Insight into the binding behavior of N-(1,3-Dimethylbutyl)-N'-phenyl-p-phenylenediamine and its quinone-metabolite with pepsin: Multidisciplinary approaches.

N-(1,3-Dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone (6PPD-Q) formed from the oxidation of N-(1,3-Dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD), have garnered worldwide concern for the acute toxicity to various organisms. Binding behaviors and their interaction differences between PEP and 6PPD/6PPD-Q are investigated by multi-spectral methods, computer simulation studies and enzymatic activity assay. The fluorescence static quenching mechanism indicated that stable PEP-6PPD/6PPD-Q complexes were spontaneously formed drove by van der Waals forces and hydrogen, these binding behaviors change polarity of the microenvironment around residues and distort enzyme structure. Molecular dynamics (MD) simulation and circular dichroism (CD) spectroscopy support that both 6PPD and 6PPD-Q induced changes in the secondary structure and ASA of active center amino acids of PEP. Meanwhile, the effect of hydrophobic forces on the stability of 6PPD/6PPD-Q-PEP complexes should not be ignored on the basis of molecular docking results. The greater fluorescence quenching in PEP-6PPD-Q system (25.36 %), compared to the PEP-6PPD system (22.62 %), is due to more fluorescent groups binding to 6PPD-Q via van der Waals forces and other intermolecular interactions. The inhibition of PEP activity by 6PPD and 6PPD-Q under the experimental conditions ranged from 2.38 %-16.75 % and 6.54 %-21.49 %, respectively, inhibition types are all mixed inhibition.

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