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 i nactivation by N-OH-AAF. The rate of DNA adduct formation resulting fr om rNAT1-9 bioactivation of [C-14]N-OH-AAF was more than 30 times grea ter than that of rNAT2-70D-catalyzed bioactivation of [C-14]N-OH-AAF. This result is consistent with substrate specificity data indicating t hat 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 inactiv ator of hamster and rat NAT1. In the presence of N-OH-AAF, both rNAT1- 9 and rNAT2-70D underwent irreversible, time-dependent inactivation th at exhibited pseudo first-order kinetics and was saturable at higher N -OH-AAF concentrations. The enzymes were partially protected from inac tivation 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 susceptibl e than rNAT2-70D to inactivation by N-OH-AAF. The kinetic parameters f or rNAT1-9 were nearly identical to values previously reported for par tially purified hamster NAT1. Partition ratios were 504 for inactivati on 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 t o inactivate each molecule of rNAT1-9 than is needed to inactivate rNA T2-70D. The partition ratio data are consistent with the finding that rNAT1-9 catalyzes a higher rate of DNA adduct formation by N-OH-AAF th an rNAT2-70D. The combined results indicate that the recombinant enzym es are catalytically and functionally identical to hamster NATs and, t herefore, will be a useful resource for studies requiring purified NAT s. BIOCHEM PHARMACOL 56;1:47-59, 1998. (C) 1998 Elsevier Science Inc.