Bernofsky C, Wanda S Y
J Biol Chem. 1982 Jun 25;257(12):6809-17.
Incubation of NADH at neutral and slightly alkaline pH leads to the gradual absorption of 1 mol of H+. This uptake of acid requires oxygen and mainly yields anomerized NAD+ (NAD+), with only minimal formation od acid-modified NADH. The overall stoichiometry of the reaction is: NADH + H+ + 1/2O2 leads to H2O + NAD+, with NADH peroxide (HO2-NADH+) serving as the intermediate that anomerizes and breaks down to give NAD+ and H2O2. The final reaction reaction mixture contains less than 0.1% of the generated H2O2, which is nonenzymically reduced by NADH. The latter reaction is inhibited by catalase, leading to a decrease in the overall rate of acid absorption, and stimulated by peroxidase, leading to an increase in the overall rate of acid absorption. Although oxygen can attack NADH at either N-1 or C-5 of the dihydropyridine ring, the attack appears to occur primarily at N-1. This assignment is based on the inability of the C-5 peroxide to anomerize, whereas the N-1 peroxide, being a quaternary pyridinium compound, can anomerize via reversible dissociation of H2O2. The peroxidase-catalyzed oxidation of NADH by H2O2 does not lead to anomerization, indicating that anomerization occurs prior to the release of H2O2. Chromatography of reaction mixtures on Dowex 1 formate shows the presence of two major and several minor neutral and cationic degradation products. One of the major products is nicotinamide, which possibly arises from breakdown of nicotinamide-1-peroxide. The other products have not been identified, but may be derived from other isomeric nicotinamide peroxides.
在中性和略碱性pH条件下孵育NADH会导致逐渐吸收1摩尔H⁺。这种酸的吸收需要氧气，主要产生异头化的NAD⁺（NAD⁺），仅生成极少量的酸修饰的NADH。该反应的总体化学计量式为：NADH + H⁺ + 1/2O₂ → H₂O + NAD⁺，其中NADH过氧化物（HO₂-NADH⁺）作为中间体进行异头化并分解生成NAD⁺和H₂O₂。最终反应混合物中生成的H₂O₂含量低于0.1%，它会被NADH非酶促还原。后一反应受到过氧化氢酶的抑制，导致酸吸收的总体速率降低，而过氧化物酶则会刺激该反应，导致酸吸收的总体速率增加。尽管氧气可以在二氢吡啶环的N-1或C-5处攻击NADH，但攻击似乎主要发生在N-1处。这一归属是基于C-5过氧化物无法进行异头化，而N-1过氧化物作为季铵吡啶化合物，可以通过H₂O₂的可逆解离进行异头化。H₂O₂在过氧化物酶催化下对NADH的氧化不会导致异头化，这表明异头化发生在H₂O₂释放之前。反应混合物在Dowex 1甲酸盐上的色谱分析显示存在两种主要的以及几种次要的中性和阳离子降解产物。主要产物之一是烟酰胺，它可能源于烟酰胺-1-过氧化物的分解。其他产物尚未鉴定，但可能来自其他异构体烟酰胺过氧化物。
