Purification and characterization of glutathione conjugate reductase: a component of the tetrachlorohydroquinone reductive dehalogenase system from Phanerochaete chrysosporium.
A membrane-bound glutathione S-transferase and a soluble glutathione conjugate reductase constitute the reductive dehalogenase system of P. chrysosporium. This enzyme system reductively removes chlorine substituents from tetrachlorohydroquinone, a metabolite of pentachlorophenol. The membrane-bound glutathione S-transferase converts tetrachlorohydroquinone to S-glutathionyltrichloro-1,4-hydroquinone, which is subsequently reduced to 3,5,6-trichlorohydroquinone by the soluble glutathione conjugate reductase (GCR). This GCR can accept glutathione, dithiothreitol, cysteine, or beta-mercaptoethanol as cosubstrates. GCR was purified to apparent homogeneity by ion-exchange and covalent chromatography. The enzyme exhibits optimum activity at pH 6.0 and 55 degrees C and appears to be a homodimer with a M(r) of approximately 60 kDa. Activity increases as the number of chlorine substituents on the hydroquinone ring is increased. GCR has an apparent K(m) of approximately 33 microM and an apparent k(cat) of approximately 3.43 s(-1) for 2-S-glutathionyl-3,5,6-trichloro-1,4-hydroquinone. Inhibitors of GCR include Cd(2+), Fe(2+), Mn(2+), iodoacetic acid, and p-chloromercuribenzoic acid, suggesting the presence of a catalytic cysteine thiol(s) at the active site. When glutathione is used as a cosubstrate, reduction of S-glutathionyltrichloro-1,4-hydroquinone is accompanied by the production of trichlorohydroquinone and oxidized glutathione in a 1:1 ratio. A mechanism for this novel enzyme is proposed.
Reddy
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GV
Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, Beaverton, Oregon 97006-8921, USA.
Gold
M H
MH
D005978:Glutathione / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D008077:Lipoproteins, LDL / Q000302:isolation & purification; D008670:Metals / Q000494:pharmacology; D010088:Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D020075:Phanerochaete / Q000201:enzymology*; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013696:Temperature
D008077:Lipoproteins, LDL; D008670:Metals; D013438:Sulfhydryl Compounds; C099252:oxidized low density lipoprotein; D010088:Oxidoreductases; C431912:glutathione conjugate reductase; D006867:Hydrolases; C078610:2,3,5,6-tetrachloro-4-hydroquinone reductive dehalogenase; D005978:Glutathione