| Tetrachlorohydroquinone (TCHQ) dehalogenase is the third enzyme in the pathway for biodegradation of pentachlorophenol by the soil bacterium Sphingobium chlorophenolicum. This enzyme catalyzes two successive reductive dehalogenations that convert TCHQ first to trichlorohydroquinone (TriCHQ) and then to 2,6-dichlorohydroquinone (DCHQ). TCHQ dehalogenase is a member of the zeta class of the glutathione S-transferase (GST) superfamily. The ancestral chemical reaction catalyzed by members of the GST superfamily is nucleophilic attack of glutathione upon an electrophilic substrate to form a glutathione conjugate. However, for TCHQ dehalogenase, this ancestral step is part of a more complex transformation. TCHQ dehalogenase can catalyze the steps leading to formation of DCHQ quite well, with a rate constant of 25 s-1. The dehalogenation reaction results in formation of a disulfide bond between glutathione and Cys 13 at the active site (ESSG). A thiol-disulfide exchange reaction that regenerates the free enzyme and forms glutathione disulfide must occur to complete turnover. This is of interest because it is not typically catalyzed by members of the GST superfamily. TCHQ dehalogenase does a poor job of catalyzing the thiol-disulfide exchange reaction. The second glutathione involved in the thiol-disulfide exchange reaction binds very weakly, and the enzyme does not alter its p Ka to improve its nucleophilicity. Furthermore, the thiol-disulfide exchange reaction is nonspecific; other thiols undergo thiol-disulfide exchange with ESSG at rates comparable to that of glutathione. We have also found that TCHQ dehalogenase is subject to severe inhibition by its aromatic substrates (TCHQ and TriCHQ). The aromatic substrates interfere with both the thiol-disulfide exchange reaction of ESSG and the dissociation of GSSG required to complete the catalytic cycle. |
