Discovery and validation of pumpkin seed protein-derived bioactive peptides targeting α-glucosidase: Insights into...

Pumpkin seed protein (PSP) is a promising precursor of bioactive peptides, but the mechanisms underlying the α-glucosidase inhibitory activity of its hydrolysates are not yet fully understood. In the present work, a previously unreported tetrapeptide with α-glucosidase inhibitory activity, YYPW, was obtained from PSP through simulated gastrointestinal digestion and in silico screening. Kinetic assays demonstrated that YYPW is a potent mixed-type inhibitor (IC₅₀ = 279.61 μmol/L). Molecular docking revealed that YYPW engages both the catalytic dyad (Asp443/Asp542) and peripheral aromatic residues via π-π stacking and hydrogen bonding. Subsequent molecular dynamics simulations indicated that this dual-site binding induces structural compaction and reduces the conformational flexibility of key catalytic loops. Multi-spectroscopic analyses (UV-Vis, fluorescence, and circular dichroism) confirmed a shift in microenvironmental polarity and a transition from α-helix to β-sheet. These findings help elucidate the inhibitory mechanism of YYPW and highlight its potential for further development in glycemic control applications.