Effects of monovalent cations and divalent metal ions on Escherichia coli selenophosphate synthetase

Ick Young Kim, Thressa C. Stadtman

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18 Citations (Scopus)

Abstract

A labile selenium donor compound, selenophosphate, is formed from selenide and ATP by selenophosphate synthetase. A divalent metal ion, Mg2+, and a monovalent cation, K+, NH4/+, or Rb+, are required for selenophosphate synthetase activity [Veres, Z., Kim, I. Y., Scholz, T. D. and Stadtman, T. C. (1994) J. Biol. Chem. 269, 10597-10603]. Na+ and Li+ are ineffective as activators and in the presence of K+ are inhibitory. Mn-ATP, although not able to replace Mg-ATP for catalytic activity, binds to the enzyme provided an active monovalent cation is present. No Mn-ATP is bound when K+ is replaced with Na+. The requirement for K+, both for Mn-ATP binding and for catalytic activity of the synthetase, indicates a specific monovalent cation- induced conformational state of the enzyme. Previously we reported that activity of the enzyme is markedly inhibited by micromolar levels of Zn2+ in the presence of millimolar levels of Mg2+ [Kim, I. Y., Veres, Z., and Stadtman, T. C. (1993) J. Biol. Chem. 268, 27020-27025]. Binding of Mn-ATP also is decreased upon addition of Zn2+, indicating that the inhibitory effect of Zn2+ is exerted at the substrate-binding step of the overall selenophosphate synthetase reaction. When a cysteine residue at position 17 or 19 is replaced with serine, Mn-ATP binding to these mutant enzymes is unaffected by Zn2+ addition. Direct involvement of these cysteine residues in the zinc binding site was shown by use of 65ZnCl2. Radioactive Zn2+ bound to wild-type enzyme and was retained after gel filtration, but under the same conditions the catalytically inactive Cys-17 mutant protein and the catalytically active Cys-19 mutant enzyme were unlabeled.

Original languageEnglish
Pages (from-to)7326-7329
Number of pages4
JournalProceedings of the National Academy of Sciences of the United States of America
Volume91
Issue number15
DOIs
Publication statusPublished - 1994 Jul 19
Externally publishedYes

Fingerprint

Monovalent Cations
Adenosine Triphosphate
Metals
Ions
Escherichia coli
Enzymes
Cysteine
Selenium Compounds
Mutant Proteins
Ligases
selenophosphate synthetase
Serine
Gel Chromatography
Zinc
Binding Sites

