Two continuous spectrophotometric assays for methionine aminopeptidase
Two continuous spectrophotometric assays for methionine aminopeptidase
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DOI：
10.1006/abio.2000.4513
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发表时间：
2000-04-10
影响因子：
2.9
通讯作者：
Pei, DH
中科院分区：
文献类型：
--
作者：
Zhou, Y;Guo, XC;Pei, DH
已开发出两种用于蛋氨酸氨基肽酶 (MetAP) 的分光光度测定法。第一种方法使用硫酯底物，其在酶促去除N-末端甲硫氨酸后产生游离硫醇基团。使用埃尔曼试剂对释放的硫醇进行定量。通过在测定反应中添加过量的埃尔曼试剂，可以在 UV-VIS 分光光度计上以连续方式方便地监测 MetAP 反应。合成了两种三肽类似物，发现它们都是大肠杆菌 MetAP 和人 MetAP2 的优良底物（对于反应性最强的底物，k(cat)/K-M = 2.8 x 10(5) M-1 s(-1)）。在第二种测定方法中，使用Met-Pro-p-硝基苯胺作为底物将MetAP反应与脯氨酰氨肽酶反应偶联。 MetAP 催化的 N 端甲硫氨酸裂解产生脯氨酰对硝基苯胺，它被凝结芽孢杆菌的脯氨酰氨肽酶快速水解，释放显色产物对硝基苯胺，这使得可以在 UV-VIS 分光光度计上在 405 nm 处连续监测 MetAP 反应。该测定已用于确定大肠杆菌和人类 MetAP 的最适 pH 值和动力学常数，以及筛选 MetAP 抑制剂。这些结果表明，当前的测定方法是用于 MetAP 动力学研究的方便、快速和灵敏的方法，也是筛选 MetAP 抑制剂的有效工具。 (C) 2000 年学术出版社。
Two spectrophotometric assays have been developed for methionine aminopeptidases (MetAPs). The first method employs a thioester substrate which, upon enzymatic removal of the N-terminal methionine, generates a free thiol group. The released thiol is quantitated using Ellman's reagent. The MetAP reaction is conveniently monitored on a UV-VIS spectrophotometer in a continuous fashion, with the addition of an excess of Ellman's reagent into the assay reaction. Two tripeptide analogues were synthesized and found to be excellent substrates of both Escherichia coli MetAP and human MetAP2 (k(cat)/K-M = 2.8 x 10(5) M-1 s(-1) for the most reactive substrate). In the second assay method, the MetAP reaction is coupled to a prolyl aminopeptidase reaction using Met-Pro-p-nitroanilide as substrate. MetAP-catalyzed cleavage of the N-terminal methionine produces prolyl-p-nitroanilide, which is rapidly hydrolyzed by the prolyl aminopeptidase from Bacillus coagulans to release a chromogenic product, p-nitroaniline, This allows the MetAP reaction to be continuously monitored at 405 nm on a UV-VIS spectrophotometer. The assays have been applied to determine the pH optima and kinetic constants for the E. coli and human MetAPs as well as to screen MetAP inhibitors. These results demonstrate that the current assays are convenient, rapid, and sensitive methods for kinetic studies of MetAPs and effective tools for screening MetAP inhibitors. (C) 2000 Academic Press.