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 YaYa and YcYc, 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 YaYa 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 YaYa isozyme, whereas two labeled peptides were present in the tryptic digest of YcYc. The Ya 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 Yc were identical with the deduced sequence of amino acids between 91 and 110 and 206 and 218. Hence, the Ya peptide and the smaller peptide purified from Yc came from the same region of the Ya and Yc subunits. This common region and a second region of the Yc subunit appear to form a portion of the active site of these two forms of glutathione S-transferase.
Hoesch
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RM
Liver Studies Unit, Veterans Administration Medical Center, San Francisco, California 94121.
Boyer
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TD
D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D001386:Azides / Q000138:chemical synthesis / Q000378:metabolism*; D001665:Binding Sites; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D005978:Glutathione / Q000138:chemical synthesis / Q000378:metabolism / Q000031:analogs & derivatives; D005982:Glutathione Transferase / Q000378:metabolism*; D007106:Immune Sera; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D010446:Peptide Fragments / Q000302:isolation & purification; D051381:Rats
D000345:Affinity Labels; D001386:Azides; D007106:Immune Sera; D007527:Isoenzymes; D010446:Peptide Fragments; C024939:S-(4-azidophenacyl)glutathione; D005982:Glutathione Transferase; D005978:Glutathione