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.
Acknowledgements
This work was supported by the Southampton Hospital Charity, Southampton General Hospital, UK; Charity registration number 1051543 [Fund no. 0182] (VW), by the National Institute of Health (NIH) [Grant R15GM086833] (MEA) and by a Texas Woman’s University Research Enhancement Grant (MEA). We thank Theresa Brown for assistance. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the article.
Declaration of interest
The authors report no declarations of interest.