Identification of the electrophilic substrate-binding site of glutathione S-transferase P by photoaffinity labeling.
We determined the electrophilic substrate-binding site of rat glutathione S-transferase P (GST-P) by photoaffinity labeling using the photosensitive compound S-[2-(2-fluoro-4-nitrophenoxy)ethyl]glutathione. This photosensitive glutathione analogue inhibited the catalytic activity in a competitive manner against both glutathione and 1-chloro-2,4-dinitrobenzene, a putative electrophilic substrate. The enzyme kinetics indicated that the photoactivatable glutathione analogue was specifically bound at the active site, which consisted of glutathione-binding (G-site) and the electrophilic substrate-binding (H-site) regions. The procedure involved the following steps: S-[2-(2-fluoro-4-nitrophenoxy)ethyl]glutathione was photochemically reacted with a purified recombinant GST-P expressed in Escherichia coli using ultraviolet irradiation for 30 min on ice. After the reaction, only the GST-P complexed with the glutathione analogue was prepared with glutathione-immobilized agarose. The GST-P covalently bound with the analogue was digested with lysyl endopeptidase (Achromobacter protease I), and the peptides were separated by high-performance liquid chromatography. Only a single major peak with appreciable absorbance at 340 nm was observed by peptide mapping. The peptide was collected and analyzed using an automated peptide sequencer (ABI 477A). Amino acid sequence analysis showed that this peptide consisted of seven amino acid residues corresponding to the sequence at positions 122-128 of GST-P (Ala-Leu-Pro-Gly-Xaa-Leu-Lys). No appreciable phenylthiohydantoin-amino acid was detected at the fifth cycle, which indicated that His126 was chemically labeled with the photosensitive glutathione analogue. It was concluded that His126 was one of the amino acid residues forming the electrophilic substrate-binding site of GST-P.
Nishihira
J
J
Central Research Institute, Hokkaido University School of Medicine, Sapporo, Japan.
Sakai
M
M
Nishi
S
S
Hatanaka
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D005982:Glutathione Transferase / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D010777:Photochemistry; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017421:Sequence Analysis; D013379:Substrate Specificity
D011994:Recombinant Proteins; D005982:Glutathione Transferase