title: The contribution of the C-terminal sequence to the catalytic activity of GST2, a human alpha-class glutathione transferase.
abstract: A plasmid vector was constructed that encodes the expression in Escherichia coli of a truncated form of GST2, a human Alpha-class glutathione transferase. The truncated enzyme, GST2del210, has 12 residues deleted from the C-terminus and has the last two residues of the new C-terminal mutated from aspartic acid and glutamic acid to histidine and glycine respectively. GST2del210 has substantially diminished specific activity with either 1-chloro-2,4-dinitrobenzene or cumene hydroperoxide as substrate. The affinity of the truncated enzyme for a GSH-agarose matrix was also diminished, but sufficient interaction remained to enable affinity purification. Inhibition of GST2del210 by bromosulphophthalein was not altered. In contrast, this truncated form was not inhibited by S-pentylglutathione, a competitive inhibitor of the wild-type GST2 isoenzyme. The results show that the C-terminal segment of the Alpha-class glutathione transferases may form a component of the hydrophobic substrate-binding site. In contrast, this region appears not to be directly involved in GSH binding and is not absolutely essential for catalytic activity.
authors.affiliation: Molecular Genetics Group, John Curtin School of Medical Research, Australian National University, Canberra, A.C.T.
authors.forename: P G
authors.initials: PG
authors.lastname: Board
authors.forename: B
authors.initials: B
authors.lastname: Mannervik
mesh_terms: D000595:Amino Acid Sequence; D002384:Catalysis; D015252:Deoxyribonucleases, Type II Site-Specific; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010446:Peptide Fragments / Q000378:metabolism*; D010957:Plasmids; D013329:Structure-Activity Relationship; D014169:Transformation, Bacterial
chemical_list: D007527:Isoenzymes; D010446:Peptide Fragments; D005982:Glutathione Transferase; C052380:endodeoxyribonuclease NcoI; D015252:Deoxyribonucleases, Type II Site-Specific