Conserved Protonation Pattern in the Extended Active Site of Human Carboxylesterase 1 and Its Impact on Enzyme Catalysis

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.