AN ACTIVE-SITE CYSTEINE OF SORGHUM LEAF NADP-MALATE DEHYDROGENASE STUDIED BY SITE-DIRECTED MUTAGENESIS

The chloroplast NADP-malate dehydrogenase is activated through the red uction of two different disulfides per subunit, The activated enzyme, as well as a permanently active mutant where all four regulatory cyste ines were replaced are still sensitive to thiol reagents, This observa tion suggested the presence of an additional important cysteine at the active site, In an attempt to identify that cysteine, site-directed m utagenesis was performed on the cDNA encoding sorghum leaf NADP-malate dehydrogenase. The replacement of Cys-175 by an alanine yielded an en zyme whose sensitivity to thiol reagents was markedly decreased wherea s its catalytic activity was enhanced, This finding suggests that Cys- 175 has no catalytic function but is located close to the active site.