Zhang_2025_Int.J.Biol.Macromol_305_141205

The emerging pollutant N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine quinone (6PPD-quinone) has attracted broad attention because of its widespread presence and harmful impacts, including hepatotoxicity and neurotoxicity. Acetylcholinesterase (AChE) is commonly used as a classical biomarker for assessing toxicity in the nervous system. Here, the interaction mechanism between AChE and 6PPD-quinone was investigated using a combination of multispectral and computational approaches, including enzyme activity assay, fluorescence thermodynamic titration, circular dichroism (CD) spectroscopy, molecular dynamics (MD) simulation, computational alanine scanning (CAS), and free energy landscape (FEL) analysis, among others. The result indicates that 6PPD-quinone spontaneously binds into the active site of AChE, thereby competitively inhibiting enzyme's activity. The interaction is primarily facilitated by hydrogen bonds and van der Waals forces, exhibiting a binding constant (K(b)) of 1.044 x 10(4) M(-1) at 298 K. The introduction of 6PPD-quinone causes a reduction in the alpha-helix content of AChE, making the structure less stable and more relaxed. Furthermore, the FEL analysis of AChE revealed that, with the presence of 6PPD-quinone, the number of global minima of AChE increased from 2 to 2-3. Additionally, Molecular docking outcomes exhibit that 6PPD-quinone interacted with tyrosine (TYR) 337, TYR124, tryptophan (TRP) 86, serine (SER) 203, glycine (GLY) 120 and other residues of AChE. CAS analysis shows binding free energy changes (deltadeltaG(binding)) of TRP86, TYR337 were 5.17 and 2.57 kcal mol(-1), respectively, highlighting their key roles in the binding process of 6PPD-quinone with AChE. The interactions of 6PPD-quinone with the TRP86 and TYR337 may be the reason for the decrease in AChE activity.