Hayashi M, Hasegawa K, Oguni Y, Unemoto T
Laboratory of Membrane Biochemistry, Faculty of Pharmaceutical Sciences, Chiba University, Japan.
Biochim Biophys Acta. 1990 Aug 17;1035(2):230-6. doi: 10.1016/0304-4165(90)90122-d.
It was found that when Escherichia coli is grown in the presence of 0.2-0.3 mM menadione (2-methyl-1,4-naphthoquinone), an FMN-dependent NADH-quinone reductase increases more than 20-fold in the cytoplasmic fraction. The menadione-induced quinone reductase was isolated from the cytoplasmic fraction of induced cells. The purified enzyme had an Mr of 24 kDa on SDS-polyacrylamide gel electrophoresis. The enzyme required flavin as a cofactor and a half-maximum activity was obtained with 0.54 microM FMN or 16.5 microM FAD. The enzyme had a broad pH optimum at pH 7.0-8.0 and reacted with NADH, but not with NADPH. The reaction followed a ping-pong mechanism and the intrinsic Km values for NADH and menadione were estimated to be 132 microM and 2.0 microM, respectively. Dicoumarol was a simple competitive inhibitor with respect to NADH with a Ki value of 0.22 microM. The electron acceptor specificity of this enzyme was very similar to that of NAD(P)H: (quinone acceptor) oxidoreductase (EC 1.6.99.2, DT-diaphorase) from rat liver. Since menadione is reduced by the two-electron reduction pathway to menadiol, the induction of this enzyme is likely to be an adaptive response of E. coli to partially alleviate the toxicity of menadione.
研究发现，当大肠杆菌在0.2 - 0.3 mM甲萘醌（2 - 甲基 - 1,4 - 萘醌）存在的情况下生长时，一种依赖黄素单核苷酸（FMN）的NADH - 醌还原酶在细胞质部分增加超过20倍。从诱导细胞的细胞质部分分离出甲萘醌诱导的醌还原酶。在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳上，纯化后的酶的相对分子质量为24 kDa。该酶需要黄素作为辅因子，在0.54 μM FMN或16.5 μM黄素腺嘌呤二核苷酸（FAD）时可获得最大活性的一半。该酶在pH 7.0 - 8.0具有较宽的最适pH值，与NADH反应，但不与NADPH反应。该反应遵循乒乓机制，NADH和甲萘醌的内在米氏常数（Km值）分别估计为132 μM和2.0 μM。双香豆素是NADH的简单竞争性抑制剂，抑制常数（Ki值）为0.22 μM。这种酶的电子受体特异性与大鼠肝脏中的NAD(P)H：（醌受体）氧化还原酶（EC 1.6.99.2，DT - 黄递酶）非常相似。由于甲萘醌通过双电子还原途径被还原为甲萘氢醌，这种酶的诱导可能是大肠杆菌的一种适应性反应，以部分减轻甲萘醌的毒性。
