Showing papers on "Monooxygenase published in 1989"
TL;DR: The lack of inhibitors specific for one or the other of the two groups of bacteria hampers the determination of their relative roles in nature.
884 citations
TL;DR: HPCN3 is a newly described cytochrome P-450 that is differentially expressed in the adult human population and that has overlapping substrate specificity compared to hPCN1 for metabolism of steroid and drug substrates.
486 citations
TL;DR: It is reported that the activities of both cytochromes depend critically on the identities of the amino acids at positions 117, 209 and 365 and, moreover, that a single mutation in which Phe 209 is substituted by Leu is sufficient to convert the specificity of P450coh from coumarin to steroid hydroxylation.
Abstract: AS a family of structurally-related enzymes, cytochrome P450 (P450) monooxygenases exhibit paradoxical characteristics: although collectively the enzymes display a broad range of substrate specificities, individually they are characterized by a high degree of substrate and product selectivity. Mouse P45015α, and P450coh, for example, which are expressed in female liver and male kidney cells1,2, catalyse 15α-hydroxylation of Δ4 3-ketone steroids, such as testosterone and 7-hydroxylation of coumarin, respectively3–6. In spite of their divergent catalytic activities, however, these enzymes differ by only 11 amino acids within their 494 residues5. To determine the structural basis of the different substrate specificities of P45015α and P450coh we therefore altered each of these 11 residues by site-directed mutagenesis, expressing the mutant cytochromes in COS-1 cells. We report that the activities of both cytochromes depend critically on the identities of the amino acids at positions 117, 209 and 365 and, moreover, that a single mutation in which Phe 209 is substituted by Leu is sufficient to convert the specificity of P450coh from coumarin to steroid hydroxylation.
381 citations
TL;DR: Site-directed mutants of cytochrome P-450cam, in which threonine-252 had been changed to alanine, valine, or serine, were employed to study the role of the hydroxy amino acid in the monooxygenase reaction, exhibited optical absorption spectra almost indistinguishable from those of the wild-type enzyme in their ferric, ferrous, oxygenated, and carbon monoxide ferrous forms.
Abstract: Site-directed mutants of cytochrome P-450cam (the cytochrome P-450 that acts as the terminal monooxygenase in the d-camphor monooxygenase system), in which threonine-252 had been changed to alanine, valine, or serine, were employed to study the role of the hydroxy amino acid in the monooxygenase reaction. The mutant enzymes were expressed in Escherichia coli and were purified by a conventional method. All the mutant enzymes in the presence of d-camphor exhibited optical absorption spectra almost indistinguishable from those of the wild-type enzyme in their ferric, ferrous, oxygenated, and carbon monoxide ferrous forms. In a reconstituted system with putidaredoxin and its reductase, the alanine enzyme consumed O2 at a rate (1100 per min per heme) comparable to that of the wild-type enzyme (1330 per min per heme), whereas the amount of exo-5-hydroxycamphor formed was less than 10% of that formed by the wild-type enzyme. About 85% of the O2 consumed was recovered as H2O2. The valine enzyme also exhibited an oxidase activity to yield H2O2 accompanied by a relative decrease in the monooxygenase activity. On the other hand, the serine enzyme exhibited essentially the same monooxygenase activity as that of the wild-type enzyme. Thus, uncoupling of O2 consumption from the monooxygenase function was produced by the substitution of an amino acid without a hydroxyl group. When binding of O2 to the ferrous forms was examined, the alanine and valine enzymes formed instantaneously an oxygenated form, which slowly decomposed to the ferric form with rates of 5.5 and 3.2 x 10(-3) sec-1 for the former and latter enzymes, respectively. Since these rates were too slow to account for the overall rates of O2 consumption, the formation of H2O2 was considered to proceed not by way of this route but through the decomposition of a peroxide complex formed by reduction of the oxygenated form by reduced putidaredoxin. Based on these findings, a possible mechanism for oxygen activation in this monooxygenase reaction has been discussed.
