Abstract: In this study, we systematically investigated the regulatory effects of two hempseed peptides (APAM and RLPA) on pancreatic lipase (PL) and cholesterol esterase (CE) activities and the molecular binding mechanism using enzyme kinetics, synergistic inhibition, fluorescence spectroscopy, isothermal titration calorimetry and molecular docking. The results showed that RLPA had stronger inhibitory activity on PL and CE with half maximal inhibitory concentration (IC50) values of (79.62 ± 3.20) and (301.27 ± 14.40) μmol/L, respectively. Kinetic analysis indicated that the inhibition mechanism of APAM on the two enzymes was competitive inhibition, whereas RLPA was a mixed-type inhibitor. Both APAM and RLPA synergistically inhibited PL and CE with orlistat at low concentrations, but the synergistic effect was weakened or even antagonized at high concentrations. Fluorescence spectroscopy results showed that both peptides could produce static quenching by binding to PL and CE, which changed the hydrophobic environment of aliphatic amino acids in the enzymes. Isothermal titration calorimetry demonstrated that the binding process between the peptides and the enzymes was spontaneous and exothermic, which was achieved mainly through hydrogen bonding and electrostatic interaction. Further analysis using molecular docking simulations showed that hydrogen bonding, salt bridges, and cation-π interactions played crucial roles in peptide-enzyme binding. This study enriches our understanding of the interaction mechanism of peptides with PL and CE, and provides a scientific basis for the development of functional foods based on hempseed protein.

Key words: lipid-digesting enzymes; inhibition mechanism; fluorescence spectroscopy; isothermal titration calorimetry; molecular modeling