Open AccessThe Catalytic Site of Chloroplastic NADP‐Dependent Malate Dehydrogenase Contains A His/Asp Pair
Author(s) -
Lemaire Martine,
MiginiacMaslow Myroslawa,
Decottignies Paulette
Publication year - 1996
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1996.00947.x
Subject(s) - malate dehydrogenase , mutant , biochemistry , cofactor , dehydrogenase , mutagenesis , site directed mutagenesis , citrate synthase , active site , chemistry , biology , enzyme , gene
Plant chloroplastic NADP–malate dehydrogenase is unique among malate dehydrogenases because of its reductive activation in the light and cofactor specificity. In this paper, the role of His229 in sorghum leaf protein has been investigated by site‐directed mutagenesis. His229 was replaced by Asn and Gln, both mutations yielding an inactive protein. The role of a conserved Asp (Asp201) as a possible partner of His229 in catalysis has been studied by the same approach. Both Asp mutants (D201A, D201N) were only slightly active and were essentially characterized by a dramatically increased K m for oxaloacetate (45–80‐fold). pH dependence of catalytic rates revealed differences between the two Asp mutants. These results demonstrate that, in sorghum leaf NADP‐dependent malate dehydrogenase, His229 is involved in catalysis in interaction with Asp201.