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The mercapturic acid pathway is a major route for the biotransformation of xenobiotic and endobiotic electrophilic compounds and their metabolites. Mercapturic acids (N-acetyl-l-cysteine S-conjugates) are formed by the sequential action of the glutathione transferases, γ-glutamyltransferases, dipeptidases, and cysteine S-conjugate N-acetyltransferase to yield glutathione S-conjugates, l-cysteinylglycine S-conjugates, l-cysteine S-conjugates, and mercapturic acids; these metabolites constitute a "mercapturomic" profile. Aminoacylases catalyze the hydrolysis of mercapturic acids to form cysteine S-conjugates. Several renal transport systems facilitate the urinary elimination of mercapturic acids; urinary mercapturic acids may serve as biomarkers for exposure to chemicals. Although mercapturic acid formation and elimination is a detoxication reaction, l-cysteine S-conjugates may undergo bioactivation by cysteine S-conjugate β-lyase. Moreover, some l-cysteine S-conjugates, particularly l-cysteinyl-leukotrienes, exert significant pathophysiological effects. Finally, some enzymes of the mercapturic acid pathway are described as the so-called "moonlighting proteins," catalytic proteins that exert multiple biochemical or biophysical functions apart from catalysis.

Arylamine N-acetyltransferases (NATs) play an important role in both the detoxification of arylamine and hydrazine drugs and the activation of arylamine carcinogens. Because the catalytic triad, Cys-His-Asp, of mammalian NATs has been shown to be essential for maintaining protein stability, rendering it impossible to assess alterations of the triad on catalysis, we explored the impact of the highly conserved proximal residue, Tyr190, which forms a direct hydrogen bond interaction with one of the triad residues, Asp122, as well as a potential pi-pi stacking interaction with the active site His107. The replacement of hamster NAT2 Tyr190 by either Phe, Ile or Ala was well tolerated and did not result in significant alterations in the overall fold of the protein. Nevertheless, stopped-flow and steady-state kinetic analysis revealed that Tyr190 was critical for maximizing the acetylation rate of NAT2 and the transacetylation rate of p-aminobenzoic acid when compared with the wild-type. Tyr190 was also shown to play an important role in determining the pKa of the active site Cys during acetylation, as well as the pH versus the rate profile for transacetylation. We hypothesized that the pH dependence was associated with global changes in the active site structure, which was revealed by the superposition of [1H, 15N] heteronuclear single quantum coherence spectra for the wild-type and Y190A. These results suggest that NAT2 catalytic efficiency is partially governed by the ability of Tyr190 to mediate the collective impact of multiple side chains on the electrostatic potential and local conformation of the active site.

Arylamines (ArNH 2) are common environmental contaminants, some of which are confirmed risk factors for cancer. Biotransformation of the amino group of arylamines involves competing pathways of oxidation and N-acetylation. Nitrosoarenes, which are products of the oxidation pathway, are electrophiles that react with cellular thiols to form sulfinamide adducts. The arylamine N-acetyltransferases, NAT1 and NAT2, catalyze N-acetylation of arylamines and play central roles in their detoxification. We hypothesized that 4-nitrosobiphenyl (4-NO-BP) and 2-nitrosofluorene (2-NO-F), which are nitroso metabolites of arylamines that are readily N-acetylated by NAT1, would be potent inactivators of NAT1 and that nitrosobenzene (NO-B) and 2-nitrosotoluene (2-NO-T), which are nitroso metabolites of arylamines that are less readily acetylated by NAT1, would be less effective inactivators. The second order rate constants for inactivation of NAT1 by 4-NO-BP and 2-NO-F were 59200 and 34500 M (-1) s (-1), respectively; the values for NO-B and 2-NO-T were 25 and 23 M (-1) s (-1). Densitometry quantification and comparisons of specific activities with those of homogeneous recombinant NAT1 showed that NAT1 constitutes approximately 0.002% of cytosolic protein in HeLa cells. Treatment of HeLa cells with 4-NO-BP (2.5 microM) for 1 h caused a 40% reduction in NAT1 activity, and 4-NO-BP (10 microM) caused a 50% loss of NAT1 activity within 30 min without affecting either glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or glutathione reductase (GR) activities. 2-NO-F (1 microM) inhibited HeLa cell NAT1 activity by 36% in 1 h, and a 10 microM concentration of 2-NO-F reduced NAT1 activity by 70% in 30 min without inhibiting GAPDH or GR. Mass spectrometric analysis of NAT1 from HeLa cells in which NAT1 was overexpressed showed that treatment of the cells with 4-NO-BP resulted in sulfinamide adduct formation. These results indicated that exposure to low concentrations of nitrosoarenes may lead to a loss of NAT1 activity, thereby compromising a critical detoxification process.

Arylamine N-acetyltransferases (NATs) are phase II xenobiotic metabolism enzymes that catalyze the detoxification of arylamines by N-acetylation and the bioactivation of N-arylhydroxylamines by O-acetylation. Endogenous and recombinant mammalian NATs with high specific activities are difficult to obtain in substantial quantities and in a state of homogeneity. This paper describes the overexpression of human wild-type NAT2 as a dihydrofolate reductase fusion protein containing a TEV protease-sensitive linker. Treatment of the partially purified fusion protein with TEV protease, followed by chromatographic purification, afforded 2.8 mg of homogeneous NAT2 from 2 L of cell culture. The kinetic specificity constants ( k cat/ K m) for N-acetylation of arylamine environmental contaminants, some of which are associated with bladder cancer risk, were determined with NAT2 and NAT1. The NAT1/NAT2 ratio of the specificity constants varied almost 1000-fold for monosubstituted and disubstituted alkylanilines containing methyl and ethyl ring substituents. 2-Alkyl substituents depressed N-acetylation rates but were more detrimental to catalysis by NAT1 than by NAT2. 3-Alkyl groups caused substrates to be preferentially N-acetylated by NAT2, and both 4-methyl- and 4-ethylaniline were better substrates for NAT1 than NAT2. NMR-based models were used to analyze the NAT binding site interactions of the alkylanilines. The selectivity of NAT1 for acetylation of 4-alkylanilines appears to be due to binding of the substituents to V216, which is replaced by S216 in NAT2. The contribution of 3-alkyl substituents to NAT2 substrate selectivity is attributed to multiple bonding interactions with F93, whereas a single bonding interaction occurs with V93 in NAT1. Unfavorable steric clashes between 2-methyl substituents and F125 of NAT1 may account for the selective NAT2-mediated N-acetylation of 2-alkylanilines; F125 is replaced by S125 in NAT2. These results provide insight into the structural basis for the substrate specificity of two NATs that play major roles in the biotransformation of genotoxic environmental arylamines.

