title: Photoaffinity labelling of the active site of the rat glutathione transferases 3-3 and 1-1 and human glutathione transferase A1-1.
abstract: The glutathione transferases (GSTs) form a group of enzymes responsible for a wide range of molecular detoxications. The photoaffinity label S-(2-nitro-4-azidophenyl)glutathione was used to study the hydrophobic region of the active site of the rat liver GST 1-1 and 2-2 isoenzymes (class Alpha) as well as the rat class-Mu GST 3-3. Photoaffinity labelling was carried out using a version of S-(2-nitro-4-azidophenyl)glutathione tritiated in the arylazido ring. The labelling occurred with higher levels of radioisotope incorporation for the Mu than the Alpha families. Taking rat GST 3-3, 1.18 (+/- 0.05) mol of radiolabel from S-(2-nitro-4-azidophenyl)glutathione was incorporated per mol of dimeric enzyme, which could be blocked by the presence of the strong competitive inhibitor, S-tritylglutathione (Ki = 1.4 x 10(-7) M). Radiolabelling of the protein paralleled the loss of enzyme activity. Photoaffinity labelling by tritiated S-(2-nitro-4-azidophenyl)glutathione on a preparative scale (in the presence and absence of S-tritylglutathione) followed by tryptic digestion and purification of the labelled peptides indicated that GST 3-3 was specifically photolabelled; the labelled peptides were sequenced. Similarly, preparative photoaffinity labelling by S-(2-nitro-4-azidophenyl)glutathione of the rat liver 1-1 isoenzyme, the human GST A1-1 and the human-rat chimaeric GST, H1R1/1, was carried out with subsequent sequencing of radiolabelled h.p.l.c.-purified tryptic peptides. The results were interpreted by means of molecular-graphics analysis to locate photoaffinity-labelled peptides using the X-ray-crystallographic co-ordinates of rat GST 3-3 and human GST A1-1. The molecular-graphical analysis indicated that the labelled peptides are located within the immediate vicinity of the region occupied by S-substituted glutathione derivatives bound in the active-site cavity of the GSTs investigated.
authors.affiliation: Department of Pharmacy, University of Manchester, U.K.
authors.forename: R J
authors.initials: RJ
authors.lastname: Cooke
authors.forename: R
authors.initials: R
authors.lastname: Björnestedt
authors.forename: K T
authors.initials: KT
authors.lastname: Douglas
authors.forename: J H
authors.initials: JH
authors.lastname: McKie
authors.forename: M D
authors.initials: MD
authors.lastname: King
authors.forename: B
authors.initials: B
authors.lastname: Coles
authors.forename: B
authors.initials: B
authors.lastname: Ketterer
authors.forename: B
authors.initials: B
authors.lastname: Mannervik
mesh_terms: D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides; D001665:Binding Sites; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D005978:Glutathione / Q000031:analogs & derivatives; D005982:Glutathione Transferase / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D008297:Male; D008969:Molecular Sequence Data; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D000345:Affinity Labels; D001386:Azides; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; C024939:S-(4-azidophenacyl)glutathione; D005982:Glutathione Transferase; D005978:Glutathione