Purpose: To investigate the structural features of wild and mutant forms of the pPAF-AH enzyme that are responsible for coronary artery disease.
Methods: Mutant variants of human pPAF-AH having either V279F, Q281R, or both were modelled and evaluated for stereo chemical and structural correctness. The 3D coordinates of substrate PAF were retrieved from the PubChem database was solvated and minimized on Discovery Studio, and docked to the wild and mutant enzyme models. The top docked pose complex was refined by MD simulation.
Results: pPAF-AH model comprises of 420 amino acids in a α/β-hydrolase fold that contains a substrate-binding hydrophobic channel with an active site pocket having a catalytic triad of Ser273, Asp296 and His351. Mutations at positions 279 and 281 are opposite one another on the middle of 12 residues long H5 helix that forms the hydrophobic core of the enzyme. V279F causes a tilt on the axis of the mutation bearing helix to avoid steric clashes with the hydrophobic residues on the β-sheets adjacent to it, inducing subtle conformational changes on the H5-β 8 loop, β 8 sheet, and the loop bearing Asp296. A cascade of conformational changes induces a change in the orientation of His351 resulting in loss of hydrogen bonded interaction with catalytic Ser273. Q281R causes a shortening of H5 and β 8, which induces conformational changes of the loops bearing Ser273 and Asp296, respectively. Simultaneous conformational changes of secondary structural elements result in the flipping of His351 causing a break in the catalytic triad. Also, there is a compromise in the substrate-binding area and volume in the mutants resulting in loss of binding to its substrate.
Conclusion: Mutant enzymes show changes at the site of the mutation, secondary motif conformations and global structural conformations that adversely affect the active site, decrease substrate channel volume and decrease stability, thereby affecting enzymatic function.

Keywords: platelet activating factor acetyl hydrolase, structure, molecular modelling, mutations, V279F, Q281R, clinical phenotype, coronary artery disease


中文翻译：

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人血浆血小板活化因子-乙酰水解酶的野生和突变形式的结构模型

目的：研究导致冠状动脉疾病的 pPAF-AH 酶的野生和突变形式的结构特征。
方法：对具有 V279F、Q281R 或两者的人类 pPAF-AH 的突变变体进行建模并评估立体化学和结构的正确性。从 PubChem 数据库中检索到的底物 PAF 的 3D 坐标在 Discovery Studio 上进行了溶剂化和最小化，并与野生和突变酶模型对接。顶部停靠的姿势复合体通过 MD 模拟进行了细化。
结果：pPAF-AH 模型由 α/β-水解酶折叠中的 420 个氨基酸组成，该折叠包含一个底物结合疏水通道和一个活性位点口袋，该口袋具有 Ser273、Asp296 和 His351 的催化三联体。位置 279 和 281 的突变在形成酶疏水核心的 12 个长 H5 螺旋的中间彼此相对。V279F 导致带有突变的螺旋轴倾斜，以避免与其相邻的 β-折叠上的疏水残基发生空间冲突，从而在 H5-β 8 环、β 8 折叠和带有 Asp296 的环上引起细微的构象变化. 一连串的构象变化导致 His351 的方向发生变化，导致与催化 Ser273 的氢键相互作用丧失。Q281R 导致 H5 和 β 8 的缩短，分别引起带有 Ser273 和 Asp296 的环的构象变化。二级结构元素的同时构象变化导致 His351 的翻转，导致催化三联体的中断。此外，突变体的底物结合面积和体积存在折衷，导致与其底物的结合丧失。
结论：突变酶在突变位点、二级基序构象和整体结构构象发生变化，对活性位点产生不利影响，降低底物通道体积，降低稳定性，从而影响酶的功能。

关键词：血小板活化因子乙酰水解酶，结构，分子模型，突变，V279F，Q281R，临床表型，冠状动脉疾病