Site-directed mutagenesis and chemical modification of cysteine residues of rat glutathione S-transferase 3-3.
Rat liver glutathione S-transferase (GST) 3-3 is composed of two identical subunits, each containing three cysteine residues, Cys-86, Cys-114 and Cys-173. We have shown previously that Cys-86 is not involved in the enzymic activity of GST 3-3 [Hsieh, Huang, Chen, Lai & Tam (1991) Biochem, J. 278, 293-297]. At 50 degrees C, iodoacetamide can inactivate the enzyme by modifying Cys-86 and Cys-114. Cys-114 can be protected against iodoacetamide inhibition by S-(dinitrophenyl)glutathione. Site-directed mutagenesis was used to construct mutants in which serine replaced one (C114S and C173S) or all three (CallS) cysteine residues. These mutants were over-expressed in Spodoptera frugiperda cells in a baculovirus system and were found to be fully active. Replacing Cys-86 or Cys-114 with alanine (C86A and C114A) does not diminish the activity of the protein. The results suggest that cysteines are not involved in the enzymic mechanism, and Cys-114 is possibly located at the active site of GST 3-3.
Chen
W L
WL
Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan.
Hsieh
J C
JC
Hong
J L
JL
Tsai
S P
SP
Tam
M F
MF
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine; D010450:Endopeptidases; D005982:Glutathione Transferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments / Q000302:isolation & purification; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity
D007527:Isoenzymes; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D005982:Glutathione Transferase; D010450:Endopeptidases; D003545:Cysteine