Inactivation of glutathione S-Transferases by nitric oxide-derived oxidants: Exploring a role for tyrosine nitration

Patrick S Y Wong, Jason P. Eiserich, Sharanya Reddy, C. Leticia Lopez, Carroll E Cross, Albert van der VlieT

Research output: Contribution to journalArticle

Abstract

Reactive intermediates derived from nitric oxide (NO) are thought to play a contributing role in dis- ase states associated with inflammation and infec-tion. We show here that glutathione S-transferases (GSTs), principal enzymes responsible for detoxifica-tion of endogenous and exogenous electrophiles, are susceptible to inactivation by reactive nitrogen spe-cies (RNS). Treatment of isolated GSTs or rat liver homogenates with either peroxynitrite, the myeloper-oxidase/hydrogen peroxide/nitrite system, or tetrani-tromethane, resulted in loss of GST activity with a concomitant increase in the formation of protein-asso-ciated 3-nitrotyrosine (NO2Tyr). This inactivation was only partially (25%) reversible by dithiothreitol, and exposure of GSTs to hydrogen peroxide orS-nitroso-glutathione was only partially inhibitory (25%) and did not result in protein nitration. Thus, irreversible modifications such as tyrosine nitration may have con-tributed to GST inactivation by RNS. Since all GSTs contain a critical, highly conserved, active-site ty-rosine residue, we postulated that this Tyr residue might present a primary target for nitration by RNS, thus leading to enzyme inactivation. To directly inves-tigate this possibility, we analyzed purified mouse liver GST-m, following nitration by several RNS, by trypsin digestion, HPLC separation, and matrix-as-sisted laser desorption/ionization-time of flight analy-sis, to determine the degree of tyrosine nitration of individual Tyr residues. Indeed, nitration was found to occur preferentially on several tyrosine residues located in and around the GST active site. However, RNS concentrations that resulted in near complete GST inactivation only caused up to 25% nitration of even preferentially targeted tyrosine residues. Hence, nitration of active-site tyrosine residues may contrib-ute to GST inactivation by RNS, but is unlikely to fully account for enzyme inactivation. Overall, our studies illustrate a potential mechanism by which RNS may promote (oxidative) injury by environmental pollut-ants in association with inflammation.

Original languageEnglish (US)
Pages (from-to)183-190
Number of pages8
JournalAnalytical Biochemistry
Volume297
Issue number2
DOIs
StatePublished - Oct 15 2001

Fingerprint

Nitration
Glutathione Transferase
Oxidants
Tyrosine
Nitric Oxide
Nitrogen
Catalytic Domain
Liver
Hydrogen Peroxide
Enzymes
Inflammation
Peroxynitrous Acid
Ants
Dithiothreitol
Nitrites
Trypsin
Ionization
Glutathione
Rats
Digestion

Keywords

  • GlutathioneS-transferase
  • In-flammation
  • Myeloperoxidase
  • Nitric oxide
  • Peroxynitrite
  • Protein oxidation
  • Tyrosine nitration

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Inactivation of glutathione S-Transferases by nitric oxide-derived oxidants : Exploring a role for tyrosine nitration. / Wong, Patrick S Y; Eiserich, Jason P.; Reddy, Sharanya; Lopez, C. Leticia; Cross, Carroll E; van der VlieT, Albert.

In: Analytical Biochemistry, Vol. 297, No. 2, 15.10.2001, p. 183-190.

Research output: Contribution to journalArticle

Wong, Patrick S Y ; Eiserich, Jason P. ; Reddy, Sharanya ; Lopez, C. Leticia ; Cross, Carroll E ; van der VlieT, Albert. / Inactivation of glutathione S-Transferases by nitric oxide-derived oxidants : Exploring a role for tyrosine nitration. In: Analytical Biochemistry. 2001 ; Vol. 297, No. 2. pp. 183-190.
@article{b7457077f6b743e4aeab7ae5ae86dba3,
title = "Inactivation of glutathione S-Transferases by nitric oxide-derived oxidants: Exploring a role for tyrosine nitration",
abstract = "Reactive intermediates derived from nitric oxide (NO) are thought to play a contributing role in dis- ase states associated with inflammation and infec-tion. We show here that glutathione S-transferases (GSTs), principal enzymes responsible for detoxifica-tion of endogenous and exogenous electrophiles, are susceptible to inactivation by reactive nitrogen spe-cies (RNS). Treatment of isolated GSTs or rat liver homogenates with either peroxynitrite, the myeloper-oxidase/hydrogen peroxide/nitrite system, or tetrani-tromethane, resulted in loss of GST activity with a concomitant increase in the formation of protein-asso-ciated 3-nitrotyrosine (NO2Tyr). This inactivation was only partially (25{\%}) reversible by dithiothreitol, and exposure of GSTs to hydrogen peroxide orS-nitroso-glutathione was only partially inhibitory (25{\%}) and did not result in protein nitration. Thus, irreversible modifications such as tyrosine nitration may have con-tributed to GST inactivation by RNS. Since all GSTs contain a critical, highly conserved, active-site ty-rosine residue, we postulated that this Tyr residue might present a primary target for nitration by RNS, thus leading to enzyme inactivation. To directly inves-tigate this possibility, we analyzed purified mouse liver GST-m, following nitration by several RNS, by trypsin digestion, HPLC separation, and matrix-as-sisted laser desorption/ionization-time of flight analy-sis, to determine the degree of tyrosine nitration of individual Tyr residues. Indeed, nitration was found to occur preferentially on several tyrosine residues located in and around the GST active site. However, RNS concentrations that resulted in near complete GST inactivation only caused up to 25{\%} nitration of even preferentially targeted tyrosine residues. Hence, nitration of active-site tyrosine residues may contrib-ute to GST inactivation by RNS, but is unlikely to fully account for enzyme inactivation. Overall, our studies illustrate a potential mechanism by which RNS may promote (oxidative) injury by environmental pollut-ants in association with inflammation.",
keywords = "GlutathioneS-transferase, In-flammation, Myeloperoxidase, Nitric oxide, Peroxynitrite, Protein oxidation, Tyrosine nitration",
author = "Wong, {Patrick S Y} and Eiserich, {Jason P.} and Sharanya Reddy and Lopez, {C. Leticia} and Cross, {Carroll E} and {van der VlieT}, Albert",
year = "2001",
month = "10",
day = "15",
doi = "10.1006/abio.2001.5325",
language = "English (US)",
volume = "297",
pages = "183--190",
journal = "Analytical Biochemistry",
issn = "0003-2697",
publisher = "Academic Press Inc.",
number = "2",