343 citations
TL;DR: Metabolism of two physiologic substrates by a human liver cytochrome P450 related to a group of "constitutive" rodent P450s believed to participate in the metabolism of endogenous compounds is shown for the first time.
172 citations
Journal Article•
TL;DR: Data indicate that specific inducible and constitutive cytochromes P-450 are involved in the metabolic activation and detoxification of the carcinogens ABP and MOCA and may be significant determinants of individual susceptibility to arylamine carcinogenesis.
Abstract: Metabolic N-oxidation and ring-oxidation of carcinogenic arylamines by hepatic cytochromes P-450 are generally regarded as critical activation and detoxification pathways, respectively. Two arylamines with known human exposure, 4-aminobiphenyl (ABP) and 4,4′-methylene-bis(2-chloroaniline) (MOCA), have been examined as substrates for 10 different purified rat hepatic cytochromes P-450 and for human liver microsomal preparations from 22 individuals. Metabolites were analyzed by high-performance liquid chromatography and flow scintillation techniques. As reported for certain other carcinogenic arylamines, the isosafrole-inducible isozyme, P-450ISF-G, had the highest catalytic activity for ABP N-oxidation (13.6 nmol/min/nmol P-450), but P-450BNF-B, P-450UT-A, P-450UT-F, and P-450PB-B also showed appreciable activity. Ring-oxidation of ABP occurred only to a minor extent. In contrast, N-oxidation of MOCA was preferentially catalyzed by the phenobarbital-inducible enzymes, P-450PB-B and P-450PB-D (9.0 and 6.6 nmol/min/nmol P-450, respectively). MOCA ring-oxidation and methylene carbon oxidation showed varied cytochrome P-450 selectivity and accounted for 14 to 79% of total oxidation products. There was a 44-fold variation in rates of ABP N-oxidation in the 22 human liver microsomal preparations, while rates of N-oxidation of MOCA varied only 8-fold. Ring/methylene carbon-oxidation of MOCA accounted for 6–19% of total oxidation products in the case of the human microsomal preparations, whereas ring-oxidation of ABP accounted for less than 7% of total oxidation. In addition, there was a strong correlation (R = 0.90) between rates of ABP N-oxidation and phenacetin O-deethylation, which is considered a human genetic polymorphism. Moreover, both the ABP N-oxidation and phenacetin O-deethylation activities of human liver microsomes showed a good correlation (R = 0.72) with the levels of cytochrome P-450 immunochemically related to rat P-450ISF-G. These data indicate that specific inducible and constitutive cytochromes P-450 are involved in the metabolic activation and detoxification of the carcinogens ABP and MOCA. Therefore, individual profiles of cytochromes P-450, affected by both environmental and genetic factors, may be significant determinants of individual susceptibility to arylamine carcinogenesis.
161 citations
TL;DR: Crustaceans are not as sensitive as fish to induction of P450 and monooxygenase activity, and some studies indicate that polycyclic aromatic hydrocarbons, but not phenobarbital-type inducers, could induce cytochrome P450 in crustaceans, whereas other studies showed no effect of either inducer type.
