Studies of Tetrachlorohydroquinone Dehalogenase

9723308 Copley四氯氢醌（TCHQ）脱卤酶发现于Sphingomonas chlorophenolica中，Sphingomonas chlorophenolica是一种革兰氏阴性土壤细菌，可完全降解五氯苯酚，一种广泛使用的木材防腐剂。该酶催化两个连续的还原脱卤反应，将TCHQ转化为三氯氢醌，然后转化为二氯氢醌。 每个步骤的还原当量由两个谷胱甘肽分子提供。研究者实验室的先前工作已经表明，TCHQ脱卤酶在进化上与谷胱甘肽S-转移酶（GST）超家族的θ类中的酶相关，并且它像GST一样催化谷胱甘肽对亲电子底物的亲核攻击以形成谷胱甘肽缀合物。 然而，TCHQ脱卤酶具有额外的能力，使其能够通过用氢取代谷胱甘肽部分将谷胱甘肽缀合物转化为还原产物。 这项研究的重点是催化的重要过程，是独特的TCHQ脱卤酶。 将鉴别催化酸和碱，并测定其pKa值。 将探讨GST中不存在的20个氨基酸插入片段的作用，该插入片段可能提供第二谷胱甘肽结合位点的一部分。 将进行预稳态动力学实验，以表征单次转换期间发生的催化事件和构象变化。 最后，将继续努力使酶结晶。 氯代有机化合物，包括许多农药，是重要的环境污染物。 其中一些化合物在环境中具有持久性，具有毒性和/或致癌性，有些化合物可能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。 这种类型的反应由称为脱卤酶的微生物酶催化。四氯对苯二酚脱卤酶催化五氯酚生物降解的关键步骤，五氯酚是一种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类广泛的解毒酶的关系，这些酶催化在某些方面相似的反应。 这项工作将导致更好地了解酶的演变，用于有毒的氯化有机化合物的生物降解。

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.