Studies on human arylamine N-acetyltransferases

In this thesis, recombinant human NAT2 was successfully overexpressed and purified to homogeneity; the substrate specificities and molecular interactions of environmental arylamines with human NAT1 and NAT2 were characterized; the in vitro and intracellular inactivation of human NAT1 and NAT2 by the nitrosoarene and N-arylhydroxamic acid metabolites of toxic and carcinogenic arylamines was investigated.;The kinetic specificity constants (kcat/K m) for N-acetylation of arylamine environmental contaminants were characterized for human NAT1 and NAT2. The dramatic effects of small alkyl substituents on the relative abilities of NAT1 and NAT2 to acetylate substituted anilines was reflected by the 1000-fold difference in the NAT1/NAT2 ratio of the specificity constants for monosubstituted and disubstituted alkylanilines containing methyl and ethyl ring substituents.;Arylamines and their N-hydroxylation products, N-arylhydroxylamines, can undergo oxidation to form nitrosoarenes in vivo. We investigated the inactivation of human NAT1 by the nitrosoarene metabolites of four environmental arylamines in vitro and in human cells. 4-Nitrosobiphenyl (4-NO-BP) and 2-nitrosofluorene (2-NO-F), which are nitroso metabolites of arylamines that are readily N-acetylated by NAT1, were found to be potent inactivators of human NAT1. 4-NO-BP is an affinity label for NAT1 (kinact/K I = 59,200 M-1s-1), whereas 2-NO-F inactivates NAT1 through an apparent bimolecular process (k2 = 34,500 M-1s-1). Glutathione (GSH) afforded only partial protection of NAT1 from inactivation by the two nitrosoarenes in vitro. Nitrosobenzene (NO-B) and 2-nitrosotoluene (2-NO-T), which are nitroso metabolites of arylamines that are less readily acetylated by NAT1, were much weaker inhibitors of NAT1. Treatment of HeLa cells with 4-NO-BP (10 muM) for 15 minutes and 60 minutes caused 39% and 58% losses of NAT1 activity, respectively, without causing a decrease in either glyceraldehyde phosphate dehydrogenase (GAPDH) or glutathione reductase (GR) activities.;It was also demonstrated that the nitrosoarene metabolites of arylamines that are efficiently N-acetylated by NAT2 are potent inactivators of NAT2 in vitro and in human cells. The second order rate constants for inactivation of NAT2 by 4-NO-BP and 2-NO-F were 80,400 M-1s-1 and 50,500 M-1s-1, respectively; the values for NO-B and 2-NO-T were 14 M-1s-1 and 16 M -1s-1. Treatment of HeLa cells with 4-NO-BP (5 muM) for 1 h caused a 23% reduction in NAT2 activity, and exposure to 2-NO-F (2.5 muM) for 1 h caused a 22% loss of NAT2 activity, without inhibiting GAPDH and GR activities. Therefore, HeLa intracellular NAT2 is less susceptible to the effects of the lower concentrations of the two nitrosoarenes than is NAT1. It is concluded that NAT1 and NAT2 are intracellular targets of the nitrosoarene metabolites of 4-aminobiphenyl and 2-aminofluorene. Low concentrations of nitrosoarenes may cause a loss of NAT1 and NAT2 activities and impair a key detoxification pathway.;We hypothesized that the relative stabilities of acetyl-enzyme intermediates influence the susceptibilities of NATs to inactivation by N-arylhydroxamic acids. The second order rate constant for inactivation of human NAT2 by N-OH-AAF was 459 M-1s-1, which was 8-fold greater than that for NAT1. Mass spectrometric analysis of both NATs after treatment with N-OH-AAF revealed that the principal adducts were sulfinamide conjugates of Cys68. Kinetic analysis revealed that the hydrolysis rate of acetyl-NAT2 was 4.7-fold greater than that of acetyl-NAT1. Thus, the more rapid inactivation of NAT2 was facilitated by the rapid hydrolysis of the Cys68 thioacetyl ester to free the Cys68 thiol group for reaction with 2-NOF. The hypothesis was further supported by the results from inactivation of human NATs by N-OH-4-AABP. Approximately 10-fold greater concentrations and longer incubation times were required for the N-arylhydroxamic acids to produce effects on intracellular NATs than were required for 2-NO-F and 4-NO-BP. HeLa NAT1 is more susceptible to the effects of the N-arylhydroxamic acids than NAT2. (Abstract shortened by UMI.)...