Establishing the Catalytic Mechanism of Human Pancreatic α-Amylase with QM/MM Methods
Establishing the Catalytic Mechanism of Human Pancreatic α-Amylase with QM/MM Methods
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DOI：
10.1021/acs.jctc.5b00222
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发表时间：
2015-06-01
影响因子：
5.5
通讯作者：
Toscano, Marirosa
中科院分区：
文献类型：
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作者：
Pinto, Gaspar P.;Bras, Natercia F.;Toscano, Marirosa
在这项工作中，我们研究了人胰腺α-淀粉酶（HPA）的催化机制。我们的目标是使用计算方法确定具有原子细节的 HPA 催化机制。我们证明 HPA 催化机制由两个步骤组成，第一步（糖基化步骤）涉及破坏糖苷键，最终形成共价中间体。第二步（去糖基化步骤）：包括添加水分子以释放酶/底物共价中间体，完成糖的水解。活性位点对溶剂非常开放。我们的机制与之前提出的机制基本上不同，参与该机制的活性位点附近有两个水分子，而不是只有一个水分子。我们还证明了三个催化氨基酸、两个天冬氨酸残基和一个谷氨酸（D197、E233 和 D300）在催化过程中的相关作用。还表明限速步骤是糖基化，其活化能与HPA的实验值一致。实验活化能为14.4 kcal mol(-1)，计算得到的活化能为15.1 kcal mol(-1)。
In this work, we studied the catalytic mechanism of human pancreatic alpha-amylase (HPA). Our goal was to determine the catalytic mechanism of HPA with atomic detail using computational methods. We demonstrated that the HPA catalytic mechanism consists of two steps, the first of which (glycosylation step) involves breaking the glycosidic bond to culminate in the formation of a covalent intermediate. The second (deglycosylation step) :consists of the addition of a water molecule to release the enzyme/substrate covalent intermediate, completing the hydrolysis of the sugar. The active site was very open to the solvent. Our mechanism basically differs from the previously proposed mechanism by having two water molecules instead of only one near the active site that participate in the mechanism. We also demonstrate the relevant role of the three catalytic amino acids, two aspartate residues and a glutamate (D197, E233, and D300), during catalysis. It was also Shown that the rate limiting step was glycosylation, and its activation energy was in agreement with experimental values obtained for HPA. The experimental activation energy was 14.4 kcal mol(-1), and the activation energy obtained computationally was 15.1 kcal mol(-1).