Glutathione S-Transferase Pi Has at Least Three Distinguishable Xenobiotic Substrate Sites Close to Its Glutathione-binding Site

Ralat, Luis A.; Colman, Roberta F.

doi:10.1074/jbc.m407445200

Cited by 46 publications

(43 citation statements)

References 44 publications

(57 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The results from SDS-PAGE confirmed the purity and identity of these enzymes: only a single band was observed for each enzyme, corresponding to the correct molecular mass of 23.5 kDa for the non-His tag wild-type and mutant class GSTs (data not shown). The total yield of GST purified by these methods was five to ten times higher than that obtained by the earlier procedures (16,17). The enzyme yield varies with the mutation; typically 100 mg/liter cell culture was obtained for the wild-type enzyme and 50 -100 mg/liter cell culture for the mutant enzymes.…”

Section: Expression and Purification Of Wild-type And Mutant Gst -mentioning

confidence: 94%

See 1 more Smart Citation

Catalytically Active Monomer of Glutathione S-Transferase π and Key Residues Involved in the Electrostatic Interaction between Subunits

Huang

Misquitta

Blond

et al. 2008

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Human glutathione transferase (GST ) has been crystallized as a homodimer, with a subunit molecular mass of ϳ23 kDa; however, in solution the average molecular mass depends on protein concentration, approaching that of monomer at <0.03 mg/ml, concentrations typically used to measure catalytic activity of the enzyme. Electrostatic interaction at the subunit interface greatly influences the dimer-monomer equilibrium of the enzyme and is an important force for holding subunits together. Arg-70, Arg-74, Asp-90, Asp-94, and Thr-67 were selected as target sites for mutagenesis, because they are at the subunit interface. R70Q, R74Q, D90N, D94N, and T67A mutant enzymes were constructed, expressed in Escherichia coli, and purified. The construct of N-terminal His tag enzyme facilitates the purification of GST , resulting in a high yield of enzyme, but does not alter the kinetic parameters or secondary structure of the enzyme. Our results indicate that these mutant enzymes show no appreciable changes in K m for 1-chloro-2,4-dinitrobenzene and have similar CD spectra to that of wild-type enzyme. However, elimination of the charges of either Arg-70, Arg-74, Asp-90, or Asp-94 shifts the dimer-monomer equilibrium toward monomer. In addition, replacement of Asp-94 or Arg-70 causes a large increase in the K m GSH , whereas substitution for Asp-90 or Arg-74 primarily results in a marked decrease in V max . The GST retains substantial catalytic activity as a monomer probably because the glutathione and electrophilic substrate sites (such as for 1-chloro-2,4-dinitrobenzene) are predominantly located within each subunit.

show abstract

Section: Expression and Purification Of Wild-type And Mutant Gst -mentioning

confidence: 94%

“…This enzyme was crystallized as a homodimer, with a subunit molecular mass of ϳ23 kDa. Each subunit contains an active site that consists of a glutathione (GSH) binding site (G site) (7, 8, 10 -12) and several xenobiotic substrate binding sites (H site) (7,8,(13)(14)(15)(16)(17)(18) adjacent to the G site.…”

mentioning

confidence: 99%

Catalytically Active Monomer of Glutathione S-Transferase π and Key Residues Involved in the Electrostatic Interaction between Subunits

Huang

Misquitta

Blond

et al. 2008

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…The purification was a modification of the previously published procedure. 44 Supernatants were then applied to a column prepacked with 60 mL S-hexylglutathione-Sepharose resin (SigmaAldrich), which was equilibrated in GSTpi Wash Buffer and washed with 1 L of GST Wash Buffer. Enzymes were eluted using GSTpi elution buffer (GSTpi wash buffer containing either 2.5 mM S-hexylglutathione or 25 mM reduced glutathione).…”

Section: Production Of Human Recombinant Gstpimentioning

confidence: 99%

GSTpi modulates JNK activity through a direct interaction with JNK substrate, ATF2

et al. 2011

Self Cite

View full text Add to dashboard Cite

Human GSTpi, an important detoxification enzyme, has been shown to modulate the activity of JNKs by inhibiting apoptosis and by causing cell proliferation and tumor growth. In this work, we describe a detailed analysis of the interaction in vitro between GSTpi and JNK isoforms (both in their inactive and active, phosphorylated forms). The ability of active JNK1 or JNK2 to phosphorylate their substrate, ATF2, is inhibited by two naturally occurring GSTpi haplotypes (Ile105/Ala114, WT or haplotype A, and Val105/Val114, haplotype C). Haplotype C of GSTpi is a more potent inhibitor of JNK activity than haplotype A, yielding 75-80% and 25-45% inhibition, respectively. We show that GSTpi is not a substrate of JNK, as was earlier suggested by others. Through binding studies, we demonstrate that the interaction between GSTpi and phosphorylated, active JNKs is isoform specific, with JNK1 being the preferred isoform. In contrast, GSTpi does not interact with unphosphorylated, inactive JNKs unless a JNK substrate, ATF2, is present. We also demonstrate, for the first time, a direct interaction: between GSTpi and ATF2. GSTpi binds with similar affinity to active JNK 1 ATF2 and to ATF2 alone. Direct binding experiments between ATF2 and GSTpi, either alone or in the presence of glutathione analogs or phosphorylated ATF2, indicate that the xenobiotic portion of the GSTpi active site and the JNK binding domain of ATF2 are involved in this interaction. Competition between GSTpi and active JNK for the substrate ATF2 may be responsible for the inhibition of JNK catalysis by GSTpi.

