The ubiquitous cofactor NADH protects against substrate‐induced inhibition of a pyridoxal enzyme

In the usual reaction catalyzed by D‐amino acid transaminase, cleavage of the alpha‐H bond is followed by the reversible transfer of the alpha‐NH 2 to a keto acid cosubstrate in a two‐step reaction mediated by the two vitamin B 6 forms pyridoxal 5′‐phosphate (PLP) and pyridoxamine 5′‐phosphate (PMP). We report here a reaction not on the main pathway, i.e., β‐decarboxylation of D‐aspartate to D‐alanine, which occurs at 0.01% the rate of the major transaminase reaction. In this reaction, β‐C‐C bond cleavage of the single substrate D‐aspartate occurs rather than the usual α‐bond cleavage in the transaminase reaction. The D‐alanine produced from D‐aspartate slowly inhibits both transaminase and decarboxylase activities, but NADH or NADPH instantaneously prevent D‐aspartate turnover and D‐alanine formation, thereby protecting the enzyme against inhibition. NADH has no effect on the enzyme spectrum itself in the absence of substrates, but it acts on the enzymeD‐aspartate complex with an apparent dissociation constant of 16 μM. Equivalent concentrations of NAD or thiols have no such effect. The suppression of β‐decarboxylase activity by NADH occurs concomitant with a reduction in the 415‐nm absorbance due to the PLP form of the enzyme and an increase at 330 nm due to the PMP form of the enzyme. α‐Ketoglutarate reverses the spectral changes caused by NADH and regenerates the active PLP form of the enzyme from the PMP form with an equilibrium constant of 10 μM. In addition to its known role in shuttling electrons in oxidation‐reduction reactions, the niacin derivative NADH may also function by preventing aberrant damaging reactions for some enzyme‐substrate intermediates. The D‐aspartate‐induced effect of NADH may indicate a slow transition between protein conformational studies if the reaction catalyzed is also slow.