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>Probing the importance of hydrogen bonds in the active site of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation
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Probing the importance of hydrogen bonds in the active site of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation
pHydrogen bonds occurring in the catalytic triad (Aspsup32/sup, Hissup64/sup and Sersup221/sup) and the oxyanion hole (Asnsup155/sup) are very important to the catalysis of peptide bond hydrolysis by serine proteases. For the subtilisin NK (nattokinase), a bacterial serine protease, construction and analysis of a three-dimensional structural model suggested that several hydrogen bonds formed by four residues function to stabilize the transition state of the hydrolysis reaction. These four residues are Sersup33/sup, Aspsup60/sup, Sersup62/sup and Thrsup220/sup. In order to remove the effect of these hydrogen bonds, four mutants (Sersup33/sup→Alasup33/sup, Aspsup60/sup→Alasup60/sup, Sersup62/sup→Alasup62/sup, and Thrsup220/sup→Alasup220/sup) were constructed by site-directed mutagenesis. The results of enzyme kinetics indicated that removal of these hydrogen bonds increases the free-energy of the transition state (ΔΔiG/isubT/sub). We concluded that these hydrogen bonds are more important for catalysis than for binding the substrate, because removal of these bonds mainly affects the ik/isubcat/sub but not the iK/isubm/sub values. A substrate, SUB1 (succinyl-Ala-Ala-Pro-Phe-ip/i-nitroanilide), was used during enzyme kinetics experiments. In the present study we have also shown the results of FEP (free-energy perturbation) calculations with regard to the binding and catalysis reactions for these mutant subtilisins. The calculated difference in FEP also suggested that these four residues are more important for catalysis than binding of the substrate, and the simulated values compared well with the experimental values from enzyme kinetics. The results of MD (molecular dynamics) simulations further demonstrated that removal of these hydrogen bonds partially releases Aspsup32/sup, Hissup64/sup and Asnsup155/sup so that the stability of the transition state decreases. Another substrate, SUB2 (H-D-Val-Leu-Lys-ip/i-nitroanilide), was used for FEP calculations and MD simulations./p
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机译：>催化三元组（Asp 32 ，His 64 和Ser 221 ）和氧阴离子孔（Asn 155 ）对于丝氨酸蛋白酶催化肽键水解非常重要。对于枯草杆菌蛋白酶NK（纳豆激酶），一种细菌丝氨酸蛋白酶，三维结构模型的构建和分析表明，由四个残基形成的几个氢键起着稳定水解反应过渡态的作用。这四个残基是Ser 33 ，Asp 60 ，Ser 62 和Thr 220 。为了消除这些氢键的作用，有四个突变体（Ser 33 →Ala 33 ，Asp 60 →Ala 60 < / sup>，Ser 62 →Ala 62 和Thr 220 →Ala 220 ）是通过site-定向诱变。酶动力学结果表明，这些氢键的去除增加了过渡态的自由能（ΔΔ G T ）。我们得出的结论是，这些氢键对催化比对底物的结合更重要，因为这些键的去除主要影响 k cat ，但不影响 K m 个值。在酶动力学实验过程中，使用了底物SUB1（琥珀酰-Ala-Ala-Pro-Phe-p-硝基苯胺）。在本研究中，我们还显示了有关这些突变枯草杆菌蛋白酶的结合和催化反应的FEP（自由能扰动）计算结果。 FEP的计算差异还表明，这四个残基对催化的作用比与底物的结合更为重要，并且模拟值与酶动力学实验值进行了比较。 MD（分子动力学）模拟的结果进一步表明，除去这些氢键会部分释放Asp 32 ，His 64 和Asn 155 过渡态的稳定性降低。另一种底物SUB2（H-D-Val-Leu-Lys- p -硝基苯胺）用于FEP计算和MD模拟。
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