title: An active-site cysteine of sorghum leaf NADP-malate dehydrogenase studied by site-directed mutagenesis.
abstract: The chloroplast 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.
authors.affiliation: Institut de Biotechnologie des Plantes, Université de Paris-Sud, Orsay, France.
authors.forename: M
authors.initials: M
authors.lastname: Lemaire
authors.forename: E
authors.initials: E
authors.lastname: Issakidis
authors.forename: E
authors.initials: E
authors.lastname: Ruelland
authors.forename: P
authors.initials: P
authors.lastname: Decottignies
authors.forename: M
authors.initials: M
authors.lastname: Miginiac-Maslow
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D003545:Cysteine / Q000502:physiology*; D004789:Enzyme Activation; D007460:Iodoacetamide / Q000494:pharmacology; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D050538:Malate Dehydrogenase (NADP+); D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D018515:Plant Leaves / Q000201:enzymology*; D006109:Poaceae / Q000201:enzymology; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology
chemical_list: D011993:Recombinant Fusion Proteins; D013439:Sulfhydryl Reagents; D008291:Malate Dehydrogenase; D050538:Malate Dehydrogenase (NADP+); D003545:Cysteine; D007460:Iodoacetamide