HALOALKENE OXIDATION BY THE SOLUBLE METHANE MONOOXYGENASE FROM METHYLOSINUS-TRICHOSPORIUM OB3B - MECHANISTIC AND ENVIRONMENTAL IMPLICATIONS
HALOALKENE OXIDATION BY THE SOLUBLE METHANE MONOOXYGENASE FROM METHYLOSINUS-TRICHOSPORIUM OB3B - MECHANISTIC AND ENVIRONMENTAL IMPLICATIONS
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DOI：
10.1021/bi00479a013
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发表时间：
1990-07-10
期刊：
影响因子：
2.9
通讯作者：
LIPSCOMB, JD
中科院分区：
文献类型：
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作者：
FOX, BG;BORNEMAN, JG;LIPSCOMB, JD
从甲基三磷菌OB3b中纯化的可溶性三蛋白组分甲烷单加氧酶能够氧化氯化、氟化和溴化烯烃，包括广泛分布的地下水污染物三氯乙烯（TCE）。氯烯烃的氧化速率被观察到与天然底物甲烷的氧化速率相当，并且比其他明确定义的生物系统所报道的氧化速率高出7000倍。发现竞争性抑制剂四氯乙烯是唯一没有翻转的氯化乙烯。然而，这似乎是由于位阻效应，而不是电子效应，或者由于三氟氯乙烯被有效氧化而缺乏可提取的质子。与4-（对硝基苄基）吡啶形成诊断加合物证明，卤代烯烃主要被环氧化氧化。随后水解产生的稳定的酸性产物被确定为主要产物。然而，在TCE、偏氯乙烯、三氟乙烯和三溴乙烯氧化过程中，分子内卤化物或氢化物迁移产生的额外醛产物收率为3-10%。对真品环氧丙烷水解反应的产物分析表明，很少或没有2,2,2-三氯乙醛（氯醛）的形成，表明在产物与酶解离之前发生了原子迁移。原子迁移产物的出现表明，底物向产物转化的中间体具有显著的阳离子特征。这种物质可以通过与活性部位高度缺电子的活性氧相互作用而产生。也有报道称微粒体细胞色素P-450将TCE氧化为TCE环氧化物和氯醛[Miller， R. E., and Guengerich， F. P. (1982) Biochemistry 21, 1090-1097]，这表明细胞色素P-450和甲烷单加氧酶利用了类似的氧化物质。在TCE氧化过程中，所有甲烷单加氧酶蛋白组分的失活都发生了依赖于转化的失活。在[1,2- 14c2]TCE的翻转过程中，每个组分的放射性标记表明，反应的扩散产物发生了共价修饰。失活率与产物形成的相关性表明，修饰物是环氧丙烷的水解产物。
The soluble, three-protein component methane monooxygenase purified from Methylosinus trichosporium OB3b is capable of oxidizing chlorinated, fluorinated, and brominated alkenes, including the widely distributed ground-water contaminant trichloroethylene (TCE). The oxidation rates for the chloroalkenes were observed to be comparable to that for methane, the natural substrate, and up to 7000-fold higher than those reported for other well-defined biological systems. The competitive inhibitor tetrachloroethylene was found to be the only chlorinated ethylene not turned over. However, this appears to be due to steric effects rather than electronic effects or the lack of an abstractable proton because of chlorotrifluoroethylene was efficiently oxidized. As evidenced by the formation of diagnostic adducts with 4-(p-nitrobenzyl)pyridine, the halogenated alkenes were oxidized predominantly by epoxidation. Stable acidic products resulting from subsequent hydrolysis were identified as the major products. However, additional aldehyde products resulting from intramolecular halide or hydride migration were observed in 3-10% yield during the oxidation of TCE, vinylidene chloride, trifluoroethylene, and tribromoethylene. Product analysis of the hydrolysis reaction of authentic TCE epoxide showed little or no 2,2,2-trichloroacetaldehyde (chloral) formation, indicating that atomic migration occurred prior to product dissociation from the enzyme. The occurrence of atomic migration products shows that an intermediate in the substrate to product conversion carries significant cationic character. Such a species could be generated through interaction with a highly electron-deficient activated oxygen in the active site. The oxidation of TCE to TCE epoxide and chloral has also been reported from microsomal cytochrome P-450 [Miller, R. E., and Guengerich, F. P. (1982) Biochemistry 21, 1090-1097], suggesting that cytochrome P-450 and methane monooxygenase utilize a similar oxidizing species. A turnover-dependent inactivation of all methane monooxygenase protein components occurred during the oxidation of TCE. Radiolabeling of each of the components during turnover of [1,2-14C2]TCE showed that covalent modification by a diffusible product of the reaction had occurred. Correlation of the rates of inactivation with product formation suggests that the modifying species is a hydrolysis product of TCE epoxide.