Tang_2026_Luminescence_41_e70573

This study investigated the molecular mechanism underlying berberine (BER)-mediated inhibition of acetylcholinesterase (AChE) and its synergistic interaction with galantamine hydrobromide (GAL). Heatmap-based dose-response analysis revealed significant synergistic inhibitory effect of BER and GAL on AChE activity at concentrations of 4.0 microM BER and 8.0 microM GAL. Enzyme kinetic analyses demonstrated that GAL, BER, and the BER GAL combination exhibited competitive, non-competitive, and mixed-type inhibition modes against AChE, respectively. Fluorescence quenching and circular dichroism spectroscopy confirmed high affinity binding of BER to AChE, predominantly stabilized by hydrophobic interactions and hydrogen bonding resulting in conformational alterations in the enzyme structure. Molecular docking simulations further indicated that BER binding affects the solvent-accessible surface area of AChE, induces microenvironmental changes within AChE, and enhances its hydrophobicity. In addition, molecular docking simulations confirmed that BER bound to the peripheral anionic site (PAS) (Trp286, Tyr341, Tyr124) of AChE inducing a more flexible enzyme conformation that facilitates more sites of BER binding. This structural loosening hinders substrate access to the catalytic active site (CAS), thereby inhibiting AChE's catalytic activity. BER mediated PAS remodeling, which enhances GAL's binding affinity for the CAS and cooperatively destabilizes the AChE structure ultimately disrupting substrate-enzyme interactions.