IDENTIFICATION OF THE ELECTROPHILIC SUBSTRATE-BINDING SITE OF GLUTATHIONE-S-TRANSFERASE-P BY PHOTOAFFINITY-LABELING

We determined the electrophilic substrate-binding site of rat glutathi one S-transferase P (GST-P) by photoaffinity labeling using the photos ensitive compound S-[2-(2-fluoro-4-nitrophenoxy)ethyl]glutathione. Thi s photosensitive glutathione analogue inhibited the catalytic activity in a competitive manner against both glutathione and 1-chloro-2,4-din itrobenzene, a putative electrophilic substrate, The enzyme kinetics i ndicated that the photoactivatable glutathione analogue was specifical ly bound at the active site, which consisted of glutathione-binding (G -site) and the electrophilic substrate-binding (H-site) regions. The p rocedure involved the following steps: S-[2-(2-fluoro-4-nitrophenoxy)e thyl]glutathione was photochemically reacted with a purified recombina nt GST-P expressed in Escherichia coli using ultraviolet irradiation f or 30 min on ice. After the reaction, only the GST-P complexed with th e glutathione analogue was prepared with glutathione-immobilized agaro se. The GST-P covalently bound with the analogue was digested with lys yl endopeptidase (Achromobacter protease I), and the peptides were sep arated by highperformance liquid chromatography. Only a single major p eak with appreciable absorbance at 340 nm was observed by peptide mapp ing. The peptide was collected and analyzed using an automated peptide sequencer (ABI 477A). Amino acid sequence analysis showed that this p eptide consisted of seven amino acid residues corresponding to the seq uence at positions 122-128 of GST-P (Ala-Leu-Pro-Gly-Xaa-Leu-Lys). No appreciable phenylthiohydantoin-amino acid was detected at the fifth c ycle, which indicated that His126 was chemically labeled with the phot osensitive glutathione analogue. It was concluded that His126 was one of the amino acid residues forming the electrophilic substrate-binding site of GST-P.