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Bioactivation of N-arylhydroxamic acids by rat hepatic N-acetyltransferase. Detection of multiple enzyme forms by
Abstract:
Enzymatic N,O-acyltransfer of carcinogenic N-arylhydroxamic acids such as N-hydroxy-2-acetylaminofluorene (N-OH-AAF) results in the production of reactive electrophiles that can bond covalently with nucleophiles and also can cause inactivation of acyltransferase activity in a mechanism-based manner. Incubation of partially purified rat hepatic N-acetyltransferases (NAT) with N-OH-AAF resulted in extensive inactivation of N-OH-AAF/4-aminoazobenzene (AAB) N,N-acetyltransferase and acetyl coenzyme A (AcCoA)/procainamide (PA) N-acetyltransferase activities, whereas AcCoA/p-aminobenzoic acid (PABA) N-acetyltransferase activity was inhibited only slightly. Affinity chromatography with Sepharose 6B 2-aminofluorene (2-AF) resulted in the separation of two NAT activities. NAT I primarily catalyzed the AcCoA-dependent acetylation of PABA; NAT II catalyzed, N,N-acetyltransfer (N-OH-AAF/AAB), AcCoA/PA N-acetyltransfer and N-OH-AAF N,O-acyltransfer (AHAT) activities. Most of the AcCoA/2-AF N-acetyltransferase activity eluted in the NAT II fraction. Results of inactivation experiments with N-OH-AAF and the NAT II fractions suggested that one NAT isozyme was responsible for catalyzing the N-OH-AAF/AAB, AcCoA/PA and N,O-acyltransfer reactions and that inactivation of NAT II correlated with the extent of covalent binding to protein. Further purification of the NAT II fractions by chromatofocusing resulted in a 1300-fold purification of the N-OH-AAF/AAB activity and the coelution of N-OH-AAF/AAB, AcCoA/PA and N,O-acyltransferase activities. These studies indicate that N,O-acyltransfer, arylhydroxamic acid-dependent N-acetylation of arylamines (N,N-acetyltransfer), and AcCoA-dependent N-acetylation of PA may be catalyzed by a common enzyme in rat liver, whereas a second enzyme is responsible for the AcCoA-dependent N-acetylation of PABA.
Insights
Carcinogenic N-arylhydroxamic acids inactivate N-acetyltransferases (NATs) during enzymatic N,O-acyltransfer. Rat liver studies reveal one NAT isozyme catalyzes multiple reactions, including N,O-acyltransfer and N-acetylation.
Area of Science:
- Biochemistry
- Enzymology
- Toxicology
Background:
- Enzymatic N,O-acyltransfer of N-arylhydroxamic acids generates reactive electrophiles.
- These electrophiles can covalently bind to nucleophiles and inactivate acyltransferase activity.
- N-arylhydroxamic acids, such as N-hydroxy-2-acetylaminofluorene (N-OH-AAF), are carcinogenic compounds.
Purpose of the Study:
- To investigate the mechanism-based inactivation of rat hepatic N-acetyltransferases (NATs) by N-OH-AAF.
- To characterize the different NAT activities and identify the specific isozymes responsible for various reactions.
- To elucidate the role of NAT isozymes in N,O-acyltransfer and N-acetylation reactions.
Main Methods:
- Partial purification of rat hepatic N-acetyltransferases (NATs).
- Enzyme activity assays including N,N-acetyltransfer, AcCoA/procainamide (PA) N-acetyltransfer, and N-OH-AAF N,O-acyltransfer (AHAT).
- Affinity chromatography using Sepharose 6B 2-aminofluorene (2-AF) and chromatofocusing for enzyme purification.
Main Results:
- N-OH-AAF extensively inactivated N-OH-AAF/4-aminoazobenzene (AAB) N,N-acetyltransferase and AcCoA/PA N-acetyltransferase activities, with minimal inhibition of AcCoA/p-aminobenzoic acid (PABA) N-acetyltransferase.
- Affinity chromatography separated NAT into two fractions: NAT I (primarily AcCoA/PABA acetylation) and NAT II (N-OH-AAF/AAB N,N-acetyltransfer, AcCoA/PA N-acetyltransfer, and AHAT).
- Further purification of NAT II demonstrated coelution of N-OH-AAF/AAB, AcCoA/PA, and N,O-acyltransferase activities, suggesting a single isozyme catalyzes these reactions.
Conclusions:
- Rat liver possesses at least two NAT isozymes with distinct substrate specificities.
- One NAT isozyme appears responsible for N,O-acyltransfer, N,N-acetyltransfer of arylamines, and AcCoA-dependent N-acetylation of PA.
- A second NAT isozyme is responsible for AcCoA-dependent N-acetylation of PABA.