An assay for Fe(II)/2-oxoglutarate-dependent dioxygenases by enzyme-coupled detection of succinate formation
An assay for Fe(II)/2-oxoglutarate-dependent dioxygenases by enzyme-coupled detection of succinate formation
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DOI：
10.1016/j.ab.2006.03.033
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发表时间：
2006-06-01
影响因子：
2.9
通讯作者：
Hausinger, RP
中科院分区：
文献类型：
--
作者：
Luo, LS;Pappalardi, MB;Hausinger, RP
铁(II)/2-氧戊二酸依赖的双加氧酶是催化多样化的非血红素铁酶家族，它们在氧化其主要底物的同时分解2-氧戊二酸共底物形成琥珀酸盐和CO。我们报告了这些酶的通用分析，使用琥珀酰辅酶a合成酶，丙酮酸激酶。和乳酸脱氢酶将产物琥珀酸的形成与还原的烟酰胺腺嘌呤二核苷酸转化为烟酰胺腺嘌呤二核苷酸偶联。我们通过测量两种细菌铁(II)/2-氧戊二酸依赖的双加氧酶的动力学参数来证明这种新方法的实用性。值得注意的是，该方法可用于研究该酶家族的生产性周转反应和非生产性“解偶联”脱羧反应。通过使用野生型和变体形式的2-氧戊二酸依赖性牛磺酸双加氧酶证明了这一点。该试验易于小型化，易于适应适合高通量筛选的格式；因此，它将是研究铁(II)/2-氧戊二酸依赖的双加氧酶的一个有价值的工具。(c) 2006爱思唯尔公司版权所有。
The Fe(II)/2-oxoglutarate-dependent dioxygenases are a catalytically diverse family of nonheme iron enzymes that oxidize their primary substrates while decomposing the 2-oxoglutarate cosubstrate to form succinate and CO,. We report a generic assay for these enzymes that uses succinyl-coenzyme A synthetase, pyruvate kinase. and lactate dehydrogenase to Couple the formation of the product succinate to the conversion of reduced nicotinamide adenine dinucleotide to nicotinamide adenine dinucleotide. We demonstrate the utility of this new method by measuring the kinetic parameters of two bacterial Fe(II)/2-oxoglutarate-dependent dioxygenases. Significantly, this method can be used to investigate both the productive turnover reactions and the nonproductive "uncoupled' decarboxylation reactions of this enzyme family. as demonstrated by Using wild-type and variant forms of 2-oxoglutarate-dependent taurine dioxygenase. This assay is amenable to miniaturization and easily adapted to a format Suitable for high-throughput screening; thus, it will be a valuable tool to study Fe(II)/2-oxoglutarate-dependent dioxygenases. (c) 2006 Elsevier Inc. All rights reserved.