Large kinetic isotope effects in methane oxidation catalyzed by methane monooxygenase: Evidence for C-H bond cleavage in a reaction cycle intermediate
Large kinetic isotope effects in methane oxidation catalyzed by methane monooxygenase: Evidence for C-H bond cleavage in a reaction cycle intermediate
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DOI：
10.1021/bi960596w
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发表时间：
1996-08-06
期刊：
影响因子：
2.9
通讯作者：
Lipscomb, JD
中科院分区：
文献类型：
--
作者：
Nesheim, JC;Lipscomb, JD
来自发孢甲基弯菌OB 3b的可溶性甲烷单加氧酶（MMO）的还原羟化酶组分（MMOH）与O-2和CH 4反应以在单周转反应中产生CH 3OH和H2O。该反应的瞬时动力学分析已经揭示了在周转期间至少五种并且可能六种中间体[Lee，S. K，，Nesheim，J，C.，& Lipscomb，J. D.（1993）J.Biol.Chem.268，21569-21577; Liu，Y.，Nesheim，J. C.，李，S.- J. M.，& J. M.（1995）生物化学杂志270？24662-24665]。一种称为化合物Q的中间体与CHI反应以产生酶结合产物。这里显示，化合物Q与CH 4反应的氘动力学同位素效应（KIE）为50-100，这是迄今为止观察到的最大效应之一。甲烷的氘代同系物的反应速率常数随着氘含量的增加而单调下降，表明大的主要同位素效应占主导地位。通过分析单次转换后的产物确定的KIE具有以下值：1：1 CH 4：CD 4（19）; CD 3 H（12）; CD 2 H 2（9）;和CH 3D（4）。通过直接观察反应性中间体和通过监测产物比率确定的KIE值都很大，与反应的氧化步骤中完全的C-H键断裂一致。然而，这两种方法测定的KIE值的差异表明，该反应比目前提出的更复杂。提出了一种改进的机制，引入了由中间甲基自由基再提取氢原子的可能性。
The reduced hydroxylase component (MMOH) of soluble methane monooxygenase (MMO) from Methylosinus trichosporium OB3b reacts with O-2 and CH4 to produce CH3OH and H2O in a single-turnover reaction, Transient kinetic analysis of this reaction has revealed at least five and probably six intermediates during The turnover [Lee, S.-K,, Nesheim, J, C., & Lipscomb, J. D. (1993) J. Biol. Chem. 268, 21569-21577; Liu, Y., Nesheim, J. C., Lee, S.-K,, & Lipscomb, J. D. (1995) J. Biol. Chem. 270? 24662-24665]. One Intermediate, termed compound Q, reacts with CHI to yield enzyme-bound product. It is shown here that the deuterium kinetic isotope effect (KIE) for the reaction of compound Q with CH4 is 50-100, which is one of the largest effects observed to date. The rate constants for the reactions of the deuterated homologs of methane decrease monotonically as the deuterium content increases, suggesting that a large primary isotope effect dominates, The KIEs determined by analyzing the products after a single turnover have the following values: 1:1 CH4:CD4 (19); CD3H (12); CD2H2 (9); and CH3D (4). The KIE values determined by directly observing the reactive intermediate and by monitoring product ratios an all large, consistent with complete C-H bond breaking in the oxygenation step of the reaction. However, the differences in the KIE values determined by these two methods suggest that the reaction is more complex than currently proposed. A modified mechanism introducing the possibility of hydrogen-atom reabstraction by an intermediate methyl radical is proposed.