Abstract: 1 The hepatopancreas is the major site of cytochrome P450-dependent xenobiotic monooxygenation in crustacean species, but the presence of monooxygenase inhibitors in hepatopancreas microsomes and cytosol from many decapod species has impeded in vitro studies Cytochrome P450 and monooxygenase activities have been reported in other crustacean organs including the antennal gland (green gland) and stomach 2 NADPH cytochrome c reductase activity is often very low (typically less than 10 nmol cytochrome c reduced/min per mg microsomal protein) in hepatopancreas microsomes from crustacean species NADPH cytochrome P450 reductase activity has not yet been detected in crustacean hepatopancreas microsomes 3 The cytochrome P450 present in hepatopancreas of several crab species and the spiny lobster has been resolved into several fractions by chromatography on DEAE-cellulose One form of cytochrome P450 from spiny lobster has been purified to 12 +/- 2 nmol/mg protein 4 Reconstitution studies with spiny lobster hepatopancreas P450 have shown that the vertebrate sex steroids, progesterone and testosterone, are excellent substrates, whereas ecdysone--the crustacean molting hormone--is not a substrate Activity was found with several xenobiotic substrates including benzphetamine, aminopyrine, benzo(a)pyrene, ethyl-, benzyl- and pentyl-phenoxazones and ethoxycoumarin Highest activities (greater than 50 nmol/min per nmol P450) were found for N-demethylation of benzphetamine and aminopyrine 5 The ability of agents which induce vertebrate cytochrome P450 to induce cytochrome P450 in crustaceans is still unclear Some studies indicate that polycyclic aromatic hydrocarbons, but not phenobarbital-type inducers, could induce cytochrome P450 in crustaceans, whereas other studies showed no effect of either inducer type Crustaceans are not as sensitive as fish to induction of P450 and monooxygenase activity
107 citations
TL;DR: Caloric restriction decreased the age-related changes in hepatic testosterone metabolism, which are associated with demasculinization of the liver, and changes in cytochrome P-450 isozyme profiles could result in altered carcinogen activation, radical formation or drug detoxication in the calorically restricted rat.
101 citations
TL;DR: It is suggested that the cytochrome P-450 isozyme(s) which is (are) orthologous to rat cyto chrome P- 450c have an important role in some of the drug oxidation reactions in cetacean liver microsomes.
91 citations
TL;DR: The low monooxygenase activities of certain species of birds may make them relatively susceptible to lipophilic environmental chemicals that are detoxified by this system, which is similar to that in mammals.
Abstract: 1. Microsomal monooxygenase systems which contain cytochrome P-450 forms as their active centres are found in birds, reptiles and amphibians. Liver provides a rich source of monooxygenases but they are also present in other tissues. 2. In the hepatic microsomes of these species, levels of cytochrome P-450 and, in most cases monooxygenase activities, are lower than are found in hepatic microsomes of mammals. 3. Amongst birds, the lowest hepatic microsomal monooxygenase activities have been reported for specialized predators (fish-eaters and raptors). 4. When birds are dosed with inducers of the 3-methylcholanthrene (MC) type, the pattern of induction is similar to that in mammals. In contrast, phenobarbitone-type inducers are less effective in birds than in mammals--in some cases having no action at all. Prochloraz is a stronger inducer in birds than in the rat; it is an inducer of mixed type in birds. 5. Partial purification of avian cytochromes P-450 indicates substantial differences in properties from those of mammals. Further differences between birds and mammals in regard to the immunochemical properties of isoforms are evident from comparative studies using Western blotting. 6. On the evidence of Western blotting and response to inducers, cytochromes of family II (P450 II) seem less well represented in birds than in mammals. 7. The low monooxygenase activities of certain species of birds may make them relatively susceptible to lipophilic environmental chemicals that are detoxified by this system.
84 citations
TL;DR: In tolerant wheat, diclofop metabolism in vivo was rapid (33% in 6 hr) and was sensitive to cytochrome P450 inhibitors (CO, tetcyclasis, and 1-aminobenzotriazole).
TL;DR: Data suggest that two distinct amino acid residues located within the amino-terminal fourth of IIB1 and IIB2 can affect substrate orientation at the active site of these phenobarbital-inducible P-450 forms.
TL;DR: Comparisons between susceptible and resistant strains by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the LPR strain showed a marked increase in the 54,400-Da protein band, suggesting this may be a cytochrome P450 isozyme involved in conferring part of the monooxygenase-mediated pyrethroid resistance.
TL;DR: The results demonstrate that human epidermis contains active membrane-bound monooxygenase(s) which preferentially generates 12-HETE from AA, exhibits a 12S stereopreference of hydroxylation, and suggests the presence of distinct mitochondrial and microsomal enzyme systems in epidermal cells.
