Institute of Theoretical and Computational Chemistry, Key Laboratory of Mesoscopic Chemistry, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China.
Phys Chem Chem Phys. 2019 Feb 27;21(9):5049-5058. doi: 10.1039/c8cp07784c.
Chymotrypsin inhibitor 2 (CI2) is a special serine protease inhibitor which can resist hydrolysis for several days with a rapid equilibrium between the Michaelis complex and acyl-enzyme intermediate. The energies and conformational changes for subtilisin-catalyzed proteolysis of CI2 were examined in this paper for the first time by employing pseudo bond ab initio QM/MM MD simulations. In the acylation reaction, a low-barrier hydrogen bond between His64 and Asp32 in the transition state together with the lack of covalent backbone constraints makes the peptide bonds of CI2 break more easily than in other serine protease inhibitors. After acyl-enzyme formation, molecular dynamics simulations showed that the access of hydrolytic water to the active site requires partial dissociation of the leaving group. However, retention of the leaving group mainly by the intra- and inter-molecular H-bonding networks hinders the access of water and retards the deacylation reaction. Instead of the dissociation constant of inhibitors, we suggest employing the free energy at the acyl-enzyme state to predict the relative hydrolysis rates of CI2 mutants, which are testified by the experimental relative hydrolysis rates.
糜蛋白酶抑制剂 2（CI2）是一种特殊的丝氨酸蛋白酶抑制剂，其米氏复合物和酰-酶中间物之间能够迅速达到平衡，在数天内抵抗水解。本文首次通过拟键从头算 QM/MM MD 模拟研究了枯草杆菌蛋白酶催化 CI2 蛋白水解的能量和构象变化。在酰化反应中，过渡态中 His64 和 Asp32 之间的低势垒氢键以及缺乏共价主链约束使得 CI2 的肽键比其他丝氨酸蛋白酶抑制剂更容易断裂。酰-酶形成后，分子动力学模拟表明，水解水进入活性位点需要部分解离离去基团。然而，离去基团主要通过分子内和分子间氢键网络的保留阻碍了水的进入并延迟了脱酰反应。我们建议用酰-酶状态下的自由能来预测 CI2 突变体的相对水解速率，而不是抑制剂的离解常数，这得到了实验相对水解速率的验证。
