Enzyme kinetics and multi-spectroscopic analyses reveal the inhibitory effect and interaction mechanism of taxifolin on urease.

Taxifolin exhibits diverse biological activities; however, its inhibitory effect on urease and the underlying molecular mechanism have not been systematically elucidated. In this study, enzyme kinetics, multi-spectroscopic techniques-including fluorescence, three-dimensional fluorescence, circular dichroism, and infrared spectroscopy-as well as scanning electron microscopy and molecular docking simulations were employed to comprehensively investigate the interaction between taxifolin and urease. The results revealed that taxifolin acts as a mixed-type inhibitor of urease and induces static quenching of its intrinsic fluorescence, with the Stern-Volmer quenching constant (Kₛᵥ) decreasing as temperature increases. Based on the calculated thermodynamic parameters (ΔG° < 0, ΔH° < 0, and ΔS° > 0), the binding process is mainly entropy-driven, accompanied by a favorable enthalpic contribution. The binding process occurred spontaneously and was stabilized by strong non-covalent interactions. Taxifolin binding significantly altered the secondary structure; the α-helix content slightly decreased to 27.5 %, the β-sheet content decreased to 15.6 %, and the random coil content decreased to 29.8 %, while the β-turn content increased to 27 %. Meanwhile, taxifolin also altered the aggregation morphology of urease. Molecular docking further confirmed that taxifolin occupies the active pocket of urease and forms stable interactions via hydrogen bonding, van der Waals forces, and hydrophobic interactions. These findings provide molecular-level evidence for the interaction between taxifolin and urease. They also establish a mechanistic basis for the development and application of flavonoid-based urease inhibitors.