Kostas P. Vatsis

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.

Replacement of active-site cysteine-436 by serine converts cytochrome P450 2B4 into an NADPH oxidase with negligible monooxygenase activity

The function of the unique axial thiolate ligand of cytochrome P450 has been investigated by mutagenesis of the active-site cysteine with other amino acids in NH(2)-truncated P450s 2B4 and 2E1. The expressed Ser-436 variant of P450 2B4 was highly purified but incurred considerable heme loss. The pyridine hemochrome spectrum of C436S is characteristic of protoporphyrin IX, and the absolute spectra display Soret maxima at 405 nm (ferric), 422 nm (ferrous), and 413 nm (ferrous CO). 2B4:C436S catalyzes the NADPH- and time-dependent formation of H(2)O(2) in the reconstituted enzyme system, with maximal rates at approximately equimolar amounts of P450 reductase and C436S hemeprotein. The 2-electron oxidase activity with saturating reductase is directly proportional to the concentration of 2B4:C436S, and the turnover is 60-70% of that of the wild-type enzyme. In contrast, the C436S variant is devoid of oxygenase activity with typical substrates such as d-benzphetamine, 1-phenylethanol, and 4-fluorophenol, and has only marginal 4-nitrophenol aromatic hydroxylation activity. H(2)O(2)-supported peroxidation of guaiacol and pyrogallol is comparable with 2B4 and mutant C436S and negligible relative to the turnover of peroxidases with these substrates. Neither 2B4 nor 2B4:C436S catalyzes H(2)O(2) decomposition. It is concluded that replacement of active-site Cys-436 by Ser converts P450 2B4 mainly into a 2-electron oxidase.

Hydroxylation of benzo[a]pyrene and binding of (-)trans 5-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene metabolites to deoxyribonucleic acid catalyzed by purified forms of rabbit liver microsomal cytochrome P-450 : Effect of 7,8-benzoflavone, butylated hydroxytoluene and ascorbic acid

The catalytic activities of hepatic microsomes from untreated, phenobarbital-treated and 3- methylcholanthrene-treated adult rabbits with respect to benzo(a)pyrene hydroxylation and the acti- vation of (-)truns-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene(( -)~ans-7,8-diol) to DNA-binding metab- olites were determined in the absence and presence of mixed-function oxidase inhibitors and compared to the corresponding activities of the individual enzyme systems. Treatment of rabbits with phenobarbital led to induction of P-450LMz and a concomitant 3-fold enhancement in microsomal benzo(a)pyrene hydroxylase activity, whereas the conversion of (-)rrans-7,8-diol to DNA-binding products was unaffected. Homogeneous phenobarbital-inducible P-450LM2 exhibited the highest activity and speci- ficity toward benzo(a)pyrene and the lowest activity toward (-)rrun.s-7,8-diol. Conversely, P-4SOLM4 was the major form of cytochrome P-450 induced in rabbit liver by 3-methylcholanthrene or p- naphthoflavone, and this was associated in microsomes with an increase in the metabolism of (-)trans- 7,8-diol but not of benzo(a)pyrene. Homogeneous P-450LM4 preferentially catalyzed the oxygenation of (-)tran.s-7,d in contrast, the activity of control microsomes with either substrate, and the activities of P-450LM4 and LM2 with benzo(a)pyrene and (-)trans-7,8-dial, respectively, were only partially or slightly decreased by 7,8-benzoflavone. Unlike 7,8_benzoflavone, butylated hydroxy- toluene inhibited benzo(a)pyrene hydroxylation only. Thus, different forms of rabbit liver microsomal cytochrome P-450 were involved in the metabolism of benzo(n)pyrene and its 7,8_dihydrodiol. The results also demonstrate that the changes in substrate specificity and inhibitor sensitivity seen in phenobarbital- and 3-methylcholanthrene-induced microsomes relative to control rabbit liver micro- somes can be accounted for by the catalytic properties of a specific form of cytochrome P-450 that prevails in these preparations, P-450LMz and LM4, respectively.

Cytochrome P450, a very hard mountain to climb: Evidence for multiple functional species of activated oxygen

Abstract Although much has been learned about cytochrome P450 as an oxygenating catalyst since its discovery as a carbon monoxide-binding pigment in liver microsomes over 40 years ago, two major problems remain unsolved. These are the role of the heme sulfur ligand and the identity of the “activated oxygen” species generated during the reduction of molecular oxygen. To address these questions, mutagenesis of pertinent amino acid residues in NH2-terminal-truncated microsomal P450s 2B4 and 2E1, has been carried out. Clones for these cytochromes with the active-site cysteine replaced by histidine, serine, or tyrosine were expressed in Escherichia coli. Despite instability, some of the resulting hemoproteins were purified. The histidine-437 mutant of P450 2E1 is reduced by NADPH in the presence of the reductase and yields H2O2 in the reconstituted system. However, no activity was detected in the oxidation of several substrates, thus indicating a functional role for the sulfur ligand. In other experiments, the active-site threonine residue that facilitates proton transfer was replaced with alanine. Changes in the rates of catalysis of aldehyde deformylation, olefin epoxidation, ipso-substitution, and other reactions by P450 2B4:T302A and 2E1:T303A mutants provided evidence for peroxo-iron, hydroperoxo-iron, and oxenoid-iron as discrete functional oxygenating species. The availability of multiple oxidants is believed to contribute to the unmatched versatility of the P450 cytochromes.