title: The role of a topologically conserved isoleucine in glutathione transferase structure, stability and function.
abstract: The common fold shared by members of the glutathione-transferase (GST) family has a topologically conserved isoleucine residue at the N-terminus of helix 3 which is involved in the packing of helix 3 against two beta-strands in domain 1. The role of the isoleucine residue in the structure, function and stability of GST was investigated by replacing the Ile71 residue in human GSTA1-1 by alanine or valine. The X-ray structures of the I71A and I71V mutants resolved at 1.75 and 2.51 A, respectively, revealed that the mutations do not alter the overall structure of the protein compared with the wild type. Urea-induced equilibrium unfolding studies using circular dichroism and tryptophan fluorescence suggest that the mutation of Ile71 to alanine or valine reduces the stability of the protein. A functional assay with 1-chloro-2,4-dinitrobenzene shows that the mutation does not significantly alter the function of the protein relative to the wild type. Overall, the results suggest that conservation of the topologically conserved Ile71 maintains the structural stability of the protein but does not play a significant role in catalysis and substrate binding.
authors.affiliation: Protein Structure-Function Research Unit, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg 2050, South Africa.
authors.forename: Ikechukwu
authors.initials: I
authors.lastname: Achilonu
authors.forename: Samantha
authors.initials: S
authors.lastname: Gildenhuys
authors.forename: Loren
authors.initials: L
authors.lastname: Fisher
authors.forename: Jonathan
authors.initials: J
authors.lastname: Burke
authors.forename: Sylvia
authors.initials: S
authors.lastname: Fanucchi
authors.forename: B Trevor
authors.initials: BT
authors.lastname: Sewell
authors.forename: Manuel
authors.initials: M
authors.lastname: Fernandes
authors.forename: Heini W
authors.initials: HW
authors.lastname: Dirr
mesh_terms: D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D005982:Glutathione Transferase / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007532:Isoleucine / Q000737:chemistry; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D056811:Unfolded Protein Response
chemical_list: D007532:Isoleucine; D005982:Glutathione Transferase