Abstract
Oxidative stress is increasingly recognized as a key mechanism in the biotransformation and/or toxicity of many xenobiotics. Human arylamine N-acetyltransferase 1 (NAT1) is a polymorphic ubiquitous phase II xenobiotic metabolizing enzyme that catalyzes the biotransformation of primary aromatic amine or hydrazine drugs and carcinogens. Functional and structural studies have shown that NAT1 catalytic activity is based on a cysteine protease-like catalytic triad, containing a reactive cysteine residue. Reactive protein cysteine residues are highly susceptible to oxidation by hydrogen peroxide (H2O2) generated within the cell. We, therefore, investigated whether human NAT1 activity was regulated by this cellular oxidant. Using purified recombinant NAT1, we show here that NAT1 is rapidly (kinact = 420 m-1.min-1) inactivated by physiological concentrations of H2O2. Reducing agents, such as reduced glutathione (GSH), reverse the H2O2-dependent inactivation of NAT1. Kinetic analysis and protection experiments with acetyl-CoA, the physiological acetyl-donor substrate of the enzyme, suggested that the H2O2-dependent inactivation reaction targets the active-site cysteine residue. Finally, we show that the reversible inactivation of NAT1 by H2O2 is due to the formation of a stable sulfenic acid group at the active-site cysteine. Our results suggest that, in addition to known genetically controlled interindividual variations in NAT1 activity, oxidative stress and cellular redox status may also regulate NAT1 activity. This may have important consequences with regard to drug biotransformation and cancer risk.
Highlights
Oxidative stress is increasingly recognized as a key ine N-acetyltransferases (NATs; EC 2.3.1.5) are phase II xenobiotic metabolizing enzymes (XME) mechanism in the biotransformation and/or toxicity of that catalyze the transfer of an acetyl moiety from acetyl-coenzyme A (CoA) many xenobiotics
In Vitro Inactivation of NAT1 by Bolus Addition of H2O2 and by Continuous Generation of H2O2 by the Glucose/Glucose Oxidase System—Several enzymes with catalytic cysteine residues have been shown to be reversibly inactivated by H2O2, a major physiological oxidant [18, 20, 23]
We cannot rule out the possibility that H2O2 modifies other cysteine residues of NAT1, these results clearly demonstrate that the catalytic active site cysteine residue of NAT1 is a target of H2O2-dependent oxidative modification, leading to reversible inactivation of the enzyme
Summary
Materials—p-Aminosalicylic acid (PAS, a NAT1-selective arylamine acceptor substrate), p-nitrophenylacetate (PNPA, acetyl donor), acetylcoenzyme A (AcCoA, acetyl donor), coenzyme A (CoA), hydrogen peroxide (H2O2), 5,5-dimethyl-1,3-cyclohexanedione (dimedone), 1,4-dithiothreitol (DTT), reduced glutathione (GSH), imidazole, lysozyme and glutathione-agarose, bovine catalase (5200 units/mg), glucose oxidase (type II from Aspergillus niger, 15,500 units/g), and -D-glucose were purchased from Sigma. We assessed the effect of bolus addition of H2O2 on NAT1 enzyme activity by incubating purified NAT1 samples (1.5 M final concentration) with various concentrations of H2O2 in 25 mM Tris-HCl, pH 7.5, 1 mM EDTA (total volume of 10 l) for 10 min at 37 °C. Samples were incubated with H2O2 (200 M final concentration) in a total volume of 20 l, as described above, and assayed Assays performed in these conditions with AcCoA or CoA alone gave 100% NAT1 activity. NAT1 (1.5 M final concentration) was first incubated with H2O2 (200 M final concentration) in the presence of 10 mM dimedone (a specific reagent of sulfenic acid) in 25 mM Tris-HCl, pH 7.5, 1 mM EDTA (10 l total volume) for 10 min at 37 °C. The membrane was washed and incubated with Supersignal reagent (Pierce) for detection
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