Exploring the effects of food by-product HMF on pepsin structure, function, and physiological activity from a binding perspective.

5-Hydroxymethylfurfural (HMF) derived from the Maillard reaction poses significant health risks including neurotoxicity and carcinogenicity. Upon ingestion with food, HMF can interact with pepsin, a pivotal enzyme in the human digestive system. This study explores their interactions and examines the impact of these interactions on the conformation and function of pepsin. The research indicates that hydrophobic forces aid in the entry of HMF into the pepsin cavity, which then spontaneously leads to the formation of complexes. This interaction results in the quenching of pepsin's fluorescence and a change in the microenvironment of aromatic amino acids. The binding constant (K) for this interaction has been determined to be 4.04 × 10 M at 310 K. Three-dimensional and circular dichroism experiments indicated that the conformation of pepsin undergoes changes when HMF is present. Specifically, molecular dynamics analysis has shown that upon binding to HMF, the conformation of pepsin becomes more compact and stable. Meanwhile, increased concentrations of HMF stimulate pepsin activity in a dose-dependent manner, likely attributable to its interaction with Asp32. The persistent hydrogen bonding between Asp32 and HMF observed during the simulation further supports the critical role of Asp32 in regulating enzyme activity. In addition, Energy decomposition analysis indicates several key amino acid residues, including Asp32, Tyr75, Phe111, and Ile120, which play significant roles in the binding process. Among these residues, Asp32 stands out as the most prominent contributor to the binding interaction with HMF.