Protonation State Insights into the Influence of Biocatalytic Function for Acetylcholinesterase Mediated by Neonicotinoids

The catalytic efficiency of acetylcholinesterase (AChE) is likely regulated by the protonation states and conformational adaptations of its catalytic residues. While neonicotinoid insecticides are recognized for impairing AChE function through neurotoxic mechanisms, the precise molecular mechanisms governing this inhibition remain poorly characterized. This investigation elucidates how structural variations among neonicotinoids modulate the protonation equilibria of Glu-202 and His-447 in AChE’s catalytic triad. Comparative analysis reveals that nitro-substituted neonicotinoids (imidacloprid, clothianidin) induce more pronounced protonation state transitions compared to their cyano-containing counterparts (thiacloprid, acetamiprid). Specifically, the strong electron-withdrawing nitro groups facilitate the conversion of Glu-202 from the deprotonation (GLU) to protonation (GLH) state and His-447 from the δ- (HID) to ε-position protonation (HIE) state through enhanced electrostatic interactions. These electronic perturbations trigger structural reorganization within the active site, evidenced by nitro group-directed residue realignment and subsequent H-bond formation. Energy decomposition analysis identifies electrostatic contributions as the primary determinant of binding affinity differences, with nitro-neonicotinoids exhibiting stronger interactions than cyano-neonicotinoids. QM/MM metadynamics reveals that substantial protonation state alterations disrupt AChE’s biocatalytic function, particularly its capacity for acetylcholine hydrolysis. Finally, SH-SY5Y-based cellular assays show that imidacloprid exhibits the strongest inhibitory effect on AChE intracellular activity, while thiacloprid and acetamiprid show weaker inhibitory effects, aligning with the computational predictions. This study provides insights into the protonation-state-induced biocatalytic function for acetylcholinesterase mediated by neonicotinoids, contributing to the assessment of exogenous ligand-induced potential ecological and human health risks.