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 k(4) and log k(H)2(O) with the pK(a) for each arylamine substrate and water displays a linear free-energy relationship in the pK(a) range from -1.7 (H(2)O) to 4.67 (PABA), with a slope of beta(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 k(4) 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 <or=5.5 to deprotonation of a tetrahedral intermediate for arylamines with pK(a) values >or=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.
Wang
Haiqing
H
Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Liu
Li
L
Hanna
Patrick E
PE
Wagner
Carston R
CR
D000105:Acetyl Coenzyme A / Q000737:chemistry* / Q000378:metabolism; D000588:Amines / Q000378:metabolism; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001191:Arylamine N-Acetyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002273:Carcinogens / Q000378:metabolism; D002384:Catalysis; D006224:Cricetinae; D006834:Hydrazines / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009596:Nitrophenols / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D015262:Xenobiotics / Q000378:metabolism
D000588:Amines; D002273:Carcinogens; D006834:Hydrazines; D009596:Nitrophenols; D015262:Xenobiotics; D000105:Acetyl Coenzyme A; C008642:4-nitrophenyl acetate; D001191:Arylamine N-Acetyltransferase