show abstract

“…For the expression of human GST pi, the WT plasmid was transformed into Escherichia coli JM105 and the cells were grown and induced for expression of GST pi [12]. For the expression of human 1-Cys Prx (Prdx 6), the WT plasmid was transformed in E. coli BL21…”

Section: Expression and Purification Of Gst Pi And 1-cys Prxmentioning

confidence: 99%

Characterization of the complex of glutathione S-transferase pi and 1-cysteine peroxiredoxin

Ralat¹,

Misquitta²,

Manevich³

et al. 2008

Archives of Biochemistry and Biophysics

Self Cite

View full text Add to dashboard Cite

Glutathione S-transferase pi has been shown to reactivate 1-cysteine peroxiredoxin (1-Cys Prx) by formation of a complex. A model of the complex was proposed based on the crystal structures of the two enzymes. We have now characterized the complex of GST pi/1-Cys Prx by determining the M w of the complex, by measuring the catalytic activity of the GST pi monomer, and by identifying the interaction sites between GST pi and 1-Cys Prx. The M w of the purified GST pi/1-Cys Prx complex is 50,200 at pH 8.0 in the presence of 2.5 mM glutathione, as measured by light scattering, providing direct evidence that the active complex is a heterodimer composed of equimolar amounts of the two proteins. In the presence of 4 M KBr, GST pi is dissociated to monomer and retains catalytic activity, but the K m value for GSH is increased substantially. To identify the peptides of GST pi that interact with 1-Cys Prx, GST pi was digested with V8 protease and the peptides were purified. The binding by 1-Cys Prx of each of four pure GST pi peptides (residues 41-85, 115-124, 131-163, and 164-197) was investigated by protein fluorescence titration. An apparent stoichiometry of 1 mol/subunit 1-Cys Prx was measured for each peptide and the formation of the heterodimer is decreased when these peptides are included in the incubation mixture. These results support our proposed model of the heterodimer. KeywordsGlutathione S-transferase pi; 1-Cys peroxiredoxin; Heterodimer Glutathione S-transferases (GSTs 1 ), which catalyze the nucleophilic attack by the thiol of glutathione on electrophilic substrates, constitute a family of enzymes important in the detoxification of xenobiotics, endogenous compounds, and the products of oxidative stress [1,2]. The pi isozyme (GST pi), crystallized as a homodimer with a subunit molecular * Corresponding author. Fax: +1 302 831 6335. rfcolman@chem.udel.edu (R.F. Colman). 1 Abbreviations used: GST, glutathione S-transferase; GST pi, pi-class glutathione S-transferase; 1-Cys Prx, 1-Cysteine peroxiredoxin; Ni-NTA, nickel-nitriloacetic acid agarose; TRIS, tris (hydroxymethyl) aminomethane; EDTA, disodium ethylenediamine tetraacetate; MES, 2-(N-morpholino) ethane-sulfonate; WT, wild-type; GSH, glutathione; CDNB, 1-chloro-2,4-dinitrobenzene; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript weight of 23,500, is of particular interest because it exhibits diverse roles in mammalian cells: it provides a defense against carcinogenesis, since it catalyzes the inactivation of known carcinogens [3]; it contributes significantly to the development of resistance to cancer chemotherapy, since GST pi levels increase in tumors and the enzyme metabolizes key anticancer drugs [4][5][6][7]; and it promotes the cellular response to oxidative stress, since GST pi has recently been reported to activate the anti-oxidant enzyme 1-Cys peroxiredoxin [8,9].1-Cys peroxiredoxin (1-Cys Prx, Prdx 6, Prx VI, and AOP2), a homodime...

show abstract

Glutathione S-Transferase Pi Has at Least Three Distinguishable Xenobiotic Substrate Sites Close to Its Glutathione-binding Site

Cited by 46 publications

References 44 publications

Catalytically Active Monomer of Glutathione S-Transferase π and Key Residues Involved in the Electrostatic Interaction between Subunits

Catalytically Active Monomer of Glutathione S-Transferase π and Key Residues Involved in the Electrostatic Interaction between Subunits

GSTpi modulates JNK activity through a direct interaction with JNK substrate, ATF2

Characterization of the complex of glutathione S-transferase pi and 1-cysteine peroxiredoxin

Contact Info

Product

Resources

About