Affinity Labeling of Glutathione S-Transferase, Isozyme 4-4, by 4-(Fluorosulfonyl)benzoic Acid Reveals Tyr115 To Be an Important Determinant of Xenobiotic Substrate Specificity

Joseph J. Barycki, Roberta F. Colman

Research output: Contribution to journalArticle

34 Citations (Scopus)

Abstract

Incubation of 4-(fluorosulfonyl)benzoic acid (4-FSB), a xenobiotic substrate analogue, with the 4-4 isozyme of rat liver glutathione S-transferase at pH 7.5 and 25°C results in a time-dependent inactivation of the enzyme. The rate of inactivation exhibits a nonlinear dependence on 4-FSB concentration from 0.50 to 7.85 mM, with kmax = 0.082 min−1 and a of 1.95 mM. Nearly 1 mol of reagent/mol of enzyme subunit is incorporated when the enzyme is maximally inactivated. Protection against incorporation and inactivation is provided by bromosulfophthalein, a competitive inhibitor with respect to the hydrophobic substrate, 1-chloro-2,4-dinitrobenzene (CDNB), suggesting that the reaction occurs in the binding site of the xenobiotic substrate. Fractionation by high-performance liquid chromatography of a tryptic digest of inactivated enzyme yields a single, modified, 14-residue peptide containing Tyr115 as the altered amino acid. Modified and control enzymes have comparable affinities for glutathione, as indicated by fluorescence titration. In contrast, as distinguished from the control enzyme, modified enzyme does not adsorb to a column of an agarose-linked Cibacron Blue derivative, indicating that it has lost its ability to bind a hydrophobic substrate analogue. These results are supported by kinetic characteristics of modified and control enzymes: upon modification of the enzyme with 4-FSB, the apparent Km for glutathione is unchanged, while the apparent Km for CDNB increases dramatically from 193 to 1690 µM. When the reaction of 4-FSB with enzyme is monitored, the final percent residual activity is found to be dependent on the substrate used in the assay: 11% for CDNB, 20% for ethacrynic acid, 2.5% for trans-stilbene oxide, and 2% for trans-4-phenyl-3-butene-2-one. Analysis of the kinetics of modified enzyme suggests that Tyr115 of glutathione S-transferase, isozyme 4-4, contributes to xenobiotic substrate binding and, when certain types of substrates are employed, is involved in catalysis.

Original languageEnglish (US)
Pages (from-to)13002-13011
Number of pages10
JournalBiochemistry
Volume32
Issue number48
DOIs
StatePublished - Jan 1 1993

Fingerprint

Xenobiotics
Substrate Specificity
Glutathione Transferase
Labeling
Isoenzymes
Substrates
Enzymes
Glutathione
4-(fluorosulfonyl)benzoic acid
Sulfobromophthalein
Ethacrynic Acid
Dinitrochlorobenzene
Kinetics
High performance liquid chromatography
Fractionation
Catalysis
Titration
Liver
Sepharose
Rats

ASJC Scopus subject areas

  • Biochemistry

Cite this

Affinity Labeling of Glutathione S-Transferase, Isozyme 4-4, by 4-(Fluorosulfonyl)benzoic Acid Reveals Tyr115 To Be an Important Determinant of Xenobiotic Substrate Specificity. / Barycki, Joseph J.; Colman, Roberta F.

In: Biochemistry, Vol. 32, No. 48, 01.01.1993, p. 13002-13011.

Research output: Contribution to journalArticle

@article{5d728c6c4283424aa77eb485296c677f,
title = "Affinity Labeling of Glutathione S-Transferase, Isozyme 4-4, by 4-(Fluorosulfonyl)benzoic Acid Reveals Tyr115 To Be an Important Determinant of Xenobiotic Substrate Specificity",
abstract = "Incubation of 4-(fluorosulfonyl)benzoic acid (4-FSB), a xenobiotic substrate analogue, with the 4-4 isozyme of rat liver glutathione S-transferase at pH 7.5 and 25°C results in a time-dependent inactivation of the enzyme. The rate of inactivation exhibits a nonlinear dependence on 4-FSB concentration from 0.50 to 7.85 mM, with kmax = 0.082 min−1 and a of 1.95 mM. Nearly 1 mol of reagent/mol of enzyme subunit is incorporated when the enzyme is maximally inactivated. Protection against incorporation and inactivation is provided by bromosulfophthalein, a competitive inhibitor with respect to the hydrophobic substrate, 1-chloro-2,4-dinitrobenzene (CDNB), suggesting that the reaction occurs in the binding site of the xenobiotic substrate. Fractionation by high-performance liquid chromatography of a tryptic digest of inactivated enzyme yields a single, modified, 14-residue peptide containing Tyr115 as the altered amino acid. Modified and control enzymes have comparable affinities for glutathione, as indicated by fluorescence titration. In contrast, as distinguished from the control enzyme, modified enzyme does not adsorb to a column of an agarose-linked Cibacron Blue derivative, indicating that it has lost its ability to bind a hydrophobic substrate analogue. These results are supported by kinetic characteristics of modified and control enzymes: upon modification of the enzyme with 4-FSB, the apparent Km for glutathione is unchanged, while the apparent Km for CDNB increases dramatically from 193 to 1690 µM. When the reaction of 4-FSB with enzyme is monitored, the final percent residual activity is found to be dependent on the substrate used in the assay: 11{\%} for CDNB, 20{\%} for ethacrynic acid, 2.5{\%} for trans-stilbene oxide, and 2{\%} for trans-4-phenyl-3-butene-2-one. Analysis of the kinetics of modified enzyme suggests that Tyr115 of glutathione S-transferase, isozyme 4-4, contributes to xenobiotic substrate binding and, when certain types of substrates are employed, is involved in catalysis.",
author = "Barycki, {Joseph J.} and Colman, {Roberta F.}",
year = "1993",
month = "1",
day = "1",
doi = "10.1021/bi00211a008",
language = "English (US)",
volume = "32",
pages = "13002--13011",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "48",

