title: The acetaminophen metabolite N-acetyl-p-benzoquinone imine (NAPQI) inhibits glutathione synthetase in vitro; a clue to the mechanism of 5-oxoprolinuric acidosis?
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.
authors.affiliation: a Department of Clinical Biochemistry , University Hospital Southampton NHS Foundation Trust , Southampton , UK.
authors.forename: Valerie
authors.initials: V
authors.lastname: Walker
authors.affiliation: b School of Pharmacy and Biomedical Sciences, University of Portsmouth , Portsmouth , UK.
authors.forename: Graham A
authors.initials: GA
authors.lastname: Mills
authors.affiliation: c Department of Chemistry and Biochemistry , Texas Woman's University , Denton , TX , USA.
authors.forename: Mary E
authors.initials: ME
authors.lastname: Anderson
authors.affiliation: d Department of Chemistry , Center for Advanced Scientific Computing and Modeling, University of North Texas , Denton , TX , USA.
authors.forename: Brandall L
authors.initials: BL
authors.lastname: Ingle
authors.affiliation: e NIHR Southampton Biomedical Research Centre, Southampton General Hospital , Southampton , UK , and.
authors.forename: John M
authors.initials: JM
authors.lastname: Jackson
authors.affiliation: f Centre for Proteomic Research and Biological Sciences, University of Southampton , Southampton , UK.
authors.forename: Charlotte L
authors.initials: CL
authors.lastname: Moss
authors.affiliation: a Department of Clinical Biochemistry , University Hospital Southampton NHS Foundation Trust , Southampton , UK.
authors.forename: Hayley
authors.initials: H
authors.lastname: Sharrod-Cole
authors.affiliation: f Centre for Proteomic Research and Biological Sciences, University of Southampton , Southampton , UK.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Skipp
mesh_terms: D000082:Acetaminophen / Q000633:toxicity; D000138:Acidosis / Q000139:chemically induced; D016227:Benzoquinones / Q000633:toxicity*; D005978:Glutathione / Q000378:metabolism; D005981:Glutathione Synthase / Q000378:metabolism*; D007097:Imines / Q000633:toxicity*
chemical_list: D016227:Benzoquinones; D007097:Imines; D000082:Acetaminophen; D005981:Glutathione Synthase; C028473:N-acetyl-4-benzoquinoneimine; D005978:Glutathione
keywords: Acetaminophen toxicity; glutathione deficiency; protein arylation; γ-glutamyl cycle