Elucidating interactions between N′-substituted p-phenylenediamine antioxidants and pepsin: Structural changes and enzymatic activity alterations

PPDs caused static fluorescence quenching of PEP. • PPDs alter the microenvironment and hydrophobicity of amino acid residues in PEP. • PPDs bind to PEP via hydrophobic interactions and hydrogen bonds. • N-substituent structural variations may affect PEP binding and subsequent functional effects. Herein, the interaction mechanisms between pepsin (PEP) and two p -phenylenediamine derivatives, DPPD and CPPD, were investigated via multi-spectroscopic, computational, and enzymatic methods. UV–vis absorption spectroscopy, fluorescence spectroscopy, and time-resolved fluorescence confirmed the formation of ground-state complexes with a static quenching mechanism. Binding constant analysis revealed a stronger, more endothermic, and entropy-driven binding of CPPD compared to DPPD, primarily driven by hydrophobic forces. Both compounds induced secondary structural changes in PEP, evidenced by an increase in β-sheet and a decrease in α-helix. Crucially, DPPD and CPPD exerted distinct effects on PEP conformation. Molecular dynamics simulations showed DPPD binding stabilized PEP's structure while CPPD increased its flexibility. Molecular surface electrostatic potential (MESP) analysis rationalized these differences: CPPD's more polarized and extensive electrostatic field, stemming from its asymmetric cyclohexyl substituent, fostered stronger interactions but greater structural perturbation. These structural changes provided a direct rationale for the observed inhibition of PEP's enzymatic activity, where DPPD, despite weaker overall binding, induced specific active-site changes causing more pronounced inhibition. The antioxidant activities of both ligands decreased upon complexation. This study elucidates how subtle ligand structural differences dictate binding modes and functional outcomes in protein-ligand systems.