Continuous spectrophotometric assays for three regulatory enzymes of the arginine biosynthetic pathway.
Continuous spectrophotometric assays for three regulatory enzymes of the arginine biosynthetic pathway.
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DOI：
10.1016/j.ab.2007.06.032
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发表时间：
2007-09
影响因子：
2.9
通讯作者：
K. Takahara;K. Akashi;A. Yokota
中科院分区：
文献类型：
--
作者：
K. Takahara;K. Akashi;A. Yokota
N-乙酰谷氨酸合酶（AGS）、N-乙酰谷氨酸激酶（AGK）和谷氨酸N-乙酰转移酶（GAT）是精氨酸合成的关键酶，精氨酸是蛋白质、多胺、尿素和一氧化氮合成的重要前体。目前可用于这三种酶的测定是费力和耗时的，并且不允许连续监测酶活性。在这里，我们建立了连续的酶测定AGS，AGK，和GAT的基础上耦合的AGS和GAT反应AGK，然后耦合的AGK反应N-乙酰谷氨酸5-磷酸还原酶（AGPR）。使用分光光度法连续监测还原型烟酰胺腺嘌呤二核苷酸磷酸的AGPR依赖性氧化速率，作为340 nm处吸光度的变化。将这些方法应用于大肠杆菌AGK、大肠杆菌E. coliAGS和Saccharomycescerevisiae GAT的偶联反应动力学参数与不连续反应动力学参数基本一致。这些偶联试验的特异性通过缺乏E. coli AGS-、E. coli AGK-和S.酿酒酵母GAT缺失突变体。此外，耦合分析使我们能够测量AGS活性从哺乳动物肝脏线粒体提取物，已知是一个重要的尿素循环的调节酶。这些偶联酶测定是快速的、高度灵敏的和可重复的。
N-Acetylglutamate synthase (AGS), N-acetylglutamate kinase (AGK), and glutamate N-acetyltransferase (GAT) are the key enzymes in the synthesis of arginine that serves as an important precursor for the synthesis of protein, polyamines, urea, and nitric oxide. Current assays available for these three enzymes are laborious and time-consuming and do not allow continuous monitoring of enzyme activities. Here we established continuous enzyme assays for AGS, AGK, and GAT based on the coupling of AGS and GAT reactions to AGK followed by coupling of the AGK reaction to N-acetylglutamate 5-phosphate reductase (AGPR). The rate of AGPR-dependent oxidation of reduced nicotinamide adenine dinucleotide phosphate was monitored continuously as a change in absorbance at 340nm using spectrophotometry. These methods were applied to kinetic analyses for Escherichia coli AGK, E. coli AGS, and Saccharomyces cerevisiae GAT, and the kinetic parameters obtained in the coupling assays showed nearly the same values as those obtained previously using discontinuous assays. The specificity of these coupled assays was confirmed by the lack of enzyme activity from extracts of E. coli AGS-, E. coli AGK-, and S. cerevisiae GAT-deletion mutants. Moreover, the coupled assay enabled us to measure AGS activity from mammalian liver mitochondrial extracts, known to be an important regulatory enzyme for the urea cycle. These coupled enzyme assays are rapid, highly sensitive, and reproducible.