Characterization of naturally occurring and recombinant human N-acetyltransferase variants encoded by NAT1.

The genotype at the NAT1* locus of an interethnic population of 38 unrelated subjects was determined by direct sequencing of 1.6-kb fragments amplified by PCR. The coding exon alone and together with the 3' noncoding exon of the wild-type (NAT1*4) and the three mutant alleles (NAT1*10, *11, and *16) detected was expressed in Escherichia coli and COS-1 cells, respectively, and the cytosolic fraction of mononuclear leukocytes from NAT1*4/*4 and NAT1*10/*10 homozygotes was also isolated. Recombinant and leukocyte cytosolic preparations were thoroughly characterized by N-acetylation activity with several NAT1-specific and -selective substrates, as well as by steady-state kinetics with varying amounts of the substrate (fixed acetyl CoA) and acetyl CoA (fixed substrate), thermodynamics, stability, and protein immunoreactivity with a polyclonal human anti-NAT1. The polyadenylation signal mutation in the 3' noncoding sequence of NAT1*10 affected none of the aforementioned parameters evaluated both with recombinant NAT1*10 and with the naturally occurring allele. Function was also unaffected by the coding and 3' noncoding exon mutations in NAT1*11. In contrast, the three extra adenosines located immediately after the sixth position of the polyadenylation signal in the 3' untranslated region of NAT1*16 ostensibly caused disruption of the predicted secondary structure of the pre-mRNA for NAT1 16, culminating in parallel 2-fold decreases in the amount and catalytic activity of NAT1 16 in COS-1 cell cytosol. This novel finding in N-acetylation pharmacogenetics clearly demonstrates a direct link between reduced catalytic activity and structural alteration in the 3' untranslated region of an NAT variant (NAT1*16) brought about by mutation.

Slow acetylation in mice is caused by a labile and catalytically impaired mutant N-acetyltransferase (NAT2 9).

Three N-acetyltransferase genes (NAT*) were detected in inbred parental and congenic mice. Direct sequencing of NAT2* and liver cytosolic N-acetylation activity determinations with NAT2-specific (p-aminobenzoic acid) and NAT2-selective (2-aminofluorene) substrates have established that the acetylator congenic A.B6 and B6.A mice are genotypically and phenotypically identical to the parental B6 ("wild-type"; rapid acetylator) and A (mutant; slow acetylator) mice, respectively, from which they originated. The apparent KM for p-aminobenzoic acid and thermal inactivation rates determined with liver cytosol from the mutant (A and B6.A) mice were 3-fold and one order of magnitude higher than the corresponding values with liver cytosol from the wild-type (B6 and A.B6) strains. Northern blotting and immunoblotting revealed hepatic NAT2 mRNA and protein bands of equal size and intensity, regardless of the NAT2* genotype or phenotype of the animals. Incubation of liver cytosol from mutant A and B6.A mice at 37 degrees C for 6 hr resulted in virtual cessation of p-aminobenzoate N-acetylation activity, whereas the steady-state level of immunoreactive NAT2 remained unchanged. The results indicate that the amino acid change (N99I) in mutant NAT2* from slow acetylator mice does not hinder the synthesis of hepatic NAT2 protein, but, rather, leads to production of a conformationally modified NAT2 molecule that resists degradation by tissue proteases but is labile and catalytically impaired.

Nomenclature for N-acetyltransferases.

A consolidated classification system is described for prokaryotic and eukaryotic N-acetyltransferases in accordance with the international rules for gene nomenclature. The root symbol (NAT) specifically identifies the genes that code for the N-acetyltransferases, and NAT* loci encoding proteins with similar function are distinguished by Arabic numerals. Allele characters, denoted by Arabic numbers or by a combination of Arabic numbers and uppercase Latin letters, are separated from gene loci by an asterisk, and the entire gene-allele symbols are italicized. Alleles at the different NAT* loci have been numbered chronologically irrespective of the species of origin. For designation of genotypes at a single NAT* locus, a slash serves to separate the alleles; in phenotype designations, which are not italicized, alleles are separated by a comma.

Promoter and transcription start site of human and rabbit butyrylcholinesterase genes.

Two kilobase segments of the 5'-untranslated regions of the human and rabbit butyrylcholinesterase (BCHE) genes were characterized. The sequences shared extensive identity except for a 333-base pair (bp) Alu repeat present only in human BCHE. One single transcription start site was found in both genes with the techniques of primer extension, amplification of the 5'-end of mRNA, and RNase protection. Cap sites in human and rabbit BCHE genes were found in strictly homologous positions. In human BCHE, the transcription start site was found 157 bp upstream of Met-28, the translation start site. Potential regulatory elements in both promoters included one AP1 site and multiple sites for topoisomerase, Oct-1 and PEA-3. Transient expression of BCHE-reporter gene constructs showed that a 194-bp fragment of the 5'-flanking region of human BCHE and a 570-bp fragment of rabbit BCHE were sufficient for promoting chloramphenicol acetyltransferase activity in HeLa cells. No consensus TATA and CAAT boxes were found. However, the sequence around the transcription start site exhibited homology with initiator elements found in other TATA-less promoters in developmentally regulated genes.

