Shanghai Engineering Research Center of Molecular Therapeutics & New Drug Development, Shanghai Key Laboratory of Green Chemistry & Chemical Process, School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200062, China.
Department of Micro/Nano Electronics, School of Electronic Information and Electrical Engineering, Shanghai Jiao Tong University, Shanghai 200240, China.
J Chem Inf Model. 2024 Oct 28;64(20):7987-7997. doi: 10.1021/acs.jcim.4c01123. Epub 2024 Oct 9.
Hydrolysis catalyzed by aspartic proteases is a crucial reaction in many biological processes. However, anchoring water molecules and unifying multiple catalytic pathways remain significant challenges. Consequently, molecular design often compromises by focusing on enhancing substrate specificity. Using our self-developed polarizable point charge (PPC) force field, we determined the significant role of polarization in the hydrolase of pepsin for the first time. To be stably anchored in the active site, the water should be intensely polarized with a charge higher than -0.94e. Induced by this polarization, the pepsin was shown to support three general base/general acid pathways, with a preference for the gemdiol-intermediate-based pathway. Consequently, we proposed the "Blade of Polarized Water Molecule" model for rational enzyme design, highlighting that the polarization of both the attacking water and the attacked carbonyl is crucial for enhancing hydrolysis. Mutants D290Q and S172P showed activity enhancements of 191.23% and 324.70%, respectively. The improved polarization of water, carbonyl, and relevant nucleophilic attack distances in the mutants reaffirmed the crucial role of polarization in improving hydrolysis. This study provides a new perspective on hydrolase analysis and modification.
天冬氨酸蛋白酶催化的水解反应是许多生物过程中的关键反应。然而，锚定水分子并统一多种催化途径仍然是重大挑战。因此，分子设计通常通过专注于增强底物特异性来妥协。使用我们自主开发的极化点电荷（PPC）力场，我们首次确定了极化在胃蛋白酶水解酶中的重要作用。为了在活性部位稳定地锚定，水分子应强烈极化，电荷高于-0.94e。这种极化诱导胃蛋白酶支持三种广义酸碱途径，偏爱基于偕二醇中间体的途径。因此，我们提出了“极性水分子刀片”模型用于合理的酶设计，强调了攻击水和被攻击羰基的极化对于增强水解的重要性。突变体 D290Q 和 S172P 的活性分别增强了 191.23%和 324.70%。突变体中水分子、羰基和相关亲核攻击距离的极化改善进一步证实了极化在提高水解中的关键作用。本研究为水解酶分析和修饰提供了新的视角。
