Insights into the interaction of tris (2,4-di-tert-butyl phenyl) phosphite (I 168) and its metabolites (24DP) with pepsin: Enzyme inhibition, structural impacts, and antioxidant interference.

This study investigated the interactions between pepsin (PEP) and the organophosphite antioxidant Irgafos 168 (I 168), its metabolite 2,4-di-tert-butylphenol (24DP), focusing on structural, functional, and antioxidant implications. UV-Vis absorption spectra, fluorescence lifetimes and Stern-Volmer quenching analyses show that I 168/24DP statically bursts PEP with high binding affinity. Thermodynamic parameters, molecular docking analyses and molecular dynamics simulations (e.g. RMSD, Rg) indicate that the spontaneous formation of the stable complexes is mainly driven by van der Waals forces and hydrophobic interactions. I 168 exhibited higher pepsin inhibition (6.97 %-27.51 %) than 24DP (5.04 %-19.29 %), with dose-dependent suppression linked to altered catalytic residue accessibility (e.g., Asp32/Asp215, Tyr75) and disrupted proton transfer efficiency. Structural analyses revealed I 168 increased PEP's flexibility and hydrophobic residue exposure, while 24DP enhanced rigidity and compactness. Antioxidant assays showed 24DP's superior DPPH scavenging capacity (9.45 %-47.35 %) compared to I 168 (2.28 %-8.00 %), though PEP significantly suppressed 24DP's antioxidant efficacy. These findings underscore the necessity of evaluating both parent compounds and metabolites in safety assessments, as metabolic processes retained biological impacts on enzymatic and antioxidant functions. The study provides critical insights into the molecular mechanisms of antioxidant-induced enzyme inhibition and highlights risks to digestive health and food safety.