Characterization of hamster recombinant monomorphic and polymorphic arylamine N-acetyltransferases: bioactivation and mechanism-based inactivation studies with N-hydroxy-2-acetylaminofluorene.
The purified hamster recombinant arylamine N-acetyltransferases (NATs), rNAT1-9 and rNAT2-70D, were characterized for their capabilities to bioactivate N-hydroxy-2-acetylaminofluorene (N-OH-AAF) to DNA binding reactants and for their relative susceptibilities to mechanism-based inactivation by N-OH-AAF. The rate of DNA adduct formation resulting from rNAT1-9 bioactivation of [14C]N-OH-AAF was more than 30 times greater than that of rNAT2-70D-catalyzed bioactivation of [14C]N-OH-AAF. This result is consistent with substrate specificity data indicating that N-OH-AAF is a much better acetyl donor for hamster NAT1 than NAT2. Previous studies indicated that N-OH-AAF is a mechanism-based inactivator of hamster and rat NAT1. In the presence of N-OH-AAF, both rNAT1-9 and rNAT2-70D underwent irreversible, time-dependent inactivation that exhibited pseudo first-order kinetics and was saturable at higher N-OH-AAF concentrations. The enzymes were partially protected from inactivation by the presence of cofactor and substrates. The limiting rate constants (ki) and dissociation constants (Ki) for inactivation by N-OH-AAF were determined. The second-order rate constants (ki/KI) were 22.1 min-1 mM-1 for rNAT1-9 and 1.0 min-l mM-1 for rNAT2-70D, indicating that rNAT1-9 is approximately 20 times more susceptible than rNAT2-70D to inactivation by N-OH-AAF. The kinetic parameters for rNAT1-9 were nearly identical to values previously reported for partially purified hamster NAT1. Partition ratios were 504 for inactivation of rNAT1-9 by N-OH-AAF and 137 for inactivation of rNAT2-70D. Thus, a turnover of almost 4 times as many N-OH-AAF molecules is required to inactivate each molecule of rNAT1-9 than is needed to inactivate rNAT2-70D. The partition ratio data are consistent with the finding that rNAT1-9 catalyzes a higher rate of DNA adduct formation by N-OH-AAF than rNAT2-70D. The combined results indicate that the recombinant enzymes are catalytically and functionally identical to hamster NATs and, therefore, will be a useful resource for studies requiring purified NATs.
Department of Pharmacology, University of Minnesota, Minneapolis 55455, USA.
K R
KR
Sticha
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Bergstrom
C R
CR
Wagner
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Hanna
D000818:Animals; D001191:Arylamine N-Acetyltransferase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D006224:Cricetinae; D018736:DNA Adducts; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006881:Hydroxyacetylaminofluorene / Q000494:pharmacology*; D007700:Kinetics; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D018736:DNA Adducts; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D006881:Hydroxyacetylaminofluorene; D001191:Arylamine N-Acetyltransferase