Kinetic Characterization of Tyrosinase-catalyzed Oxidation of Four Polyphenols
Kinetic Characterization of Tyrosinase-catalyzed Oxidation of Four Polyphenols
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酪氨酸酶催化四种多酚氧化的动力学表征
DOI：
10.1007/s11596-020-2186-0
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发表时间：
2020-04-01
影响因子：
2.4
通讯作者：
Huang, Jian-geng
中科院分区：
文献类型：
--
作者：
Liu, Wan-yu;Zou, Cong-ming;Huang, Jian-geng
绿原酸、隐绿原酸、新绿原酸、咖啡酸等酚类化合物广泛存在于水果、蔬菜和中药中，具有广泛的生物活性。酪氨酸酶在食品工业中起着至关重要的作用，但最近的研究提出了尚未探索的临床应用方面。酪氨酸酶催化四种多酚的氧化及其潜在机制尚不清楚。在目前的工作中，我们研究了酪氨酸酶催化氧化四种感兴趣的多酚的动力学性质。为了测定不稳定邻醌产物，建立了3-甲基-2-苯并噻唑啉酮腙（MBTH）的分析方法。研究了不同多酚存在下对酶活性影响的最佳孵育时间、缓冲液pH、温度和酶浓度。在最终优化的条件下，考察了四种多酚的动力学和底物特异性。动力学数据表明，酪氨酸酶与其异构体和咖啡酸相比，对绿原酸具有最大的底物亲和力。绿原酸的催化效率是其他3种多酚的8 ~ 15倍。分子对接研究表明，绿原酸在外周位点的紧密结合可能是对绿原酸具有特异性的主要原因。鉴于此，合理设计高亲和力的酪氨酸酶抑制剂可以同时结合Cu位点和外周位点。本研究将为食品工业和卫生保健中酚酸的选择提供依据。
Phenolic compounds such as chlorogenic acid, cryptochlorogenic acid, neochlorogenic acid and caffeic acid are widely distributed in fruits, vegetables and traditional Chinese medicines with a wide range of biological activities. Tyrosinase plays a critical role in the food industry, but recent studies have proposed unexplored aspects of clinical application. Tyrosinase-catalyzed oxidation of four polyphenols as well as its underlying mechanism remains unclear. In the current work, we investigated the kinetic properties of tyrosinase-catalyzed oxidation of the four polyphenols of interest. To measure the unstable o-quinone products, an analytical method using 3-methyl-2-benzothiazolinone hydrazone (MBTH) was established. The optimal incubation time, buffer pH, temperature and enzyme concentration for the enzyme activity in the presence of each polyphenol of interest were investigated. Under the final optimized conditions, the kinetics and substrate specificity of four polyphenols were examined. Kinetic data showed that tyrosinase had the greatest substrate affnity to chlorogenic acid compared with its isomers and caffeic acid. The catalytic effciency with chlorogenic acid was 8- to 15-fold higher than that with the other 3 polyphenols. Molecular docking study demonstrated that the tight binding of chlorogenic acid at the peripheral site should be the major reason for the specifcity to chlorogenic acid. In light of this, the rational design of high-affnity inhibitors against tyrosinase may focus on the binding of both the Cu site and peripheral site. This study will supply a basis for the selection of phenolic acids in food industry and health care.