Keywords

  • ATP
  • manganese
  • zinc inhibition

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

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title = "Effects of monovalent cations and divalent metal ions on Escherichia coli selenophosphate synthetase",
abstract = "A labile selenium donor compound, selenophosphate, is formed from selenide and ATP by selenophosphate synthetase. A divalent metal ion, Mg2+, and a monovalent cation, K+, NH4/+, or Rb+, are required for selenophosphate synthetase activity [Veres, Z., Kim, I. Y., Scholz, T. D. and Stadtman, T. C. (1994) J. Biol. Chem. 269, 10597-10603]. Na+ and Li+ are ineffective as activators and in the presence of K+ are inhibitory. Mn-ATP, although not able to replace Mg-ATP for catalytic activity, binds to the enzyme provided an active monovalent cation is present. No Mn-ATP is bound when K+ is replaced with Na+. The requirement for K+, both for Mn-ATP binding and for catalytic activity of the synthetase, indicates a specific monovalent cation- induced conformational state of the enzyme. Previously we reported that activity of the enzyme is markedly inhibited by micromolar levels of Zn2+ in the presence of millimolar levels of Mg2+ [Kim, I. Y., Veres, Z., and Stadtman, T. C. (1993) J. Biol. Chem. 268, 27020-27025]. Binding of Mn-ATP also is decreased upon addition of Zn2+, indicating that the inhibitory effect of Zn2+ is exerted at the substrate-binding step of the overall selenophosphate synthetase reaction. When a cysteine residue at position 17 or 19 is replaced with serine, Mn-ATP binding to these mutant enzymes is unaffected by Zn2+ addition. Direct involvement of these cysteine residues in the zinc binding site was shown by use of 65ZnCl2. Radioactive Zn2+ bound to wild-type enzyme and was retained after gel filtration, but under the same conditions the catalytically inactive Cys-17 mutant protein and the catalytically active Cys-19 mutant enzyme were unlabeled.",
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N2 - A labile selenium donor compound, selenophosphate, is formed from selenide and ATP by selenophosphate synthetase. A divalent metal ion, Mg2+, and a monovalent cation, K+, NH4/+, or Rb+, are required for selenophosphate synthetase activity [Veres, Z., Kim, I. Y., Scholz, T. D. and Stadtman, T. C. (1994) J. Biol. Chem. 269, 10597-10603]. Na+ and Li+ are ineffective as activators and in the presence of K+ are inhibitory. Mn-ATP, although not able to replace Mg-ATP for catalytic activity, binds to the enzyme provided an active monovalent cation is present. No Mn-ATP is bound when K+ is replaced with Na+. The requirement for K+, both for Mn-ATP binding and for catalytic activity of the synthetase, indicates a specific monovalent cation- induced conformational state of the enzyme. Previously we reported that activity of the enzyme is markedly inhibited by micromolar levels of Zn2+ in the presence of millimolar levels of Mg2+ [Kim, I. Y., Veres, Z., and Stadtman, T. C. (1993) J. Biol. Chem. 268, 27020-27025]. Binding of Mn-ATP also is decreased upon addition of Zn2+, indicating that the inhibitory effect of Zn2+ is exerted at the substrate-binding step of the overall selenophosphate synthetase reaction. When a cysteine residue at position 17 or 19 is replaced with serine, Mn-ATP binding to these mutant enzymes is unaffected by Zn2+ addition. Direct involvement of these cysteine residues in the zinc binding site was shown by use of 65ZnCl2. Radioactive Zn2+ bound to wild-type enzyme and was retained after gel filtration, but under the same conditions the catalytically inactive Cys-17 mutant protein and the catalytically active Cys-19 mutant enzyme were unlabeled.

AB - A labile selenium donor compound, selenophosphate, is formed from selenide and ATP by selenophosphate synthetase. A divalent metal ion, Mg2+, and a monovalent cation, K+, NH4/+, or Rb+, are required for selenophosphate synthetase activity [Veres, Z., Kim, I. Y., Scholz, T. D. and Stadtman, T. C. (1994) J. Biol. Chem. 269, 10597-10603]. Na+ and Li+ are ineffective as activators and in the presence of K+ are inhibitory. Mn-ATP, although not able to replace Mg-ATP for catalytic activity, binds to the enzyme provided an active monovalent cation is present. No Mn-ATP is bound when K+ is replaced with Na+. The requirement for K+, both for Mn-ATP binding and for catalytic activity of the synthetase, indicates a specific monovalent cation- induced conformational state of the enzyme. Previously we reported that activity of the enzyme is markedly inhibited by micromolar levels of Zn2+ in the presence of millimolar levels of Mg2+ [Kim, I. Y., Veres, Z., and Stadtman, T. C. (1993) J. Biol. Chem. 268, 27020-27025]. Binding of Mn-ATP also is decreased upon addition of Zn2+, indicating that the inhibitory effect of Zn2+ is exerted at the substrate-binding step of the overall selenophosphate synthetase reaction. When a cysteine residue at position 17 or 19 is replaced with serine, Mn-ATP binding to these mutant enzymes is unaffected by Zn2+ addition. Direct involvement of these cysteine residues in the zinc binding site was shown by use of 65ZnCl2. Radioactive Zn2+ bound to wild-type enzyme and was retained after gel filtration, but under the same conditions the catalytically inactive Cys-17 mutant protein and the catalytically active Cys-19 mutant enzyme were unlabeled.

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