}

TY - JOUR

T1 - Inactivation of glutathione S-Transferases by nitric oxide-derived oxidants

T2 - Exploring a role for tyrosine nitration

AU - Wong, Patrick S Y

AU - Eiserich, Jason P.

AU - Reddy, Sharanya

AU - Lopez, C. Leticia

AU - Cross, Carroll E

AU - van der VlieT, Albert

PY - 2001/10/15

Y1 - 2001/10/15

N2 - Reactive intermediates derived from nitric oxide (NO) are thought to play a contributing role in dis- ase states associated with inflammation and infec-tion. We show here that glutathione S-transferases (GSTs), principal enzymes responsible for detoxifica-tion of endogenous and exogenous electrophiles, are susceptible to inactivation by reactive nitrogen spe-cies (RNS). Treatment of isolated GSTs or rat liver homogenates with either peroxynitrite, the myeloper-oxidase/hydrogen peroxide/nitrite system, or tetrani-tromethane, resulted in loss of GST activity with a concomitant increase in the formation of protein-asso-ciated 3-nitrotyrosine (NO2Tyr). This inactivation was only partially (25%) reversible by dithiothreitol, and exposure of GSTs to hydrogen peroxide orS-nitroso-glutathione was only partially inhibitory (25%) and did not result in protein nitration. Thus, irreversible modifications such as tyrosine nitration may have con-tributed to GST inactivation by RNS. Since all GSTs contain a critical, highly conserved, active-site ty-rosine residue, we postulated that this Tyr residue might present a primary target for nitration by RNS, thus leading to enzyme inactivation. To directly inves-tigate this possibility, we analyzed purified mouse liver GST-m, following nitration by several RNS, by trypsin digestion, HPLC separation, and matrix-as-sisted laser desorption/ionization-time of flight analy-sis, to determine the degree of tyrosine nitration of individual Tyr residues. Indeed, nitration was found to occur preferentially on several tyrosine residues located in and around the GST active site. However, RNS concentrations that resulted in near complete GST inactivation only caused up to 25% nitration of even preferentially targeted tyrosine residues. Hence, nitration of active-site tyrosine residues may contrib-ute to GST inactivation by RNS, but is unlikely to fully account for enzyme inactivation. Overall, our studies illustrate a potential mechanism by which RNS may promote (oxidative) injury by environmental pollut-ants in association with inflammation.

AB - Reactive intermediates derived from nitric oxide (NO) are thought to play a contributing role in dis- ase states associated with inflammation and infec-tion. We show here that glutathione S-transferases (GSTs), principal enzymes responsible for detoxifica-tion of endogenous and exogenous electrophiles, are susceptible to inactivation by reactive nitrogen spe-cies (RNS). Treatment of isolated GSTs or rat liver homogenates with either peroxynitrite, the myeloper-oxidase/hydrogen peroxide/nitrite system, or tetrani-tromethane, resulted in loss of GST activity with a concomitant increase in the formation of protein-asso-ciated 3-nitrotyrosine (NO2Tyr). This inactivation was only partially (25%) reversible by dithiothreitol, and exposure of GSTs to hydrogen peroxide orS-nitroso-glutathione was only partially inhibitory (25%) and did not result in protein nitration. Thus, irreversible modifications such as tyrosine nitration may have con-tributed to GST inactivation by RNS. Since all GSTs contain a critical, highly conserved, active-site ty-rosine residue, we postulated that this Tyr residue might present a primary target for nitration by RNS, thus leading to enzyme inactivation. To directly inves-tigate this possibility, we analyzed purified mouse liver GST-m, following nitration by several RNS, by trypsin digestion, HPLC separation, and matrix-as-sisted laser desorption/ionization-time of flight analy-sis, to determine the degree of tyrosine nitration of individual Tyr residues. Indeed, nitration was found to occur preferentially on several tyrosine residues located in and around the GST active site. However, RNS concentrations that resulted in near complete GST inactivation only caused up to 25% nitration of even preferentially targeted tyrosine residues. Hence, nitration of active-site tyrosine residues may contrib-ute to GST inactivation by RNS, but is unlikely to fully account for enzyme inactivation. Overall, our studies illustrate a potential mechanism by which RNS may promote (oxidative) injury by environmental pollut-ants in association with inflammation.

KW - GlutathioneS-transferase

KW - In-flammation

KW - Myeloperoxidase

KW - Nitric oxide

KW - Peroxynitrite

KW - Protein oxidation

KW - Tyrosine nitration

UR - http://www.scopus.com/inward/record.url?scp=85007624583&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85007624583&partnerID=8YFLogxK

U2 - 10.1006/abio.2001.5325

DO - 10.1006/abio.2001.5325

M3 - Article

AN - SCOPUS:85007624583

VL - 297

SP - 183

EP - 190

JO - Analytical Biochemistry

JF - Analytical Biochemistry

SN - 0003-2697

IS - 2

ER -