An active‐site cysteine of sorghum leaf NADP‐malate dehydrogenase studied by site‐directed mutagenesis

FEBS Letters

Decottignies, Paulette¹  Issakidis, Emmanuelle¹  Ruelland, Eric¹  Miginiac-Maslow, Myroslawa¹  Lemaire, Martine¹ 

[1] Institut de Biotechnologie des Plantes, URA 1128 CNRS, Bât. 630, Université de Paris-Sud, 91405 Orsay Cedex, France

关键词: Malate dehydrogenase;    Thioredoxin;    Light-activation;    Disulfide mutagenesis;    Site-directed mutagenesis;    DTT;    dithioreitol;    NADP-MDH;    NADP-dependent malate dehydrogenase;    OAA;    oxaloacetate;    PMSF;    phenylmethylsulfonyl fluoride;    WT;    wild-type;   

DOI  :  10.1016/0014-5793(96)00153-6

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown