Insight into the Catalytic Mechanism of GH11 Xylanase: Computational Analysis of Substrate Distortion Based on a Neutron Structure
Insight into the Catalytic Mechanism of GH11 Xylanase: Computational Analysis of Substrate Distortion Based on a Neutron Structure
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DOI：
10.1021/jacs.0c02148
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发表时间：
2020-10-21
影响因子：
15
通讯作者：
Smith, Jeremy C.
中科院分区：
文献类型：
--
作者：
Ishida, Toyokazu;Parks, Jerry M.;Smith, Jeremy C.
木聚糖酶分解生物质的反应机理仍然是争论的主题。为了阐明该机制，我们研究了GH 11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。利用最近的中子晶体结构，揭示了相关残基的质子化状态，我们使用从头算量子力学/分子力学（QM/MM）计算，以确定详细的反应机理的糖基化步骤。特别是，我们的重点是有争议的问题，是否oxocarbenium离子中间体形成的反应途径。计算结果支持双位移方案中基本保持机制的有效性。用QM/MMCCSD（T）/6-31（+）G**//MP2/6- 31+G**/AMBER计算，该反应的估计自由能垒类似于18 kcal/mol，糖基化的速率决定步骤是质子从酸性Glu 177转移后糖苷键的断裂。氧碳正离子中间体的估计寿命（几十ps的顺序）和次级动力学同位素效应表明，没有积累的反应路径上的中间体，虽然中间体可以瞬时形成。在酶-底物（ES）复合物中，木糖残基在-1亚位点的碳水化合物结构具有相当扭曲（歪斜）的几何形状，并且当形成氧碳正离子中间体时，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定向参与初始质子转移的残基。由于催化残基的精细排列，该酶可以加速糖基化反应而不付出重组能量代价。
The reaction mechanism of biomass decomposition by xylanases remains 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 crystal structure, which revealed the protonation states of relevant residues, we used ab initio quantum mechanics/ molecular mechanics (QM/MM) calculations to determine the detailed reaction mechanism of the glycosylation step. 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 within a double-displacement scheme. The estimated free energy barrier of this reaction is similar to 18 kcal/mol with QM/MMCCSD(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) and 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 enzyme-substrate (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 halfchair conformation when the oxocarbenium ion intermediate is formed. The major catalytic role of the protein environment is to orient residues that take part in the initial proton transfer. Because of a fine alignment of catalytic residues, the enzyme can accelerate the glycosylation reaction without paying a reorganization energy penalty.