Studies on arylamine N-acetyltransferases

Arylamine N-acetyltransferases (NATs) catalyze the N-acetylation of arylamines. NATs also catalyze the bioactivation of N-arylhydroxylamines by O-acetylation and the bioactivation of N-arylhydroxamic acids by N,O-acetyltransfer. The transacetylation reaction occurs through a ping-pong bi-bi mechanism. The 3D structures of prokaryotic NATs have revealed a Cys-His-Asp catalytic triad, which is strictly conserved across all known NATs (Cys68, His107, and Asp122 in mammalian NATs). In this thesis, the catalytic mechanism of hamster NAT2 was probed by active site modification, site-directed mutagenesis, pre-steady state, and steady state kinetic studies. The results support a thiolate-imidazolium ion pair (Cys-S--His-ImH+) mechanism for the enzyme acetylation step. The acetyl-enzyme deacetylation step relies on nucleophilic attack of the enzyme thiol ester by an arylamine substrate, followed by deprotonation by the active site imidazole, His-107, functioning as a general base catalyst. Human NAT1 was overexpressed in E. coli as a mutant dihydrofolic acid reductase fusion protein with a thrombin-sensitive linker. Kinetic characterization of human NAT1 suggests that human NAT1 and hamster NAT2 have similar but distinct kinetic properties with certain substrates, and that folic acid, at least in the non-polyglutamate form, may not have an effect on human NAT1 activity in vivo. Treatment of hamster NAT1, hamster NAT2, and human NAT1 with the carcinogenic hydroxamic acid, N-hydroxy-4-acetylaminobiphenyl (N-OH-4-AABP), caused irreversible inactivation of the NATs. Mass spectrometric analysis demonstrated that the inactivations result from reaction of 4-nitrosobiphenyl with Cys68 of each enzyme. Kinetic analysis of the NAT inactivations revealed that hamster NAT I is much more efficiently inactivated in the presence of N-OH-4-AABP then either hamster NAT2 or human NAT1, and that the relative sensitivities of the NATs to inactivation are consistent with the relative half-lives of the acetylated enzymes. The results support a process of 4-nitrosobiphenyl formation that involves NAT-catalyzed deacetylation of N-OH-4-AABP to yield N-hydroxy-4-aminobiphenyl (N-OH-4-ABP), followed by oxidation of N-OH-4-ABP to 4-nitrosobiphenyl.