Residue 219 Impacts on the Dynamics of the C-Terminal Region in Glutathione Transferase A1-1:  Implications for Stability and Catalytic and Ligandin Functions

Abstract

The C-terminal region in class alpha glutathione transferases (GSTs) modulates the catalytic and nonsubstrate ligand binding functions of these enzymes. Except for mouse GST A1-1 (mGST A1-1), the structures of class alpha GSTs have a bulky aliphatic side chain topologically equivalent to Ile219 in human GST A1-1 (hGST A1-1). In mGST A1-1, the corresponding residue is an alanine. To investigate the role of Ile219 in determining the conformational dynamics of the C-terminal region in hGST A1-1, the residue was replaced by alanine. The substitution had no effect on the global structure of hGST A1-1 but did reduce the conformational stability of the C-terminal region of the protein. This region could be stabilized by ligands bound at the active site. The catalytic behavior of hGST A1-1 was significantly compromised by the I219A mutation as demonstrated by reduced enzyme activity, increased K(m) for the substrates glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB), and reduced catalytic efficiencies. Inhibition studies also indicated that the binding affinities for product and substrate analogues were dramatically decreased. The affinity of the mutant for GSH was, however, only slightly increased, indicating that the G-site was unaltered by the mutation. The binding affinity and stoichiometry for the anionic dye 8-anilino-1-naphthalene sulfonate (ANS) was also not significantly affected by the I219A mutation. However, the lower DeltaC(p) for ANS binding to the mutant (-0.34 kJ/mol per K compared with -0.84 kJ/mol per K for the wild-type protein) suggests that ANS binding to the mutant results in the burial of less hydrophobic surface area. Fluorescence data also indicates that ANS bound to the mutant is more prone to quenching by water. Overall, the data from this study, together with the structural details of the C-terminal region in mGST A1-1, show that Ile219 is an important structural determinant of the stability and dynamics of the C-terminal region of hGST A1-1.

Molecular Basis for the Selectivity of the Mammalian Bombesin Peptide, Neuromedin B, for Its Receptor

“…There is almost no information on the molecular mechanisms that determine the importance of the presence of an isoleucine in the different Gprotein-coupled receptors for high-affinity ligand interaction. Isoleucine is particularly important for hydrophobic interactions, which could contribute either to receptor protein folding and/or conformational receptor change or to receptorligand interaction (Mosebi et al, 2003). In NMBR, the isoleucine 199 is located adjacent to Cys 198 in EC3, which is presumed to form a disulfide bridge with Cys 116 in EC2, which is important in determining EC3 conformation.…”

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

“…It is only when the active site becomes occupied by ligand that the C-terminal region becomes stabilized and localized on the surface of the protein (5-7, 16, 19, 44). Tertiary interactions at the interfaces between the C-terminal region, protein, and bound ligand, therefore, play a major role in stabilizing the region (16,17), consistent with the helix-stabilizing effect of TFE. Disrupting the conserved N-capping motif of helix 9 by the D209G mutation substantially reduces the intrinsic ability of its sequence to form a helix.…”

“…hydrophobic residue at position 219 contributes significantly toward the stability of the region in apo and complexed hGST A1-1 (17).…”

“…Active Site Tyrosinate Formation-The ionization of the catalytic Tyr 9 in the active site of hGST A1-1 was determined over the pH range 5.5-9.0 by UV absorbance difference spectroscopy as described (17). The concentration of tyrosinate ion formation was calculated using a molar extinction coefficient of 2350 M Ϫ1 ⅐cm Ϫ1 and the pK a of Tyr 9 determined as described (15,33).…”

“…Isothermal Titration Calorimetry-Isothermal titration calorimetry with a VP-ITC calorimeter from MicroCal (Northampton, MA) was employed to determine the thermodynamic parameters of ANS⅐hGST A1-1 complex formation in 20 mM phosphate buffer, pH 6.5, as described (17,18). Total heats were obtained by titrating 0.04 mM D209G hGST A1-1 (subunit concentration) with 5-l aliquots of 4.1 mM ANS.…”