THE CATALYTIC ROLE OF ASPARTATE IN THE ACTIVE-SITE OF GLUTAMATE-DEHYDROGENASE

A putative catalytic aspartyl residue, Asp-165, in the active site of clostridial glutamate dehydrogenase has been replaced with serine by s ite-directed mutagenesis. The mutant enzyme is efficiently overexpress ed in Escherichia coli as a soluble protein and can be successfully pu rified by the dye-ligand chromatographic procedure normally employed f or the wild-type enzyme. By several criteria, including circular dichr oism spectrum, sulphydryl reactivity with Ellman's reagent, crystalliz ation and mobility in non-denaturing electrophoresis, the enzyme appea rs to be correctly folded. NAD(+) protects the D165S mutant against mo dification by Ellman's reagent, suggesting unimpaired binding of coenz yme. In standard assays the specific activity is decreased 10(3)-fold in the reductive amination reaction and 10(5)-fold for oxidative deami nation. Kinetic studies show that apparent K-m values for NADH and 2-o xoglutarate are almost unchanged. The large reduction in the reaction rate coincides with a weakening of the affinity for ammonium ion (K-m > 300 mM, compared with 60 mM for the wild-type). The data are entirel y consistent with the direct involvement of D165 in catalysis rather t han in the binding of coenzyme or 2-oxoglutarate.