Enzyme kinetic properties of human recombinant arylamine n-acetyltransferase 2 allotypic variants expressed in Escherichia coli

Abstract

Arylamine N-acetyltransferase (NAT2) catalyses the N-acetylation of primary arylamine and hydrazine drugs and chemicals. N-Acetylation is subject to polymorphism, and humans can be categorized as either fast or slow acetylators according to their ability to N-acetylate certain arylamine substrates in vivo. Genetic variants at the polymorphic NAT2 locus have been described. We expressed five of the most common NAT2 variants (NAT2 4, NAT2 5A, NAT2 5B, NAT2 6A and NAT2 7B) in Escherichia coli as a convenient source of the human variants. The apparent K_m values (at 100 μM acetyl CoA as co-substrate) of the different NAT2 variants for sulphamethazine, dapsone, p-anisidine, 2-aminofluorene, procainamide and isoniazid were determined. Data show that the apparent K_m of the slow variant NAT2 7B for the arylamine sulphamethazine was 10-fold lower than all the other allotypes. The apparent K_m for the structurally related sulphone antibiotic dapsone was 5-fold lower for the slow variant NAT2 7B when compared with the wild-type NAT2 4. These results indicate that the NAT2 7B specific amino acid substitution, Gly286-Glu, is important in promoting the binding of sulphamethazine and dapsone to the active site.