Arylamine N-acetyltransferase I (NAT1) is a phase II enzyme that acetylates a wide range of arylamine and hydrazine substrates. The NAT1 gene is located on chromosome 8 and shares homology to NAT genes found in most mammalian species. Gene expression occurs from at least two promoters and a number of tissue-specific transcripts have been identified. The gene is polymorphic with most mutations identified to date producing an unstable protein that is subject to polyubiquitination. The NAT1 protein contains a catalytic triad similar to a number of cysteine proteases and transglutaminases. NAT1 is widely distributed in the body, but the only endogenous substrate identified to date is the folate catabolite p-aminobenzoylglutamate. Recent links between NAT1 genotypes and susceptibility to spina bifida suggests that the enzyme has an important role in folate homeostasis.

Arylamine N-acetyltransferases (NATs) catalyze an acetyl group transfer from AcCoA to primary arylamines, hydrazines, and hydrazides and play a very important role in the metabolism and bioactivation of drugs, carcinogens, and other xenobiotics. The reaction follows a ping-pong bi-bi mechanism. Structure analysis of bacterial NATs revealed a Cys-His-Asp catalytic triad that is strictly conserved in all known NATs. Previously, we have demonstrated by kinetic and isotope effect studies that acetylation of the hamster NAT2 is dependent on a thiolate-imidazolium ion pair (Cys-S(-)-His-ImH(+)) and not a general acid-base catalysis. In addition, we established that, after formation of the acetylated enzyme intermediate, the active-site imidazole, His-107, is likely deprotonated at physiological pH. In this paper, we report steady-state kinetic studies of NAT2 with two acetyl donors, acetyl coenzyme A (AcCoA) and p-nitrophenyl acetate (PNPA), and four arylamine substrates. The pH dependence of k(cat)/K(AcCoA) exhibited two inflection points at 5.32 +/- 0.13 and 8.48 +/- 0.24, respectively. The pK(a) at 5.32 is virtually identical with the previously reported pK(a) of 5.2 for enzyme acetylation, reaffirming that the first half of the reaction is catalyzed by a thiolate-imidazolium ion pair in the active site. The inflection point at 8.48 indicates that a pH-sensitive group on NAT2 is involved in AcCoA binding. A Brønsted plot constructed by the correlation of log k(4) and log k(H)2(O) with the pK(a) for each arylamine substrate and water displays a linear free-energy relationship in the pK(a) range from -1.7 (H(2)O) to 4.67 (PABA), with a slope of beta(nuc) = 0.80 +/- 0.1. However, a further increase of the pK(a) from 4.67 (PABA) to 5.32 (anisidine) resulted in a 2.5-fold decrease in the k(4) value. Analysis of the pH-k(cat)/K(PABA) profile revealed a pK(a) of 5.52 +/- 0.14 and a solvent kinetic isotope effect (SKIE) of 2.01 +/- 0.04 on k(cat)/K(PABA). Normal solvent isotope effects of 4.8 +/- 0.1, 3.1 +/- 0.1, and 3.2 +/- 0.1 on the k(cat)/K(b) for anisidine, pABglu, and PNA, respectively, were also determined. These observations are consistent with a deacetylation mechanism dominated by nucleophilic attack of the thiol ester for arylamines with pK(a) valuesor=5.5 to deprotonation of a tetrahedral intermediate for arylamines with pK(a) valuesor=5.5. The general base is likely His-107 because the His-107 to Gln and Asn mutants were found to be devoid of catalytic activity. In contrast, an increase in pH-dependent hydrolysis of the acetylated enzyme was not observed over a pH range of 5.2-7.5. On the basis of these observations, a catalytic mechanism for the acetylation of arylamines by NAT2 is proposed.

Human arylamine N-acetyltransferase 1 (NAT1) has been overexpressed in E. coli as a mutant dihydrofolic acid reductase (DHFR) fusion protein with a thrombin sensitive linker. An initial DEAE anion-exchange chromatography resulted in partial purification of the fusion protein. The fusion protein was cleaved with thrombin, and human rNAT1 was purified with a second DEAE column. A total of 8 mg of human rNAT1 from 2 1 of cell culture was purified to homogeneity with this methodology. Arylamine substrate specificities were determined for human rNATI and hamster rNAT2. With both NATs, the second order rate constants (k(cat)/ Kmb) for p-aminobenzoic acid (PABA) and 2-aminofluorene (2-AF) were several thousand-fold higher than those for procainamide (PA), consistent with the expected substrate specificities of the enzymes. However, p-aminosalicylic acid (PAS), previously reported to be a human NAT1 and hamster NAT2 selective substrate, exhibits 20-fold higher specificity for hamster rNAT2 (k(cat)/Kmb 3410 microM(-1) s(-1)) than for human rNAT1 (k(cat)/Kmb 169.4 microM(-1) s(-1)). p-aminobenzoyl-glutamic acid (pABglu) was acetylated 10-fold more efficiently by human rNAT1 than by hamster rNAT2. Inhibition studies of human rNAT1 and hamster rNAT2 revealed that folic acid and methotrexate (MTX) are competitive inhibitors of both the unacetylated and acetylated forms of the enzymes, with K(I) values in 50 - 300 micro range. Dihydrofolic acid (DHF) was a much poorer inhibitor of human rNAT1 than of hamster rNAT2. The combined results demonstrate that human rNAT1 and hamster rNAT2 have similar but distinct kinetic properties with certain substrates, and suggest that folic acid, at least in the non-polyglutamate form, may not have an effect on human NAT1 activity in vivo.

Arylamine N-acetyltransferases (NAT; EC 2.3.1.5) catalyse the transfer of acetyl groups from acetylCoA to xenobiotics, including drugs and carcinogens. The enzyme is found extensively in both eukaryotes and prokaryotes, yet the endogenous roles of NATs are still unclear. In order to study the properties of eukaryotic NATs, high-throughput substrate and inhibitor screens have been developed using pure soluble recombinant Syrian hamster NAT2 (shNAT2) protein. The assay can be used with a wide range of compounds and was used to determine substrate specificity of shNAT2. We describe the expression and characterisation of shNAT2 and also purified recombinant human NAT1 and NAT2, including the use of the assay to explore the substrate specificities of each of the enzymes. Hamster NAT2 has similar substrate specificity to human NAT1, acetylating para-aminobenzoate but not arylhydrazine and hydralazine compounds. The overlapping but distinct substrate-specific activity profiles of human NAT1 and NAT2 were clearly observed from the screen. Naturally occurring compounds were tested as substrates or inhibitors of shNAT2 and succinylCoA was found to be a potent inhibitor of shNAT2.

