Interactions of betulinic acid with pepsin, trypsin, and α-chymotrypsin: A multi-spectroscopic and molecular simulation study

Betulinic acid (BA) is a naturally occurring pentacyclic triterpenoid with diverse pharmacological activities. This study systematically investigated the binding mechanisms of BA with pepsin (PEP), trypsin (TRY), and α-chymotrypsin (CHY). Steady-state and synchronous fluorescence, circular dichroism (CD), Fourier transform infrared (FTIR) spectroscopy, molecular docking, and molecular dynamics (MD) simulations were employed to characterize the interactions, fluorescence quenching, and conformational changes. BA quenched the intrinsic fluorescence of all three enzymes in a concentration- and temperature-dependent manner; PEP underwent dynamic quenching, whereas TRY and CHY exhibited static or mixed-mode quenching. Binding analyses revealed a single dominant site on each enzyme, with the highest affinity observed for CHY. Thermodynamic analysis indicated that hydrophobic interactions predominantly drove BA–PEP binding, whereas electrostatic interactions and hydrogen bonding were critical for BA–TRY and BA–CHY complexation. FTIR confirmed that BA bound to all three enzymes via synergistic hydrogen bonding and hydrophobic interactions, inducing secondary structural alterations. Molecular docking revealed that BA stably occupied the active sites of each enzyme, forming enzyme-specific binding modes. MD simulations (RMSD, Rg, SASA, hydrogen bond analysis, and MMPBSA) validated the dynamic stability of the complexes, with binding free energies of −13.90, −20.14, and −23.08 kcal/mol for BA–PEP, BA–TRY, and BA–CHY, respectively, consistent with the experimental affinity trend. These findings elucidate the molecular mechanisms by which BA modulates digestive proteases, providing insights into its gastrointestinal behavior and bioactivity.