Arfman N, Hektor H J, Bystrykh L V, Govorukhina N I, Dijkhuizen L, Frank J
Department of Microbiology, University of Groningen, The Netherlands.
Eur J Biochem. 1997 Mar 1;244(2):426-33. doi: 10.1111/j.1432-1033.1997.00426.x.
Oxidation of C1-C4 primary alcohols in thermotolerant Bacillus methanolicus strains is catalyzed by an NAD-dependent methanol dehydrogenase (MDH), composed of ten identical 43,000-Mr subunits. Each MDH subunit contains a tightly, but non-covalently, bound NAD(H) molecule, in addition to 1 Zn2+ and 1-2 Mg2+ ions. The NAD(H) cofactor is oxidized and reduced by formaldehyde and methanol, respectively, while it remains bound to the enzyme. Incubation of MDH with methanol and exogenous NAD (coenzyme) results in reduction of this NAD coenzyme. Both NAD species are not exchanged during catalysis. NAD thus plays two different and important roles in the MDH-catalyzed reaction, with the bound NAD cofactor acting as primary electron acceptor and the NAD coenzyme being responsible for reoxidation of the reduced cofactor. MDH obeys a ping-pong type reaction mechanism, which is consistent with such a temporary parking of reducing equivalents at the MDH-bound cofactor. Spectral studies show that, in the presence of exogenous NAD and Mg2+ ions, MDH interacts with a previously identified 50,000-Mr activator protein. The activator protein appears to facilitate the oxidation of the reduced NADH cofactor of MDH, which results in a strongly increased turnover rate of MDH.
耐热甲醇芽孢杆菌菌株中 C1 - C4 伯醇的氧化由一种依赖 NAD 的甲醇脱氢酶（MDH）催化，该酶由十个相同的 43,000 道尔顿亚基组成。每个 MDH 亚基除含有 1 个 Zn2 + 和 1 - 2 个 Mg2 + 离子外，还含有一个紧密但非共价结合的 NAD(H) 分子。NAD(H) 辅因子分别被甲醛和甲醇氧化和还原，同时它仍与酶结合。将 MDH 与甲醇和外源 NAD（辅酶）一起孵育会导致该 NAD 辅酶的还原。在催化过程中两种 NAD 形式都不会交换。因此，NAD 在 MDH 催化的反应中发挥两种不同且重要的作用，结合的 NAD 辅因子作为主要电子受体，而 NAD 辅酶负责还原辅因子的再氧化。MDH 遵循乒乓型反应机制，这与还原当量在 MDH 结合的辅因子处的这种暂时储存相一致。光谱研究表明，在外源 NAD 和 Mg2 + 离子存在下，MDH 与先前鉴定的 50,000 道尔顿激活蛋白相互作用。激活蛋白似乎促进了 MDH 还原的 NADH 辅因子的氧化，这导致 MDH 的周转速率大幅提高。
