title: Mutational analysis of human glutathione transferase A2-2 identifies structural elements supporting high activity with the prodrug azathioprine.
abstract: Glutathione transferase (GST) A2-2 is the human enzyme displaying the highest catalytic activity with the prodrug azathioprine (Aza). The reaction releases pharmacologically active 6-mercaptopurine by displacing the imidazole moiety from the Aza molecule. The GST-catalyzed reaction is of medical significance, since high rates of Aza activation may lead to adverse side effects in treated patients. The present study involves structure-activity relationships in GST A2-2 variants. Chimeric GSTs were previously generated by DNA shuffling and two peptide segments, one N-terminal and one C-terminal, were identified as primary determinants of Aza activity. The segments contain several residues of the substrate-binding H-site and their significance for supporting high Aza activity was investigated. Substitution of the corresponding two small regions in the low-activity human GST A3-3 or rat GST A3-3 by the human GST A2-2 segments generated chimeras with ∼10-fold enhanced Aza activity. The H-site residues Met208 and Leu213 in the C-terminal segment of GST A2-2 were mutated to produce a library with all possible residue combinations. At a calculated 93% library coverage, all of the 1880 mutants examined showed wild-type or decreased Aza activity, even though some retained activities with alternative substrates, further emphasizing the importance of this region for the targeted activity.
authors.affiliation: Department of Biochemistry and Organic Chemistry,  Uppsala University, BMC, Box 576, SE-75123 Uppsala,  Sweden.
authors.forename: Olof
authors.initials: O
authors.lastname: Modén
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.forename: Bengt
authors.initials: B
authors.lastname: Mannervik
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001379:Azathioprine / Q000737:chemistry / Q000494:pharmacology*; D001665:Binding Sites; D002384:Catalysis; D005982:Glutathione Transferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007166:Immunosuppressive Agents / Q000737:chemistry / Q000494:pharmacology*; D007700:Kinetics; D009154:Mutation; D011355:Prodrugs / Q000737:chemistry / Q000494:pharmacology*; D051381:Rats; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D007166:Immunosuppressive Agents; D011355:Prodrugs; D005982:Glutathione Transferase; D001379:Azathioprine