β Subunit Glu-185 of Escherichia coli H+-ATPase (ATP synthase) is an essential residue for cooperative catalysis

Hiroshi Omote, Phi Le Nga Phi Le, M. Y. Park, M. Maeda, M. Futai

Research output: Contribution to journalArticle

31 Citations (Scopus)

Abstract

Glu-β185 of the Escherichia coil H+-ATPase (ATP synthase) β subunit was replaced by 19 different amino acid residues. The rates of multisite (steady state) catalysis of all the mutant membrane ATPases except Asp-β185 were less than 0.2% of the wild type one; the Asp-β185 enzyme exhibited 15% (purified) and 16% (membrane-bound) ATPase activity. The purified inactive Cys-β185 F1-ATPase recovered substantial activity after treatment with iodoacetate in the presence of MgCl2; maximal activity was obtained upon the introduction of about 3 mol of carboxymethyl residues/mol of F1. The divalent cation dependences of the S-carboxymethyl-β185 and Asp-β185 ATPase activities were altered from that of the wild type. The Asp-β185, Cys- β185, S-carboxymethyl-β185, and Gln-β185 enzymes showed about 130, 60, 20, and 50% of the wild type unisite catalysis rates, respectively. The S- carboxymethyl-β185 and Asp-β85 enzymes showed altered divalent cation sensitivities, and the S-carboxymethyl-β185 enzyme showed no Mg2+ inhibition. Unlike the wild type, the two mutant enzymes showed low sensitivities to azide, which stabilizes the enzyme Mg-ADP complex. These results suggest that Glu-β185 may form a Mg2+ binding site, and its carboxyl moiety is essential for catalytic cooperativity. Consistent with this model, the bovine glutamate residue corresponding to Glu-β185 is located close to the catalytic site in the higher order structure (Abrahams, J.P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621- 628).

Original languageEnglish
Pages (from-to)25656-25660
Number of pages5
JournalJournal of Biological Chemistry
Volume270
Issue number43
DOIs
Publication statusPublished - 1995
Externally publishedYes

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Proton-Translocating ATPases
Catalysis
Escherichia coli
Adenosine Triphosphate
Enzymes
Adenosine Triphosphatases
Divalent Cations
Iodoacetates
Membranes
Escherichia
Magnesium Chloride
Azides
Adenosine Diphosphate
Glutamic Acid
Catalytic Domain
Binding Sites
Amino Acids

ASJC Scopus subject areas

  • Biochemistry

Cite this

β Subunit Glu-185 of Escherichia coli H+-ATPase (ATP synthase) is an essential residue for cooperative catalysis. / Omote, Hiroshi; Nga Phi Le, Phi Le; Park, M. Y.; Maeda, M.; Futai, M.

In: Journal of Biological Chemistry, Vol. 270, No. 43, 1995, p. 25656-25660.

Research output: Contribution to journalArticle

Omote, Hiroshi ; Nga Phi Le, Phi Le ; Park, M. Y. ; Maeda, M. ; Futai, M. / β Subunit Glu-185 of Escherichia coli H+-ATPase (ATP synthase) is an essential residue for cooperative catalysis. In: Journal of Biological Chemistry. 1995 ; Vol. 270, No. 43. pp. 25656-25660.
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title = "β Subunit Glu-185 of Escherichia coli H+-ATPase (ATP synthase) is an essential residue for cooperative catalysis",
abstract = "Glu-β185 of the Escherichia coil H+-ATPase (ATP synthase) β subunit was replaced by 19 different amino acid residues. The rates of multisite (steady state) catalysis of all the mutant membrane ATPases except Asp-β185 were less than 0.2{\%} of the wild type one; the Asp-β185 enzyme exhibited 15{\%} (purified) and 16{\%} (membrane-bound) ATPase activity. The purified inactive Cys-β185 F1-ATPase recovered substantial activity after treatment with iodoacetate in the presence of MgCl2; maximal activity was obtained upon the introduction of about 3 mol of carboxymethyl residues/mol of F1. The divalent cation dependences of the S-carboxymethyl-β185 and Asp-β185 ATPase activities were altered from that of the wild type. The Asp-β185, Cys- β185, S-carboxymethyl-β185, and Gln-β185 enzymes showed about 130, 60, 20, and 50{\%} of the wild type unisite catalysis rates, respectively. The S- carboxymethyl-β185 and Asp-β85 enzymes showed altered divalent cation sensitivities, and the S-carboxymethyl-β185 enzyme showed no Mg2+ inhibition. Unlike the wild type, the two mutant enzymes showed low sensitivities to azide, which stabilizes the enzyme Mg-ADP complex. These results suggest that Glu-β185 may form a Mg2+ binding site, and its carboxyl moiety is essential for catalytic cooperativity. Consistent with this model, the bovine glutamate residue corresponding to Glu-β185 is located close to the catalytic site in the higher order structure (Abrahams, J.P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621- 628).",
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T1 - β Subunit Glu-185 of Escherichia coli H+-ATPase (ATP synthase) is an essential residue for cooperative catalysis

