title: Chemical modification at subunit 1 of rat kidney Alpha class glutathione transferase with 2,3,5,6-tetrachloro-1,4-benzoquinone: close structural connectivity between glutathione conjugation activity and non-substrate ligand binding.
abstract: 2, 3, 5, 6-Tetrachloro-1, 4-benzoquinone (TCBQ) is a metabolite of pentachlorophenol known to react with cysteines of glutathione transferases (GSTs). TCBQ treatment of rat kidney rGSTA1-2 and rGSTA1-1 abolishes 70-80% conjugation of glutathione (GSH) to 1-chloro-2, 4-dinitrobenzene and results in strongly correlated quenching of intrinsic fluorescence of Trp-20 (R>0.96). rGSTA2-2 is only inhibited by 25%. Approximately 70% (rGSTA1-1) and 60% (rGSTA1-2) conjugation activity is abolished at TCBQ: GST stoichiometries near 1:1. The inactivation follows a Kitz/Wilson model with K(D) of 4.77+/-2.5microM for TCBQ and k(3) for inactivation of 0.036+/-0.01min(-1). A single tryptic peptide labelled with TCBQ was isolated from kidney rGSTA1-2 containing Cys-17 which we identify as the site of modification. Treatment with more than stoichiometric amounts of TCBQ modified other residues but resulted in only modest further inhibition of catalysis. We interpret these findings in terms of localised steric effects on the relatively rigid alpha-helix 1 adjacent to the catalytic site of subunit 1 possibly affecting the Alpha class-specific alpha-helix 9 which acts as a "lid" on the hydrophobic part of the active site. Homology modelling of rGSTA1-1 modified at Cys-17 of one subunit revealed only modest structural perturbations in the second subunit and tends to exclude global structural effects.
authors.affiliation: Proteomics Research Group, Department of Biochemistry, University College Cork, Lee Maltings, Prospect Row, Mardyke, Cork, Ireland.
authors.forename: Siobhan M
authors.initials: SM
authors.lastname: O'Sullivan
authors.forename: Ronan M
authors.initials: RM
authors.lastname: McCarthy
authors.forename: Melissa A
authors.initials: MA
authors.lastname: Vargo
authors.forename: Roberta F
authors.initials: RF
authors.lastname: Colman
authors.forename: David
authors.initials: D
authors.lastname: Sheehan
mesh_terms: D000818:Animals; D020134:Catalytic Domain; D002703:Chloranil / Q000494:pharmacology*; D005978:Glutathione / Q000378:metabolism*; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D007668:Kidney / Q000201:enzymology*; D008024:Ligands; D008297:Male; D008958:Models, Molecular; D021122:Protein Subunits; D051381:Rats; D017208:Rats, Wistar
chemical_list: D007527:Isoenzymes; D008024:Ligands; D021122:Protein Subunits; D002703:Chloranil; D005982:Glutathione Transferase; C119995:glutathione S-transferase alpha; D005978:Glutathione