Abstract
Arylamine N-acetyltransferases catalyze the transfer of acetyl groups from the endogenous cofactor acetyl coenzyme A (AcCoA) to arylamine (N-acetylation) and N-hydroxy-arylamine (O-acetylation) acceptors. Humans express two arylamine N-acetyltransferase isozymes (NAT1 and NAT2) which catalyze both N- and O-acetylation but differ in genetic regulation, substrate selectivity, and expression in human tissues. We investigated recombinant human NAT1 and NAT2 expressed in an Escherichia coli JM105 and Schizosaccharomyces pombe expression systems as well as in Chinese hamster ovary (CHO) cells to assess the relative affinity of AcCoA for human NAT1 and NAT2. NAT1 and NAT2 affinity for AcCoA was higher for recombinant human NAT1 than NAT2 when catalyzing N-acetylation of aromatic amine carcinogens 2-aminofluroene (AF), 4-aminobiphenyl (ABP), and β-naphthylamine (BNA) and the metabolic activation of N-hydroxy-2-aminofluorene (N-OH-AF) and N-hydroxy-4-aminobiphenyl (N-OH-ABP) via O-acetylation. These results suggest that AcCoA level may influence differential rates of arylamine carcinogen metabolism catalyzed by NAT1 and NAT2 in human tissues. Affinity was higher for NAT2 than for NAT1 using N-OH-AF and N-OH-ABP as substrate consistent with a larger active site for NAT2. In conclusion, following recombinant expression in bacteria, yeast, and CHO cells, we report significant differences in affinity between human NAT1 and NAT2 for its required co-factor AcCoA, as well as for N-hydroxy-arylamines activated via O-acetylation. The findings provide important information to understand the relative contribution of human NAT1 vs NAT2 towards N-acetylation and O-acetylation reactions in human hepatic and extrahepatic tissues.
Highlights
N-acetyltransferase 1 (NAT1) and 2 (NAT2) catalyze the N-acetylation of carcinogenic arylamines
We investigated recombinant human NAT1 and NAT2 expressed in an Escherichia coli JM105 and Schizosaccharomyces pombe expression systems as well as in Chinese hamster ovary (CHO)
NAT1 and NAT2 affinity for acetyl coenzyme A (AcCoA) was higher for recombinant human NAT1 than NAT2 when catalyzing N-acetylation of aromatic amine carcinogens AF, ABP, and BNA and the metabolic activation of N-hydroxy-AF and N-hydroxy-ABP via O-acetylation (Figure 1)
Summary
N-acetyltransferase 1 (NAT1) and 2 (NAT2) catalyze the N-acetylation of carcinogenic arylamines. Previous determinations of human NAT1 and NAT2 affinity for AcCoA were conducted for recombinant NAT2 expressed in bacteria (Hein et al, 1993) and recombinant NAT1 expressed in yeast (Zhu and Hein, 2008) with substrates specific for NAT1and NAT2-catalyzed N-acetylation. Arylamine carcinogens such as 2-aminofluorene (AF), 4-aminobiphenyl (ABP) and β -naphthylamine (BNA) undergo N-acetylation catalyzed by human NAT1 and NAT2 following recombinant expression in bacteria (Hein et al, 1993) or yeast (Leggett et al, 2021) and are more appropriate to use for comparing human NAT1 and NAT2 affinity for AcCoA. NAT1 “appeared” to be more selective for the N-hydroxy derivatives of carboxylic arylamine carcinogens (Minchin et al, 1992; Hein et al, 1993), this has not to our knowledge been the focus of a more robust investigation comparing their substrate affinities for human NAT1 and NAT2
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