Arylamine N-acetyltransferases (NATs) catalyze an acetyl group transfer from acetyl coenzyme A (AcCoA) to arylamines, hydrazines, and their N-hydroxylated arylamine metabolites. The recently determined three-dimensional structures of prokaryotic NATs have revealed a cysteine protease-like Cys-His-Asp catalytic triad, which resides in a deep and hydrophobic pocket. This catalytic triad is strictly conserved across all known NATs, including hamster NAT2 (Cys-68, His-107, and Asp-122). Treatment of NAT2 with either iodoacetamide (IAM) or bromoacetamide (BAM) at neutral pH rapidly inactivated the enzyme with second-order rate constants of 802.7 +/- 4.0 and 426.9 +/- 21.0 M(-1) s(-1), respectively. MALDI-TOF and ESI mass spectral analysis established that Cys-68 is the only site of alkylation by IAM. Unlike the case for cysteine proteases, no significant inactivation was observed with either iodoacetic acid (IAA) or bromoacetic acid (BAA). Pre-steady state and steady state kinetic analysis with p-nitrophenyl acetate (PNPA) and NAT2 revealed a single-exponential curve for the acetylation step with a second-order rate constant of (1.4 +/- 0.05) x 10(5) M(-1) s(-1), followed by a slow linear rate of (7.85 +/- 0.65) x 10(-3) s(-1) for the deacetylation step. Studies of the pH dependence of the rate of inactivation with IAM and the rate of acetylation with PNPA revealed similar pK(a)(1) values of 5.23 +/- 0.09 and 5.16 +/- 0.04, respectively, and pK(a)(2) values of 6.95 +/- 0.27 and 6.79 +/- 0.25, respectively. Both rates reached their maximum values at pH 6.4 and decreased by only 30% at pH 9.0. Kinetic studies in the presence of D(2)O revealed a large inverse solvent isotope effect on both inactivation and acetylation of NAT2 [k(H)(inact)/k(D)(inact) = 0.65 +/- 0.02 and (k(2)/K(m)(acetyl))(H)/(k(2)/K(m)(acetyl))(D) = 0.60 +/- 0.03], which were found to be identical to the fractionation factors (Phi) derived from proton inventory studies of the rate of acetylation at pL 6.4 and 8.0. Substitution of the catalytic triad Asp-122 with either alanine or asparagine resulted in the complete loss of protein structural integrity and catalytic activity. From these results, it can be concluded that the catalytic mechanism of NAT2 depends on the formation of a thiolate-imidazolium ion pair (Cys-S(-)-His-ImH(+)). However, in contrast to the case with cysteine proteases, a pH-dependent protein conformational change is likely responsible for the second pK(a), and not deprotonation of the thiolate-imidazolium ion. In addition, substitutions of the triad aspartate are not tolerated. The enzyme appears, therefore, to be engineered to rapidly form a stable acetylated species poised to react with an arylamine substrate.

Arylamine N-acetyltransferases (NATs) catalyze a variety of biotransformation reactions, including N-acetylation of arylamines and O-acetylation of arylhydroxylamines. Chemical modification of hamster recombinant NAT2 with 2-(bromoacetylamino)fluorene (Br-AAF) and bromoacetanilide revealed that Br-AAF is an affinity label for the enzyme whereas bromoacetanilide inactivates NAT2 through a bimolecular alkylation process. Electrospray ionization quadrupole time-of-flight mass spectrometry analysis of Br-AAF-treated NAT2 showed that a single molecule of 2-acetylaminofluorene had been adducted. Peptide sequencing with tandem mass spectrometry identified the catalytically essential Cys68 as the alkylated amino acid. Br-AAF exhibits similar affinity for hamster NAT1 and NAT2, but is a more effective inactivator of NAT1 because, subsequent to the formation of a reversible enzyme-Br-AAF complex, the rate of alkylation of NAT1 is greater than the rate of alkylation of NAT2. Bromoacetanilide alkylates Cys68 and, to a lesser extent, Cys237 of NAT2; it does not exhibit significant selectivity for either NAT1 or NAT2.

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.

BACKGROUND Clinical symptoms of prostatitis, prostatodynia, and benign prostatic hyperplasia are relieved by the pollen extract cernilton, and the water-soluble fraction of this extract selectively inhibits growth of some prostate cancer cells. A cyclic hydroxamic acid, DIBOA, has been isolated from this extract and mimics its cell growth-inhibitory properties, but the specificity of DIBOA for inhibition of prostate cell growth has not been reported. METHODS The in vitro growth inhibitory effects of DIBOA and nine structurally related compounds on DU-145 prostate cancer cells, MCF-7 breast cancer cells, and COS-7 monkey kidney cells were determined by treatment of the cells with various concentrations of the compounds for 2–6 days. RESULTS The compounds exhibited a wide range of potencies, but none of them exhibited selective inhibition of DU-145 cell growth. MCF-7 cells were more sensitive to DIBOA than either DU-145 cells or COS-7 cells. 3,4-dihydroquinoline-2(1H)-one, compound (4), and 1-hydroxy-6-chloro-3,4-dihydroquinolin-2(1H)-one, compound (7), selectively inhibited MCF-7 cell growth at a concentration of 10 μg/ml. 1-hydroxy-3,4-dihydroquinolin-2(1H)-one, compound (3), and compound 7 were the most potent inhibitors of DU-145 cell growth. Treatment of DU-145 cells with 3 (100 μg/ml) substantially decreased the number of viable cells within 2 days, and no viable cells remained in the culture by day 4. CONCLUSIONS It is unlikely that DIBOA, compound (1), is responsible for the selective growth inhibition of prostate cancer cells by the water-soluble fraction of the pollen extract cernilton. Cell morphology results indicate that the growth-inhibitory effects of DIBOA and structurally related agents on DU-145 cells are due to their ability to cause cell death. Prostate 34:92–99, 1998. © 1998 Wiley-Liss, Inc.

N-Acetylation is a major pathway in the metabolism of hydrazine and arylamine drugs, and has been associated with carcinogen bioactivation. Monomorphic hamster liver N -acetyltransferase (NAT1) cDNA was cloned from hamster liver cells by reverse transcriptase-coupled polymerase chain reaction. The determined nucleotide sequence was identical to that reported for NAT1 . The NAT1 coding region was subcloned into the pG1 yeast expression vector, but cell extracts provided only transient acetyltransferase activity. In addition, cDNA was subcloned into the expression vectors pFLAG-ATS and pFLAG-MAC, The latter vectors encoded a tac promoter and appended a low-molecular weight (1 kDa) hydrophilic FLAG; marker peptide to the amino terminus of NAT1. Unexpectedly, periplasmic export of FLAG ATS -NAT1 by the ompA signal peptide of pFLAG-ATS proved to be detrimental to enzyme activity. High acetyltransferase activity, however, was obtained when the fusion protein was expressed in the cytosol. Enzyme purified to homogeneity by immunoaffinity chromatography exhibited substrate specificity comparable to that of the hamster-derived protein.

The N-carbobenzyloxy derivative of 2,3-dioxindole (N-Cbz-isatin) (1) has been synthesized and shown to be a slow binding inhibitor of chymotrypsin. Compound 1 does not inhibit porcine pancreatic elastase. The N-tert-butoxycarbonyl derivative of 2,3-dioxindole (2) is a weak inhibitor of chymotrypsin and does not inhibit porcine pancreatic elastase.

