Institute of Biological Sciences, Federal University of Pará , Belém, PA 66075-110, Brazil.
Institute of Exact and Natural Sciences, Federal University of Pará , Belém, PA 66075-110, Brazil.
J Phys Chem B. 2017 Sep 21;121(37):8626-8637. doi: 10.1021/acs.jpcb.7b05063. Epub 2017 Sep 8.
Enolpyruvyl transfer from phosphoenolpyruvate (PEP) to the hydroxyl group of shikimate-5-OH-3-phosphate (S3P) is catalyzed by 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase in a reaction that involves breaking the C-O bond of PEP. Catalysis involves an addition-elimination mechanism with the formation of a tetrahedral intermediate (THI). Experiments have elucidated the mechanism of THI formation and breakdown. However, the catalytic action of EPSP synthase and the individual roles of catalytic residues Asp313 and Glu341 remains unclear. We have used a hybrid quantum mechanical/molecular mechanical (QM/MM) approach to explore the free energy surface in a reaction catalyzed by EPSP synthase. The Glu341 was the most favorable acid/base catalyst. Our results indicate that the protonation of PEP C3 precedes the nucleophilic attack on PEP C2 in the addition mechanism. Also, the breaking of the C-O bond of THI to form an EPSP cation intermediate must occur before proton transfer from PEP C3 to Glu341 in the elimination mechanism. Analysis of the FES supports cationic intermediate formation during the reaction catalyzed by EPSP synthase. Finally, the computational model indicates a proton transfer shift (Hammond shift) from Glu341 to C3 for an enzyme-based reaction with the shifted transition state, earlier than in the reference reaction in water.
烯醇丙酮酸基从磷酸烯醇丙酮酸 (PEP) 转移到莽草酸-5-磷酸-3-羟基 (S3P) 的羟基由 5-烯醇丙酮酸基莽草酸 3-磷酸 (EPSP) 合酶催化，该反应涉及打破 PEP 的 C-O 键。催化涉及一个加成消除机制，形成四面体中间体 (THI)。实验已经阐明了 THI 形成和分解的机制。然而，EPSP 合酶的催化作用以及催化残基 Asp313 和 Glu341 的作用仍不清楚。我们使用了混合量子力学/分子力学 (QM/MM) 方法来探索 EPSP 合酶催化的反应的自由能表面。Glu341 是最有利的酸碱催化剂。我们的结果表明，在加成机制中，PEP C3 的质子化先于对 PEP C2 的亲核攻击。此外，在消除机制中从 PEP C3 到 Glu341 的质子转移之前，THI 的 C-O 键必须断裂以形成 EPSP 阳离子中间体。FES 的分析支持 EPSP 合酶催化反应中阳离子中间体的形成。最后，计算模型表明，对于基于酶的反应，质子转移从 Glu341 转移到 C3 发生转移过渡态，早于在水中的参考反应。
