Structural analysis of human alpha-class glutathione transferase A1-1 in the apo-form and in complexes with ethacrynic acid and its glutathione conjugate

Cameron, Alexander D.; Sinning, Irmgard; L’Hermite, Guillaume; Olin, B.; Board, Philip G.; Mannervik, Bengt; Jones, T. Alwyn

doi:10.1016/s0969-2126(01)00206-4

Cited by 174 publications

(277 citation statements)

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“…5, lane A) or whole embryo (Fig. 5, lane B) purified using the GSH affinity column were within the 22-29 kDa range; this is consistent with the range of molecular masses reported for avian GSTs (24-30 kDa) (Bardai et al, 2006;Cameron et al, 1995;Hsieh et al, 1999). Thus, the enzymes purified from whole embryo and embryonic eye using the GSH affinity column are GST-type enzymes.…”

Section: Enzyme Purification Of Quail Embryo Gstssupporting

confidence: 83%

“…Enzyme-catalyzed GTN biotransformation assays for in vitro embryo homogenates were run in triplicate using 6-mL glass septum-sealed vials, flushed with argon to create anaerobic conditions, with constant agitation in the dark at 37 °C for 2 h. Each 1 mL sample contained: GTN (30 µmol), cytosol (5 mg protein) or enzyme preparation (0.50 mg protein), GSH (100 µM), enzyme inhibitor, either ethacrynic acid (200 µM), an &,'-unsaturated ketone that inhibits the enzyme by binding to the cysteinyl residue in the active site (Cameron et al, 1995;Bryant et al, 2011), or diphenyliodonium chloride (100 µM), an inhibitor of flavoenzymes (Bhushan et al, 2003) and Buffer A. Controls were prepared by omitting enzyme or GSH from the reaction mixture.…”

Section: Analysis Of Enzyme-catalyzed Gtn Metabolismmentioning

confidence: 99%

“…4). Ethacrynic acid (EA), a GST inhibitor (Cameron et al, 1995;Tew et al, 1997), was used as a biochemical tool to determine if in the metabolism of GTN. It should be noted that the studies described above used crude whole cytosol containing soluble proteins and co-factors.…”

Section: Enzyme-catalyzed Gtn Biotransformationmentioning

confidence: 99%

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Glyceryl trinitrate metabolism in the quail embryo by the glutathione S-transferases leads to a perturbation in redox status and embryotoxicity

Bardai

Hales

Sunahara

2013

Comparative Biochemistry and Physiology Part B: Biochemistry an

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/npsi/ctrl?lang=en http://nparc.cisti-icist.nrc-cnrc.gc.ca/npsi/ctrl?lang=fr Access and use of this website and the material on it are subject to the Terms and Conditions set forth at http://nparc.cisti-icist.nrc-cnrc.gc.ca/npsi/jsp/nparc_cp.jsp?lang=en NRC Publications Archive Archives des publications du CNRCThis publication could be one of several versions: author's original, accepted manuscript or the publisher's version. / La version de cette publication peut être l'une des suivantes : la version prépublication de l'auteur, la version acceptée du manuscrit ou la version de l'éditeur. For the publisher's version, please access the DOI link below./ Pour consulter la version de l'éditeur, utilisez le lien DOI ci-dessous.http://dx.doi.org/10. 1016/j.cbpb.2013.04.001 Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 165, 3, pp. 153-164, 2013-04-12 Glyceryl trinitrate metabolism in the quail embryo by the glutathione Stransferases leads to a perturbation in redox status and embryotoxicity Bardai, Ghalib K.; Hales, Barbara F.; Sunahara, Geoffrey I. induces malformations that were associated in previous studies with an increase in protein nitration.Increased nitration suggests metabolism of GTN by the embryo. The goals of this study were to characterize the enzymes and co-factors required for GTN metabolism by quail embryos, and to determine the effects of in ovo treatment with N-acetyl cysteine (NAC), a precursor of glutathione (GSH), on GTN embryotoxicity. GTN treatment of quail embryo resulted in an increase in nitrite, a 2 decrease in total GSH, and an increase in the ratio of NADP+/NADPH, indicating that redox balance may be compromised in exposed embryos. Glutathione S-transferases (GSTs; EC 2.5.1.18) purified from the whole embryo (K m 0.84 mM; V max 36 µM/min) and the embryonic eye (K m 0.20 mM; V max 30 µM/min) had GTN-metabolizing activity (1436 and 34 nmol/min/mg, respectively); the addition of ethacrynic acid, an inhibitor of GST activity, decreased GTN metabolism. Peptide sequencing of the GST isozymes indicated that alpha-or mu-type GSTs in the embryo and embryonic eye had GTN metabolizing activity. NAC co-treatment partially protected against the effects of GTN exposure. Thus, GTN denitration by quail embryo GSTs may represent a key initial step in the developmental toxicity of GTN.

