Thermodynamics of the ligandin function of human class Alpha glutathione transferase A1-1: energetics of organic anion ligand binding

Sayed, Yasien; Hornby, Judith A. T.; Lopez, Marimar; Dirr, Heini W.

doi:10.1042/bj3630341

Cited by 31 publications

(45 citation statements)

References 42 publications

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“…The binding curve fits well to a model describing one binding site per subunit with a stoichiometry of one ANS molecule per site consistent with that reported for the wild-type protein (18). The affinity of hGST A1-1 for ANS is not significantly affected by the D209G mutation (K d ϭ 52 M) when compared with that of the wild-type protein (K d ϭ 65 M) (18) and the I219A mutant (K d ϭ 47 M) (17).…”

Section: Asp-209 the N-cap Residue Of Helix 9 -supporting

confidence: 53%

“…The affinity of hGST A1-1 for ANS is not significantly affected by the D209G mutation (K d ϭ 52 M) when compared with that of the wild-type protein (K d ϭ 65 M) (18) and the I219A mutant (K d ϭ 47 M) (17). ANS does, however, bind more tightly to these enzymes forms than to the Phe-222 deletion mutant (K d ϭ 105 M) (18), indicating the importance of this residue in the binding of ligands at the H-site.…”

Section: Asp-209 the N-cap Residue Of Helix 9 -mentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

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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

Dirr

Little

Kuhnert

et al. 2005

Journal of Biological Chemistry

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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.

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Section: Asp-209 the N-cap Residue Of Helix 9 -supporting

confidence: 53%

Section: Asp-209 the N-cap Residue Of Helix 9 -mentioning

confidence: 99%

mentioning

confidence: 99%

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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

Dirr

Little

Kuhnert

et al. 2005

Journal of Biological Chemistry

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“…The isoenzyme hGSTA1-1 exhibits wide ligand-binding specificities since the size, shape and high flexibility of the H-site make it suitable for accommodating a wide variety of large hydrophobic ligands [7,8]. Neither dieldrin nor spiromesifen bears any strong structural resemblance to the substrates/inhibitors found in the crystal structures of the enzyme available in PDB (Fig.…”

Section: Delineation Of the Hgsta1-1 Pesticide Binding Site By Kinetimentioning

confidence: 99%

“…GSTs also exhibit a ligand binding ('ligandin') function that can facilitate the binding of numerous hydrophobic and amphiphatic compounds in a nonsubstrate manner into a distinct site (this site is termed L-site). Binding of such ligands results in the inhibition of GST catalytic activity [7][8][9][10][11].…”

Section: Introductionmentioning

confidence: 99%

Inhibition of human glutathione transferases by pesticides: Development of a simple analytical assay for the quantification of pesticides in water

Chronopoulou

Papageorgiou

Markoglou

et al. 2012

Journal of Molecular Catalysis B: Enzymatic

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a b s t r a c tGlutathione transferases (GSTs; EC 2.5.1.18) form a group of multifunctional enzymes that are involved in phase II cellular detoxification mechanism. Here, screening of the inhibition potency of a wide range of pesticides toward selected human GST isoenzymes (hGSTA1-1, hGSTP1-1, hGSTT2-2 and hGSTO1-1) was carried out. hGSTA1-1 was found more susceptible to inhibition by pesticides than other isoenzymes. The insecticides dieldrin and spiromesifen were identified as potent reversible inhibitors toward hGSTA1-1 with IC 50 values equal to 17.9 ± 1.7 M and 12.1 ± 3.4 M, respectively. Based on in silico docking analysis and kinetic inhibition studies it was concluded that dieldrin and spiromesifen bind specifically to the enzyme presumably at a distinct position that partially overlaps with both the G-and H-site. The ability of dieldrin and spiromesifen to inhibit hGSTA1-1 activity was exploited for the development of analytical quantification assays for these two pesticides. Linear calibration curves were obtained for dieldrin and spiromesifen, with useful concentration in the range of 0-10 M. The reproducibility of the assay response, expressed by relative standard deviation, was in the order of 4.1% (N = 28). The method was successfully applied to the determination of these pesticides in real water samples without sample preparation steps.

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A thermodynamic characterization of the interaction of 8‐anilino‐1‐naphthalenesulfonic acid with native globular proteins: the effect of the ligand dimerization in the analysis of the binding isotherms

Andújar‐Sánchez

Jara‐Pérez

Cobos

et al. 2011

J of Molecular Recognition

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8-Anilino-1-naphthalenesulfonic acid (ANS) is a popular fluorescence probe, broadly used for the analysis of proteins, but the nature of its interaction with proteins and the high increase in the fluorescence intensity that takes place upon such process are still unclear. In the last few years, isothermal titration calorimetry has been used to characterize the nature of the interaction of this dye with proteins. The analysis of the binding isotherms of these studies has not considered the dimerization equilibrium of ANS, which is pH dependent, and it can result in serious errors in the data analysis. In the present work we have developed a suitable data analysis by which this process is taken into account. To study the binding of the dye to proteins at different pH values, we have used the Abl-SH3 domain. Our results suggest that at pH 3 and 5, where the dimerization of the ANS is important, electrostatic interactions are significant for the binding of ANS to the Abl-SH3 domain. However, at pH 7, ANS behaves mostly as monomer and the interaction with the protein is mainly hydrophobic. The pH dependent behavior of the ANS binding to proteins can be explained in terms of ionization states of both, the protein and the ANS.

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Thermodynamics of the ligandin function of human class Alpha glutathione transferase A1-1: energetics of organic anion ligand binding

Cited by 31 publications

References 42 publications

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

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

Inhibition of human glutathione transferases by pesticides: Development of a simple analytical assay for the quantification of pesticides in water

A thermodynamic characterization of the interaction of 8‐anilino‐1‐naphthalenesulfonic acid with native globular proteins: the effect of the ligand dimerization in the analysis of the binding isotherms

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