Deciphering the inhibitory mechanism of genistein on xanthine oxidase in vitro

• Genistein reversibly inhibits xanthine oxidase (XO) in a competitive manner.
• The interaction is mainly driven by hydrophobic interactions and hydrogen bonds.
• Genistein interacts with several residues located within the active pocket of XO.
• Genistein induces the conformational changes of XO.

Genistein (Gen), widely distributed in soybean, is proved to be important in homeostasis in the human body. Herein, the inhibitory mechanism of Gen against xanthine oxidase (XO) was studied through multispectroscopic methods and molecular simulation. The inhibition kinetics showed that Gen competitively inhibited XO with an inhibition constant of (1.39 ± 0.11) μM by competing with xanthine for binding to the active site of XO. Fluorescence titration study suggested that the fluorescence quenching mechanism of XO was static, resulting from the formation of a Gen–XO complex at one fold site. The calculated thermodynamic parameters revealed that the interaction process was driven mainly by hydrophobic interactions and hydrogen bonds with affinity of (5.24 ± 0.02) × 104 L mol− 1. Conformational analyses demonstrated that the microenvironment and the secondary structure of XO were changed upon binding of Gen. The molecular docking displayed that Gen bound to the active cavity of XO by interacting with the surrounding amino acid residues (Leu648, Phe649, Glu802, Ser876, Glu879, Arg880, Phe914, Phe1009, Thr1010 and Phe1013). Thus, the inhibition may be attributed to the insertion of Gen into the active site of XO occupying the catalytic center of the enzyme to avoid entry of the substrate and inducing conformational changes of XO (more compact), which was further unfavorable for forming the active cavity and further reduced the landing and oxidation of substrate. This study may offer novel insights into the inhibition mechanism of Gen on XO.

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