Studies of Tetrachlorohydroquinone Dehalogenase

9723308 Copley四氯对苯二酚（TCHQ）脱盐酶存在于绿酚藻单胞菌中，这是一种革兰氏阴性土壤细菌，可以完全降解广泛使用的木材防腐剂五氯酚。该酶催化两个连续的还原脱卤反应，将TCHQ转化为三氯对苯二酚，然后再转化为二氯对苯二酚。每一步的还原等价物由两分子谷胱甘肽提供。研究者实验室之前的工作表明，TCHQ脱卤酶在进化上与谷胱甘肽s -转移酶（GST）超家族中的θ类酶相关，并且与GST一样，它催化谷胱甘肽对亲电底物的亲核攻击，形成谷胱甘肽偶联物。然而，TCHQ脱卤酶具有额外的能力，允许它通过用氢取代谷胱甘肽基部分将谷胱甘肽偶联物转化为还原产物。本研究主要关注TCHQ脱卤酶特有的催化重要过程。催化酸和碱将被识别，并确定其pKa值。在gst中不存在的20个氨基酸插入物的作用，可能提供第二谷胱甘肽结合位点的一部分，将被探索。预稳态动力学实验将进行表征催化事件和构象变化发生在一个单一的翻转。最后，将继续努力使酶结晶。包括许多农药在内的氯化有机化合物是重要的环境污染物。其中一些化合物在环境中是持久性的，有毒和/或致癌的，有些可能会破坏人类和野生动物的内分泌功能。这些化合物的生物降解需要去除氯原子。这种反应是由一种叫做脱卤酶的微生物酶催化的。四氯对苯二酚脱卤酶是催化五氯酚生物降解的关键步骤，五氯酚是广泛使用的木材防腐剂。研究者将研究这种酶的机制及其与一大类解毒酶的关系，这些解毒酶催化的反应在某些方面是相似的。这项工作将使人们对有毒氯化有机化合物生物降解酶的进化有更深入的了解。

9723308 Copley Tetrachlorohydroquinone (TCHQ) dehalogenase is found in Sphingomonas chlorophenolica, a Gram-negative soil bacterium that completely degrades pentachlorophenol, a widely used wood preservative. This enzyme catalyzes two successive reductive dehalogenations to convert TCHQ to trichlorohydroquinone and then to dichlorohydroquinone. The reducing equivalents for each step are provided by two molecules of glutathione. Previous work in the investigator's laboratory has shown that TCHQ dehalogenase is evolutionarily related to enzymes in the theta class of the glutathione S-transferase (GST) superfamily and that it, like the GSTs, catalyzes the nucleophilic attack of glutathione upon an electrophilic substrate to form a glutathione conjugate. However, TCHQ dehalogenase has additional capabilities that allow it to convert glutathione conjugates to reduced products by replacement of the glutathionyl moiety with a hydrogen. This study focuses on the catalytically important processes that are unique to TCHQ dehalogenase. Catalytic acids and bases will be identified and their pKa values determined. The role of a 20-amino acid insert that is not present in GSTs and that may provide part of a second glutathione binding site will be explored. Pre-steady state kinetic experiments will be carried out to characterize catalytic events and conformational changes taking place during a single-turnover. Finally, efforts to crystallize the enzyme will continue. Chlorinated organic compounds, including many pesticides, are important environmental pollutants. A number of these compounds are persistent in the environment, toxic and/or carcinogenic, and some may disrupt endocrine function in humans and wildlife. Biodegradation of these compounds requires removal of chlorine atoms. This type of reaction is catalyzed by microbial enzymes called dehalogenases. Tetrachlorohydroquinone dehalogenase catalyzes a key step in the biodegradation of pentachlorophenol, a widely used wood preservative. Th e investigator will study the mechanism of this enzyme and its relationship to a broad class of detoxification enzymes that catalyze reactions that are similar in some respects. This work will lead to a greater understanding of the evolution of enzymes for biodegradation of toxic chlorinated organic compounds.