AU - Omote, Hiroshi

AU - Nga Phi Le, Phi Le

AU - Park, M. Y.

AU - Maeda, M.

AU - Futai, M.

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N2 - Glu-β185 of the Escherichia coil H+-ATPase (ATP synthase) β subunit was replaced by 19 different amino acid residues. The rates of multisite (steady state) catalysis of all the mutant membrane ATPases except Asp-β185 were less than 0.2% of the wild type one; the Asp-β185 enzyme exhibited 15% (purified) and 16% (membrane-bound) ATPase activity. The purified inactive Cys-β185 F1-ATPase recovered substantial activity after treatment with iodoacetate in the presence of MgCl2; maximal activity was obtained upon the introduction of about 3 mol of carboxymethyl residues/mol of F1. The divalent cation dependences of the S-carboxymethyl-β185 and Asp-β185 ATPase activities were altered from that of the wild type. The Asp-β185, Cys- β185, S-carboxymethyl-β185, and Gln-β185 enzymes showed about 130, 60, 20, and 50% of the wild type unisite catalysis rates, respectively. The S- carboxymethyl-β185 and Asp-β85 enzymes showed altered divalent cation sensitivities, and the S-carboxymethyl-β185 enzyme showed no Mg2+ inhibition. Unlike the wild type, the two mutant enzymes showed low sensitivities to azide, which stabilizes the enzyme Mg-ADP complex. These results suggest that Glu-β185 may form a Mg2+ binding site, and its carboxyl moiety is essential for catalytic cooperativity. Consistent with this model, the bovine glutamate residue corresponding to Glu-β185 is located close to the catalytic site in the higher order structure (Abrahams, J.P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621- 628).

AB - Glu-β185 of the Escherichia coil H+-ATPase (ATP synthase) β subunit was replaced by 19 different amino acid residues. The rates of multisite (steady state) catalysis of all the mutant membrane ATPases except Asp-β185 were less than 0.2% of the wild type one; the Asp-β185 enzyme exhibited 15% (purified) and 16% (membrane-bound) ATPase activity. The purified inactive Cys-β185 F1-ATPase recovered substantial activity after treatment with iodoacetate in the presence of MgCl2; maximal activity was obtained upon the introduction of about 3 mol of carboxymethyl residues/mol of F1. The divalent cation dependences of the S-carboxymethyl-β185 and Asp-β185 ATPase activities were altered from that of the wild type. The Asp-β185, Cys- β185, S-carboxymethyl-β185, and Gln-β185 enzymes showed about 130, 60, 20, and 50% of the wild type unisite catalysis rates, respectively. The S- carboxymethyl-β185 and Asp-β85 enzymes showed altered divalent cation sensitivities, and the S-carboxymethyl-β185 enzyme showed no Mg2+ inhibition. Unlike the wild type, the two mutant enzymes showed low sensitivities to azide, which stabilizes the enzyme Mg-ADP complex. These results suggest that Glu-β185 may form a Mg2+ binding site, and its carboxyl moiety is essential for catalytic cooperativity. Consistent with this model, the bovine glutamate residue corresponding to Glu-β185 is located close to the catalytic site in the higher order structure (Abrahams, J.P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621- 628).

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