Escherichia coli mutant SELD enzymes: The cysteine 17 residue is essential for selenophosphate formation from ATP and selenide

Ick Young Kim, Zsuzsa Veres, Thressa C. Stadtman

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Abstract

Synthesis of a labile selenium donor compound, selenophosphate, from selenide and ATP by the Escherichia coli SELD enzyme was reported previously from this laboratory. From the gene sequence, SELD is a 37-kDa protein that contains 7 cysteine residues, 2 of which are located at positions 17 and 19 in the sequence -Gly-Ala-Cys-Gly-Cys-Lys-Ile- (Leinfelder, W., Forchhammer, K., Veprek, B., Zehelein, E., and Böck, A. (1990) Proc. Natl. Acad. Sci. U.S.A. 73, 543-547). Inactivation of the enzyme by alkylation with iodoacetamide indicated that at least 1 cysteine residue in the protein is essential for enzyme activity. To test the possibility that the Cys17 and/or Cys19 residue might be essential, these were changed to serine residues by site-specific mutagenesis. The biological activities of the wild type and mutant proteins were studied using E. coli MB08 (selD-) transformed with plasmids containing the selD genes. The plasmid containing the Cys17-mutated gene failed to complement MB08, whereas the Cys19-mutated gene was indistinguishable from wild type. The mutant proteins, like the wild type enzyme, bound to an ATP-agarose matrix, showing that their affinities for ATP were unimpaired. Selenide-dependent formation of AMP from ATP was abolished by mutation of Cys17, but the Cys19 mutation had no effect on the ability of the enzyme to catalyze the reaction. These results indicate that Cys17 has an essential role in the catalytic process that leads to the formation of selenophosphate from ATP and selenide.

Original languageEnglish
Pages (from-to)19650-19654
Number of pages5
JournalJournal of Biological Chemistry
Volume267
Issue number27
Publication statusPublished - 1992 Sep 25
Externally publishedYes

Fingerprint

Escherichia coli
Cysteine
Genes
Adenosine Triphosphate
Mutant Proteins
Enzymes
Plasmids
Iodoacetamide
Mutagenesis
Selenium Compounds
Alkylation
Enzyme activity
Adenosine Monophosphate
Selenium
Bioactivity
Mutation
Serine
Site-Directed Mutagenesis
Proteins
selenophosphate

ASJC Scopus subject areas

  • Biochemistry

Cite this

Escherichia coli mutant SELD enzymes : The cysteine 17 residue is essential for selenophosphate formation from ATP and selenide. / Kim, Ick Young; Veres, Zsuzsa; Stadtman, Thressa C.

In: Journal of Biological Chemistry, Vol. 267, No. 27, 25.09.1992, p. 19650-19654.

Research output: Contribution to journalArticle

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