A Mechanistic Investigation of the Thiol−Disulfide Exchange Step in the Reductive Dehalogenation Catalyzed by Tetrachlorohydroquinone Dehalogenase

Abstract

Tetrachlorohydroquinone dehalogenase catalyzes the reductive dehalogenation of tetrachloro- and trichlorohydroquinone to give 2,6-dichlorohydroquinone in the pathway for degradation of pentachlorophenol by Sphingobium chlorophenolicum. Previous work has suggested that this enzyme may have originated from a glutathione-dependent double bond isomerase such as maleylacetoacetate isomerase or maleylpyruvate isomerase. While some of the elementary steps in these two reactions may be similar, the final step in the dehalogenation reaction, a thiol-disulfide exchange reaction that removes glutathione covalently bound to Cys13, certainly has no counterpart in the isomerization reaction. The thiol-disulfide exchange reaction does not appear to have been evolutionarily optimized. There is little specificity for the thiol; many thiols react at high rates. TCHQ dehalogenase binds the glutathione involved in the thiol-disulfide exchange reaction very poorly and does not alter its pK(a) in order to improve its nucleophilicity. Remarkably, single-turnover kinetic studies show that the enzyme catalyzes this step by approximately 10000-fold. This high reactivity requires an as yet unidentified protonated group in the active site.

Structures of Ternary Complexes of BphK, a Bacterial Glutathione S-Transferase That Reductively Dechlorinates Polychlorinated Biphenyl Metabolites

“…This mechanism is similar to the better supported mechanism of TCHQ dehalogenase (26,36) and can be divided into two half-reactions demarcated by the formation of a mixed disulfide between the enzyme and GSH. The first halfreaction was proposed to proceed via one of two routes: the addition of GS Ϫ at carbon 3 of HOPDA followed by elimination of Cl Ϫ or the tautomerization of the enol(ate) followed by an S N 2 nucleophilic attack by GS Ϫ at carbon 3 to release Cl Ϫ .…”

“…For example, TCHQ dehalogenase binds the second GSH with a K d value of 19 mM (36). In addition, the TCHQ substrate inhibited the formation of GSSG, suggesting that the aromatic substrate has higher affinity for the H-subsite (36). Thus, the observation of HOPDA binding to BphK in the presence of excess GSH is not surprising based on analogy between these enzymes.…”

“…The pK a of 3-Cl HOPDA in solution is 6.1 (24) but may be perturbed by binding to the enzyme. Finally, the proposed mechanism for the first half-reaction of BphK differs from that of TCHQ dehalo- genase, which involves chloride elimination from the aromatic ring followed by GSH attack (36). The BphK-(GSH) 2 structure gives insight into the second half-reaction to regenerate the resting enzyme.…”

A Glutathione S -Transferase Catalyzes the Dehalogenation of Inhibitory Metabolites of Polychlorinated Biphenyls

“…Catabolite transformations include dehalogenation and carbon-carbon double bond isomerization (45). One of the better-studied bacterial GSTs is tetrachlorohydroquinone (TCHQ) dehalogenase from Sphingobium chlorophenolicum which catalyzes the reductive dehalogenation of TCHQ to dichlorohydroquinone via trichlorohydroquinone (23,24,46) and appears to have been recently recruited for the degradation of pentachlorophenol (PCP) (15).…”

Expression of a soluble form of iodotyrosine deiodinase for active site characterization by engineering the native membrane protein from Mus musculus

“…Reductive dehalogenation is an alternative common to anaerobes but rarely detected in aerobes. [6][7][8][9] Two exceptions are key to human health, and both relate to the synthesis and regulation of the thyroid hormone thyroxine (3,3 0 ,5,5 0 -tetraiodothyronine).…”