Abstract
Methomyl is one of the most important carbamates that has caused potential hazardous effects on both human beings and the environment. Here, we systematically investigated the hydrolysis mechanism of methomyl catalyzed by esterase PestE using molecular dynamics simulations (MD) and quantum mechanics/molecular mechanics (QM/MM) calculations. The hydrolysis mechanism involves two elementary steps: (Ⅰ) serine-initiated nucleophilic attack and (Ⅱ) C-O bond cleavage. Our work elicits the atomic level details of the hydrolysis mechanism and free energy profiles along the reaction pathway. The Boltzmann-weighted average potential barriers are 19.1 kcal/mol and 7.5 kcal/mol for steps Ⅰ and Ⅱ, respectively. We identified serine-initiated nucleophilic attack as the rate determining-step. The deep learning-based k(cat) prediction model indicated that the barrier of the rate-determining step is 15.4 kcal/mol, which is in good agreement with the calculated results using Boltzmann-weighted average method. We have elucidated the importance of the protein-substrate interactions and the roles of the key active site residues during the hydrolysis process through noncovalent interactions analysis and electrostatic potential (ESP) analysis. The results provide practical value for achieving efficient degradation of carbamates by hydrolases.