The Role of Local Environment and Flexibility in the Catalytic Power of Malate and 3Phopsphoglycerate Dehydrogenases

Malate dehydrogenase and 3phosphoglycerate dehydrogenase each catalyze a proton abstraction from the hydroxyl group of a tetrahedral carbon bearing the hydroxyl group involved in proton abstraction as well as a hydrogen involved in hydride transfer. The crystal structures of both enzymes indicate that the catalytic base in each case is a histidine [H220 in gMDH and H292 in 3pGDH] and that the the basicity of the imidazole group is enhanced by a carboxyl group [D193 in gMDH and E269 in 3pGDH. Despite these similarities, the enzymes exhibit widely different efficiencies although experimental evidence suggests that the rate limiting step is hydride transfer in each enzyme. Mutation of either the histidine or the carboxyl group has very different effects with much greater impact observed in gMDH than 3pGDH. We are using a systematic analysis of the computed chemical and physical properties of both the active site cavity and subunit interfaces and QM‐MM approaches to analyze the reaction pathways. These approaches are being combined with direct experimental approaches including dynamic light scattering to look at the strength of the subunit interfaces, hydrogen deuterium exchange techniques to examine local flexibility and fluorescence techniques [using either the single tryptophan in 3pGDH or a series of tryptophan containing mutants of gMDH] to look at ligand induced conformational changes with the goal of establishing whether local chemical environment or flexibility difference is the major contributor to the activity differences between these two enzymes. This work is funded by NSF Grant MCB 0448905 to EB