N-Acetoxyarylamines are reactive metabolites that are produced from N-arylhydroxamic acids by N-arylhydroxamic acid N,O-acyltransferase and by the acetyl coenzyme A-dependent O-acetylation of N-arylhydroxylamines. Solvolytic decomposition of 7-acetyl-2-(N-acetoxyamino)fluorene (3) afforded 7-acetyl-2-aminofluorene (4) and 7,7'-diacetyl-2,2'-azofluorene (5). Solvolysis of 3 in the presence of 2'-deoxyguanosine resulted in formation of N-(deoxyguanosin-8-yl)-7-acetyl-2-aminofluorene (2), along with smaller yields of 4 and 5 than were obtained in the absence of 2'-deoxyguanosine. The inclusion of nitrene trapping agents, such as piperylene, triethyl phosphite, 1-chloro-4-nitrosobenzene, and oxygen, in the reaction medium with 3 and 2'-deoxyguanosine caused a reduction in the yields of 2, 4, and 5. Additionally, products that would be expected to be formed through reaction with an arylnitrene intermediate were obtained when triethyl phosphite, 1-chloro-4-nitrosobenzene, and oxygen were included in the incubations with 3 and 2'-deoxyguanosine. The results support the proposal that a nitrene intermediate is involved in the formation of 2, 4, and 5 from 3.

The N-carbobenzyloxy derivative of 2,3-dioxindole (N-Cbz-isatin) (1) has been synthesized and shown to be a slow binding inhibitor of chymotrypsin. Compound 1 does not inhibit porcine pancreatic elastase. The N-tert-butoxycarbonyl derivative of 2,3-dioxindole (2) is a weak inhibitor of chymotrypsin and does not inhibit porcine pancreatic elastase.

Human arylamine N-acetyltransferases (NATs) are expressed as two polymorphic isoforms, NAT1 and NAT2, that have toxicologically significant functions in the detoxification of xenobiotic arylamines by N-acetylation and in the bioactivation of N-arylhydroxylamines by O-acetylation. NAT1 also catalyzes the N-acetylation of 4-aminobenzoylglutamic acid, a product of folic acid degradation, and is associated with endogenous functions in embryonic development. On the basis of earlier studies with hamster NAT1, hamster NAT2, and human NAT1, we proposed that human NAT2 would be more susceptible than NAT1 to inactivation by N-arylhydroxamic acid metabolites of arylamines. Kinetic analyses of the inactivation of recombinant NAT1 and NAT2 by the N-arylhydroxamic acid, N-hydroxy-2-acetylaminofluorene (N-OH-AAF), as well as the inactivation of NAT2 by N-hydroxy-4-acetylaminobiphenyl (N-OH-4-AABP), resulted in second-order inactivation rate constants (k(inact)/K(I)) that were several fold greater for NAT2 than for NAT1. Mass spectrometric analysis showed that inactivation of NAT2 in the presence of the N-arylhydroxamic acids was due to formation of a sulfinamide adduct with Cys68. Treatment of HeLa cells with N-OH-4-AABP and N-OH-AAF revealed that the compounds were less potent inactivators of intracellular NAT activity than the corresponding nitrosoarenes, but unexpectedly, the hydroxamic acids caused a significantly greater loss of NAT1 activity than of NAT2 activity. Nitrosoarenes are the electrophilic products responsible for NAT inactivation upon interaction of the enzymes with N-arylhydroxamic acids, as well as being metabolic products of arylamine oxidation. Treatment of recombinant NAT2 with the nitrosoarenes, 4-nitrosobiphenyl (4-NO-BP) and 2-nitrosofluorene (2-NO-F), caused rapid and irreversible inactivation of the enzyme by sulfinamide adduct formation with Cys68, but the k(inact)/K(I) values for inactivation of recombinant NAT2 and NAT1 did not indicate significant selectivity for either isoform. Also, the IC(50) values for inactivation of HeLa cell cytosolic NAT1 and NAT2 by 4-NO-BP were similar, as were the IC(50) values obtained with 2-NO-F. Treatment of HeLa cells with low concentrations (1-10 microM) of either 4-NO-BP or 2-NO-F resulted in preferential and more rapid loss of NAT1 activity than NAT2 activity. Because of its wide distribution in human tissues and its early expression in developing tissues, the apparent high sensitivity of intracellular NAT1 to inactivation by reactive metabolites of environmental arylamines may have important toxicological consequences.

Enzymatic N,O-acyltransfer of carcinogenic N-arylhydroxamic acids such as N-droxy-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 mechanismbased 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 AcCoAdependent N-acetylation of PABA.

Arylamine N-acetyltransferases (NATs) catalyze the acetylation of arylamines, a key step in the detoxification of many carcinogens. The determinants of NAT substrate specificity are not known, yet this knowledge is required to understand why NAT enzymes acetylate some arylamines, but not others. Here, we use NMR spectroscopy and homology modeling to reveal the structural determinants of arylamine acetylation by NATs. In particular, by using chemical shift perturbation analysis, we have identified residues that play a critical role in substrate binding and catalysis. This study reveals why human NAT1 acetylates the sunscreen additive p-aminobenzoic acid and tobacco smoke carcinogen 4-aminobiphenyl, but not o-toluidine and other arylamines linked to bladder cancer. Our results represent an important step toward predicting whether arylamines present in new products can be detoxified by mammalian NATs.

Arylamine N-acetyltransferases (NAT1 and NAT2) acetylate and detoxify arylamine carcinogens. Humans harboring certain genetic variations within the NAT genes exhibit increased likelihood of developing various cancer types, especially urinary bladder cancer. Such DNA polymorphisms result in protein products with reduced cellular activity, which is proposed to be due to their constitutive ubiquitylation and enhanced proteasomal degradation. To identify the properties that lead to the reduced cellular activity of certain NAT variants, we introduced one such polymorphism into the human NAT1 ortholog hamster NAT2. The polymorphism chosen was human NAT1*17, which results in the replacement of R64 with a tryptophan residue, and we demonstrate this substitution to cause hamster NAT2 to be constitutively ubiquitylated. Biophysical characterization of the hamster NAT2 R64W variant revealed that its overall protein structure and thermostability are not compromised. In addition, we used steady-state kinetics experiments to demonstrate that the R64W mutation does not interfere with NAT catalysis in vitro. Hence, the constitutive ubiquitylation of this variant is not caused by its inability to be acetylated. Instead, we demonstrate this mutation to cause the hamster NAT2 protein to aggregate in vitro and in vivo. Importantly, we tested and confirmed that the R64W mutation also causes human NAT1 to aggregate in cultured cells. By using homology modeling, we demonstrate that R64 is located at a peripheral location, which provides an explanation for how the NAT protein structure is not significantly disturbed by its mutation to tryptophan. Altogether, we provide fundamental information on why humans harboring certain NAT variants exhibit reduced acetylation capabilities.

Arylamine N-acetyltransferases (NAT; EC 2.3.1.5) catalyse the transfer of acetyl groups from acetylCoA to xenobiotics, including drugs and carcinogens. The enzyme is found extensively in both eukaryotes and prokaryotes, yet the endogenous roles of NATs are still unclear. In order to study the properties of eukaryotic NATs, high-throughput substrate and inhibitor screens have been developed using pure soluble recombinant Syrian hamster NAT2 (shNAT2) protein. The assay can be used with a wide range of compounds and was used to determine substrate specificity of shNAT2. We describe the expression and characterisation of shNAT2 and also purified recombinant human NAT1 and NAT2, including the use of the assay to explore the substrate specificities of each of the enzymes. Hamster NAT2 has similar substrate specificity to human NAT1, acetylating para-aminobenzoate but not arylhydrazine and hydralazine compounds. The overlapping but distinct substrate-specific activity profiles of human NAT1 and NAT2 were clearly observed from the screen. Naturally occurring compounds were tested as substrates or inhibitors of shNAT2 and succinylCoA was found to be a potent inhibitor of shNAT2.

