Multispectral method combined with molecular modelling to investigate the binding mechanisms of DBP and DIBP on pepsin

Abstract

The binding mechanisms of dibutyl phthalate (DBP) and diisobutyl phthalate (DIBP) with pepsin (PEP) were investigated by multispectral method and computational techniques (molecular docking, molecular dynamics, surface electrostatic potential), as well as cytotoxicity assay. Fluorescence studies revealed that both DBP and DIBP had a static bursting effect on PEP, forming a stable complex. Furthermore, the formation of PEP-DBP/DIBP complexes was observed to occur spontaneously (ΔG < 0), driven by the interactions of hydrogen bonding and van der Waals forces. PEP exhibits a higher binding constant Ka of DBP (3.30 × 104 L·mol−1) compared to that of DIBP (2.37 × 104 L·mol−1). The higher stability of PEP-DBP complexes can be attributed to the different branching structures of DBP and DIBP, this observation was further supported by the larger negative surface area of DBP (125.90 Å2) in the ESP analysis, and were aligned with the MD simulations results of more stable RMSD and lower Rg value in presence of DBP. Computational analysis revealed that both DBP and DIBP exhibited binding interactions with the fluorescent groups (Tyr 75, Phe 111, and Phe 117) by van der Waals forces. DBP/DIBP binds to the Asp 32 and Asp 215 motifs of the catalytic activity center of PEP. Combined with the lower Rg values, CD tests suggested that the addition of DBP/DIBP caused the rise of α-Helix content thereby compacted the conformation structure of PEP into a compact shape. In addition, DBP generated more hydrogen bonds with PEP compared to DIBP, also as evidence to the higher stability of EPE-DBP. Finally, the IC50 of DBP and DIBP on Caco-2 cells were determined to be 5.16 × 10-4 mol·L-1 and 1.37 × 10-3 mol·L-1, respectively confirming the higher level of toxicity of DBP. The present study elucidates the effect and binding mechanism of DBP/DIBP on PEP at the molecular level, providing fundamental data for the impact and potential risk assessment of PAEs on the human digestive system.