}

TY - JOUR

T1 - Affinity Labeling of Glutathione S-Transferase, Isozyme 4-4, by 4-(Fluorosulfonyl)benzoic Acid Reveals Tyr115 To Be an Important Determinant of Xenobiotic Substrate Specificity

AU - Barycki, Joseph J.

AU - Colman, Roberta F.

PY - 1993/1/1

Y1 - 1993/1/1

N2 - Incubation of 4-(fluorosulfonyl)benzoic acid (4-FSB), a xenobiotic substrate analogue, with the 4-4 isozyme of rat liver glutathione S-transferase at pH 7.5 and 25°C results in a time-dependent inactivation of the enzyme. The rate of inactivation exhibits a nonlinear dependence on 4-FSB concentration from 0.50 to 7.85 mM, with kmax = 0.082 min−1 and a of 1.95 mM. Nearly 1 mol of reagent/mol of enzyme subunit is incorporated when the enzyme is maximally inactivated. Protection against incorporation and inactivation is provided by bromosulfophthalein, a competitive inhibitor with respect to the hydrophobic substrate, 1-chloro-2,4-dinitrobenzene (CDNB), suggesting that the reaction occurs in the binding site of the xenobiotic substrate. Fractionation by high-performance liquid chromatography of a tryptic digest of inactivated enzyme yields a single, modified, 14-residue peptide containing Tyr115 as the altered amino acid. Modified and control enzymes have comparable affinities for glutathione, as indicated by fluorescence titration. In contrast, as distinguished from the control enzyme, modified enzyme does not adsorb to a column of an agarose-linked Cibacron Blue derivative, indicating that it has lost its ability to bind a hydrophobic substrate analogue. These results are supported by kinetic characteristics of modified and control enzymes: upon modification of the enzyme with 4-FSB, the apparent Km for glutathione is unchanged, while the apparent Km for CDNB increases dramatically from 193 to 1690 µM. When the reaction of 4-FSB with enzyme is monitored, the final percent residual activity is found to be dependent on the substrate used in the assay: 11% for CDNB, 20% for ethacrynic acid, 2.5% for trans-stilbene oxide, and 2% for trans-4-phenyl-3-butene-2-one. Analysis of the kinetics of modified enzyme suggests that Tyr115 of glutathione S-transferase, isozyme 4-4, contributes to xenobiotic substrate binding and, when certain types of substrates are employed, is involved in catalysis.

AB - Incubation of 4-(fluorosulfonyl)benzoic acid (4-FSB), a xenobiotic substrate analogue, with the 4-4 isozyme of rat liver glutathione S-transferase at pH 7.5 and 25°C results in a time-dependent inactivation of the enzyme. The rate of inactivation exhibits a nonlinear dependence on 4-FSB concentration from 0.50 to 7.85 mM, with kmax = 0.082 min−1 and a of 1.95 mM. Nearly 1 mol of reagent/mol of enzyme subunit is incorporated when the enzyme is maximally inactivated. Protection against incorporation and inactivation is provided by bromosulfophthalein, a competitive inhibitor with respect to the hydrophobic substrate, 1-chloro-2,4-dinitrobenzene (CDNB), suggesting that the reaction occurs in the binding site of the xenobiotic substrate. Fractionation by high-performance liquid chromatography of a tryptic digest of inactivated enzyme yields a single, modified, 14-residue peptide containing Tyr115 as the altered amino acid. Modified and control enzymes have comparable affinities for glutathione, as indicated by fluorescence titration. In contrast, as distinguished from the control enzyme, modified enzyme does not adsorb to a column of an agarose-linked Cibacron Blue derivative, indicating that it has lost its ability to bind a hydrophobic substrate analogue. These results are supported by kinetic characteristics of modified and control enzymes: upon modification of the enzyme with 4-FSB, the apparent Km for glutathione is unchanged, while the apparent Km for CDNB increases dramatically from 193 to 1690 µM. When the reaction of 4-FSB with enzyme is monitored, the final percent residual activity is found to be dependent on the substrate used in the assay: 11% for CDNB, 20% for ethacrynic acid, 2.5% for trans-stilbene oxide, and 2% for trans-4-phenyl-3-butene-2-one. Analysis of the kinetics of modified enzyme suggests that Tyr115 of glutathione S-transferase, isozyme 4-4, contributes to xenobiotic substrate binding and, when certain types of substrates are employed, is involved in catalysis.

UR - http://www.scopus.com/inward/record.url?scp=0027769771&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0027769771&partnerID=8YFLogxK

U2 - 10.1021/bi00211a008

DO - 10.1021/bi00211a008

M3 - Article

C2 - 8241154

AN - SCOPUS:0027769771

VL - 32

SP - 13002

EP - 13011

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 48

ER -