The locally denatured state of glutathione S-transferase A1-1: transition state analysis of ligand-dependent formation of the C-terminal helix.
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.
Nieslanik
B S
BS
Department of Medicinal Chemistry, University of Washington, Seattle 98195-7610, USA.
Dietze
E C
EC
Atkins
W M
WM
Trong
I L
IL
Adman
E
E
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
D007527:Isoenzymes; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D005982:Glutathione Transferase; D005978:Glutathione