Rodríguez-Aparicio L B, Reglero A, Martínez-Blanco H, Luengo J M
Departamento Interfacultativo de Bioquimica y Biologia Molecular, Universidad de León, Spain.
Biochim Biophys Acta. 1991 Mar 4;1073(2):431-3. doi: 10.1016/0304-4165(91)90153-8.
Phenylacetyl-CoA ligase (AMP-forming) from Pseudomonas putida is a newly described enzyme (Martinez-Blanco, H., Reglero, A., Rodriguez-Aparicio, L.B. and Luengo, J.M. (1990) J. Biol. Chem. 265, 7084-7090) specifically involved in the catabolism of phenylacetic acid. This enzyme catalyzes the formation of phenylacetyl-CoA in the presence of ATP, CoA, Mg2+ and phenylacetic acid. A rapid method of assaying this enzyme in partially purified preparations has been developed by coupling this reaction with adenylate kinase, pyruvate kinase and kinase and lactate dehydrogenase. The rate of phenylacetyl-CoA formation was measured indirectly by monitoring fluorometrically the NADH oxidation at 340 nm (excitation at 340 nm and analysis of the emitted light at 465 nm). The advantage of this method of assay over others (colorimetric, HPLC and spectrophotometric) is discussed.
来自恶臭假单胞菌的苯乙酰辅酶A连接酶（生成AMP）是一种新描述的酶（马丁内斯-布兰科，H.，雷格勒罗，A.，罗德里格斯-阿帕里西奥，L.B.和伦戈，J.M.（1990年）《生物化学杂志》265卷，7084 - 7090页），专门参与苯乙酸的分解代谢。该酶在ATP、辅酶A、Mg2 +和苯乙酸存在的情况下催化苯乙酰辅酶A的形成。通过将此反应与腺苷酸激酶、丙酮酸激酶和乳酸脱氢酶偶联，已开发出一种在部分纯化制剂中测定该酶的快速方法。通过荧光监测340 nm处NADH的氧化（在340 nm激发并在465 nm分析发射光）间接测量苯乙酰辅酶A的形成速率。讨论了这种测定方法相对于其他方法（比色法、高效液相色谱法和分光光度法）的优势。
