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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.

Most fungal glutathione transferases (GSTs) do not fit easily into any of the previously characterised classes by immunological, sequence or catalytic criteria. In contrast to the paucity of studies on GSTs cloned or isolated from fungal sources, a screen of databases revealed 67 GST-like sequences from 21 fungal species. Comparison by multiple sequence alignment generated a dendrogram revealing five clusters of GST-like proteins designated clusters 1, 2, EFIBgamma, Ure2p and MAK16, the last three of which have previously been related to the GST superfamily. Surprisingly, a relatively small number of fungal GSTs belong to mainstream classes and the previously-described fungal Gamma class is not widespread in the 21 species studied. Representative crystal structures are available for the EFIBgamma and Ure2p classes and the domain structures of representative sequences are compared with these. In addition, there are some "orphan" sequences that do not fit into any previously-described class, but show similarity to genes implicated in fungal biosynthetic gene clusters. We suggest that GST-like sequences are widespread in fungi, participating in a wide range of functions. They probably evolved by a process similar to domain "shuffling".