A CONTINUOUS SPECTROPHOTOMETRIC ASSAY FOR 5-AMINOLEVULINATE SYNTHASE THAT UTILIZES SUBSTRATE CYCLING
A CONTINUOUS SPECTROPHOTOMETRIC ASSAY FOR 5-AMINOLEVULINATE SYNTHASE THAT UTILIZES SUBSTRATE CYCLING
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DOI：
10.1006/abio.1995.1217
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发表时间：
1995-04-10
影响因子：
2.9
通讯作者：
FERREIRA, GC
中科院分区：
文献类型：
--
作者：
HUNTER, GA;FERREIRA, GC
本文介绍了一种测定5-氨基乙酰丙酸合成酶活性的连续分光光度法。该测定基于将5-氨基乙酰丙酸合酶产生辅酶A与α-酮戊二酸脱氢酶还原NAD（+）偶联，并监测340 nm处吸光度的增加。NAD（+）的还原与5-氨基乙酰丙酸的形成是化学计量的。甘氨酸和琥珀酰辅酶A的动力学参数与测量5-氨基乙酰丙酸形成的其他测定所报告的动力学参数相似。在α-酮戊二酸脱氢酶反应中琥珀酰辅酶A的再生有助于确定该底物亚饱和浓度下的初始速率。该测定将允许快速积累动力学数据，并有助于5-氨基乙酰丙酸合酶及其重组突变体的机理分析。(C)出版社：Academic Press
A continuous spectrophotometric assay for determining 5-aminolevulinic acid synthase activity is described. The assay is based upon coupling the production of coenzyme A by 5-aminolevulinic acid synthase to the reduction of NAD(+) by alpha-ketoglutarate dehydrogenase and monitoring the increase in absorbance at 340 nm. Reduction of NAD(+) is stoichoimetric with formation of 5-aminolevulinic acid. Kinetic parameters for glycine and succinyl-CoA are similar to those reported for other assays which measure the formation of 5-aminolevulinic acid. Regeneration of succinyl-CoA in the alpha-ketoglutarate dehydrogenase reaction facilitates determination of initial rates at subsaturating concentrations of this substrate. This assay will permit the rapid accumulation of kinetic data and aid in mechanistic analyses of both 5-aminolevulinic acid synthase and its recombinant mutants. (C) 1995 Academic Press, Inc.