Hebbard Carleigh F F, Wang Yan, Baker Catherine J, Morrissey James H
Department of Biochemistry and §College of Medicine, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
Biomacromolecules. 2014 Aug 11;15(8):3190-6. doi: 10.1021/bm500872g. Epub 2014 Jul 15.
Inorganic polyphosphates, linear polymers of orthophosphate, occur naturally throughout biology and have many industrial applications. Their biodegradable nature makes them attractive for a multitude of uses, and it would be important to understand how polyphosphates are turned over enzymatically. Studies of inorganic polyphosphatases are, however, hampered by the lack of high-throughput methods for detecting and quantifying rates of polyphosphate degradation. We now report chromogenic and fluorogenic polyphosphate substrates that permit spectrophotometric monitoring of polyphosphate hydrolysis and allow for high-throughput analyses of both endopolyphosphatase and exopolyphosphatase activities, depending on assay configuration. These substrates contain 4-nitrophenol or 4-methylumbelliferone moieties that are covalently attached to the terminal phosphates of polyphosphate via phosphoester linkages formed during reactions mediated by EDAC (1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide). This report identifies Nudt2 as an inorganic polyphosphatase and also adds to the known coupling chemistry for polyphosphates, permitting facile covalent linkage of alcohols with the terminal phosphates of inorganic polyphosphate.
无机多聚磷酸盐是正磷酸盐的线性聚合物，在整个生物界中天然存在，并具有许多工业应用。它们的可生物降解特性使其在众多用途中颇具吸引力，了解多聚磷酸盐如何通过酶促作用进行周转非常重要。然而，无机多聚磷酸酶的研究受到缺乏用于检测和量化多聚磷酸盐降解速率的高通量方法的阻碍。我们现在报告了生色和荧光多聚磷酸盐底物，这些底物允许通过分光光度法监测多聚磷酸盐的水解，并根据测定配置对内切多聚磷酸酶和外切多聚磷酸酶活性进行高通量分析。这些底物含有4-硝基苯酚或4-甲基伞形酮部分，它们通过在由EDAC（1-乙基-3-（3-（二甲基氨基）丙基）碳二亚胺）介导的反应中形成的磷酸酯键与多聚磷酸盐的末端磷酸共价连接。本报告确定Nudt2为一种无机多聚磷酸酶，同时也增加了已知的多聚磷酸盐偶联化学方法，允许醇与无机多聚磷酸盐的末端磷酸进行简便的共价连接。
