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• W2079943300 abstract "A library of active-site mutants has been constructed by targeting selected amino acid residues in human glutathione transferase (GST) A1-1 for random mutagenesis. The mutated residues are suitably positioned for interaction with the second, electrophilic substrate, in particular chloronitrobenzene derivatives undergoing SNAr reactions. DNA representing the GST A1-1 mutant library was fused with DNA encoding gene III protein, a component of the coat of filamentous phage. Phage display was used for affinity selection of GST A1-1 mutants with altered catalytic properties. The affinity ligand used was the σ-complex of 1,3,5-trinitrobenzene and glutathione immobilized to Sepharose. The complex was designed to mimic the transition state of SNAr reactions catalyzed by GSTs. The selection system is based on the combination of affinity for the σ-complex as well as the ability to promote its formation, thus mimicking two salient features of the assumed catalytic mechanism for the SNAr reactions. Many of the GST A1-1 mutants selected and analyzed contained an aromatic amino acid residue in one of the mutated positions, suggesting favorable interactions with the trinitrocyclohexadienate moiety of the affinity ligand. A mutant C36 was selected for more detailed studies. Its catalytic efficiency with several chloronitrobenzene substrates was 20−90-fold lower than that of wild-type GST A1-1, but fully comparable to naturally evolved GSTs of different classes, providing a 105-fold rate enhancement over the uncatalyzed reaction. In the conjugation of ethacrynic acid, a Michael addition reaction, mutant C36 was 13-fold more efficient than the wild-type enzyme. Within experimental error, the quotient between the KF values for wild-type GST A1-1 and mutant C36 is the same as that between the kcat/KM values determined with 1-chloro-2,4-dinitrobenzene for the two enzyme forms. This result indicates that σ-complex formation is rate-limiting for the catalyzed reaction. Thus, the principle of transition-state stabilization as a component of catalysis has been successfully exploited in affinity selection of catalytically competent GST A1-1 mutants. This mechanism-based procedure also selects for the ability to promote σ-complex formation, and serves as a probe of the catalytic mechanism." @default.
• W2079943300 created "2016-06-24" @default.
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• W2079943300 date "1997-09-01" @default.
• W2079943300 modified "2023-09-27" @default.
• W2079943300 title "Mechanism-Based Phage Display Selection of Active-Site Mutants of Human Glutathione Transferase A1-1 Catalyzing S_NAr Reactions" @default.
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• W2079943300 doi "https://doi.org/10.1021/bi9702952" @default.
• W2079943300 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9287168" @default.
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