Arylamine N-acetyltransferases (NATs) are expressed in most mammalian tissues. NATs catalyze the N-acetylation of primary arylamines, the O-acetylation of N-arylhydroxylamines, and the N,O-transacetylation of N-arylhydroxamic acids. The latter two reactions result in formation of reactive, electrophilic N-acetoxyarylamines, which are considered to be the ultimate carcinogenic metabolites of certain environmental and dietary arylamines. Incubation of various N-(aryl)acetohydroxamic acids, such as N-hydroxy-2-acetylaminofluorene (N-OH-AAF) with hamster NAT1, results in time-dependent, concentration-dependent, and kinetically first-order irreversible inactivation of the enzyme. N-OH-AAF also causes in vivo inactivation of NAT1. The purpose of this research was to investigate the molecular mechanism of NAT1 inactivation by identifying the amino acid residues that undergo covalent modification upon NAT1-catalyzed bioactivation of N-OH-AAF and by characterizing the chemical structures of the adducts. Electrospray ionization quadrupole time-of-flight mass spectrometric analysis of NAT1 that had been incubated with N-OH-AAF revealed that the mass of the major adduct (+195 Da) was consistent with a (2-fluorenyl)sulfinamide modification. The major adduct underwent hydrolysis to yield a protein with a molecular mass that corresponded to a sulfinic acid-modified NAT1. Treatment of NAT1 with 2-nitrosofluorene resulted in a modification (+195 Da) that was identical in mass to that obtained with N-OH-AAF-inactivated enzyme. Matrix-assisted laser desorption-ionization quadrupole time-of flight tandem mass spectrometric (MALDI Q-TOF MS/MS) analysis revealed that the modified residue was the catalytically essential Cys68. MALDI Q-TOF MS/MS sequencing of peptides from protease digests of inactivated NAT1 also identified two minor adducts at Tyr17 and Tyr186, each of which was covalently conjugated with 2-aminofluorene. Thus, the mechanism of inactivation of NAT1 by N-OH-AAF involves NAT1-catalyzed deacetylation to afford N-hydroxy-2-aminofluorene, which after oxidative conversion to 2-nitrosofluorene, forms a sulfinamide adduct by reacting with Cys68. GSH had little effect on the inactivation of NAT1 by N-OH-AAF, although high concentrations of cysteine attenuated both the extent of inactivation and the sulfinamide adduct formation.

Kinetic analysis of the inactiviation of hamster NAT1 by 2-(bromoacetylamino)fluorene (Br-AAF) and bromoacetanilide revealed that Br-AAF is an active site directed affinity label whereas bromoacetanilide acts as a bimolecular alkylating agent. ESI MS analysis of NAT1 treated with Br-AAF showed that a single molecule of 2-acetylaminofluorene had been incorporated. Proteolysis with pepsin followed by sequencing of adducted peptides by ESI MS/MS identified the modified residue as the catalytically essential Cys-68. ESI Q-TOF MS analysis of NAT1 that had been treated with bromoacetanilide resulted in identification of a monoadducted protein as the primary product and a diadducted protein as a minor product. Pepsin digestion of bromoacetanilide-inactivated NAT1 and sequencing by ESI MS/MS identified Cys-68 as the primary site of adduct formation. Additional proteolysis of the bromoacetanilide-treated NAT1 led to the identification of a second modified peptide which was adducted at Cys-44. The data reveal substantial differences in the interactions of small hydrophobic alkylating reagents with hamster NAT1.

N-Acetyltransferases (NATs) are enzymes that catalyze the detoxification and/or bioactivation of a variety of xenobiotics. Rapid kinetic, biophysical, structural, and bioactivation studies on NATs require quantities of purified enzyme capable of being obtained only through recombinant DNA technology. This laboratory has previously developed a protein expression and purification system in which NATs are expressed as proteins fused to a FLAG octapeptide followed by a thrombin-cleavage site to allow liberation of the rNAT. Typically, however, only 0.5-1.5 mg of the recombinant NAT's could be readily purified in a single isolation sequence by immunoaffinity chromatography. Therefore, the expression system was modified by inserting the L54F dihydrofolate reductase (DHFR) mutant gene sequence between the FLAG octapeptide and the thrombin-cleavage site. Expression was carried out with TOPP3 Escherichia coli cells. The new purification methodology utilizes the unique pH dependence of binding to a methotrexate (MTX)-affinity column by the L54F DHFR mutant. Unfortunately, the affinity chromatography strategy did not work satisfactorily. Although the specific activity of the purified rNAT2 was comparable to that of NAT2 obtained from hamster tissue, only 3% of the activity was recovered. The apparent cause of the low recovery is the unanticipated irreversible binding of rNAT2 to MTX. Ion-exchange chromatography was investigated as an alternative purification method. An initial DEAE anion-exchange column resulted in partial purification of the fusion protein. The fusion protein was cleaved with thrombin and reapplied to a DEAE anion-exchange column. The second DEAE column resulted in not only the separation of rNAT2-70D from FLAG-L54F DHFR, but also the purification of rNAT2-70D to near homogeneity. Application of the nearly homogeneous rNAT2-70D to a gel-filtration column resulted in recovery of homogeneous protein. The ion-exchange method of purifying rNAT2-70D is inexpensive and simple and yields more than 8 mg of pure enzyme from 1 liter of cell culture.

Arylamine N-acetyltransferases (NATs) are cytosolic enzymes that play important roles in the detoxification and activation of xenobiotic arylamines and their metabolites. Vinyl fluorenyl ketone (VFK) is a selective and potent active site-directed irreversible inhibitor of rat liver monomorphic NAT. The present study demonstrated that VFK is an active site-directed affinity label for hamster liver monomorphic NAT, but is a much less effective inactivator of the polymorphic N-acetyltransferase isozyme. The potency, irreversibility and selectivity of VFK make it a potentially valuable tool for characterization of NATs that exhibit acetyl donor specificity similar to that of hamster monomorphic NAT.

Publisher Summary Acetyltransferases play a central role in the metabolic disposition, detoxication, and bioactivation of a diverse group of drugs—carcinogens and other xenobiotics. Acetylation is a major metabolic pathway for primary aromatic amines (arylamines, ArNH) and hydrazines. In earlier times, acetylation was the principal mechanism for termination of the tuberculostatic action of isoniazid. The importance of human acetylation capacity is further illustrated by the propensity of phenotypically slow acetylator patients to experience methemoglobinemia or drug-induced lupus erythematosus or both when treated either with arylamine drugs or with various agents that contain the hydrazine moiety. The mutagenicity and carcinogenicity of arylamines along with thedocumented environmental and dietary exposure to these agents have stimulated numerous investigations of arylamine metabolism. Thus, the premise that formation of unreactive arylamides by N-acetylation of arylamines is primarily a detoxification process and that oxidative Nhydroxylation is a toxification reaction is supported by a substantial body of evidence. However, the demonstration that arylamine N- acetyltransferases are versatile enzymes that can catalyze the conversion of both N-arylhydroxylamines and N-arylhydroxamic acids to reactive, electrophilic metabolites has broadened the scope of research on acetyltransferases, which are now included among those conjugation systems that play important roles in the bioactivation of xenobiotics.

