Enzymatic in situ analysis by 1H-NMR of the hydrogen transfer stereospecificity of NAD(P)+-dependent dehydrogenases.
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 alpha-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 be tested, 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.
Nakajima
N
N
Department of Food and Nutrition, Okayama Prefectural Junior College.
Nakamura
K
K
Esaki
N
N
Tanaka
H
H
Soda
K
K
D000591:Amino Acid Isomerases / Q000378:metabolism; D003903:Deuterium; D005969:Glutamate Dehydrogenase / Q000378:metabolism; D005971:Glutamates / Q000096:biosynthesis; D018698:Glutamic Acid; D006859:Hydrogen / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D009252:NADPH Dehydrogenase / Q000032:analysis / Q000378:metabolism*; D013237:Stereoisomerism
D005971:Glutamates; D018698:Glutamic Acid; D009249:NADP; D006859:Hydrogen; D003903:Deuterium; D005969:Glutamate Dehydrogenase; D009247:NADH, NADPH Oxidoreductases; D009252:NADPH Dehydrogenase; D000591:Amino Acid Isomerases; C062292:glutamate racemase