Studies of Glutamate Dehydrogenase

Abstract

Specific interaction between α‐NADH and glutamate dehydrogenase is demonstrated by difference spectroscopy, circular dichroism and fluorescence measurements. Quantitative binding studies in the preparative ultracentrifuge yield six identical α‐NADH binding sites per oligomer with a dissociation constant of 20 μM. Evidence for six to eight additional, very weak α‐NADH binding sites is presented. Excess ADP prevents the binding of α‐NADH to the tight binding sites, indicating competition of the two nucleotides. In the enzymatic reaction, α‐NADH is inactive as a coenzyme; however, like ADP, it is an activator of the reductive amination reaction of 2‐oxoglutarate. α‐NADH reduces the self‐inhibition of high β‐NADH concentrations and thus agin parallels the effects caused by ADP. Contrary to β‐NADH or β‐NADH plus GTP, α‐NADH or α‐NADH plus GTP does not affect the dissociation‐association equiilibrium of glutamate dehydrogenase. The results are consistent with the assumption of six binding sites for α‐NADH, which are identical with the ADP binding sites and most probably the six non‐active β‐NADH binding sites.