Characterization of hamster recombinant monomorphic and polymorphic arylamine N-acetyltransferases. Bioactivation and mechanism-based inactivation studies with n-hydroxy-2-acetylaminofluorene

Kristina R.K. Sticha, Carl P. Bergstrom, Carston R Wagner, Patrick E. Hanna

Research output: Contribution to journalArticle

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Abstract

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 (k(i)) and dissociation constants (K(I)) for inactivation by N-OH-AAF were determined. The second-order rate constants (k(i)/K(I)) were 22.1 min-1 mM-1 for rNAT1-9 and 1.0 min-1 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. Copyright (C) 1998 Elsevier Science, Inc.

Original languageEnglish (US)
Pages (from-to)47-59
Number of pages13
JournalBiochemical Pharmacology
Volume56
Issue number1
DOIs
StatePublished - Jul 1 1998

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Arylamine N-Acetyltransferase
2-Acetylaminofluorene
Cricetinae
Acetyltransferases
DNA Adducts
hydroxide ion
Rate constants
Molecules
Substrates
Enzymes
Substrate Specificity

Keywords

  • Arylamine N-acetyltransferase
  • Bioactivation
  • Mechanism-based inactivation
  • N-hydroxy-2-acetylaminofluorene
  • Recombinant NATs

Cite this

Characterization of hamster recombinant monomorphic and polymorphic arylamine N-acetyltransferases. Bioactivation and mechanism-based inactivation studies with n-hydroxy-2-acetylaminofluorene. / Sticha, Kristina R.K.; Bergstrom, Carl P.; Wagner, Carston R; Hanna, Patrick E.

In: Biochemical Pharmacology, Vol. 56, No. 1, 01.07.1998, p. 47-59.

Research output: Contribution to journalArticle

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abstract = "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 (k(i)) and dissociation constants (K(I)) for inactivation by N-OH-AAF were determined. The second-order rate constants (k(i)/K(I)) were 22.1 min-1 mM-1 for rNAT1-9 and 1.0 min-1 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. Copyright (C) 1998 Elsevier Science, Inc.",
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T1 - Characterization of hamster recombinant monomorphic and polymorphic arylamine N-acetyltransferases. Bioactivation and mechanism-based inactivation studies with n-hydroxy-2-acetylaminofluorene

AU - Sticha, Kristina R.K.

AU - Bergstrom, Carl P.

AU - Wagner, Carston R

AU - Hanna, Patrick E.

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N2 - 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 (k(i)) and dissociation constants (K(I)) for inactivation by N-OH-AAF were determined. The second-order rate constants (k(i)/K(I)) were 22.1 min-1 mM-1 for rNAT1-9 and 1.0 min-1 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. Copyright (C) 1998 Elsevier Science, Inc.

AB - 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 (k(i)) and dissociation constants (K(I)) for inactivation by N-OH-AAF were determined. The second-order rate constants (k(i)/K(I)) were 22.1 min-1 mM-1 for rNAT1-9 and 1.0 min-1 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. Copyright (C) 1998 Elsevier Science, Inc.

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KW - Bioactivation

KW - Mechanism-based inactivation

KW - N-hydroxy-2-acetylaminofluorene

KW - Recombinant NATs

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