Cysteine-86 is not needed for the enzymic activity of glutathione S-transferase 3-3.
Recombinant glutathione S-transferase 3-3 expressed in Spodoptera frugiperda (SF9) cells with the use of a baculovirus expression system was modified with 1 mM-iodoacetamide. Amino acid analysis indicated that 0.79 +/- 0.15 cysteine residue was modified per enzyme subunit. The S-carbaminomethylated protein retains the GSH-conjugating activity. Glutathione S-transferase 3-3 modified with iodo[14C]acetamide was digested with Achromobacter proteinase I and the resulting peptides were separated by h.p.l.c. The modified peptides were pooled and further digested with Staphylococcus aureus V8 proteinase. Isotope-labelled peptides were isolated and collected for N-terminal sequence analysis. By this procedure, cysteine-86 was identified as the major S-carbaminomethylated residue. Verification of this findings came from the use of site-directed mutagenesis in which this cysteine was replaced by serine (C86S mutant). The C86S mutant is enzymically active. Therefore cysteine-86 is not needed for the conjugation of GSH with electrophilic compounds on glutathione S-transferase 3-3.
Hsieh
J C
JC
Institute of Molecular Biology, Academia Sinica, Nankang Taipei, Taiwan.
Huang
S C
SC
Chen
W L
WL
Lai
Y C
YC
Tam
M F
MF
D000595:Amino Acid Sequence; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D004247:DNA / Q000235:genetics; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007460:Iodoacetamide / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012697:Serine Endopeptidases; D013329:Structure-Activity Relationship; D014162:Transfection
D010446:Peptide Fragments; D011994:Recombinant Proteins; D004247:DNA; D005982:Glutathione Transferase; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; C036813:lysyl endopeptidase; D003545:Cysteine; D007460:Iodoacetamide