title: Site-directed mutagenesis of amino acid residues involved in the glutathione binding of human glutathione S-transferase P1-1.
abstract: The four residues of human glutathione S-transferase P1-1 whose counterparts were indicated by X-ray crystallography to reside in the GSH-binding site of pig glutathione S-transferase P1-1 were individually replaced with threonine or alanine by site-directed mutagenesis to obtain mutants R13T, K44T, Q51A, and Q64A. The kinetic parameters, susceptibilities to an inhibitor, S-hexyl-GSH, and affinities for GSH-Sepharose of the latter were compared with those of the wild-type enzyme, and pKa of the thiol group of GSH bound in R13T was shown to be equivalent to that in the wild type. From the results, Lys44, Gln51, and Gln64 were deduced to contribute to the binding of GSH. On the other hand, Arg13 seems to be essential for the enzymatic activity as mainly involved in the construction of a proper structure of the active site.
authors.affiliation: Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Japan.
authors.forename: K H
authors.initials: KH
authors.lastname: Kong
authors.forename: H
authors.initials: H
authors.lastname: Inoue
authors.forename: K
authors.initials: K
authors.lastname: Takahashi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005978:Glutathione / Q000378:metabolism*; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013552:Swine; D014961:X-Ray Diffraction
chemical_list: D007527:Isoenzymes; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D005982:Glutathione Transferase; D005978:Glutathione