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Abstract：
Recently major advances were gained on the designed proteins aimed to generate biomolecular mimics of proteases. Although such enzyme-like catalysts must still suffer refinements for improving the catalytic activity, at the moment, they represent a good example of artificial enzymes to be tested in different fields. Herein, a de novo designed homo-heptameric peptide assembly (CC-Hept) where the esterase activity towards p-nitro-phenylacetate was obtained for introduction of the catalytic triad (Cys-His-Glu) into the hydrophobic matrix, is the object of the present combined molecular dynamics and quantum mechanics/molecular mechanics investigation. Constant pH Molecular Dynamics simulations on the apoform of CC-Hept suggested that the Cys residues are present in the protonated form. Molecular dynamics (MD) simulations of the enzyme-substrate complex evidenced the attitude of the enzyme-like system to retain water molecules, necessary in the hydrolytic reaction, in correspondence of the active site, represented by the Cys-His-Glu triad on each of the seven chains, without significant structural perturbations. A detailed reaction mechanism of esterase activity of CC-Hept-Cys-His-Glu was investigated on the basis of the quantum mechanics/molecular mechanics calculations employing a large quantum mechanical (QM) region of the active site. The proposed mechanism is consistent with available esterases kinetics and structural data. The roles of the active site residues were also evaluated. The deacylation phase emerged as the rate-determining step, in agreement with esterase activity of other natural proteases.
摘要：
最近在旨在产生蛋白酶的生物分子模拟物的设计蛋白质上获得了重大进展。尽管此类酶样催化剂仍必须进行改进以提高催化活性，此刻,它们代表了在不同领域进行测试的人工酶的一个很好的例子。在这里，从头设计的同源七聚体肽组装体（CC-Hept），其中获得了对硝基苯乙酸的酯酶活性，用于将催化三联体（Cys-His-Glu）引入疏水基质中，是当前组合的分子动力学和量子力学/分子力学研究的对象。恒定pH对CC-Hept脱位形式的分子动力学模拟表明，Cys残基以质子化形式存在。酶-底物复合物的分子动力学（MD）模拟证明了类酶系统保留水分子的态度，在水解反应中所需的，与活动站点相对应，由七个链条中的每个链条上的Cys-His-Glu三合会代表，没有显著的结构扰动。基于使用活性位点的大量子力学（QM）区域的量子力学/分子力学计算，研究了CC-Hept-Cys-His-Glu酯酶活性的详细反应机理。所提出的机制与可用的酯酶动力学和结构数据一致。还评估了活性位点残基的作用。脱酰阶段是速率决定步骤，与其他天然蛋白酶的酯酶活性一致。
