Nuclear magnetic resonance studies on pyridine dinucleotides: Carbon-13 assignments and structure determination of NADHX and the primary acid product of NADH

Abstract

The 13C spectra of β-NADH, NADHX, and the primary acid product of NADH were obtained and assigned. The conversion of the NADHX isomers to the two isomers of NADH acid product is demonstrated through the use of 13C-enriched compounds. The structure of NADHX is assigned as β-6-hydroxy-1,4,5,6-tetrahydronicotinamide adenine dinucleotide and the structures of the primary acid products of NADH are assigned as α- O 2′-6B-cyclotetrahydronicotinamide adenine dinucleotide and α- O 2′-6A-cyclotetrahydronicotinamide adenine dinucleotide. The structures of NADHX and the major isomer of the primary acid product, derived from studies of model compounds, are consistent with those proposed by Oppenheimer and Kaplan [ Biochemistry (1974) 13, 4675, 4685] . However, the spectra of 13C-enriched primary acid product also demonstrated the existence of the A isomer which was not observed in the latter 1H study. The A and B isomers were found to exist in the same ratio even when the primary acid product was formed directly from NADHX. This observation is discussed in terms of the previously proposed mechanism for the acid decomposition of NADH.