Catalytic mechanism of hamster arylamine N-acetyltransferase 2

Arylamine N-acetyltransferases (NATs) catalyze an acetyl group transfer from AcCoA to primary arylamines, hydrazines, and hydrazides and play a very important role in the metabolism and bioactivation of drugs, carcinogens, and other xenobiotics. The reaction follows a ping-pong bi-bi mechanism. Structure analysis of bacterial NATs revealed a Cys-His-Asp catalytic triad that is strictly conserved in all known NATs. Previously, we have demonstrated by kinetic and isotope effect studies that acetylation of the hamster NAT2 is dependent on a thiolate-imidazolium ion pair (Cys-S^--His-ImH ⁺) and not a general acid-base catalysis. In addition, we established that, after formation of the acetylated enzyme intermediate, the active-site imidazole, His-107, is likely deprotonated at physiological pH. In this paper, we report steady-state kinetic studies of NAT2 with two acetyl donors, acetyl coenzyme A (AcCoA) and p-nitrophenyl acetate (PNPA), and four arylamine substrates. The pH dependence of k_cat/K_AcCoA exhibited two inflection points at 5.32 ± 0.13 and 8.48 ± 0.24, respectively. The pK_a at 5.32 is virtually identical with the previously reported pK_a of 5.2 for enzyme acetylation, reaffirming that the first half of the reaction is catalyzed by a thiolate-imidazolium ion pair in the active site. The inflection point at 8.48 indicates that a pH-sensitive group on NAT2 is involved in AcCoA binding. A Brønsted plot constructed by the correlation of log k₄ and log k_H2O with the pK_a for each arylamine substrate and water displays a linear free-energy relationship in the pK_a range from -1.7 (H₂O) to 4.67 (PABA), with a slope of β_nuc = 0.80 ± 0.1. However, a further increase of the pK_a from 4.67 (PABA) to 5.32 (anisidine) resulted in a 2.5-fold decrease in the k₄ value. Analysis of the pH-k_cat/K_PABA profile revealed a pK_a of 5.52 ± 0.14 and a solvent kinetic isotope effect (SKIE) of 2.01 ± 0.04 on k_cat/K_PABA. Normal solvent isotope effects of 4.8 ± 0.1, 3.1 ± 0.1, and 3.2 ± 0.1 on the k _cat/K_b for anisidine, pABglu, and PNA, respectively, were also determined. These observations are consistent with a deacetylation mechanism dominated by nucleophilic attack of the thiol ester for arylamines with pK_a values ≤5.5 to deprotonation of a tetrahedral intermediate for arylamines with pK_a values ≥5.5. The general base is likely His-107 because the His-107 to Gln and Asn mutants were found to be devoid of catalytic activity. In contrast, an increase in pH-dependent hydrolysis of the acetylated enzyme was not observed over a pH range of 5.2-7.5. On the basis of these observations, a catalytic mechanism for the acetylation of arylamines by NAT2 is proposed.