title: Replacement surgery with unnatural amino acids in the lock-and-key joint of glutathione transferase subunits.
abstract: Proteins contain amino acid residues essential to structure and function. Ribosomal protein synthesis is typically limited to the 20 amino acids of the genetic code, but posttranslational chemical modifications can greatly expand the diversity of side chain functionalities. In this investigation, a natural aromatic residue in the lock-and-key joint at the subunit interface of the dimeric glutathione transferase P1-1 was replaced by an S-alkylcysteine residue to give a functional enzyme. Introduction of Cys in the key position inactivates the enzyme, but subsequent alkylation of this residue enhances the catalytic efficiency up to 27,000-fold. Combinatorial modification of Cys by a mixture of reagents facilitated identification of an n-butyl group as the most efficient activator. Alkylation also enhanced binding affinity for active-site ligands and stabilized the enzyme against chemical denaturation and thermal inactivation.
authors.affiliation: Department of Biochemistry and Organic Chemistry, Biomedical Center, Uppsala University, Box 576, SE-751 23 Uppsala, Sweden.
authors.forename: Usama M
authors.initials: UM
authors.lastname: Hegazy
authors.forename: Ulf
authors.initials: U
authors.lastname: Hellman
authors.forename: Bengt
authors.initials: B
authors.lastname: Mannervik
mesh_terms: D000478:Alkylation; D019943:Amino Acid Substitution; D002073:Butanes / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain; D003545:Cysteine / Q000378:metabolism; D051549:Glutathione S-Transferase pi / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006847:Hydrocarbons, Iodinated / Q000378:metabolism; D013058:Mass Spectrometry; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering / Q000379:methods*; D017433:Protein Structure, Secondary; D014443:Tyrosine / Q000378:metabolism
chemical_list: D002073:Butanes; D006847:Hydrocarbons, Iodinated; D014443:Tyrosine; C019435:1-iodobutane; C014055:methyl iodide; C496556:GSTP1 protein, human; D051549:Glutathione S-Transferase pi; D003545:Cysteine