Evidence for binding of NAD dimers to NAD-dependent dehydrogenases

The binding of dimers of nicotinamide adenine dinucleotide, (NAD)2, to lactate, malate and alcohol dehydrogenase has been studied by the fluorescence quenching technique. While the alcohol dehydrogenase shows a low binding ability, malate and lactate dehydrogenases have been found to bind (NAD)2 in a specific way with high affinity. Malate dehydrogenase binds (NAD)2 more than NADH. All three dehydrogenases are inhibited by (NAD)2, which behaves as a competitive inhibitor with respect to both NAD+ and NADH. The results show that (NAD)2 is bound to the nucleotide-specific binding site of the dehydrogenases. (NAD)2 was found to stoichiometrically react with ferricyanide at variance with NADH. The specific interactions with the NAD-dependent dehydrogenases and the ability to enter in monoelectronic redox cycles suggest possible physiological roles for (NAD)2.