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Insight into the Catalytic Mechanism of GH11 Xylanase: Computational Analysis of Substrate Distortion based on a Neutron Structure
Journal of the American Chemical Society ( IF 15.0 ) Pub Date : 2020-09-22 , DOI: 10.1021/jacs.0c02148
Toyokazu Ishida 1 , Jerry M Parks 2 , Jeremy C Smith 2
Affiliation  

The reaction mechanism of xylanase biomass decomposition enzymes has been the subject of debate. To clarify the mechanism we investigated the glycosylation step of GH11 xylanase, an enzyme that catalyzes the hydrolysis of lignocellulosic hemicellulose (xylan). Making use of a recent neutron structure, which identified the protonation states of relevant residues, we use ab initio QM/MM methodology to determine the detailed reaction mechanism of the glycosylation. In particular, our focus is on the controversial question of whether or not an oxocarbenium ion intermediate is formed on the reaction pathway. The calculations support the validity of a basic retaining mechanism with a double displacement scheme. The estimated free energy barrier of this reaction is ~18 kcal/mol with QM/MM-CCSD(T)/6-31(+)G**//MP2/6-31+G**/AMBER calculations, and the rate-determining step of the glycosylation is scission of the glycosidic bond after proton transfer from the acidic Glu177. The estimated lifetime of the oxocarbenium ion intermediate (on the order of tens of ps) as well as the secondary kinetic isotope effect suggest that there is no accumulation of this intermediate on the reaction path, although the intermediate can be transiently formed. In the ES complex, the carbohydrate structure of the xylose residue at the -1 subsite has a rather distorted (skewed) geometry, and this xylose unit at the active site has an apparent half-chair conformation when the oxocarbenium ion intermediate is formed. The major catalytic role of the protein environment is to appropriately fix and align residues that take part in the initial proton transfer. Due to a fine alignment of catalytic residues, the enzyme can accelerate the glycosylation reaction without paying a reorganization energy penalty.

中文翻译:

深入了解 GH11 木聚糖酶的催化机制:基于中子结构的底物畸变计算分析

木聚糖酶生物质分解酶的反应机理一直是争论的主题。为了阐明机制,我们研究了 GH11 木聚糖酶的糖基化步骤,该酶催化木质纤维素半纤维素 (木聚糖) 的水解。利用最近的中子结构,该结构确定了相关残基的质子化状态,我们使用 ab initio QM/MM 方法来确定糖基化的详细反应机制。特别是,我们的重点是在反应途径上是否形成氧代碳鎓离子中间体这一有争议的问题。计算支持双位移方案的基本保持机构的有效性。通过 QM/MM-CCSD(T)/6-31(+)G**//MP2/6-31+G**/AMBER 计算,该反应的估计自由能垒为 ~18 kcal/mol,糖基化的速率决定步骤是从酸性 Glu177 质子转移后糖苷键的断裂。氧碳鎓离子中间体的估计寿命(大约几十 ps)以及二次动力学同位素效应表明,该中间体没有在反应路径上积累,尽管中间体可以瞬时形成。在 ES 复合物中,-1 亚位点木糖残基的碳水化合物结构具有相当扭曲(偏斜)的几何形状,当氧碳鎓离子中间体形成时,活性位点的木糖单元具有明显的半椅构象。蛋白质环境的主要催化作用是适当地固定和对齐参与初始质子转移的残基。由于催化残基的精细排列,
更新日期:2020-09-22
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