N-Acetoxyarylamines are reactive metabolites that lead to arylamine adduct formation with biological macromolecules. A series of 7-substituted-N-hydroxy-2-acetylaminofluorenes were converted to reactive N-acetoxyarylamines by enzymatic N,O-acyltransfer in the presence of DNA. The N-arylhydroxamic acid substrates that contained electronegative 7-substituents formed greater amounts of DNA adducts than either the unsubstituted compound (N-OH-AAF) or those analogs that contained electron-donating groups in the 7-position. Glutathione did not decrease the rates of DNA adduct formation, but other nucleophiles, such as potassium O-ethylxanthate, thiourea and N-acetylmethionine, inhibited adduct formation by the 7-Br-substituted compound (7-Br-N-OH-AAF) and the unsubstituted parent compound (N-OH-AAF). Nucleophiles, reducing agents (e.g. ascorbic acid) and spin-trapping agents had minimal effect on DNA adduct formation by the bioactivated form of 7-acetyl-2-(N-hydroxy-acetylamino)fluorene (7-Ac-N-OH-AAF). Triethylphosphite, an agent that reacts with aryl nitrenes, caused a concentration-dependent reduction in the amount of DNA adduct formed subsequent to bioactivation of 7-Ac-N-OH-AAF, but did not influence adduct formation when N-OH-AAF and 7-Br-N-OH-AAF were the substrates. The results indicate that a change in the reaction mechanism(s) responsible for DNA adduct formation occurred when the strongly electronegative acetyl group was incorporated into the 7-position of N-OH-AAF. It is proposed that a nitrene intermediate is involved in the formation of covalent adducts with DNA when 7-Ac-N-OH-AAF is activated by N,O-acyltransfer.

Exponentially growing TK6 human lymphoblasts were exposed to either 0-50 microM N-hydroxy-2-acetylaminofluorene (N-OH-AAF) or 0-10 microM 7-acetyl-N-hydroxy-2-acetylaminofluorene (7-acetyl-N-OH-AAF) in both the absence and presence of a partially purified preparation of hamster-liver N-arylhydroxamic acid N,O-acyltransferase (AHAT). Neither N-arylhydroxamic acid was toxic to the lymphoblasts, nor mutagenic at the thymidine kinase (tk) locus, in the absence of AHAT over the concentration range examined. In the presence of AHAT, an enzyme that activates N-arylhydroxamic acids to electrophilic N-acetoxyarylamine intermediates, both compounds caused toxicity and mutagenicity in TK6 cells. The 7-acetyl-N-OH-AAF was approximately 10-fold more toxic and mutagenic than the unsubstituted N-OH-AAF. These data demonstrate that metabolism of these N-arylhydroxamic acids, presumably to N-acetoxyarylamine intermediates by AHAT, is a key event in the biological activity of these agents. In addition, the presence of electron-withdrawing 7-acetyl substituent that is thought to stabilize N-acetoxy intermediates, appears to enhance the biological activity of the unsubstituted N-OH-AAF.

Two forms of hamster hepatic arylamine N -acetyltransferase (NAT; EC 2.3.1.5), designated NAT I and NAT II, were purified 200- to 300-fold by sequential 35–50% ammonium sulfate fractionation, Sephadex G-100 gel filtration chromatography, AAB affinity chromatography, DEAE ion exchange chromatography, and P-200 gel filtration chromatography. Treatment of either NAT I or NAT II with N -ethylmaleimide (NEM), a cysteine selective reagent, caused a concentration-dependent loss of enzymatic activities. Acetyl coenzyme A (AcCoA) protected NAT I against inactivation by NEM, whereas both 2-acetylaminofluorene (2-AAF) and AcCoA protected NAT II against inactivation. Incubation of either NAT I or NAT II with phenylglyoxal (PG), an arginine selective reagent, caused a time-dependent and a concentration-dependent loss of both NAT I and NAT II activities; the inactivations followed pseudo first-order kinetics. The reaction order with respect to PG was approximately two for each enzyme, consistent with the expected stoichiometry for the reaction of PG with arginine. The presence of AcCoA provided full protection of NAT I against inactivation by PG. However, neither AcCoA nor 2-AAF provided protection of NAT II against inactivation by PG. Diethylpyrocarbonate (DEPC), a histidine selective reagent, caused time-dependent and concentration-dependent pseudo first-order inactivation of both NAT I and NAT II. Neither AcCoA nor products of NAT-catalyzed reactions protected NAT I and NAT II against inactivation by DEPC. These results suggest that cysteine, arginine and histidine residues are essential to the catalytic activity of both NAT I and NAT II; the cysteine(s) is located at or near the binding site of NAT I and NAT II, and the arginine residue appears to be located in the AcCoA binding site of NAT I. In contrast, the essential arginine residue(s) of NAT II and the essential histidine residue(s) of both NAT I and NAT II are not likely to reside in the binding site of the enzymes.

Affinity chromatography of an ammonium sulfate precipitate obtained from rat hepatic cytosol resulted in the separation of two fractions of N -acetyltransferase (NAT) activity. NAT I catalyzed the S -acetylcoenzyme A (AcCoA)-dependent acetylation of p -aminobenzoic acid (PABA); NAT II catalyzed the N -hydroxy-2-acetylaminonuorene (N-OH-AAF)-dependent acetylation of 4-aminoazobenzene (AAB) ( N , N -acetyltransferase), the AcCoA-dependent acetylation of procainamide (PA), and the N -arylhydroxamic acid N , O -acyltransferase (AHAT) activity that results in the conversion of N-OH-AAF and related hydroxamic acids to electrophilic reactants. 1-(Fluoren-2-yl)-2-propen-1-one (vinyl fluorenyl ketone, VFK) was shown to be a potent and irreversible inactivator of NAT II activities. A 200-fold higher concentration of VFK was required to inactivate NAT I activity than was required for inactivation of NAT II activities. Similar selectivity in the inactivation of the isozymes was observed when experiments were conducted with enzyme preparations that contained both NAT I and NAT II activities. The presence of substrates and products of the NAT II-catalyzed reactions such as AcCoA, 2-acetylaminofluorene (2-AAF), and N -acetyl-4-aminoazobenzene (N-Ac-AAB) protected NAT II from the inactivating effects of VFK, providing evidence that VFK is an active site directed inhibitor (affinity label) of NAT II. Studies with 1-(fluoren-2-yl)-2-propan-1-one (EFK), an analogue of VFK in which the α, β-unsaturated vinyl ketone group of VFK has been replaced with an ethyl ketone group, demonstrated that the conjugated ketone of VFK is required for inactivation of enzyme activity. The results of these studies suggest that agents such as VFK should have utility as probes of acetyltransferase multiplicity and in the investigation of the active site topography of the enzymes.

