Abstract
The proton magnetic resonance spectra of the dihydronicotinamide ring of αNADH 3 3 αNAD + and αNADH, the nicotinamide adenine dinucleotides in which the nicotinamide-ribosidic linkage has an α configuration; (4D)αNAD +, αNAD + with a deuterium label at the C4 position; αNADD B, βNADD B, α and βNADH with a deuterium label in the C4B position of the dihydronicotinamide ring; Y.ADH, yeast alcohol dehydrogenase. and the nicotinamide ring of αNAD + are reported and the proton absorptions assigned. The absolute assignment of the C4 methylene protons of αNADH is based on the generation of specifically deuterium-labeled (pro-S) B-deuterio-αNADH from enzymatically prepared B-deuterio-βNADH. The C4 proton absorption of αNAD + is assigned by oxidation of B-deuterio-αNADH by the A specific, yeast alcohol dehydrogenase to yield 4-deuterio-αNAD +. The epimerization of either αNADH or βNADH yields an equilibrium ratio of approximately 9:1 βNADH to αNADH. The rate of epimerization of αNADH to βNADH at 38 °C in 0.05, pH 7.5, phosphate buffer is 3.1 × 10 −3 min −1, corresponding to a half-life of 4 hr. Four related dehydrogenases, yeast and horse liver alcohol dehydrogenase and chicken M 4 and H 4 lactate dehydrogenase, are shown to oxidize αNADH to αNAD + at rates three to four orders of magnitude slower than for βNADH. By using specifically labeled B-deuterio-αNADH the enzymatic oxidation by yeast alcohol dehydrogenase has been shown to occur with the identical stereospecificity as the oxidation of βNADH. The nonenzymatic epimerization of αNADH to βNADH and the enzymatic oxidation αNADH are discussed as a possible source of αNAD + in vivo.
Published Version
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