Huang_2025_J.Chem.Inf.Model_65_8794

Human carboxylesterase 1 (hCES1), a crucial serine hydrolase, plays extensive roles in human metabolic processes. Its catalytic center exhibits structural similarities to cholinesterases (AChE and BChE) from the Type-B carboxylesterase/lipase family, featuring hallmark elements such as the catalytic triad and oxyanion hole. Previous studies on AChE and BChE have demonstrated that a protonated glutamate residue within the extended active site is essential for forming a water-centered hydrogen bond network that stabilizes the catalytic triad. However, the hydrogen bond network surrounding hCES1's catalytic triad is more complex, incorporating additional glutamate residues compared to cholinesterases. The protonation states of these glutamates and their precise roles in enzymatic catalysis remain unclear, necessitating further investigation. In this study, we systematically investigated the protonation states of key glutamate residues within hCES1's extended active site and their functional impacts using conventional molecular dynamics simulations, constant pH molecular dynamics simulations, and thermodynamic integration calculations. Our results reveal that protonation of E220 and E246 is critical for maintaining the stability of the water-centered hydrogen bond network, thereby stabilizing the catalytic triad and ensuring catalytic efficiency. Conversely, deprotonation of these residues induces electrostatic repulsion that disrupts the hydrogen bond network and disorders the catalytic triad. Moreover, structural analysis and sequence alignment indicate that this water-centered extended active site and its associated protonation pattern represent a conserved structural motif across the Type-B carboxylesterase/lipase family, rather than being unique to hCES1. These findings provide novel insights into the catalytic mechanism of hCES1 and establish a theoretical foundation for engineering serine hydrolases with analogous catalytic architectures.