title: Evidence for the involvement of tryptophan 38 in the active site of glutathione S-transferase P.
abstract: Glutathione S-transferase P (GST-P) exists as a homodimeric form and has two tryptophan residues, Trp28 and Trp38, in each subunit. In order to elucidate the role of the two tryptophan residues in catalytic function, we examined intrinsic fluorescence of tryptophan residues and effect of chemical modification by N-bromosuccinimide (NBS). The quenching of intrinsic fluorescence was observed by the addition of S-hexylglutathione, a substrate analogue, and the enzymatic activity was totally lost when single tryptophan residue was oxidized by NBS. To identify which tryptophan residue is involved in the catalytic function, each tryptophan was changed to histidine by site-directed mutagenesis. Trp28His GST-P mutant enzyme showed a comparable enzymatic activity with that of the wild type one. Trp38His mutant neither was bound to S-hexylglutathione-linked Sepharose nor exhibited any GST activity. These findings indicate that Trp38 is important for the catalytic function and substrate binding of GST-P.
authors.affiliation: Second Department of Biochemistry, School of Medicine, Hokkaido University, Sapporo, Japan.
authors.forename: J
authors.initials: J
authors.lastname: Nishihira
authors.forename: T
authors.initials: T
authors.lastname: Ishibashi
authors.forename: M
authors.initials: M
authors.lastname: Sakai
authors.forename: S
authors.initials: S
authors.lastname: Nishi
authors.forename: T
authors.initials: T
authors.lastname: Kumazaki
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D001974:Bromosuccinimide / Q000494:pharmacology; D003001:Cloning, Molecular; D005982:Glutathione Transferase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
chemical_list: D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014364:Tryptophan; D005982:Glutathione Transferase; D001974:Bromosuccinimide