title: C-terminal region amino acid substitutions contribute to catalytic differences between murine class alpha glutathione transferases mGSTA1-1 and mGSTA2-2 toward anti-diol epoxide isomers of benzo[c]phenanthrene.
abstract: The molecular basis for catalytic differences between structurally closely related murine class alpha glutathione (GSH) transferases mGSTA1-1 and mGSTA2-2 in the GSH conjugation of anti-diol epoxide isomers of benzo[c]phenanthrene (anti-B[c]PDE) was investigated. GSH conjugation of both (-)- and (+)-enantiomers of anti-B[c]PDE was observed in the presence of mGSTA1-1 (60 and 40% GSH conjugation, respectively), whereas mGSTA2-2 exhibited a preference for the (-)-anti-isomer (>97%). In addition, the specific activity of mGSTA2-2 toward the (-)-anti-B[c]PDE isomer was relatively higher than that of mGSTA1-1. The amino acid sequences of mGSTA1-1 and mGSTA2-2 differ at 10 positions that are distributed in three sections. Section I contains amino acid residues in positions 65 and 95; section II contains residues in positions 157, 162, and 169, and section III contains residues in positions 207, 213, 218, 221, and 222. Enzyme activity measurements with chimeras of mGSTA1-1 and mGSTA2-2 revealed that amino acid substitutions in section III account for their differential enantioselectivity and catalytic activity toward anti-B[c]PDE. Site-directed mutagenesis of amino acid residues in section III of mGSTA2-2 with corresponding residues of mGSTA1-1 followed by activity measurements of the wild type and mutated enzymes indicates that leucine 207 and phenylalanine 221 may be critical for the high catalytic activity of mGSTA2-2 toward (-)-anti-B[c]PDE. Molecular modeling studies demonstrated that the active site of mGSTA1-1 accommodates both enantiomers of anti-B[c]PDE, whereas the (-)-anti-isomer interacts more favorably with active site residues in mGSTA2-2. The results of this study clearly indicate that amino acid substitutions in the C-terminal region contribute to catalytic differences between mGSTA1-1 and mGSTA2-2 with respect to anti-B[c]PDE.
authors.affiliation: Department of Pharmacology and University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.
authors.forename: A
authors.initials: A
authors.lastname: Pal
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; D002273:Carcinogens / Q000737:chemistry*; D002384:Catalysis; D004789:Enzyme Activation / Q000235:genetics; D005978:Glutathione / Q000737:chemistry / Q000235:genetics; D005982:Glutathione Transferase / Q000737:chemistry* / Q000235:genetics; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics; D010616:Phenanthrenes / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D013237:Stereoisomerism; D013329:Structure-Activity Relationship
chemical_list: D002273:Carcinogens; D007527:Isoenzymes; D010446:Peptide Fragments; D010616:Phenanthrenes; D011993:Recombinant Fusion Proteins; C112500:glutathione S-transferase 9.5-1; C112501:glutathione S-transferase 9.5-2; D005982:Glutathione Transferase; D005978:Glutathione; C002615:benzo(c)phenanthrene