Structural heterogeneity of Caucasian N-acetyltransferase at the NAT1 gene locus.

The human N-acetylation polymorphism is a genetic trait phenotypically reflected by differences in N-acetyltransferase (NAT) activity with therapeutic agents (rapid and slow acetylation), but a genetic invariability in N-acetylation of some arylamine drugs is also known. There are two highly similar human NAT genes: NAT1 is thought to encode a genetically invariant protein, whereas NAT2 has conclusively been shown to represent a polymorphic locus. This study demonstrates the presence of discrete NAT1 structural variants among Caucasians. These were detected by direct sequencing of 1.6-kilobase NAT1 fragments generated by the polymerase chain reaction with liver and leukocyte DNA from 13 subjects of established acetylator phenotype and NAT2 genotype. A prominent alteration in one of the variants was obliteration of the consensus polyadenylation signal (AATAAA-->AAAAAA). Several mutations were discernible in all regions of the second variant allele, including silent (codon 153) and nonsilent (Ser-214-->Ala) substitutions in the coding region and deletion of nine bases from an AT-rich segment in the 3' untranslated region. One-half of the unrelated subjects were either homozygous or heterozygous for the mutant NAT1 alleles, both of which obeyed a Mendelian inheritance pattern. These novel results unambiguously show that human NAT1, like NAT2, is a polymorphic locus.

Molecular genetic basis of rapid and slow acetylation in mice.

The molecular genetic basis of N-acetylation polymorphism has been investigated in inbred mouse models of the human acetylation polymorphism. Two genomic clones, Nat1 and Nat2, were isolated from a C57BL/6J (B6) mouse (rapid acetylator) genomic library. The Nat1 and Nat2 genes both have intronless coding regions of 870 nucleotides and display greater than 47% deduced amino acid similarity with human, rabbit, and chicken N-acetyltransferases. Amplification of Nat1 and Nat2 from A/J (A) mouse (slow acetylator) genomic DNA by the polymerase chain reaction and subsequent sequencing revealed that Nat1 was identical in B6 and A mice, whereas Nat2 contained a single nucleotide change from adenine in B6 to thymine in A mice. This nucleotide substitution changes the deduced amino acid at position 99 from asparagine in B6 to isoleucine in A mice. Hydropathy analysis revealed that this amino acid change alters the hydropathy of the flanking peptide segment in NAT2 from hydrophilic in the B6 mouse to hydrophobic in the A mouse. The amino acid change occurs in a region of the gene where no polymorphism has yet been reported in human or rabbit NAT2 and may represent an important structural domain for N-acetyltransferase activity. Nat1 and Nat2 have the same 5' to 3' orientation in the B6 mouse; the two genes are separated by approximately 9 kilobases, with Nat1 located 5' of Nat2.

Diverse point mutations in the human gene for polymorphic N-acetyltransferase.

Classification of humans as rapid or slow acetylators is based on hereditary differences in rates of N-acetylation of therapeutic and carcinogenic agents, but N-acetylation of certain arylamine drugs displays no genetic variations. Two highly homologous human genes for N-acetyltransferase (NAT; arylamine acetyltransferase, acetyl CoA:arylamine N-acetyltransferase, EC 2.3.1.5), NAT1 and NAT2, presumably code for the genetically invariant and variant NAT proteins, respectively. In the present investigation, 1.9-kilobase human genomic EcoRI fragments encoding NAT2 were generated by the polymerase chain reaction with liver and leukocyte DNA from seven subjects phenotyped as homozygous and heterozygous acetylators. Direct sequencing revealed multiple point mutations in the coding region of two distinct NAT2 variants. One of these was derived from leukocytes of a slow acetylator and was distinguished by a silent mutation (codon 94) and a separate G----A transition (position 590) leading to replacement of Arg-197 by Gln; the mutated guanine was part of a CpG dinucleotide and a Taq I site. The second NAT2 variant originated from liver with low N-acetylation activity. It was characterized by three nucleotide transitions giving rise to a silent mutation (codon 161), accompanied by obliteration of the sole Kpn I site, and two amino acid substitutions: Thr for Ile (codon 114) and Arg for Lys (codon 268). Heterozygosity was detected in three NAT2 samples: two were heterozygous for the rapid and one of the allelic variants, and the third was a compound heterozygote of both mutant alleles. The results show conclusively that the genetically variant NAT is encoded by NAT2.

