Localization of a portion of the active site of two rat liver glutathione S-transferases using a photoaffinity label

The glutathione S-transferases are a family of dimeric enzymes that catalyze the reaction between GSH and a variety of electrophiles. Two closely related isozymes, referred to as Y(a)Y(a) and Y(c)Y(c), were purified from rat liver. A radiolabeled azido derivative of glutathione (S-(p-azidophenacyl)[³H]glutathione) was prepared and used to label covalently the active site of the above two glutathione S-transferases. The noncovalently bound affinity label was a competitive inhibitor of glutathione S-transferase Y(a)Y(a) toward both 1-chloro-2,4-dinitrobenzene and GSH. The covalently labeled enzymes no longer bound to a GSH-affinity column, and covalent labeling was reduced in the presence of GSH and S-(dinitrophenyl)glutathione. These results suggest that the affinity label was binding at the active site. The covalently labeled enzymes were digested with trypsin, and the labeled peptides were purified by HPLC and then sequenced. A single-labeled peptide was identified in the tryptic digest of the Y(a)Y(a) isozyme, whereas two labeled peptides were present in the tryptic digest of Y(c)Y(c). The Y(a) peptide sequence was identical with the published deduced sequence of amino acids between residues 212 and 218 and the sequences of the two peptides purified from Y(c) were identical with the deduced sequence of amino acids between 91 and 110 and 206 and 218. Hence, the Y(a) peptide and the smaller peptide purified from Y(c) came from the same region of the Y(a) and Y(c) subunits. This common region and a second region of the Y(c) subunit appear to form a portion of the active site of these two forms of glutathione S-transferase.