Fluorescent analogs of UDP-glucose and their use in characterizing substrate binding by toxin A from Clostridium difficile
Fluorescent analogs of UDP-glucose and their use in characterizing substrate binding by toxin A from Clostridium difficile
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DOI：
10.1046/j.1432-1033.2002.03013.x
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发表时间：
2002-07-01
期刊：
影响因子：
--
通讯作者：
Feig, AL
中科院分区：
文献类型：
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作者：
Bhattacharyya, S;Kerzmann, A;Feig, AL
尿苷-5 '-二磷酸-1-α-D-葡萄糖（UDP-Glc）是葡糖基转移酶使用的常见底物，包括某些细菌毒素，例如来自艰难梭菌的毒素A和B。已经制备了UDP-Glc的荧光类似物用于我们对梭菌毒素的研究。这些化合物与常用于探测ATP依赖性酶的化学性质的甲基邻氨基苯甲酰-ATP化合物有关。过量的甲基靛红酸酐与UDP-Glc在水溶液中的反应主要产生甲基邻氨基苯甲酰-UDP-Glc（MUG）的2'和3'异构体。由于2'和3'异构体容易相互转化，因此通过HPLC将该异构体混合物与其他异构体产物共纯化，并通过NMR、荧光和质谱方法的组合进行表征。TcdA在不存在Mg 2+的情况下以15 +/-2 μ m的亲和力相对于UDP-Glc竞争性地结合MUG。目前还没有证据表明，荧光底物类似物是由毒素在葡糖基转移酶或葡糖基水解酶反应。使用竞争测定，在不存在Mg 2+的情况下，UDP-Glc的亲和力被确定为45+/-10 μ m。UDP-Glc和Mg 2+的结合与Mg 2+的亲和力在90-600 μ m的范围内高度耦合，这取决于实验条件。这些结果意味着金属离子的重要作用之一可能是在转移酶化学反应开始之前稳定酶-底物复合物。
Uridine-5'-diphospho-1-alpha-D-glucose (UDP-Glc) is a common substrate used by glucosyltransferases, including certain bacterial toxins such as Toxins A and B from Clostridium difficile . Fluorescent analogs of UDP-Glc have been prepared for use in our studies of the clostridial toxins. These compounds are related to the methylanthraniloyl-ATP compounds commonly used to probe the chemistry of ATP-dependent enzymes. The reaction of excess methylisatoic anhydride with UDP-Glc in aqueous solution yields primarily the 2' and 3' isomers of methylanthraniloyl-UDP-Glc (MUG). As the 2' and 3' isomers readily interconvert, this isomeric mixture was copurified by HPLC away from the other isomeric products, and was characterized by a combination of NMR, fluorescence and mass spectrometric methods. TcdA binds MUG competitively with respect to UDP-Glc with an affinity of 15 +/- 2 mum in the absence of Mg2+ . There is currently no evidence that the fluorescent substrate analog is turned over by the toxin in either glucosyltransferase or glucosylhydrolase reactions. Using a competition assay, the affinity of UDP-Glc was determined to be 45+/-10 mum in the absence of Mg2+ . The binding of UDP-Glc and Mg2+ are highly coupled with Mg2+ affinities in the range of 90-600 mum, depending on the experimental conditions. These results imply that one of the significant roles of the metal ion might be to stabilize the enzyme-substrate complex prior to initiation of the transferase chemistry.