Reactions of reduced nicotinamide adenine dinucleotide in acid: studies by reversed-phase high-pressure liquid chromatography.

Reversed-phase high-pressure liquid chromatography was used to isolate acid breakdown products of reduced nicotinamide adenine dinucleotide (NADH) and products produced when NADH breakdown is catalyzed by glyceraldehyde-3-phosphate dehydrogenase (G-3-PD). Chromatographic and UV spectral data on these and related products support a mechanism for NADH acid degradation involving hydroxy addition at the nicotinamide C-6 followed by cyclization of the ring and the adjacent ribose moiety. G-3-PD is shown to catalyze a reaction in which two products are formed which are also intermediates in the acid degradation of NADH (alpha- and beta-6-hydroxynicotinamide products). Formation of the major acid products fits a three-step, first-order mechanism curve, making it possible to calculate the rate constants k2 and k3 as well as the previously determined k1.