Abstract: We investigated the enzymatic mechanisms responsible for AA oxygenation in homogenous cell suspensions obtained by trypsinization of epidermis from healthy subjects Cell incubation with AA (03-150 microM) invariably resulted in the predominant generation of a compound identified as 12-hydroxyeicosatetraenoic acid (12-HETE) by HPLC and by both negative-ion chemical ionization and electron-impact mass spectrometry Maximal amounts of 12-HETE were 126 +/- 21 pmol/10(6) cells (+/- SE), and concentration-response curves yielded half-maximal levels for 12-HETE similar to PGE2 at 2 microM AA Two epoxyeicosatrienoic acids derived from AA were also identified Stereochemical analysis by chiral-phase chromatography demonstrated that the epidermal cell 12-HETE was a mixture of the 12S- and 12R-hydroxy isomers in a molar ratio varying from 2:1 to 8:1 among subjects Subcellular fractionation into 12,000 g pellet (containing mitochondria) and 100,000 g supernatant (cytosol) and pellet (microsome) demonstrated that greater than 99% of the 12-HETE was generated by enzymatic activity distributed equally in the two pellets Both mitochondrial and microsomal activities were increased upon addition of NADPH and were inhibited by carbon monoxide, but the molar ratio of 12S/12R-HETE was threefold greater in microsomal than in mitochondrial fractions The results demonstrate that human epidermis contains active membrane-bound monooxygenase(s) which preferentially generates 12-HETE from AA, exhibits a 12S stereopreference of hydroxylation, and suggests the presence of distinct mitochondrial and microsomal enzyme systems in epidermal cells
TL;DR: Data indicate that multiple forms of P450 exist in the black swallowtail midgut and that they are differentially induced by xanthotoxin, which exerts much of its toxic effects when photoactivated by ultraviolet light.
Abstract: Xanthotoxin is a phototoxic allomone found in many of the host plants of the black swallowtail,Papilio polyxenes (Lepidoptera: Papilionidae). When added to the diet of final instar larvae, xanthotoxin can induce the cytochrome P450 monooxygenase (P450) activity in midgut microsomes by which it is detoxified. Induction is dose-dependent, increasing sevenfold when larvae feed on parsley treated topically with xanthotoxin at 0.5 or 1.0% fresh weight. Although xanthotoxin exerts much of its toxic effects when photoactivated by ultraviolet light, induction of P450 activity did not differ in the presence or absence of ultraviolet light. Despite a 4.7-fold induction of xanthotoxin-metabolizing P450 activity, total P450 content measured in the same microsomal samples did not increase significantly. These data indicate that multiple forms of P450 exist in the black swallowtail midgut and that they are differentially induced by xanthotoxin.
TL;DR: Efficient regulation prevents chloroaromatics from being misrouted into the unproductive meta-cleavage pathway and shows constitutive production of six enzymes of the 2,4-D pathway, which were known to be localized in at least three different transcriptional units.
Abstract: Alicaligenes eutrophus JMP 134 is able to grow on 2,4-dichloro-, 4-chloro-2-methyl- and 2-methylphenoxy acetic acid. The unsubstituted phenoxyacetic acid, however, is no growth substrate due to very poor induction of the 2,4-D monooxygenase. Spontaneous mutants of Alcaligenes eutrophus JMP 134 capable of growth with phenoxyacetic acid were selected on agar plates. One of these mutants, designated Alcaligenes eutrophus JMP 134-1, shows constitutive production of six enzymes of the 2,4-D pathway, which were known to be localized in at least three different transcriptional units. A common regulatory gene is postulated to be mutated. 2,4-Dichloro-, 4-chloro-2-methyl- and 2-methylphenoxyacetic acid were the inducers of the enzymes of the “chloroaromatic pathway” in Alcaligenes eutrophus JMP 134. Phenol and 2-methylphenol, metabolites of the degradation of phenoxyacetic acid and 2-methylphenoxyacetic acid, were shown to be inducers of the meta-cleavage pathway, whereas 2,4-dichlorophenol and 4-chloro-2-methylphenol were not. Thus efficient regulation prevents chloroaromatics from being misrouted into the unproductive meta-cleavage pathway. Because 2,4-dichloro-and 4-chloro-2-methylphenol did not show any induction potential, they were growth substrates only for the mutant strain JMP 134-1.
