title: Amino acid substitutions at positions 207 and 221 contribute to catalytic differences between murine glutathione S-transferase Al-1 and A2-2 toward (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrene.
abstract: We have previously identified a novel Alpha class murine glutathione (GSH) S-transferase isoenzyme (designated mGSTAl-2) which is exceptionally efficient in catalyzing the GSH conjugation of (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE], the ultimate carcinogen of widespread environmental pollutant benzo[a]pyrene. Furthermore, we have demonstrated that the Al-type subunit of this isoenzyme is significantly more active toward (+)-anti-BPDE than the other subunit (mGSTA2). To establish the basis for catalytic differences between mGSTAl and mGSTA2, which differ in their primary structures by 10 amino acids [distributed in three sections (I-III) as clusters of two (residues 65 and 95), three (residues 157, 162, and 169), and five (residues 207, 213, 218, 221, and 222) amino acids], three chimeric enzymes were expressed and tested for their activity toward (+)-anti-BPDE. These studies revealed that amino acid substitution(s) in section III determined the high catalytic activity of mGSTAl. Molecular modeling studies suggested that amino acid substitutions at positions 207 and/or 221, but not at positions 213, 218, and 222, may be responsible for such a difference. To test this possibility, amino acids at positions 207 and 221 of mGSTAl were mutated with the equivalent residues of mGSTA2. Kinetic analysis of the wild type and the mutant enzymes revealed that both methionine-207 and isoleucine-221 are critical for higher activity of mGSTA1-1 toward (+)-anti-BPDE compared with that of mGSTA2-2.
authors.affiliation: Cancer Research Laboratory, Mercy Hospital of Pittsburgh, Pennsylvania 15219, USA.
authors.forename: H
authors.initials: H
authors.lastname: Xia
authors.forename: Y
authors.initials: Y
authors.lastname: Gu
authors.forename: S S
authors.initials: SS
authors.lastname: Pan
authors.forename: X
authors.initials: X
authors.lastname: Ji
authors.forename: S V
authors.initials: SV
authors.lastname: Singh
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001580:Benzopyrenes / Q000378:metabolism*; D002384:Catalysis; D004789:Enzyme Activation / Q000235:genetics; D005260:Female; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D008805:Mice, Inbred A; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013237:Stereoisomerism
chemical_list: D001580:Benzopyrenes; D007527:Isoenzymes; D011994:Recombinant Proteins; C039385:7,8-dihydroxy-9,10-epoxy-8-methyl-7,8,9,10-tetrahydrobenzo(a)pyrene; C112500:glutathione S-transferase 9.5-1; C112501:glutathione S-transferase 9.5-2; D005982:Glutathione Transferase