Chen_2019_Chemosphere_236_124365

Nitroaromatic compounds (NACs) are widely distributed in the environment and are considered toxic or carcinogenic. However, little attention has been paid to the binding interactions between NACs and biomacromolecules (e.g., proteins). Here we investigated the effects of three model NACs, nitrobenzene (NB), 1,3-dinitrobenzene (DNB), and 1,3,5-trinitrobenzene (TNB), on the activity of acetylcholinesterase (AChE). The presence of NACs (up to 0.5mM) effectively suppressed the AChE-catalyzed hydrolysis of acetylthiocholine iodide, with the suppression effect increasing with the nitro-group substitution (TNB>DNB>NB). Consistently, the UV absorption of AChE at 206nm arising from the skeleton structure decreased by the addition NACs, and the decrease exhibited the same compound sequence, reflecting the perturbing interactions with the skeleton enzyme structure. However, no changes were made on the secondary structure of AChE, as evidenced by the circular dichroism analysis. The fluorescence quenching analysis of AChE demonstrated that NB and DNB interacted with both tryptophan (Trp) and tyrosine (Tyr) residues, whereas TNB interacted only with Trp. The UV absorption and fluorescence quenching analyses both reflected that the interactions with the non-skeleton aromatic amino acids were weak. (1)H NMR analysis confirmed the strong pi-pi coupling interactions between TNB and model Trp. Molecular simulation indicated that the DNB or TNB molecule was sandwiched between Trp84 and Phe330 at the catalytic site via pi-pi coupling interactions. The findings highlight the importance of specific interactions of NACs with proteins to cause them to malfunction.