Abstract
We have established a simple procedure for the in situ analysis of stereospecificity of an NAD (P)-dependent dehydrogenase for C-4 hydrogen transfer of NAD (P) H by means of glutamate racemase [EC 5. 1. 13] and glutamate dehydrogenase [EC 1. 4. 1. 3]. Glutamate racemase inherently catalyzes the exchange of α-H of glutamate with 2H during racemization in 2H2O. When the reactions of glutamate racemase and glutamate dehydrogenase, which is pro-S specific for the C4-H transfer of NAD (P) H, are coupled in 2H2O, [4S-2H]-NAD (P) H is exclusively produced. Therefore, if 1H is fully retained at C-4 of NAD (P) +after incubation of a reaction mixture containing both the enzymes and a dehydrogenase to betested, the stereospecificity of the dehydrogenase is the same as that of glutamate dehydrogenase. When the C4-H of NAD (P) +is exchanged with 2H, the enzyme to be examined is different from glutamate dehydrogenase in stereospecificity. Thus, we can readily determine the stereospecificity by 1H-NMR measurement of NAD (P) +without isolation of the coenzymes and products.
