Catalytic mechanism of hamster arylamine N-acetyltransferase 2

Haiqing Wang, Li Liu, Patrick E. Hanna, Carston R Wagner

Research output: Contribution to journalArticle

40 Citations (Scopus)

Abstract

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 kcat/KAcCoA exhibited two inflection points at 5.32 ± 0.13 and 8.48 ± 0.24, respectively. The pKa at 5.32 is virtually identical with the previously reported pKa 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 kH2O with the pKa for each arylamine substrate and water displays a linear free-energy relationship in the pKa range from -1.7 (H2O) to 4.67 (PABA), with a slope of βnuc = 0.80 ± 0.1. However, a further increase of the pKa from 4.67 (PABA) to 5.32 (anisidine) resulted in a 2.5-fold decrease in the k4 value. Analysis of the pH-kcat/KPABA profile revealed a pKa of 5.52 ± 0.14 and a solvent kinetic isotope effect (SKIE) of 2.01 ± 0.04 on kcat/KPABA. Normal solvent isotope effects of 4.8 ± 0.1, 3.1 ± 0.1, and 3.2 ± 0.1 on the k cat/Kb 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 pKa values ≤5.5 to deprotonation of a tetrahedral intermediate for arylamines with pKa 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.

Original languageEnglish (US)
Pages (from-to)11295-11306
Number of pages12
JournalBiochemistry
Volume44
Issue number33
DOIs
StatePublished - Aug 23 2005

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Arylamine N-Acetyltransferase
Acetylation
Acetyl Coenzyme A
Isotopes
Cricetinae
4-Aminobenzoic Acid
Acetyltransferases
Kinetics
Enzymes
Hydrazines
Ions
Deprotonation
Xenobiotics
Substrates
Sulfhydryl Compounds
Metabolism
Carcinogens
Catalysis
Free energy
Hydrolysis

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Catalytic mechanism of hamster arylamine N-acetyltransferase 2. / Wang, Haiqing; Liu, Li; Hanna, Patrick E.; Wagner, Carston R.

In: Biochemistry, Vol. 44, No. 33, 23.08.2005, p. 11295-11306.

Research output: Contribution to journalArticle

Wang, Haiqing ; Liu, Li ; Hanna, Patrick E. ; Wagner, Carston R. / Catalytic mechanism of hamster arylamine N-acetyltransferase 2. In: Biochemistry. 2005 ; Vol. 44, No. 33. pp. 11295-11306.
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N2 - 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 kcat/KAcCoA exhibited two inflection points at 5.32 ± 0.13 and 8.48 ± 0.24, respectively. The pKa at 5.32 is virtually identical with the previously reported pKa 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 kH2O with the pKa for each arylamine substrate and water displays a linear free-energy relationship in the pKa range from -1.7 (H2O) to 4.67 (PABA), with a slope of βnuc = 0.80 ± 0.1. However, a further increase of the pKa from 4.67 (PABA) to 5.32 (anisidine) resulted in a 2.5-fold decrease in the k4 value. Analysis of the pH-kcat/KPABA profile revealed a pKa of 5.52 ± 0.14 and a solvent kinetic isotope effect (SKIE) of 2.01 ± 0.04 on kcat/KPABA. Normal solvent isotope effects of 4.8 ± 0.1, 3.1 ± 0.1, and 3.2 ± 0.1 on the k cat/Kb 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 pKa values ≤5.5 to deprotonation of a tetrahedral intermediate for arylamines with pKa 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.

AB - 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 kcat/KAcCoA exhibited two inflection points at 5.32 ± 0.13 and 8.48 ± 0.24, respectively. The pKa at 5.32 is virtually identical with the previously reported pKa 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 kH2O with the pKa for each arylamine substrate and water displays a linear free-energy relationship in the pKa range from -1.7 (H2O) to 4.67 (PABA), with a slope of βnuc = 0.80 ± 0.1. However, a further increase of the pKa from 4.67 (PABA) to 5.32 (anisidine) resulted in a 2.5-fold decrease in the k4 value. Analysis of the pH-kcat/KPABA profile revealed a pKa of 5.52 ± 0.14 and a solvent kinetic isotope effect (SKIE) of 2.01 ± 0.04 on kcat/KPABA. Normal solvent isotope effects of 4.8 ± 0.1, 3.1 ± 0.1, and 3.2 ± 0.1 on the k cat/Kb 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 pKa values ≤5.5 to deprotonation of a tetrahedral intermediate for arylamines with pKa 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.

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