Duine J A, Frank J, Westerling J
Biochim Biophys Acta. 1978 Jun 9;524(2):277-87. doi: 10.1016/0005-2744(78)90164-x.
(1) A method for the isolation of methanol dehydrogenase (alcohol:(acceptor) oxidoreductase, EC 1.1.99.8) from Hyphomicrobium X is decribed. The purified enzyme was resolved by polyacrylamide gel electrophoresis into one main and two minor active bands. Iron and manganese were the only detected metals in the enzyme preparation. (2) The substrate, methanol, was oxidized to formic acid by a stoichiometric amount of artificial electron acceptor. During the reaction, no free formaldehyde could be detected. Other primary alcohols were oxidized to the corresponding aldehydes were a poor substrate or no substrate at all. (3) Some new and efficient one-electron acceptors were found. With these electron acceptors, the enzyme had a high pH optimum and ammonia was still required in the assay system. (4) ESR spectroscopy showed the presence of an enzyme-bound organic free radical. With X-band ESR the signal had a peak-to-peak linewidth of about 0.7 mT. The signal was further resolved by Q-band ESR and the values gparallel = 2.0024 and gperpendicular = 2.0056 were derived. (5) Under denaturing conditions the ESR signal and enzymatic activity disappeared at the same time as fluorescence appeared. Enzymatic activity is not restored when extracted cofactor and apoenzyme are brought together under normal conditions. Some properties of the unusual prosthetic group are presented in a preliminary form.
(1) 描述了一种从X型生丝微菌中分离甲醇脱氢酶（醇：（受体）氧化还原酶，EC 1.1.99.8）的方法。纯化后的酶通过聚丙烯酰胺凝胶电泳分离为一条主要活性带和两条次要活性带。在酶制剂中检测到的唯一金属是铁和锰。(2) 底物甲醇被化学计量的人工电子受体氧化成甲酸。反应过程中未检测到游离甲醛。其他伯醇被氧化成相应的醛，但作为底物效果不佳或根本不是底物。(3) 发现了一些新型高效的单电子受体。使用这些电子受体时，该酶具有较高的最适pH值，并且测定系统中仍需要氨。(4) 电子顺磁共振光谱显示存在与酶结合的有机自由基。使用X波段电子顺磁共振，该信号的峰峰线宽约为0.7 mT。通过Q波段电子顺磁共振进一步解析该信号，并得出g平行 = 2.0024和g垂直 = 2.0056的值。(5) 在变性条件下，电子顺磁共振信号和酶活性同时消失，同时出现荧光。在正常条件下将提取的辅因子和脱辅基酶重新组合时，酶活性无法恢复。以初步形式介绍了这种特殊辅基的一些特性。
