Guo Cuixia, Hu Yiling, Yang Chunyu, Nanjaraj Urs Ankanahalli N, Zhang Yan
Tianjin Key Laboratory for Modern Drug Delivery & High-Efficiency, Collaborative Innovation Center of Chemical Science and Engineering, School of Pharmaceutical Science and Technology, Tianjin University, Tianjin, 300072, China.
Tianjin Key Laboratory for Modern Drug Delivery & High-Efficiency, Collaborative Innovation Center of Chemical Science and Engineering, School of Pharmaceutical Science and Technology, Tianjin University, Tianjin, 300072, China.
Anal Biochem. 2018 May 1;548:109-114. doi: 10.1016/j.ab.2018.02.013. Epub 2018 Feb 27.
The Fe(II)/2-oxoglutarate-dependent dioxygenases (2-OGDs) catalyze the oxidation of substrates ranging from small molecules to large biomolecules with concomitant oxidation of co-substrate (2-oxoglutarate) into succinate. In the present study, we reported a coupled colorimetric assay that can be generally applied to measure the activities of all members of 2-OGDs family. Succinyl-CoA synthetase is employed as the coupling enzyme to transform succinate produced from 2-OGDs catalysis to form succinyl-CoA with concomitant hydrolysis of ATP to form ADP and orthophosphate. Orthophosphate can be quantitated by reacting it with molybdic acid forming a blue pigment. As a proof of concept, kinetic parameters of ectoine hydroxylase obtained using this method are compared to a traditional time- and labor-consuming HPLC based method. As 2-OGDs family enzymes are important drug targets due to their impressive versatility in catalyzing numerous oxidative reactions that are still very challenging using synthetic chemistry, colorimetric method detailed in the manuscript has the potential to enable the practice of high throughput drug screening for 2-OGDs.
依赖Fe(II)/2-氧代戊二酸的双加氧酶（2-OGDs）催化从小分子到大型生物分子等多种底物的氧化反应，同时将共底物（2-氧代戊二酸）氧化为琥珀酸。在本研究中，我们报道了一种比色法偶联测定，它可普遍用于测量2-OGDs家族所有成员的活性。琥珀酰辅酶A合成酶用作偶联酶，将2-OGDs催化产生的琥珀酸转化为琥珀酰辅酶A，同时ATP水解形成ADP和正磷酸盐。正磷酸盐可通过与钼酸反应形成蓝色色素进行定量。作为概念验证，将使用该方法获得的依克多因羟化酶的动力学参数与传统的耗时且费力的基于高效液相色谱的方法进行比较。由于2-OGDs家族酶在催化众多氧化反应方面具有令人印象深刻的多功能性，而这些反应使用合成化学仍然极具挑战性，因此本文详述的比色法有潜力用于2-OGDs的高通量药物筛选实践。
