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J. Biol. Chem., Vol. 280, Issue 20, 19480-19487, May 20, 2005

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A Conserved N-capping Motif Contributes Significantly to the Stabilization and Dynamics of the C-terminal Region of Class Alpha Glutathione S-Transferases^*

Heini W. Dirr, Tessa Little, Diane C. Kuhnert, and Yasien Sayed

From the Protein Structure-Function Research Programme, School of Molecular and Cell Biology, University of the Witwatersrand, Jan Smuts Ave., Johannesburg 2050, South Africa

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.

Received for publication, December 3, 2004 , and in revised form, March 8, 2005.

^* This work was supported by the University of the Witwatersrand, the South African National Research Foundation (Grant 205359), and the Wellcome Trust (Grant 060799). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Tel.: 27-11-717-6352; Fax: 27-11-717-6351; E-mail: heinid{at}gecko.biol.wits.ac.za.

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