N-acetyltransferases (NATs; EC 2.3.1.5) utilize acetyl Co-A in acetylation conjugation reactions. This is the preferred route of conjugating aromatic amines (R-NH2, converted to aromatic amides R-NH-COCH3) and hydrazines (R-NH-NH2, converted to R-NH-NH-COCH3). Aliphatic amines are not substrates for NAT. The basic reaction is

Acetyl-CoA + an arylamine = CoA + an N- acetylarylamine

NATs are cytosolic and in humans, 2 isoforms are expressed, NAT1 and NAT2. A third isoform, NATP, is a pseudogene and is not expressed. The NAT2 gene contains mutations that decrease NAT2 activity. This mutations was first seen as slow acetylation compared to the normal, fast acetylation of the antituberculosis drug isoniazid. Incidence of the slow acetylator phenotype is high in Middle Eastern populations (70%), average (50%) in Europeans, Americans and Australians and low in Asians (<25% in Chinese, Japanese and Koreans). N-acetylation and methylation pathways differ from other conjugation pathways in that they mask an amine with a nonionizable group so that the conjugates are less water soluble than the parent compound. However, certain N-acetlylations facilitate urinary excretion.
N-acetylation occurs in two sequential steps via a ping-pong Bi-Bi mechanism. In the first step, the acetyl group from acetyl-CoA is transferred to a cysteine residue in NAT, with consequent release of coenzyme-A. In the second step, the acetyl group is released from the acetylated NAT to the substrate, subsequently regenerating the enzyme.