THE CATALYTIC SITE OF CHLOROPLASTIC NADP-DEPENDENT MALATE-DEHYDROGENASE CONTAINS A HIS ASP PAIR/

Plant chloroplastic NADP-malate dehydrogenase is unique among malate d ehydrogenases because of its reductive activation in the light and cof actor specificity. In this paper, the role of His229 in sorghum leaf p rotein 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 i n catalysis has been studied by the same approach. Both Asp mutants (D 201A, D201N) were only slightly active and were essentially characteri zed 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-depende nt malate dehydrogenase, His229 is involved in catalysis in interactio n with Asp201.