title: The locally denatured state of glutathione S-transferase A1-1: transition state analysis of ligand-dependent formation of the C-terminal helix.
abstract: On the basis of available x-ray structures, A-class glutathione S-transferases (GSTs) contain at their C-termini a short alpha-helix that provides a 'lid' over the active site in the presence of the reaction products, glutathione-conjugates. However, in the ligand-free enzyme this helix is disordered and crystallographically invisible. An aromatic cluster including Phe-10, Phe-220, and the catalytic Tyr-9 within the C-terminal strand control the order of this helix. Here, preliminary x-ray crystallographic analyses of the wild type and F220Y rGSTA1-1 in the presence of GSH are described. Also, a transition state analysis is presented for ligand-dependent formation of the helix, based on variable temperature stopped-flow fluorescence. Together, the results suggest that the ligand-dependent ordering of the C-terminal strand occurs with a transition state that is highly desolvated, but with few intramolecular hydrogen bonds or electrostatic interactions. However, substitutions at Phe-220 modulate the activation parameters through interactions with the side chain of Tyr-9.
authors.affiliation: Department of Medicinal Chemistry, University of Washington, Seattle 98195-7610, USA.
authors.forename: B S
authors.initials: BS
authors.lastname: Nieslanik
authors.forename: E C
authors.initials: EC
authors.lastname: Dietze
authors.forename: W M
authors.initials: WM
authors.lastname: Atkins
authors.forename: I L
authors.initials: IL
authors.lastname: Trong
authors.forename: E
authors.initials: E
authors.lastname: Adman
mesh_terms: D003196:Computer Graphics; D018360:Crystallography, X-Ray; D004926:Escherichia coli; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism; D005982:Glutathione Transferase / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D046911:Macromolecular Substances; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D007527:Isoenzymes; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D005982:Glutathione Transferase; D005978:Glutathione