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.