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Biochemistry. 2002 Jan 29;41(4):1315-22.

Characterization of the initial steps in the reductive dehalogenation catalyzed by tetrachlorohydroquinone dehalogenase.

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Department of Molecular, Cellular, and Developmental Biology and Cooperative Institute for Research in Environmental Sciences, Campus Box 216, University of Colorado at Boulder, Boulder, Colorado 80309, USA.


Tetrachlorohydroquinone dehalogenase catalyzes the reductive dehalogenation of tetrachlorohydroquinone and trichlorohydroquinone during the degradation of pentachlorophenol by Sphingbium chlorophenolicum. Remarkably, the same active site catalyzes the glutathione-dependent isomerization of a double bond in maleylacetone (an analogue of maleylacetoacetate and maleylpyruvate) [Anandarajah, K. et al. (2000) Biochemistry 39, 5303-5311]. The mechanism of the initial steps in the reaction has been probed using the C13S mutant enzyme, which catalyzes the reaction only to the point at which Cys13 is required. The reaction proceeds by a rapid equilibrium random sequential kinetic mechanism. Substrate analogues that lack a second hydroxyl group cannot be turned over to products, although they can bind to the active site. The rate of the reaction is strongly influenced by the number of electron-withdrawing substituents on the substrate. These findings are consistent with a mechanism that begins with ketonization of the deprotonated substrate to form 2,3,5,6-tetrachloro-4-hydroxycyclohexa-2,4-dienone, followed by 1,4-elimination of HCl to from trichlorobenzoquinone. Subsequently, trichlorobenzoquinone is attacked by glutathione to form a glutathione conjugate that, in the absence of Cys13, decomposes to a mixture of products, either at the active site or after release into solution. Possible similarities between this mechanism and the mechanism for isomerization of maleylacetoacetate and maleylpyruvate are discussed.

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