Michaliszyn G A, Wing S S, Meighen E A
J Biol Chem. 1977 Nov 10;252(21):7495-9.
A NAD(P)H:flavin oxidoreductase, which produces FMNH2, one of the substrates for the luciferase reaction in bioluminescent bacteria, has been purified with the aid of affinity chromatography on epsilon-aminohexanoyl-FMN-Sepharose. The purified enzyme, isolated from Beneckea harveyi, had a specific activity of 89 mumol of NADH oxidized/min/mg of protein at 23 degrees in the presence of saturating FMN and NADH and appeared homogeneous by several criteria on polyacrylamide gel electrophoresis. A molecular weight of 24,000 was estimated both by gel filtration and and sodium dodecyl sulfate gel electrophoresis indicating that the enzyme is composed of a single polypeptide chain. Kinetic studies showed that the higher specificity of the enzyme for NADH than NADPH and for riboflavin and FMN than FAD was primarily due to variations in the Michaelis constants for the different substrates. Initial velocity studies with all pairs of substrates gave intersecting patterns supporting a sequential mechanism for the NAD(P)H:flavin oxidoreductase.
一种能产生FMNH₂的NAD(P)H：黄素氧化还原酶已借助ε-氨基己酰-FMN-琼脂糖亲和层析法得到纯化。FMNH₂是发光细菌中荧光素酶反应的底物之一。从哈维贝内克氏菌中分离出的纯化酶，在23℃、FMN和NADH饱和存在的情况下，其比活性为每分钟每毫克蛋白质氧化89微摩尔NADH，并且通过聚丙烯酰胺凝胶电泳的几个标准来看显得均一。通过凝胶过滤和十二烷基硫酸钠凝胶电泳估计其分子量为24,000，这表明该酶由一条单一的多肽链组成。动力学研究表明，该酶对NADH的特异性高于NADPH，对核黄素和FMN的特异性高于FAD，这主要是由于不同底物的米氏常数存在差异。对所有底物对进行的初速度研究给出了相交模式，支持NAD(P)H：黄素氧化还原酶的有序机制。