Structure of the gene for human butyrylcholinesterase. Evidence for a single copy.

We have isolated five genomic clones for human butyrylcholinesterase (BChE), using cDNA probes encoding the catalytic subunit of the hydrophilic tetramer [McTiernan et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 6682-6686]. The BChE gene is at least 73 kb long and contains four exons. Exon 1 contains untranslated sequences and two potential translation initiation sites at codons -69 and -47. Exon 2 (1525 bp) contains 83% of the coding sequence for the mature protein, including the N-terminal and the active-site serine, and a third possible translation initiation site (likely functional), at codon -28. Exon 3 is 167 nucleotides long. Exon 4 (604 bp) codes for the C-terminus of the protein and the 3' untranslated region where two polyadenylation signals were identified. Intron 1 is 6.5 kb long, and the minimal sizes of introns 2 and 3 are estimated to be 32 kb each. Southern blot analysis of total human genomic DNA is in complete agreement with the gene structure established by restriction endonuclease mapping of the genomic clones: this strongly suggests that the BChE gene is present in a single copy.

Studies on covalent binding of (-)trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene metabolites to cytochromes P-450 LM2 and LM4 and NADPH-cytochrome P-450 reductase.

1. Metabolism of 14C-labelled benzo[a]pyrene (-)trans-7,8-dihydrodiol to protein- and DNA-binding products in a reconstituted enzyme system proceeds 5 to 10 times faster with rabbit cytochrome P-450 LM4 than with LM2. 2. Either cytochrome converts the substrate to ethyl acetate- and water-soluble metabolites, identified by h.p.l.c. Water-soluble metabolites comprise 78% of the total products with cytochrome P-450 LM2, but only 50% of those formed by LM4. The relative proportion of the two types of metabolites is differentially affected by certain modifiers such as 7,8-benzoflavone. 3. Half of the radioactivity in the aqueous phase of reaction mixtures containing cytochrome P-450 LM4 represents (-)trans-7,8-diol metabolites in complex primarily with NADPH and phosphate. The remaining water-soluble products are bound covalently to proteins in the reconstituted system. 4. Polyacrylamide gel electrophoresis, autoradiography, and measurement of the radioactivity in individual bands indicate that a larger fraction of metabolites is bound to cytochrome P-450 LM4 than to NADPH-cytochrome P-450 reductase, and only marginal binding to cytochrome P-450 LM2 is seen. Metabolite binding to added DNA is likewise substantially greater in magnitude when cytochrome P-450 LM4, as opposed to LM2, catalyses (-)trans-7,8-diol oxygenation. Thus, the degree of metabolite binding to monoxygenase proteins and to DNA correlates well with the catalytic activity of cytochrome P-450 LM4 and LM2 towards (-)trans-7,8-diol. 5. DNA causes a dramatic enhancement in the activity of cytochrome P-450 LM4 with (-)trans-7,8-diol, indicating that the cytochrome and/or the reductase may be functionally impaired by metabolites of this substrate. Such an effect may alter the balance between detoxication and activation of the carcinogenic benzo[a]pyrene.

Hydroxylation of prostaglandins by inducible isozymes of rabbit liver microsomal cytochrome P-450. Participation of cytochrome b5.

The hydroxylation of prostaglandin (PG) E1, PGE2, and PGA1 was investigated in a reconstituted rabbit liver microsomal enzyme system containing phenobarbital-inducible isozyme 2 or 5,6-benzoflavone-inducible isoenzyme 4 of P-450, NADPH-cytochrome P-450 reductase, phosphatidylcholine, and NADPH. Significant metabolism of prostaglandins by isozyme 2 occurred only in the presence of cytochrome b5. Under these conditions, PGE1 hydroxylation was linear with time (up to 45 min) and protein concentration, and maximal rates were obtained with a 1:1:2 molar ratio of reductase: cytochrome b5:P-450LM2. Moreover, P-450LM2 catalyzed the conversion of PGE1, PGE2, and PGA1 to the respective 19- and 20-hydroxy metabolites in a ratio of about 5:1, and displayed comparable activities toward the three prostaglandins based on the total products formed in 60 min. Apocytochrome b5 or ferriheme could not substitute for intact cytochrome b5, while reconstitution of apocytochrome b5 with ferriheme led to activities similar to those obtained with the native cytochrome. Isozyme 4 of P-450 differed markedly from isozyme 2 in that it catalyzed prostaglandin hydroxylation at substantial rates in the absence of cytochrome b5, was regiospecific for position 19 of all three prostaglandins, and had an order of activity of PGA1 greater than PGE1 greater than PGE2. P-450LM4 preparations from untreated and induced animals had similar activities with PGE1 and PGE2, respectively. Addition of cytochrome b5 resulted in a 20 to 30% increase in the rate of PGE1 hydroxylation and an appreciably greater enhancement in the extent of all the P-450LM4-catalyzed reactions, the stimulation being greatest with PGE2 (3-fold) and least with PGA1 (1.6-fold). Cytochrome b5 was thus required for maximal metabolism of all three prostaglandins, but did not alter the regiospecificity or the order of activity of P-450 isozyme 4 with the individual substrates. In the presence of cytochrome b5, the prostaglandin hydroxylase activities of isozyme 4 were two to six times higher than those of isozyme 2.

