Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, SE-10691 Stockholm, Sweden.
Biochemistry. 2011 Mar 8;50(9):1505-13. doi: 10.1021/bi101493p. Epub 2011 Feb 2.
The reaction mechanism of mycolic acid cyclopropane synthase is investigated using hybrid density functional theory. The direct methylation mechanism is examined with a large model of the active site constructed on the basis of the crystal structure of the native enzyme. The important active site residue Glu140 is modeled in both ionized and neutral forms. We demonstrate that the reaction starts via the transfer of a methyl to the substrate double bond, followed by the transfer of a proton from the methyl cation to the bicarbonate present in the active site. The first step is calculated to be rate-limiting, in agreement with experimental kinetic results. The protonation state of Glu140 has a rather weak influence on the reaction energetics. In addition to the natural reaction, a possible side reaction, namely a carbocation rearrangement, is also considered and is shown to have a low barrier. Finally, the energetics for the sulfur ylide proposal, which has already been ruled out, is also estimated, showing a large energetic penalty for ylide formation.
采用杂化密度泛函理论研究了分枝菌酸环丙烷合酶的反应机制。利用基于天然酶晶体结构构建的大型活性位点模型，考察了直接甲基化机制。重要的活性位点残基Glu140 分别以离子化和中性形式建模。我们证明，反应首先通过甲基转移到底物双键，然后通过甲基阳离子将质子从活性位点中的碳酸氢盐转移。第一步被计算为限速步骤，与实验动力学结果一致。Glu140 的质子化状态对反应能学的影响相当微弱。除了自然反应之外，还考虑了一种可能的副反应，即碳正离子重排，结果表明其势垒较低。最后，还估计了已经被排除的硫叶立德提议的能量学，表明叶立德形成的能量学惩罚很大。
