化学
基质(水族馆)
双加氧酶
催化作用
酶
组合化学
水解
生物分子
高通量筛选
生物化学
有机化学
生物
生态学
作者
Cuixia Guo,Yiling Hu,Chunyu Yang,Ankanahalli N. Nanjaraj Urs,Yan Zhang
标识
DOI:10.1016/j.ab.2018.02.013
摘要
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.
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