Tyrosinase is a ubiquitous enzyme that plays an essential role in the production of melanin. Effective inhibitors of tyrosinase have extensive applications in the medical, cosmetic and food industries. In this study, a combination of enzyme kinetics, ultraviolet (UV)‐visible absorption, fluorescence spectroscopic techniques and a computational simulation method was used to characterize the inhibitory mechanism of 7,8,4´‐trihydroxyflavone on tyrosinase. 7,8,4´‐Trihydroxyflavone was found to strongly inhibit the oxidation of l‐DOPA by tyrosinase with an IC₅₀ value of 10.31 ± 0.41 μM. The inhibitory mechanism was determined to be reversible and non‐competitive with a K_i of 9.50 ± 0.40 μM. The UV absorption spectra showed that 7,8,4´‐trihydroxyflavone could chelate with copper ions and form a complex with tyrosinase. The intrinsic fluorescence of tyrosinase was quenched by 7,8,4´‐trihydroxyflavone through a static quenching mechanism. 7,8,4´‐Trihydroxyflavone was found to occupy a single binding site with a binding constant of 7.50 ± 1.20 × 10⁴ M⁻¹ at 298 K. The conformation of tyrosinase changed, and the microenvironment became more hydrophilic after 7,8,4´‐trihydroxyflavone binding. Thermodynamics parameters indicated that the binding was a spontaneous process and involved hydrogen bonds and van der Waals forces. The binding distance was evaluated to be 4.54 ± 0.05 nm. Docking simulation analysis further authenticated that 7,8,4´‐trihydroxyflavone could form hydrogen bonds with the residues His244 and Met280 within the tyrosinase active site. Our results will contribute to further understanding of the inhibitory mechanisms of 7,8,4´‐trihydroxyflavone against tyrosinase and will facilitate future screening for tyrosinase inhibitors.

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7,8,4′-三羟基黄酮对酪氨酸酶活性和构象的影响：光谱学和对接研究

酪氨酸酶是一种普遍存在的酶，在黑色素的产生中起着至关重要的作用。有效的酪氨酸酶抑制剂在医学，化妆品和食品工业中具有广泛的应用。在这项研究中，结合酶动力学，紫外可见吸收，荧光光谱技术和计算模拟方法来表征7,8,4′-三羟基黄酮对酪氨酸酶的抑制机理。发现7,8,4′-三羟基黄酮能强烈抑制酪氨酸酶降解l- DOPA，IC ₅₀值为10.31± 0.41μM 。抑制机制被确定为可逆且与K _i不竞争为9.50±0.40μM。紫外线吸收光谱表明，7,8,4′-三羟基黄酮可能与铜离子螯合，并与酪氨酸酶形成复合物。酪氨酸酶的固有荧光通过静态猝灭机理被7,8,4′-三羟基黄酮猝灭。发现7,8,4′-三羟基黄酮占据单个结合位点，结合常数为7.50±1.20×10 ⁴  M ^-1在298 K时，酪氨酸酶的构象发生改变，并且在7,8,4′-三羟基黄酮结合后微环境变得更加亲水。热力学参数表明结合是一个自发过程，涉及氢键和范德华力。结合距离被评估为4.54±0.05nm。对接模拟分析进一步验证了7,8,4′-三羟基黄酮可以与酪氨酸酶活性位点内的His244和Met280残基形成氢键。我们的结果将有助于进一步了解7,8,4′-三羟基黄酮对酪氨酸酶的抑制机制，并将有助于将来筛选酪氨酸酶抑制剂。