Molecular Insights Into Copper‐Induced Structural and Functional Alterations of Key Soil Enzymes: Multispectral and In Vivo Evidence

Heavy metal‐induced environmental degradation has become a pressing concern. Soil enzymes are the products of plant, animal, and microbial activities in the soil. Participating in diverse soil biochemical processes, their activity levels serve as sensitive indicators of biochemical process intensity in soil ecosystems. In this paper, the toxic effects of Cu 2+ on glutamate dehydrogenase (L‐GDH), copper/zinc‐superoxide dismutase (Cu/Zn‐SOD), and acid phosphatase (ACP) were investigated under simulated physiological conditions. Fluorescence spectra and lifetime measurements showed that Cu 2+ reduced the fluorescence intensity and hydrophobicity and altered the protein conformation of L‐GDH, Cu/Zn‐SOD, and ACP; the intrinsic fluorescence of L‐GDH and Cu/Zn‐SOD underwent dynamic quenching, whereas ACP underwent static quenching. Enzyme activity assays revealed that ACP was the most susceptible to Cu 2+ inhibition among the three enzymes, showing a clear dose‐dependent effect. UV–Vis and CD spectroscopy showed that Cu 2+ loosened the protein backbone structure and induced peptide chain extension and protein unfolding. The changes of enzyme activity in Eisenia fetida were measured after exposure to Cu 2+ at different times and concentrations. Overall, this study provides a foundation for understanding the effects of Cu 2+ on the structure and function of soil enzymes, revealing the potential effects of heavy metals on enzymes.