“Blade of\nPolarized Water Molecule”\nIs the Key to Hydrolase Catalysis Regulation

Hydrolysis catalyzed by aspartic proteases is a crucial\nreaction\nin many biological processes. However, anchoring water molecules and\nunifying multiple catalytic pathways remain significant challenges.\nConsequently, molecular design often compromises by focusing on enhancing\nsubstrate specificity. Using our self-developed polarizable point\ncharge (PPC) force field, we determined the significant role of polarization\nin the hydrolase of pepsin for the first time. To be stably anchored\nin the active site, the water should be intensely polarized with a\ncharge higher than −0.94e. Induced by this polarization, the\npepsin was shown to support three general base/general acid pathways,\nwith a preference for the gemdiol-intermediate-based pathway. Consequently,\nwe proposed the “Blade of Polarized Water Molecule”\nmodel for rational enzyme design, highlighting that the polarization\nof both the attacking water and the attacked carbonyl is crucial for\nenhancing hydrolysis. Mutants D290Q and S172P showed activity enhancements\nof 191.23% and 324.70%, respectively. The improved polarization of\nwater, carbonyl, and relevant nucleophilic attack distances in the\nmutants reaffirmed the crucial role of polarization in improving hydrolysis.\nThis study provides a new perspective on hydrolase analysis and modification.