Nicotinamide mononucleotide (NMN), a key Nicotinamideadeninedinucleotide (NAD+) precursor, has attracted attention for anti-aging potential. As an oral bioactive molecule, NMN inevitably interacts with digestive enzymes in the gastrointestinal tract, which may affect its bioavailability, stability and physiological function. Nevertheless, the interactions between NMN and digestive enzymes have not been systematically characterized. This study investigated NMN binding with three digestive enzymes (trypsin, pepsin, and α-amylase) using multi-spectroscopic techniques, molecular docking, and molecular dynamics (MD) simulations. Fluorescence quenching analysis showed NMN induced static quenching of all three enzymes through spontaneous binding. Thermodynamic analysis indicated hydrophobic interactions, supplemented by hydrogen bonding, as primary driving forces. Molecular docking demonstrated NMN was embedded in the hydrophobic cavity and stabilized with the surrounding residues through hydrogen bonds and hydrophobic interaction. Synchronous fluorescence, UV-Vis absorption, circular dichroism (CD), and resonance light scattering (RLS) analyses showed NMN altered aromatic residue microenvironments and induced conformational rearrangements. MD simulations confirmed structural stabilization of NMN-enzyme complexes, particularly the NMN-pepsin system, exhibiting the lowest root mean square deviation (RMSD) and smaller radius of gyration (Rg). Functional assays revealed concentration-dependent inhibition of digestive enzyme activities, with pepsin showing strongest susceptibility. Additionally, antioxidant capacities decreased after complex formation, while anti-inflammatory activity was enhanced. These findings provide mechanistic insights into the interaction between NMN and digestive enzymes under in vitro conditions, which is helpful in clarifying the roles of NMN in digestion, transport, and metabolism processes. They have significant guiding significance for drug design and development.
