Identification of the nonsubstrate steroid binding site of rat liver glutathione s-transferase, isozyme 1-1, by the steroid affinity label, 3β- (Iodoacetoxy)dehydroisoandrosterone

Joseph J. Barycki, Roberta F. Colman

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Abstract

3β-(Iodoacetoxy) dehydroisoandrosterone (3β-IDA), an analogue of the electrophilic substrate, Δ5-androstene-3,17-dione, as well as an analogue of several other steroid inhibitors of glutathione S-transferase, was tested as an affinity label of rat liver glutathione S-transferase, isozyme 1-1. A time-dependent loss of enzyme activity is observed upon incubation of 3β- IDA with the enzyme. The rate of enzyme inactivation exhibits a nonlinear dependence on 3β-IDA concentration, yielding an apparent K(i) of 21 μM. Upon complete inactivation of the enzyme, a reagent incorporation of approximately 1 mol/mol of enzyme subunit or 2 mol/mol of enzyme dimer is observed. Protection against inactivation and incorporation is afforded by alkyl glutathione derivatives and nonsubstrate steroid ligands such as 17β- estradiol-3,17-disulfate but, surprisingly, not by Δ5-androstene-3,17- dione or any other electrophilic substrate analogues tested. These results suggest that the site of reaction is within the nonsubstrate steroid binding site of the enzyme, which is distinguishable from the electrophilic substrate binding site, near the active site of the enzyme. Two cysteine residues, Cys17 and Cys111, are modified in nearly equal amounts, despite an average reagent incorporation of 1 mol/mol enzyme subunit. Isolation of enzyme sub-units indicates the presence of unmodified, singly labeled, and doubly labeled subunits, consistent with mutually exclusive modification of cysteine residues across enzyme subunits; i.e., modification of Cys111 on subunit A prevents modification of Cys111 on subunit B and similarly for Cys17. Molecular modeling analysis suggests that Cys17 and Cys111 are located in the non substrate steroid binding site, within the cleft between the subunits of the dimeric enzyme.

Original languageEnglish (US)
Pages (from-to)16-31
Number of pages16
JournalArchives of Biochemistry and Biophysics
Volume345
Issue number1
DOIs
StatePublished - Sep 1 1997

Fingerprint

Affinity Labels
Transferases
Glutathione Transferase
Liver
Isoenzymes
Glutathione
Rats
Steroids
Binding Sites
Enzymes
Substrates
Cysteine
3-(iodoacetoxy)dehydroisoandrosterone
Molecular modeling
Enzyme activity
Dimers

Keywords

  • 17β-estradiol- 3,17-disulfate
  • Affinity labeling
  • Glutathione S.transferase
  • Steroids

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

Cite this

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title = "Identification of the nonsubstrate steroid binding site of rat liver glutathione s-transferase, isozyme 1-1, by the steroid affinity label, 3β- (Iodoacetoxy)dehydroisoandrosterone",
abstract = "3β-(Iodoacetoxy) dehydroisoandrosterone (3β-IDA), an analogue of the electrophilic substrate, Δ5-androstene-3,17-dione, as well as an analogue of several other steroid inhibitors of glutathione S-transferase, was tested as an affinity label of rat liver glutathione S-transferase, isozyme 1-1. A time-dependent loss of enzyme activity is observed upon incubation of 3β- IDA with the enzyme. The rate of enzyme inactivation exhibits a nonlinear dependence on 3β-IDA concentration, yielding an apparent K(i) of 21 μM. Upon complete inactivation of the enzyme, a reagent incorporation of approximately 1 mol/mol of enzyme subunit or 2 mol/mol of enzyme dimer is observed. Protection against inactivation and incorporation is afforded by alkyl glutathione derivatives and nonsubstrate steroid ligands such as 17β- estradiol-3,17-disulfate but, surprisingly, not by Δ5-androstene-3,17- dione or any other electrophilic substrate analogues tested. These results suggest that the site of reaction is within the nonsubstrate steroid binding site of the enzyme, which is distinguishable from the electrophilic substrate binding site, near the active site of the enzyme. Two cysteine residues, Cys17 and Cys111, are modified in nearly equal amounts, despite an average reagent incorporation of 1 mol/mol enzyme subunit. Isolation of enzyme sub-units indicates the presence of unmodified, singly labeled, and doubly labeled subunits, consistent with mutually exclusive modification of cysteine residues across enzyme subunits; i.e., modification of Cys111 on subunit A prevents modification of Cys111 on subunit B and similarly for Cys17. Molecular modeling analysis suggests that Cys17 and Cys111 are located in the non substrate steroid binding site, within the cleft between the subunits of the dimeric enzyme.",
keywords = "17β-estradiol- 3,17-disulfate, Affinity labeling, Glutathione S.transferase, Steroids",
author = "Barycki, {Joseph J.} and Colman, {Roberta F.}",
year = "1997",
month = "9",
day = "1",
doi = "10.1006/abbi.1997.0244",
language = "English (US)",
volume = "345",
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TY - JOUR