Kinetics of reduction of purified liver microsomal cytochrome P-450 in the reconstituted enzyme system studied by stopped flow spectrophotometry.

Stopped flow studies were undertaken to examine the kinetics of reduction of 5,6-benzoflavone-inducible P-450 LM4 by NADPH in the presence of NADPH-cytochrome P-450 reductase and phospholipid under anaerobic CO at 25 degrees C. The reaction exhibited biphasic kinetics irrespective of NADPH concentration or of the molar ratio of reductase to P-450 LM4. The apparent first order rate constants for the fast and slow phases were determined to be 0.9 to 1.0 and 0.25 s-1, respectively. With the reductase and P-450 LM4 present in equimolar amounts, the total amount of P-450 LM4 reduced increased linearly with NADPH concentration; the titration gave a stoichiometry of 2 mol of NADPH per mol of reductase-cytochrome complex. The NADPH concentration had no appreciable effect on the magnitude of the first order rate constants for the fast and slow phases. The kinetics obtained in the presence of benzphetamine were essentially indistinguishable from those seen in the absence of this substrate, while the amount of P-450 LM4 reduced in the fast phase, but not the rate constant for this phase, decreased when phospholipid was omitted from the reaction mixture. Nearly maximal rates of NADPH oxidation by P-450 LM2 OR LM4 were obtained with a molar ratio of reductase to P-450 LM of 1.0. Benzphetamine enhanced the oxidation of NADPH by P-450 LM2 but had no effect on the activity of P-450 LM4. Rates of NADPH oxidation in the presence of P-450 LM2 and LM4 decreased by 80 and 40%, respectively, when phospholipid was omitted from the reconstituted enzyme system. These studies provide evidence for the formation of a catalytically functional 1:1 complex between the reductase and P-450 LM4, and indicate that P-450 LM2 and LM4 differ in their dependence on phospholipid.

Regio- and stereoselectivity of various forms of purified cytochrome P-450 in the metabolism of benzo[a]pyrene and (-) trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene as shown by product formation and binding to DNA.

Highly purified cytochromes P-450(LM2) and P-450(LM4) and partially purified P-450(LM1), P-450(LM3b), and P-450(LM7) from rabbit liver microsomes exhibit different catalytic activities in the metabolism of benzo[a]pyrene (BzP) and (-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene [(-)trans-7,8-diol] in a reconstituted enzyme system. The two highly purified cytochromes also exhibit differences in the activation of BzP and (-)trans-7,8-diol to intermediates that bind to DNA, as well as in the stereoselective conversion of (-)trans-7,8-diol to the highly mutagenic and carcinogenic diol-epoxides r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10- tetrahydrobenzo[a]pyrene (diol-epoxide I) and r - 7,t - 8 - dihydroxy - c - 9,10 - oxy - 7,8,9,10 - tetrahydrobenzo[a]pyrene (diol-epoxide II). P-450(LM2) is more active than P-450(LM4) in the metabolism of BzP and in its conversion to products that bind to DNA. In contrast, P-450(LM4) is more active than P-450(LM2) in the metabolism of (-)trans-7,8-diol and in its conversion to products that bind to DNA. The ratio of activity (percent substrate metabolized) with BzP relative to that with (-)trans-7,8-diol is 21 for P-450(LM2) and 0.3 for P-450(LM4); P-450(LM1), P-450(LM3b), and P-450(LM7) gave intermediate ratios. Marked stereoselectivity in the oxygenation of the (-)trans-7,8-diol to the highly mutagenic and putatively carcinogenic diol-epoxides I and II was observed with P-450(LM4), whereas the other preparations showed less selectivity. The ratio of diolepoxide I to diol-epoxide II ranges from 0.3 for P-450(LM7) to 11 for P-450(LM4). The substrate specificity and regio- and stereo-selectivity of the different forms of cytochrome P-450 may regulate the balance between activation and detoxification pathways of BzP and therefore determine the susceptibility of individual tissues, strains, and species to the carcinogenic action of BzP.