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Section: Enzyme Purification Of Quail Embryo Gstssupporting

confidence: 83%

Section: Analysis Of Enzyme-catalyzed Gtn Metabolismmentioning

confidence: 99%

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Glyceryl trinitrate metabolism in the quail embryo by the glutathione S-transferases leads to a perturbation in redox status and embryotoxicity

Bardai

Hales

Sunahara

2013

Comparative Biochemistry and Physiology Part B: Biochemistry an

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“…The binding of BSP to the human GST A1-1 was monitored by the quenching of the intrinsic protein fluorescence (excitation at 280 nm or 295 nm, emission 325 nm) and by the quenching of the AEDANS label covalently attached to Cys111. Cys111 is located in a short turn between helices A4 and A5 [23,24], and alkylation of this residue with IAEDANS does not alter the specific activity of the enzyme (with respect to CDNB as the electrophilic substrate) or the conformational stability of the enzyme [25]. It does however alter the affinity of the protein for the anionic dye ANS in that the dyes dissociation constant for the enzyme increases from 15 µM to 40 µM.…”

Section: Resultsmentioning

confidence: 99%

Aflatoxin B1 and sulphobromophthalein binding to the dimeric human glutathione S‐transferase A1‐1 : a fluorescence spectroscopic analysis

Sluis‐Cremer

Wallace

Burke

et al. 1998

European Journal of Biochemistry

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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...

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“…The structures of unliganded human GSTAI-1 and GST from !j 1998 International Union of Crystallography Printed in Great Britain -all rights reserved Sch&tosoma japonica have been published recently (Cameron et al, 1995;McTigue et al, 1995). For the mu class, only the liganded structures of the rat GST3-3 (currently named the rGSTMI-1) (Ji et al, 1992;: Xiao et al, 1996 and a mutant form of human hGSTM2-2 (F214W) (Raghunathan et al, 1994) have been studied.…”

Section: Introductionmentioning

confidence: 99%

Expression, crystallization and preliminary X-ray analysis of ligand-free human glutathione S-transferase M2–2

Patskovska

Fedorov

Patskovsky

et al. 1998

Acta Crystallogr D Biol Cryst

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Human glutathione-S-transferase M2-2 (hGSTM2-2) was expressed in Escherichia coli and purified by GSH-affinity chromatography. The recombinant enzyme and thc protein isolated from human tissue were indistinguishablc based on physicochemical, enzymatic and immunological criteria. The catalytically active dimeric hGSTM2-2 was crystallized without GSH or other active-site ligands in two crystal forms. Diffraction from form A crystals extends to 2.5 A and is consistent with the °space group P2~ (a =-53.9, b = 81.5, c = 55.6 ,~, /3 = 109.26 A) with two monomers in the asymmetric unit. Diffraction from form B crystals extends to 3 A and is consistent with a space group P21212~ (a = 57.2, b = 80.7, c = 225.9 A) with two dimers in the asymmetric unit. This is the first report of ligand-free mu-class GST crystals, and a comparison with liganded complexes will provide insight into thc structural conscquences of substrate binding which are thought to be important for catalysis.

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Structural analysis of human alpha-class glutathione transferase A1-1 in the apo-form and in complexes with ethacrynic acid and its glutathione conjugate

Cited by 174 publications

References 39 publications

Glyceryl trinitrate metabolism in the quail embryo by the glutathione S-transferases leads to a perturbation in redox status and embryotoxicity

Glyceryl trinitrate metabolism in the quail embryo by the glutathione S-transferases leads to a perturbation in redox status and embryotoxicity

Aflatoxin B1 and sulphobromophthalein binding to the dimeric human glutathione S‐transferase A1‐1 : a fluorescence spectroscopic analysis

Expression, crystallization and preliminary X-ray analysis of ligand-free human glutathione S-transferase M2–2

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