Studies of Glutamate Dehydrogenase.

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 again 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 equilibrium 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. [ABSTRACT FROM AUTHOR]