Active binding mechanism to superoxide dismutase and toxicological implication for environmentally prevalent phthalates and their hydrolytic products: Coupling in vitro bioassay with molecular dynamics simulation.

Phthalic acid esters (PAEs) are widely used as plasticizers to improve the flexibility and durability of plastics. However, their environmental presence poses risks by inducing oxidative stress and contributing to metabolic syndrome. Despite being linked to various diseases, the mechanisms by which PAEs disrupt antioxidant enzymes, particularly superoxide dismutase (SOD), are not well understood. This study investigated the molecular interactions between PAEs, their metabolites, and SOD using bioassays and theoretical simulations. The results showed that key metabolites, monophthalates (MAEs) and phthalic acid (PA), strongly inhibited SOD activity, with potency increasing as side chain length decreased. In contrast, PAEs caused minor changes in SOD activity. The inhibition resulted from tight binding of MAEs and PA to the residues in the enzyme's bottom cavity. PAEs and metabolites induced significant structural changes in the secondary structures, catalytic channel, and hydrogen bond network, destabilizing the protein and impairing its function. A strong correlation between SOD inhibition and Gibbs free binding energies at Arg141 was observed. Arg141 and allelic residues can serve as biomarkers for early warnings of oxidative stress. This study improves our understanding of oxidative stress mechanisms caused by PAEs and emphasizes the need for better risk management of phthalate exposure.