TL;DR: The cofactor requirement and CMP-Neu5Ac substrate specificity are identical to those of the enzyme in high-speed supernatants of pig submandibular gland, suggesting that this is a common route of Neu5Gc biosynthesis.
Abstract: The finding that N-glycoloylneuraminic acid (Neu5Gc) in pig submandibular gland is synthesized by hydroxylation of the sugar nucleotide CMP-Neu5Ac [Shaw & Schauer (1988) Biol. Chem. Hoppe-Seyler 369, 477-486] prompted us to investigate further the biosynthesis of this sialic acid in mouse liver. Free [14C]Neu5Ac, CMP-[14C]Neu5Ac and [14C]Neu5Ac glycosidically bound by Gal alpha 2-3- and Gal alpha 2-6-GlcNAc beta 1-4 linkages to fetuin were employed as potential substrates in experiments with fractionated mouse liver homogenates. The only substrate to be hydroxylated was the CMP-Neu5Ac glycoside. The product of the reaction was identified by chemical and enzymic methods as CMP-Neu5Gc. All of the CMP-Neu5Ac hydroxylase activity was detected in the high-speed supernatant fraction. The hydroxylase required a reduced nicotinamide nucleotide [NAD(P)H] coenzyme and molecular oxygen for activity. Furthermore, the activity of this enzyme was enhanced by exogenously added Fe2+ or Fe3+ ions, all other metal salts tested having a negligible or inhibitory influence. This hydroxylase is therefore tentatively classified as a monooxygenase. The cofactor requirement and CMP-Neu5Ac substrate specificity are identical to those of the enzyme in high-speed supernatants of pig submandibular gland, suggesting that this is a common route of Neu5Gc biosynthesis. The relevance of these results to the regulation of Neu5Gc expression in sialoglycoconjugates is discussed.
TL;DR: The HepG2 human hepatoma cell line appears to be suitable for the study of many parameters of metabolism and action of ABZ and other structurally related compounds in humans.
Abstract: The metabolism of albendazole (ABZ), a benzimidazole anthelminthic, was studied in either microsomal preparations of human liver biopsies or cultured human hepatoma cell lines. Metabolites were analyzed by HPLC. Our data show that microsomes from human biopsies and two human cell lines, HepG2 and Hep3B, oxidize the drug to the sulfoxide very efficiently, whereas the third cell line tested, SK-HEP-1, does not. Both cytochrome P-450 dependent monooxygenases and favin-containing monooxygenases appear to be involved in human ABZ metabolism. Using the cell line displaying the highest ABZ-metabolizing activity, HepG2, the cytotoxic and the inducing effects of the parent drug ABZ and of two primary metabolites, the sulfoxide and the sulfone were studied. These three chemicals provoked a rise in mitotic index resulting from cell division blockage at the prophase or at the metaphase (ABZ metabolites) stage, and ABZ was more cytotoxic than its metabolites. With regard to enzyme-inducing effects, our data clearly demonstrate that the sulfoxide and, to a lesser degree, the sulfone are potent inducers of some drug metabolizing enzymes (i.e., cytochrome P-488 dependent monooxygenases and UDP glucuronyltransferase), whereas ABZ fails to increase and even slightly decreases these enzymatic activities. In conclusion, the HepG2 human hepatoma cell line appears to be suitable for the study of many parameters of metabolism and action of ABZ and other structurally related compounds in humans.
