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The acetaminophen metabolite N-acetyl-p-benzoquinone imine (NAPQI) inhibits glutathione synthetase in vitro; a clue to the mechanism of 5-oxoprolinuric acidosis?

The acetaminophen metabolite N-acetyl-p-benzoquinone imine (NAPQI) inhibits glutathione synthetase in vitro; a clue to the mechanism of 5-oxoprolinuric acidosis?
The acetaminophen metabolite N-acetyl-p-benzoquinone imine (NAPQI) inhibits glutathione synthetase in vitro; a clue to the mechanism of 5-oxoprolinuric acidosis?
1. Metabolic acidosis due to accumulation of l-5-oxoproline is a rare, poorly understood, disorder associated with acetaminophen treatment in malnourished patients with chronic morbidity. l-5-Oxoprolinuria signals abnormal functioning of the γ-glutamyl cycle, which recycles and synthesises glutathione. Inhibition of glutathione synthetase (GS) by N-acetyl-p-benzoquinone imine (NAPQI) could contribute to 5-oxoprolinuric acidosis in such patients. We investigated the interaction of NAPQI with GS in vitro.

2. Peptide mapping of co-incubated NAPQI and GS using mass spectrometry demonstrated binding of NAPQI with cysteine-422 of GS, which is known to be essential for GS activity. Computational docking shows that NAPQI is properly positioned for covalent bonding with cysteine-422 via Michael addition and hence supports adduct formation.

3. Co-incubation of 0.77 μM of GS with NAPQI (25-400 μM) decreased enzyme activity by 16-89%. Inhibition correlated strongly with the concentration of NAPQI and was irreversible.

4. NAPQI binds covalently to GS causing irreversible enzyme inhibition in vitro. This is an important novel biochemical observation. It is the first indication that NAPQI may inhibit glutathione synthesis, which is pivotal in NAPQI detoxification. Further studies are required to investigate its biological significance and its role in 5-oxoprolinuric acidosis.
0049-8254
164-175
Walker, Valerie
e42b352f-5bdd-4ee0-adcd-0ce0fca21a71
Mills, Graham A.
3f99eb9e-1c6c-44ec-ac67-361a6421830a
Anderson, Mary E.
beb84b6c-c7bb-404e-88d2-7d0e5359cc97
Ingle, Brandall L.
483773c9-7384-4c43-a006-13378fcd8357
Moss, Charlotte L.
ae96895f-5c4a-49cd-a771-53bbd6f1d5fb
Jackson, John M.
56d28442-62b1-4425-96c6-27c321dfd310
Sharrod-Cole, Hayley
be0a0c19-adee-4826-8e85-02eef9a99945
Skipp, Paul
1ba7dcf6-9fe7-4b5c-a9d0-e32ed7f42aa5
Walker, Valerie
e42b352f-5bdd-4ee0-adcd-0ce0fca21a71
Mills, Graham A.
3f99eb9e-1c6c-44ec-ac67-361a6421830a
Anderson, Mary E.
beb84b6c-c7bb-404e-88d2-7d0e5359cc97
Ingle, Brandall L.
483773c9-7384-4c43-a006-13378fcd8357
Moss, Charlotte L.
ae96895f-5c4a-49cd-a771-53bbd6f1d5fb
Jackson, John M.
56d28442-62b1-4425-96c6-27c321dfd310
Sharrod-Cole, Hayley
be0a0c19-adee-4826-8e85-02eef9a99945
Skipp, Paul
1ba7dcf6-9fe7-4b5c-a9d0-e32ed7f42aa5

Walker, Valerie, Mills, Graham A., Anderson, Mary E., Ingle, Brandall L., Moss, Charlotte L., Jackson, John M., Sharrod-Cole, Hayley and Skipp, Paul (2017) The acetaminophen metabolite N-acetyl-p-benzoquinone imine (NAPQI) inhibits glutathione synthetase in vitro; a clue to the mechanism of 5-oxoprolinuric acidosis? Xenobiotica, 47 (2), 164-175. (doi:10.3109/00498254.2016.1166533).

Record type: Article

Abstract

1. Metabolic acidosis due to accumulation of l-5-oxoproline is a rare, poorly understood, disorder associated with acetaminophen treatment in malnourished patients with chronic morbidity. l-5-Oxoprolinuria signals abnormal functioning of the γ-glutamyl cycle, which recycles and synthesises glutathione. Inhibition of glutathione synthetase (GS) by N-acetyl-p-benzoquinone imine (NAPQI) could contribute to 5-oxoprolinuric acidosis in such patients. We investigated the interaction of NAPQI with GS in vitro.

2. Peptide mapping of co-incubated NAPQI and GS using mass spectrometry demonstrated binding of NAPQI with cysteine-422 of GS, which is known to be essential for GS activity. Computational docking shows that NAPQI is properly positioned for covalent bonding with cysteine-422 via Michael addition and hence supports adduct formation.

3. Co-incubation of 0.77 μM of GS with NAPQI (25-400 μM) decreased enzyme activity by 16-89%. Inhibition correlated strongly with the concentration of NAPQI and was irreversible.

4. NAPQI binds covalently to GS causing irreversible enzyme inhibition in vitro. This is an important novel biochemical observation. It is the first indication that NAPQI may inhibit glutathione synthesis, which is pivotal in NAPQI detoxification. Further studies are required to investigate its biological significance and its role in 5-oxoprolinuric acidosis.

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More information

Accepted/In Press date: 13 March 2016
e-pub ahead of print date: 18 April 2016
Published date: February 2017

Identifiers

Local EPrints ID: 416122
URI: http://eprints.soton.ac.uk/id/eprint/416122
ISSN: 0049-8254
PURE UUID: 86988fdd-e57f-4cb4-9742-d8e3add4f311
ORCID for Paul Skipp: ORCID iD orcid.org/0000-0002-2995-2959

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Date deposited: 05 Dec 2017 17:30
Last modified: 16 Mar 2024 02:41

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Contributors

Author: Valerie Walker
Author: Graham A. Mills
Author: Mary E. Anderson
Author: Brandall L. Ingle
Author: Charlotte L. Moss
Author: John M. Jackson
Author: Hayley Sharrod-Cole
Author: Paul Skipp ORCID iD

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