PHOTOAFFINITY-LABELING OF THE ACTIVE-SITE OF THE RAT GLUTATHIONE TRANSFERASE-3-3 AND TRANSFERASE-1-1 AND HUMAN GLUTATHIONE TRANSFERASE A1-1

The glutathione transferases (GSTs) form a group of enzymes responsibl e for a wide range of molecular detoxications. The photoaffinity label S-(2-nitro-4-azidophenyl)glutathione was used to study the hydrophobi c region of the active site of the rat liver GST 1-1 and 2-2 isoenzyme s (class Alpha) as well as the rat class-Mu GST 3-3. Photoaffinity lab elling was carried out using a version of S-(2-nitro-4-azidophenyl)glu tathione 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 competit ive inhibitor, S-tritylglutathione (K-i = 1.4 x 10(-7) M). Radiolabell ing of the protein paralleled the loss of enzyme activity. Photoaffini ty labelling by tritiated S-(2-nitro-4-azidophenyl)glutathione on a pr eparative scale (in the presence and absence of S-tritylglutathione) f ollowed by tryptic digestion and purification of the labelled peptides indicated that GST 3-3 was specifically photolabelled; the labelled p eptides were sequenced. Similarly, preparative photoaffinity labelling by S-(2-nitro-4-azidophenyl)glutathione of the rat liver 1-1 isoenzym e, the human GST A1-1 and the human-rat chimaeric GST, H1R1/1, was car ried 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 pho to affinity-labelled peptides using th e X-ray-crystallo graphic co-ordinates of rat GST 3-3 and human GST A1 -1. The molecular-graphical analysis indicated that the labelled pepti des are located within the immediate vicinity of the region occupied b y S-substituted glutathione derivatives bound in the active-site cavit y of the GSTs investigated.