TL;DR: It was found that the amino acid residue differences between enzyme from P. fluorescence and aeruginosa do marginally change some observed transient state kinetic parameters, even though the structure of the enzyme shows they have no direct role in catalysis.
Journal Article•
TL;DR: It is demonstrated that flavin-containing monooxygenase can be involved in the detoxication of pyrrolizidine alkaloids via N-oxidation, but the relative contribution of flavin and cytochromes P-450 may be species and tissue dependent.
Abstract: The pyrrolizidine alkaloid, senecionine, is N-oxidized by purified pig liver flavin-containing monooxygenase but not by purified rabbit lung flavin-containing monooxygenase. The activity of the pig liver enzyme toward senecionine was linear with time and amount of enzyme. The oxygenation was not due to some indirect mechanism, such as O2- release from the enzyme, as scavengers of activated oxygen had no effect on product formation. The Km of purified pig liver flavin-containing monooxygenase for senecionine was 0.3 mM. Although senecionine is a substrate for the pig liver enzyme, studies performed with rat liver microsomes suggest that, in this species, cytochromes P-450 catalyze the majority of senecionine-N-oxidation. These experiments included inhibition by chemical inhibitors of P-450, treatment of the microsomes with elevated temperatures, inhibition by anti-NADPH-cytochrome P-450 reductase antibody, the effect of dexamethasone on N-oxidation, and relative amounts of flavin-containing monooxygenase determined by immunoquantitation. These results demonstrate that flavin-containing monooxygenase can be involved in the detoxication of pyrrolizidine alkaloids via N-oxidation, but the relative contribution of flavin-containing monooxygenase and cytochromes P-450 may be species and tissue dependent.
Journal Article•
TL;DR: The stereochemistry of the cytochrome P-450(P-450)-dependent oxidation of styrene to styrene 7,8-oxide (SO) enantiomers was evaluated with rat hepatic microsomes and with individual rat liver P- 450 enzymes in reconstituted monooxygenase systems.
Abstract: The stereochemistry of the cytochrome P-450(P-450)-dependent oxidation of styrene to styrene 7,8-oxide (SO) enantiomers was evaluated with rat hepatic microsomes and with individual rat liver P-450 enzymes in reconstituted monooxygenase systems. The stereoselectivity of the monooxygenase reaction with styrene was determined by high-performance liquid chromatographic analysis of the glutathione conjugates formed quantitatively from SO, the product of the monooxygenase reaction. Hepatic microsomes from control rats oxidized styrene at a rate of 13.1 +/- 4.5 nmol/min/nmol of P-450 and with a ratio of the amount of the (R)-styrene 7,8-oxide enantiomer to the amount of the (S)-styrene 7,8-oxide enantiomer (R/S) SO ratio of 0.65 +/- 0.1. These values were determined under incubation conditions in which epoxide hydrolase activity was inhibited by cyclohexene oxide, and at least 95% of the SO formed was converted enzymatically to glutathione conjugates. Treatment of rats i.p. with phenobarbital (PB) or beta-naphthoflavone (beta NF) caused changes in both parameters. Whereas the rates of oxidation in hepatic microsomes prepared from PB-treated rats was unchanged at 15.4 +/- 7.5 nmol/min/nmol of P-450 and decreased in hepatic microsomes from beta NF-treated rats to 9.4 +/- 2.8 nmol/min/nmol of P-450, the preference for formation of the R-enantiomer increased as the R/S ratio changed to 0.92 +/- 0.1 for PB and 1.25 +/- 0.1 for beta NF.(ABSTRACT TRUNCATED AT 250 WORDS)
Journal Article•
TL;DR: The findings suggest that 3-methylindole is metabolized to alkylating, electrophilic intermediates preferentially by the homologues of "phenobarbital-inducible" isozymes to cytochrome P-450 in pulmonary microsomes, rather than by the polycyclic aromatic hydrocarbon-Inducible isoz enzymes.
