βAla-156/βThr-157) were constructed, and their properties were studied extensively. The βSer-156 mutant was active in ATP synthesis and had ∼1.5-fold higher membrane ATPase activity than the wild type. Other mutants were defective in ATP synthesis, had 1 in their membranes as the wild type. Purified mutant enzymes (βAla-155, βSer-155, βAla-156, and βCys-156) showed low rates of multisite (1 values of the mutant enzymes for unisite catalysis were lower than that of the wild type: not detectable with the βAla-156 and βCys-156 enzymes and 102-fold lower with the βAla-155 and βSer-155 enzymes. The βThr-156 → Ala or Cys enzyme showed an altered response to Mg2+, suggesting that βThr-156 may be closely related to Mg2+ binding. These results suggest that βLys-155 and βThr-156 are essential for catalysis and are possibly located in the catalytic site, although βThr-156 could be replaced by a serine residue.",
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