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An active‐site cysteine of sorghum leaf NADP‐malate dehydrogenase studied by site‐directed mutagenesis
Author(s) -
Lemaire Martine,
Issakidis Emmanuelle,
Ruelland Eric,
Decottignies Paulette,
Miginiac-Maslow Myroslawa
Publication year - 1996
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(96)00153-6
Subject(s) - cysteine , active site , malate dehydrogenase , biochemistry , site directed mutagenesis , mutagenesis , alanine , enzyme , chemistry , biology , mutant , amino acid , gene
The chlorplast NADP‐malate dehydrogenase is activated through the reduction of two different disulfides per subunit. The activated enzyme, as well as a permanently active mutant where all four regulatory cysteines were replaced are still sensitive to thiol reagents. This observation suggested the presence of an additional important cysteine at the active site. In an attempt to identify that cysteine, site‐directed mutagenesis was performed on the cDNA encoding sorghum leaf NADP‐malate dehydrogenase. The replacement of Cys‐175 by an alanine yielded an enzyme whose sensitivity to thiol reagents was markedly decreased whereas its catalytic activity was enhanced. This finding suggests that Cys‐175 has no catalytic function but is located close to the active site.