title: Mutagenic analysis of conserved arginine residues in and around the novel sulfate binding pocket of the human Theta class glutathione transferase T2-2.
abstract: The human Theta class glutathione transferase GSTT2-2 has a novel sulfatase activity that is not dependent on the presence of a conserved hydrogen bond donor in the active site. Initial homology modeling and the crystallographic studies have identified three conserved Arg residues that contribute to the formation of (Arg107 and Arg239), and entry to (Arg242), a sulfate binding pocket. These residues have been individually mutated to Ala to investigate their potential role in substrate binding and catalysis. The mutation of Arg107 had a significant detrimental effect on the sulfatase reaction, while the Arg242 mutation caused only a small reduction in sulfatase activity. Surprisingly, the Arg239 had an increased activity resulting from a reduction in stability. Thus, Arg239 appears to play a role in maintaining the architecture of the active site. Electrostatic calculations performed on the wild-type and mutant forms of the enzyme are in good agreement with the experimental results. These findings, along with docking studies, suggest that prior to conjugation, the location of 1-menaphthyl sulfate, a model substrate for the sulfatase reaction, is approximately midway between the position ultimately occupied by the naphthalene ring of 1-menaphthylglutathione and the free sulfate. It is further proposed that the Arg residues in and around the sulfate binding pocket have a role in electrostatic substrate recognition.
authors.affiliation: John Curtin School of Medical Research, Australian National University, Canberra ACT.
authors.forename: J U
authors.initials: JU
authors.lastname: Flanagan
authors.forename: J
authors.initials: J
authors.lastname: Rossjohn
authors.forename: M W
authors.initials: MW
authors.lastname: Parker
authors.forename: P G
authors.initials: PG
authors.lastname: Board
authors.forename: G
authors.initials: G
authors.lastname: Chelvanayagam
mesh_terms: D001120:Arginine / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D055672:Static Electricity; D013431:Sulfates / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D013431:Sulfates; D001120:Arginine; C491107:GSTT2 protein, human; D005982:Glutathione Transferase