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.

The binding interactions between dimeric human class alpha glutathione S-transferase A1-1 (GST A1-1) and aflatoxin B1 or sulphobromophthalein (BSP) were characterised. Aflatoxin B1 binds to GST A1-1 with a stoichiometry of 1.1 mol/mol of dimeric enzyme. The binding interaction, which can be described by a hyperbolic saturation isotherm (K d ϭ 8Ϯ 2 µM), does not induce major structural changes in the enzyme, nor does it inhibit enzymatic activity. The average distance between the single tryptophan residue (Trp20) of GST A1-1 and protein-bound aflatoxin B1 was calculated to be 22.7 Å by means of fluorescence resonance energy transfer. The aflatoxin-binding region, according to this calculated distance, was determined to be located in the dimer interface cleft near the crystallographic two-fold axis. Hill-plot analyses suggest that a positive co-operative interaction exists between BSP and the dimeric GST A1-1 (h ϭ 1.6 Ϯ0.1 ; K′ ϭ 14Ϯ0.6 µM). The binding of BSP induces a conformational change in the enzyme which is accompanied by a decrease in the molecular flexibility and in the solvent-accessible properties of the enzyme's Trp20 residue. Site-directed mutagenesis of Trp20 (Trp20→Phe) confirms that this residue is situated in the binding environment and although it is not essential for BSP binding, it is involved in the interaction. Furthermore, the structural change associated with BSP binding alters the hyperbolic character of the glutathione saturation curve. This indicates that there may also be a cooperative interaction between glutathione and BSP or that BSP binding induces asymmetric functioning of the two enzyme subunits so that they become unequal in catalytic activity.Keywords : glutathione S-transferase ; aflatoxin B1; sulphobromophthalein; co-operative binding.Glutathione S-transferases (GSTs) are a family of multifunctional enzymes found in all vertebrates, plants, insects, nematodes, yeast and aerobic bacteria. They constitute a complex supergene family that collectively metabolises a broad variety of potentially toxic xenobiotics and reactive endogenous compounds resulting from oxidative metabolism. GSTs are an integral part of the phase-I detoxification mechanism and primarily catalyse the nucleophilic addition of the thiol of reduced glutathione (γ-glutamyl-cysteinyl-glycine) to electrophilic centres in a wide variety of hydrophobic electrophiles, including alkyl and aryl halides, epoxides, quinones and activated alkenes [1]. The dimeric cytosolic GSTs exhibit multiple enzyme forms that, according to primary structure and molecular recognition, can be grouped into one of six species independent gene classes (alpha, kappa, mu, pi, theta and sigma). In addition to their catalytic activities, GSTs also function as ligand binding proteins and thereby facilitate the intracellular storage of a variety of hydrophobic non-substrate compounds, including hormones, metabolites and drugs [2]. The binding of these compounds to GSTs prevents the build up of apolar molecules at lipophilic sites such as membranes...

Canonical glutathione (GSH) transferases are dimeric proteins with subunits composed of an N-terminal GSH binding region (domain 1) and a C-terminal helical region (domain 2). The stabilities of several GSH transferase dimers are dependent upon two groups of interactions between domains 1 and 2 of opposing subunits: a hydrophobic ball-and-socket motif and a buried charge cluster motif. In rGSTM1-1, these motifs involve residues F56 and R81, respectively. The structural basis for the effects of mutating F56 to different residues on dimer stability and function has been reported (Codreanu et al. (2005) Biochemistry 44, 10605-10612). Here, we show that the simultaneous disruption of both motifs in the F56S/R81A mutant causes complete dissociation of the dimer to a monomeric protein on the basis of gel filtration chromatography and multiple-angle laser light scattering. The fluorescence and far-UV CD properties of the double mutant as well as the kinetics of amide H/D exchange along the polypeptide backbone suggest that the monomer has a globular structure that is similar to a single subunit in the native protein. However, the mutant monomer has severely impaired catalytic activity, suggesting that the dimer interface is vital for efficient catalysis. Backbone amide H/D exchange kinetics in the F56S and F56S/R81A mutants indicate that a reorganization of the loop structure between helix alpha2 and strand beta3 near the active site is responsible for the decreased catalytic activity of the monomer. In addition, the junction between the alpha4 and alpha5 helices in F56S/R81R shows decreased H/D exchange, indicating another structural change that may affect catalysis. Although the native subunit interface is important for dimer stability, urea-induced unfolding of the F56S/R81A mutant suggests that the interface is not essential for the thermodynamic stability of individual subunits. The H/D exchange data reveal a possible molecular basis for the folding cooperativity observed between domains 1 and 2.

Cytosolic class pi glutathione transferase P1-1 (GSTP1-1) is associated with drug resistance and proliferative pathways because of its catalytic detoxification properties and ability to bind and regulate protein kinases. The native wild-type protein is homodimeric, and whereas the dimeric structure is required for catalytic functionality, a monomeric and not dimeric form of class pi GST is reported to mediate its interaction with and inhibit the activity of the pro-apoptotic enzyme c-Jun N-terminal kinase (JNK) [Adler, V., et al. (1999) EMBO J. 18, 1321-1334]. Thus, the existence of a stable monomeric form of wild-type class pi GST appears to have physiological relevance. However, there are conflicting accounts of the subunit's intrinsic stability since it has been reported to be either unstable [Dirr, H., and Reinemer, P. (1991) Biochem. Biophys. Res. Commun. 180, 294-300] or stable [Aceto, A., et al. (1992) Biochem. J. 285, 241-245]. In this study, the conformational stability of GSTP1-1 was re-examined by equilibrium folding and unfolding kinetics experiments. The data do not demonstrate the existence of a stable monomer but that unfolding of hGSTP1-1 proceeds via an inactive, nativelike dimeric intermediate in which the highly dynamic helix 2 is unfolded. Furthermore, molecular modeling results indicate that a dimeric GSTP1-1 can bind JNK. According to the available evidence with regard to the stability of the monomeric and dimeric forms of GSTP1-1 and the modality of the GST-JNK interaction, formation of a complex between GSTP1-1 and JNK most likely involves the dimeric form of the GST and not its monomer as is commonly reported.