Abstract: The cytochrome P-450 suicide substrates 1-aminobenzotriazole (ABT) and alpha-methylbenzylaminobenzotriazole (alpha MB) were used as probes to examine the participation of cytochrome P-450 monooxygenases in the metabolism and covalent binding of 3-methylindole. ABT was a potent inactivator of 3-methylindole turnover and covalent binding of [methyl-14C]3-methylindole to protein in goat lung microsomal incubations. Both covalent binding and 3-methylindole turnover were decreased approximately 50% at 0.01 mM and 100% at 0.1 mM concentrations of ABT. The effects of ABT indicated that toxicity, as related to covalent binding, was directly dependent upon cytochrome P-450 catalysis. The inactivation of 3-methylindole turnover was greater with a 0.01 mM concentration of the isozyme-selective inhibitor alpha MB, 74% as compared with 47% for ABT. alpha MB (0.01 mM) decreased benzphetamine N-demethylase activity by 82% but decreased 7-ethoxyresorufin O-deethylase activity by only 28%. Thus, both 3-methylindole metabolism and benzphetamine oxidation were selectively inactivated by alpha MB. These findings suggest that 3-methylindole is metabolized to alkylating, electrophilic intermediates preferentially by the homologues of "phenobarbital-inducible" isozymes (presumably forms 2 and 5 in analogy to rabbit lung isozymes) to cytochrome P-450 in pulmonary microsomes, rather than by the polycyclic aromatic hydrocarbon-inducible isozymes.
TL;DR: Results and Western blot analysis indicated that rat skin microsomes contain both monooxygenase systems associated with cytochromes P- 450IIB1 and P-450IA1 and that both the isoforms involved in conjugation of phenols and bilirubin were present in skinmicrosomes.
Journal Article•
TL;DR: The substrate specificity and alkyl group selectivity in the metabolism of nitrosamines by cytochrome P-450 isozymes are demonstrated.
Abstract: Metabolic activation may be a key step in determining the tissue specificity of carcinogenic nitrosamines. In previous work, we characterized P450IIE1 (an acetone/ethanol-inducible form of cytochrome P-450) as the major enzyme for the metabolic activation of N-nitrosodimethylamine. In this work, we investigated the metabolism of other N-nitrosodialkylamines in rat liver microsomes and in reconstituted monooxygenase systems containing purified cytochrome P-450 isozymes. The enzyme specificities in the metabolism of N-nitrosoethylmethylamine and N-nitrosodiethylamine were similar to those of N-nitrosodimethylamine; i.e., these substrates were more efficiently metabolized by acetone- or ethanol-induced microsomes than by other types of microsomes. However, substituting one methyl group with a benzyl or butyl group, as in N-nitrosobenzylmethylamine or N-nitrosobutylmethylamine (NBMA), substantially changed the enzyme specificity. P450IIE1 efficiently catalyzed the demethylation but not the debutylation of NBMA, whereas P450IIB1 (a phenobarbital-inducible form) efficiently catalyzed both the debutylation and demethylation reactions. In the demethylation of NBMA by P450IIE1, the addition of cytochrome b5 markedly increased the activity at low but not at high substrate concentrations, suggesting a decrease in Km value. This effect, however, was not observed in the debutylation of NBMA by P450IIE1 or P450IIB1, and in the demethylation of NBMA by P450IIB1. These studies demonstrate the substrate specificity and alkyl group selectivity in the metabolism of nitrosamines by cytochrome P-450 isozymes.
TL;DR: A variety of organic compounds such as carbon tetrachloride, halothane, gentian violet, and benznidazole are reductively metabolized to free radicals by liver microsomal cytochrome P-450, characterized by a maximum loss of cell viability at oxygen partial pressures (PO2) between 1 and 10 mm Hg.
