FORCED EVOLUTION OF GLUTATHIONE-S-TRANSFERASE TO CREATE A MORE EFFICIENT DRUG DETOXICATION ENZYME

Glutathione S-transferases (EC 2.5.1.18) in mammalian cells catalyze t he conjugation, and thus, the detoxication of a structurally diverse g roup of electrophilic environmental carcinogens and alkylating drugs, including the antineoplastic nitrogen mustards, We proposed that struc tural alteration of the nonspecific electrophile-binding site would pr oduce mutant enzymes with increased efficiency for detoxication of a s ingle drug and that these mutants could serve as useful somatic transg enes to protect healthy human cells against single alkylating agents u sed in cancer chemotherapy protocols, Random mutagenesis of three regi ons (residues 9-14, 102-112, and 210-220), which together compose the glutathione S-transferase electrophile-binding site, followed by selec tion of Escherichia coli expressing the enzyme library with the nitrog en mustard mechlorethamine (20-500 mu M), yielded mutant enzymes that showed significant improvement in catalytic efficiency for mechloretha mine conjugation (up to W-fold increase in k(cat) and up to 6-fold inc rease in k(cat)/K-m) and that confer up to 31-fold resistance, which i s 9-fold greater drug resistance than that conferred by the wild-type enzyme, The results suggest a general strategy for modification of dru g- and carcinogen-metabolizing enzymes to achieve desired resistance i n both prokaryotic and eukaryotic plant and animal cells.