Valentine A M, LeTadic-Biadatti M H, Toy P H, Newcomb M, Lippard S J
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
J Biol Chem. 1999 Apr 16;274(16):10771-6. doi: 10.1074/jbc.274.16.10771.
Radical clock substrate probes were used to assess the viability of a discrete substrate radical species in the mechanism of hydrocarbon oxidation by the soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath). New substituted cyclopropane probes were used with very fast ring-opening rate constants and other desirable attributes, such as the ability to discriminate between radical and cationic intermediates. Oxidation of these substrates by a reconstituted sMMO system resulted in no rearranged products, allowing an upper limit of 150 fs to be placed on the lifetime of a putative radical species. This limit strongly suggests that there is no such substrate radical intermediate. The two enantiomers of trans-1-methyl-2-phenyl-cyclopropane were prepared, and the regioselectivity of their oxidation to the corresponding cyclopropylmethanol and cyclopropylphenol products was determined. The results are consistent with selective orientation of the two enantiomeric substrates in the hydrophobic cavity at the active site of sMMO, specific models for which were examined by molecular modeling.
自由基时钟底物探针被用于评估来自荚膜甲基球菌（巴斯德菌株）的可溶性甲烷单加氧酶（sMMO）催化烃类氧化机制中离散底物自由基物种的活性。新型取代环丙烷探针具有非常快的开环速率常数以及其他理想特性，比如能够区分自由基中间体和阳离子中间体。用重组sMMO系统氧化这些底物不会产生重排产物，这使得假定自由基物种的寿命上限为150飞秒。这一限制有力地表明不存在这样的底物自由基中间体。制备了反式-1-甲基-2-苯基环丙烷的两种对映体，并确定了它们氧化为相应环丙基甲醇和环丙基苯酚产物的区域选择性。结果与两种对映体底物在sMMO活性位点疏水腔内的选择性取向一致，通过分子建模研究了其具体模型。