The mechanism-based inactivation (suicide inactivation) by N-hydroxyphenacetin (NHP) of N-arylhydroxamic acid N,O-acyltransferase (AHAT) and p-aminobenzoic acid N-acetyltransferase (PABA NAT) activities of a partially purified hamster liver preparation was investigated. The inactivation of both enzyme activities was irreversible, but a partial protection of PABA NAT could be achieved by inclusion of the nucleophile cysteine in the incubation mixture; cysteine did not reduce the extent of inactivation of AHAT by NHP. Hepatic AHAT and PABA NAT activities were separated by affinity chromatography, and the resolved enzyme activities were subjected to incubation in the presence of NHP, N-hydroxy-2-acetamidofluorene (N-OH-AAF), and N-hydroxy-4-acetamidobiphenyl (N-OH-AABP); AHAT, but not PABA NAT, was inactivated by NHP, N-OH-AAF and N-OH-AABP. Incubation of hamster heptic PABA NAT with radiolabeled N-OH-AAF resulted in the formation of only 15% as much fluorenylamine-tRNA adduct as was formed when N-OH-AAF was bioactivated with hamster hepatic AHAT. Hamster intestinal AHAT and PABA NAT activities also were resolved by affinity chromatography; the intestinal AHAT fractions were much more effective than the PABA NAT fractions in bioactivating N-OH-AAF. These results demonstrate that hamster liver and intestine contain at least two arylamine transacetylating activities, one of which is much more effective than the other in the bioactivation of toxic and carcinogenic N-arylhydroxamic acids.

N -Hydroxy-2-acetamidofluorene ( N -OH-AAF), a carcinogenic N -arylhydroxamic acid, is a selective and irreversible inhibitor of arylamine N -acetyltransferase (NAT) activity in vitro . The present study demonstrates that intraperitoneal administration of ( N -OH-AAF) to hamsters caused an irreversible reduction of the hepatic transacetylase activity that catalyzes the transfer of the acetyl group from NOH-AAF to 4-aminoazobenzene (AAB), but did not affect the acetyl coenzyme A (CoASAc) dependent NAT that is responsible for acetylation of p-aminobenzoic acid (PABA). A 40% loss of N -OHAAF:AAB transacetylase activity occurred 4 hr after administration of 50 mg/kg of N -OH-AAF. To determine whether biotransformation of N -OH-AAF is a factor in determining its ability to inactivate N -OH-AAF:AAF transacetylase activity in vivo , the enzyme-inducing agent phenobarbital and the esterase/acylamidase inhibitor bis( p -nitrophenyl)phosphate (BNPP) were administered to the animals prior to the administration of N -OH-AAF. The loss of N -OH-AAF:AAB transacetylase activity was prevented by treatment of the animals with either phenobarbital or with BNPP. The ability of the esterase/acylamidase inhibitor, BNPP, to prevent the N -OH-AAF-mediated loss of transacetylase activity indicates that, in contrast to the inactivation process in vitro , esterase-catalyzed deacetylation of N -OH-AAF may be required for transacetylase inactivation in vivo . It is proposed that in vivo the endogenous acetyl donor, CoASAc, acetylates the enzyme and prevents the deacetylation of N -OHAAF by NAT, thereby impeding the N -OH-AAF-mediated inactivation process, but facilitating enzyme inactivation by N -hydroxy-2-aminofluorene. The latter proposal was supported by the demonstration that CoASAc inhibited the in vitro inactivation of N -OH-AAF:AAB transacetylase activity by N -OHAAF.

N-Arylhydroxamic acid N,O-acyltransferase (AHAT) catalyzes the transfer of the N-acetyl group from N-arylhydroxamic acids to arylamines. In the absence of an arylamine acceptor, AHAT catalyzes the conversion of N-arylhydroxamic acids to reactive electrophilic intermediates that become irreversibly bound to cellular nucleophiles, including those present on AHAT itself. As part of an investigation of the AHAT-catalyzed bioactivation process, a series of 7-substituted analogues of N-hydroxy-2-acetamidofluorene (1) was synthesized and evaluated in vitro as substrates and inactivators of a partially purified hamster hepatic AHAT preparation. All of the compounds functioned as acetyl donors in the AHAT-catalyzed transacetylation of 4-aminoazobenzene (AAB) and all of them were inactivators of AHAT. The inactivation process exhibited apparent first-order kinetics, and the 7-methoxy compound exhibited the largest inactivation rate constant. Quantitative structure-activity analysis provided support for the concept that positively charged species are involved in the inactivation of AHAT by this series of compounds. Results of experiments in which nucleophilic trapping agents such as glutathione, cysteine, methionine, guanosine phosphate, and tRNA were included in incubation mixtures with AHAT and the N-arylhydroxamic acids indicated that electrophiles which diffuse away from the enzyme active site participate in the inactivation process.

Both N-hydroxy-2-acetamidofluorene (N-OH-AAF) and the heterocyclic analogue, 2-(N-hydroxyacetamido)carbazole (N-OH-AAC), were shown to be mechanism-based irreversible inhibitors (suicide inhibitors) of partially purified rat hepatic N-acetyltransferase (NAT) activity. Although N-OH-AAC exhibited an apparent first-order inactivation rate constant (ki) that was 7-fold lower than that of N-OH-AAF, their relative ki/KD values indicate that N-OH-AAC was the more potent and efficient inactivator of transacetylase activity. Inactivation of NAT activity by these N-arylhydroxamic acids appeared to involve contributions by electrophiles that react with the enzyme subsequent to release from the active site and by electrophiles that remain complexed with the active site. The irreversible nature of the enzyme inactivation was demonstrated by the failure to recover transacetylase activity upon either extensive dialysis or gel filtration of preparations that had been subjected to incubation with N-OH-AAF or N-OH-AAC. The use of the nucleophile N-acetylmethionine to trap the electrophilic reactants formed in the transacetylase-catalyzed bioactivation process resulted in a lower rate and extent of formation of methylthio adducts with N-OH-AAC than with N-OH-AAF. The results of this study indicate that N-OH-AAF and N-OH-AAC have been potential for use as tools in the investigation of rat hepatic transacetylases.

