A conserved N-capping motif contributes significantly to the stabilization and dynamics of the C-terminal region of class Alpha glutathione S-transferases.

Helix 9, the major structural element in the C-terminal region of class Alpha glutathione transferases, forms part of the active site of these enzymes where its dynamic properties modulate both catalytic and ligandin functions. A conserved aspartic acid N-capping motif for helix 9 was identified by sequence alignments of the C-terminal regions of class Alpha glutathione S-transferases (GSTs) and an analysis by the helix-coil algorithm AGADIR. The contribution of the N-capping motif to the stability and dynamics of the region was investigated by replacing the N-cap residue Asp-209 with a glycine in human glutathione S-transferase A1-1 (hGST A1-1) and in a peptide corresponding to its C-terminal region. Far-UV circular dichroism and AGADIR analyses indicate that, in the absence of tertiary interactions, the wild-type peptide displays a low intrinsic tendency to form a helix and that this tendency is reduced significantly by the Asp-to-Gly mutation. Disruption of the N-capping motif of helix 9 in hGST A1-1 alters the conformational dynamics of the C-terminal region and, consequently, the features of the H-site to which hydrophobic substrates (e.g. 1-chloro-2,4-dinitrobenzene (CDNB)) and nonsubstrates (e.g. 8-anilino-1-naphthalene sulfonate (ANS)) bind. Isothermal calorimetric and fluorescence data for complex formation between ANS and protein suggest that the D209G-induced perturbation in the C-terminal region prevents normal ligand-induced localization of the region at the active site, resulting in a less hydrophobic and more solvent-exposed H-site. Therefore, the catalytic efficiency of the enzyme with CDNB is diminished due to a lowered affinity for the electrophilic substrate and a lower stabilization of the transition state.