A QM/MM study of the racemization of vinylglycolate catalyzed by mandelate racemase enzyme.
A QM/MM study of the racemization of vinylglycolate catalyzed by mandelate racemase enzyme.
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DOI：
10.1021/ja002879o
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发表时间：
2001-01
影响因子：
15
通讯作者：
M. Garcia-Viloca;González-Lafont A;J. M. Lluch
中科院分区：
文献类型：
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作者：
M. Garcia-Viloca;González-Lafont A;J. M. Lluch
实验假设的(S)-乙烯基乙醇酸酯和(R)-乙烯基乙醇酸酯在扁豆酸消旋酶催化下的相互转化机理包括一个相当对称的两步过程，该过程通过一个二阴离子烯醇中间体形成，该中间体是在乙烯基乙醇酸酯的α质子被碱性酶残基提取后形成的，然后由另一个残基再复制。这个反应背后具有挑战性的问题是酶如何设法稳定这样一个中间体，也就是说，它如何降低质子的高pK(a)以使反应发生。本文进行的QM/MM模拟表明，催化作用是基于在反应过程中底物上形成的负电荷的稳定。我们已经确定了三种不同的反应机制，从底物-酶复合物的不同准简并结构开始。在其中两种方法中，稳定作用是通过催化质子转移来实现的，从而避免了重阴离子中间体的形成，而且它们涉及六个步骤，而不是实验提出的两个步骤。相反，第三种机制在质子提取过程中通过一种由质子化酶残基协同方法稳定的重阴离子。沿着这些机制理论上发现的势能势垒与乙烯基乙醇酸酯和扁豆酸酯外消旋化的实验自由能势垒在质量上是一致的。Gln317突变Glu317对反应动力学影响的理论研究揭示了Glu317在天然酶中形成的氢键的催化作用，因为只有一种较慢的机制能够在突变体的活性位点产生外消旋。然而，我们发现这个氢键在我们的模型中并不是LBHB。
The experimentally postulated mechanism for the interconversion between (S)-vinylglycolate and (R)-vinylglycolate catalyzed by mandelate racemase enzyme consists of a two-step quite symmetric process through a dianionic enolic intermediate that is formed after the abstraction of the alpha-proton of vinylglycolate by a basic enzymatic residue and is then reprotonated by another residue. The challenging problem behind this reaction is how the enzyme manages to stabilize such an intermediate, that is, how it lowers enough the high pK(a) of the alpha-proton for the reaction to take place. The QM/MM simulations performed in this paper indicate that catalysis is based on the stabilization of the negative charge developed on the substrate along the reaction. We have identified three different reaction mechanisms starting from different quasi-degenerate structures of the substrate-enzyme complex. In two of them the stabilizing role is done by means of a catalytic proton transfer that avoids the formation of a dianionic intermediate, and they involve six steps instead of the two experimentally proposed. On the contrary, the third mechanism passes through a dianionic species stabilized by the concerted approach of a protonated enzymatic residue during the proton abstraction. The potential energy barriers theoretically found along these mechanisms are qualitatively in good agreement with the experimental free energy barriers determined for racemization of vinylglycolate and mandelate. The theoretical study of the effect of the mutation of Glu317 by Gln317 in the kinetics of the reaction reveals the important role in the catalysis of the hydrogen bond formed by Glu317 in the native enzyme, as only one of the mechanisms, the slower one, is able to produce the racemization in the active site of the mutant. However, we have found that this hydrogen bond is not an LBHB within our model.