Acetylation of aromatic cysteine conjugates by recombinant human N-acetyltransferase 8

Abstract

1. The mercapturic acid (MA) pathway is a metabolic route for the processing of glutathione conjugates to MA (N-acetylcysteine conjugates). An N-acetyltransferase enzyme, NAT8, catalyzes the transfer of an acetyl group from acetyl-CoA to the cysteine amino group, producing a MA, which is excreted in the urine. We expressed human NAT8 in HEK293T cells and developed an HPLC-MS method for the quantitation of the S-aryl-substituted cysteine conjugates and their MA.

2. We measured the activity of the enzyme for acetylation of benzyl-, 4-nitrobenzyl-, and 1-menaphthylcysteine substrates.

3. NAT8 catalyzed the acetylation of all three cysteine conjugates with similar Michaelis–Menten kinetics.

• Benzylcysteine
• 4-nitrobenzylcysteine
• mercapturic acid pathway
• NAT8
• N-acetyltransferase
• 1-menaphthylcysteine

Acknowledgements

We thank Dr. Veiga-Da-Cunha for providing the pEF6-NAT8 expression plasmid and for helpful advice. We gratefully acknowledge the assistance of Dr. Dyanne Brewer and Dr. Armen Charchoglyan (Advanced Analysis Center, University of Guelph) for LC-MS analysis. Undergraduate students Rachael McNeilly, Caio Gomes de Barros Martins, and Christena Watts assisted with the experiments. We thank Alex Michaelides (Chemistry) and Arthur Rocha for synthesis of 4-nitrobenzylcysteine and its MA derivative. P.D.J. wishes to thank Karen Barker (an undergraduate student at the time) for helpful discussions that stimulated this line of research.

Declaration of interest

This research was funded by a grant provided from the Natural Sciences and Engineering Research Council of Canada.