N-Hydroxylation is an obligate step in the bioactivation of carcinogenic aryl amides. Previous reports from this laboratory demonstrated that variation of the 4' substituent of trans-4-acetamidostilbene (1) has a marked effect on the rate of its in vitro microsomal N-hydroxylation. In order to further investigate the effects of electronegative and aliphatic substituents, the 4'-CN, 4'-CH3, 4'-C(CH3)3, and 4'-CF3 analogues of 1 were synthesized and subjected to metabolic transformation by hamster hepatic microsomes. Each compound was synthesized in radiolabeled form, and the metabolites were identified and quantified by TLC, mass spectrometry, and liquid scintillation counting. The Vmax for N-hydroxylation of the 4'-CN analogue was 24% and the Km was 11% of that of 1. The glycolamide was a minor metabolite of the 4'-CN compound. The principal metabolite of the 4'-CH3 compound was the 4'-CH2OH derivative, the N-hydroxylated product being formed in small quantities. Similarly, the 4'-C(CH3)3 analogue was metabolized to yield trans-4'-[2-(hydroxymethyl)-2-propyl]-4-acetamidostilbene (26) along with trace quantities of the hydroxamic acid. The 4'-CF3 substrate yielded small amounts of the N-hydroxylated material as the only detectable metabolite. Thus, introduction of a 4' substituent into 1 resulted in a decreased rate of N-hydroxylation for all compounds studied. The reduction in N-hydroxylation depends on both the physicochemical properties of the 4' substituent and upon the susceptibility of the substituent to metabolic oxidation.

N-(4-Phenylcyclohexyl)acetohydroxamic acid and a series of N-(phenylalkyl)acetohydroxamic acids were synthesized and evaluated as substrates for partially purified rat and hamster hepatic arylhydroxamic acid N,O-acyltransferase systems (AHAT). The compounds were assayed for their abilities to function as acetyl donors in the AHAT-mediated transacetylation of 4-aminoazobenzene and for their abilities to participate in the AHAT-mediated conversion of N-arylhydroxylamines to electrophilic intermediates that form methylthio adducts upon reaction with N-acetylmethionine. None of the newly synthesized compounds displayed significant activity in either of the assays. The results of this study indicate that acetohydroxamic acids that have the nitrogen atom of the hydroxamic acid group attached directly to aliphatic or cycloalkyl groups are not likely to serve as substrates or inhibitors of AHAT.

Stereoselective syntheses of trans- and cis-3-amino-5-phenyl-1-(2-pyridyl)pyrrolidine (21 and 24) are reported. Evidence for the relative stereochemistries of 21 and 24 was obtained by preparation of the bicyclic lactams exo- and endo-7-phenyl-1,4-diazabicyclo[3.2.1.]octan-3-one (16 and 19) from precursors of 21 and 24.

The effect of halogen substituents placed at the 4' position of trans-4-acetamidostilbene (1, AAS) to alter the pattern of biotransformation and thus the mutagenicity of these derivative was evaluated by comparing the mutagenic effects of 1 on Salmonella typhimurium TA-100 with the corresponding 4'-F (2), 4'-Cl (3), and 4'-Br (4) analogues. The mutagenic properties of trans-4-(N-hydroxyacetamido)stilbene (5) and its 4'-F (6), 4'-Cl (7), and 4'-Br (8) derivatives were also evaluated in this system. Both the amides (1-4) and hydroxamic acids (5-8) required the presence of a metabolic activating system prepared from hamster liver in order to produce a mutagenic effect. All of these compounds were mutagenic to A-100. Their mutagenic potencies were markedly influenced by the 4'-halogen substituents, the relative mutagenic potencies of the amides being 2 (4'-F) greater than 1 (4'-H), 3 (4'Cl) greater than 4 (4'-Br), while the hydroxamic acids followed the order of 1 (4'-H) greater than 2 (4'-F) greater than 3 (4'-Cl), 4 (4'-Br).

Carcinogenic arylhydroxamic acids (N-hydroxy-2-acetylamino-fluorene, N-hydroxy-4-acetylaminobiphenyl, and trans -N-hydroxy-4-acetyl-aminostilbene) are irreversible inhibitors of rat and hamster hepatic N,O-acyltransferase. The kinetic characteristics of this inhibition are consistent with suicide inactivation of the enzyme. This finding appears to be the first example of carcinogens serving as suicide substrates for an enzyme system which is responsible for their bioactivation.

N-Arylhydroxamic acid N,O-acyltransferase (AHAT) is a cytosolic enzyme system that is capable of converting toxic and carcinogenic N-arylhydroxamic acids into electrophilic reactants and of catalyzing the transacetylation of arylamines. The role of the N-hydroxyl group in promoting AHAT-catalyzed transacetylation of arylamines was investigated by the synthesis and biochemical evaluation of a series of o-hydroxyaryl amides and N-arylglycolamides. Several of these compounds are metabolites of carcinogenic aryl amides in vivo. 3-Hydroxy-4-acetamidobiphenyl (8) was weakly effective as an acetyl donor when partially purified preparations of hamster or rat hepatic AHAT were used to catalyze the transacetylation of 4-aminoazobenzene. 1-Hydroxy-2-acetamidofluorene (1), 3-hydroxy-2-acetamidofluorene (2), 2-glycolamidofluorene (3), 4-glycolamidobiphenyl (9), and trans-4-glycolamidostilbene (5) were less effective acyl donors than 4-acetamidobiphenyl (7) itself. The compounds were also assayed for their abilities to participate in the AHAT-catalyzed conveydroxy-2-acetamidofluorene (1), 3-hydroxy-2-acetamidofluorene (2), 2-glycolamidofluorene (3), 4-glycolamidobiphenyl (9), and trans-4-glycolamidostilbene (5) were less effective acyl donors than 4-acetamidobiphenyl (7) itself. The compounds were also assayed for their abilities to participate in the AHAT-catalyzed conveydroxy-2-acetamidofluorene (1), 3-hydroxy-2-acetamidofluorene (2), 2-glycolamidofluorene (3), 4-glycolamidobiphenyl (9), and trans-4-glycolamidostilbene (5) were less effective acyl donors than 4-acetamidobiphenyl (7) itself. The compounds were also assayed for their abilities to participate in the AHAT-catalyzed conversion of N-arylhydroxylamines to electrophilic intermediates that form methylthio adducts upon reaction with N-acetylmethionine. None of the compounds exhibited more than 4% of the activity of the prototype compound, N-hydroxy-4-acetamidobiphenyl (10). These results indicate that the presence of an hydroxyl group on the ring position ortho to the amide group or on the alpha-position of the acyl group is not sufficient to confer significant acyltransferase activity with AHAT.

The synthesis of a series of 12 N-(2-fluorenyl)hydroxamic acids, N-(2-fluorenyl)-N-hydroxyureas, and N-(2-fluorenyl)-N-hydroxycarbamates is reported. The compounds were evaluated for their ability to serve as substrates for a partially purified hamster hepatic arylhydroxamic acid N,O-acyltransferase preparation. Transacylating activity was measured spectrophotometrically with 4-aminoazobenzene as the acyl group acceptor, and electrophile-generating activity was quantified by the N-acetylmethionine trapping assay. Only the N-acetyl, N-propionyl, and N-methoxyacetyl derivatives exhibited relatively high levels of activity as measured by either of the assay methods. These results are generally consistent with previously reported conclusions regarding the steric and electronic characteristics of acyl groups that are required for activation by this enzyme system. N,O-Acyltransferase inactivation by N-hydroxy-2-acetamidofluorene depressed the bioactivation of the N-acetyl compound to a greater extent than either the N-propionyl or N-methyloxyacetyl derivative.