T1 - Identification of the nonsubstrate steroid binding site of rat liver glutathione s-transferase, isozyme 1-1, by the steroid affinity label, 3β- (Iodoacetoxy)dehydroisoandrosterone

AU - Barycki, Joseph J.

AU - Colman, Roberta F.

PY - 1997/9/1

Y1 - 1997/9/1

N2 - 3β-(Iodoacetoxy) dehydroisoandrosterone (3β-IDA), an analogue of the electrophilic substrate, Δ5-androstene-3,17-dione, as well as an analogue of several other steroid inhibitors of glutathione S-transferase, was tested as an affinity label of rat liver glutathione S-transferase, isozyme 1-1. A time-dependent loss of enzyme activity is observed upon incubation of 3β- IDA with the enzyme. The rate of enzyme inactivation exhibits a nonlinear dependence on 3β-IDA concentration, yielding an apparent K(i) of 21 μM. Upon complete inactivation of the enzyme, a reagent incorporation of approximately 1 mol/mol of enzyme subunit or 2 mol/mol of enzyme dimer is observed. Protection against inactivation and incorporation is afforded by alkyl glutathione derivatives and nonsubstrate steroid ligands such as 17β- estradiol-3,17-disulfate but, surprisingly, not by Δ5-androstene-3,17- dione or any other electrophilic substrate analogues tested. These results suggest that the site of reaction is within the nonsubstrate steroid binding site of the enzyme, which is distinguishable from the electrophilic substrate binding site, near the active site of the enzyme. Two cysteine residues, Cys17 and Cys111, are modified in nearly equal amounts, despite an average reagent incorporation of 1 mol/mol enzyme subunit. Isolation of enzyme sub-units indicates the presence of unmodified, singly labeled, and doubly labeled subunits, consistent with mutually exclusive modification of cysteine residues across enzyme subunits; i.e., modification of Cys111 on subunit A prevents modification of Cys111 on subunit B and similarly for Cys17. Molecular modeling analysis suggests that Cys17 and Cys111 are located in the non substrate steroid binding site, within the cleft between the subunits of the dimeric enzyme.

AB - 3β-(Iodoacetoxy) dehydroisoandrosterone (3β-IDA), an analogue of the electrophilic substrate, Δ5-androstene-3,17-dione, as well as an analogue of several other steroid inhibitors of glutathione S-transferase, was tested as an affinity label of rat liver glutathione S-transferase, isozyme 1-1. A time-dependent loss of enzyme activity is observed upon incubation of 3β- IDA with the enzyme. The rate of enzyme inactivation exhibits a nonlinear dependence on 3β-IDA concentration, yielding an apparent K(i) of 21 μM. Upon complete inactivation of the enzyme, a reagent incorporation of approximately 1 mol/mol of enzyme subunit or 2 mol/mol of enzyme dimer is observed. Protection against inactivation and incorporation is afforded by alkyl glutathione derivatives and nonsubstrate steroid ligands such as 17β- estradiol-3,17-disulfate but, surprisingly, not by Δ5-androstene-3,17- dione or any other electrophilic substrate analogues tested. These results suggest that the site of reaction is within the nonsubstrate steroid binding site of the enzyme, which is distinguishable from the electrophilic substrate binding site, near the active site of the enzyme. Two cysteine residues, Cys17 and Cys111, are modified in nearly equal amounts, despite an average reagent incorporation of 1 mol/mol enzyme subunit. Isolation of enzyme sub-units indicates the presence of unmodified, singly labeled, and doubly labeled subunits, consistent with mutually exclusive modification of cysteine residues across enzyme subunits; i.e., modification of Cys111 on subunit A prevents modification of Cys111 on subunit B and similarly for Cys17. Molecular modeling analysis suggests that Cys17 and Cys111 are located in the non substrate steroid binding site, within the cleft between the subunits of the dimeric enzyme.

KW - 17β-estradiol- 3,17-disulfate

KW - Affinity labeling

KW - Glutathione S.transferase

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