Abstract: A variety of organic compounds such as carbon tetrachloride, halothane, gentian violet, and benznidazole are reductively metabolized to free radicals by liver microsomal cytochrome P-450. Due to the fact that this metabolic activation occurs at a site where O2 usually becomes activated during the monooxygenase cycle, there is a competition between these xenobiotics and O2 for electrons. Reductive activation occurs at a maximal rate under anaerobic conditions. Cell injury may be directly produced by the reactive metabolites. It may also result from lipid peroxidation induced by interaction of the free radicals with polyunsaturated fatty acids of membrane phospholipids. For polyhalogenated alkanes the latter process is most likely the one leading to cell death. When loss of cell viability is mediated by lipid peroxidation, a complex O2 dependence may result, characterized by a maximum loss of cell viability at oxygen partial pressures (PO2) between 1 and 10 mm Hg. These PO2 values are found in the centrilobular area of the liver lobule.
TL;DR: The purification and some molecular, kinetic and spectral properties of this flavoenzyme which catalyzes the hydroxylation and reduction of 2-aminobenzoyl-CoA to an unknown non-aromatic compound are described.
Abstract: A novel aerobic mechanism of 2-aminobenzoate metabolism was proposed in a denitrifying Pseudomonas species. 2-Aminobenzoic acid is activated in a coenzyme-A-ligase reaction to 2-aminobenzoyl-CoA and this intermediate is dearomatized by a unique enzyme, tentatively named 2-aminobenzoyl-CoA monooxygenase/reductase. This paper describes the purification and some molecular, kinetic and spectral properties of this flavoenzyme which catalyzes the hydroxylation and reduction of 2-aminobenzoyl-CoA to an unknown non-aromatic compound. 2-Aminobenzoyl-CoA monooxygenase/reductase was purified 25-fold to a specific activity of 25 mumol.min-1.mg-1 protein using ammonium sulfate precipitation, DEAE-cellulose anion-exchange, hydroxylapatite and Mono Q FPLC anion-exchange chromatography. Superose 6 gel filtration for estimation of molecular mass resulted in one symmetrical protein peak corresponding to a molecular mass of 170 kDa. Several experimental data suggest that the protein is probably an alpha 2 dimer; however, it may exist in three dimeric forms, alpha alpha, alpha alpha' and alpha' alpha', where alpha' may be a subunit with a different conformation. Approximately 2 mol noncovalently bound FAD/mol enzyme was found, which in the absence of O2 was reduced by NADH. The enzyme was specific for the substrates 2-aminobenzoyl-CoA (Km less than or equal to 25 microM) and O2 (Km less than or equal to 5 microM), but less specific for the reduced pyridine nucleotides NADH (Km = 42 microM) or NADPH [Km = 500 microM; Vmax (NADH)/Vmax (NADPH) = 1.7:1]. The turnover number was 4250 min-1. The enzyme also reduced N-ethylmaleimide and maleimide with NAD(P)H. The substrate, the products and the reaction stoichiometry are described in two following papers.
Journal Article•
TL;DR: The effects of cytochrome P-450 inhibitors and positive effectors for the flavin-containing monooxygenase as well as on studies with the purified enzyme suggest that a role for this enzyme in the stereoselective first-pass metabolism of verapamil is suggested.
Abstract: The chemical and enzymatic N-oxygenation of verapamil was investigated. Verapamil N-oxide is readily synthesized by chemical means. It is not indefinitely stable, however, and undergoes Cope-type elimination to produce 3,4-dimethoxystyrene and a hydroxylamine. The major stable metabolite observed during the metabolism of verapamil with rat and hog liver microsomes and purified flavin-containing monooxygenase is 3,4-dimethoxystyrene. 3,4-Dimethoxystyrene is formed at a rate 4 times that of nor-verapamil. Studies suggest that N-oxygenation is catalyzed largely by the flavin-containing monooxygenase and N-demethylation is catalyzed by cytochrome P-450. This conclusion is based on the effects of cytochrome P-450 inhibitors and positive effectors for the flavin-containing monooxygenase as well as on studies with the purified enzyme. In the presence of rat and hog liver microsomes, significant stereoselectivity in N-oxygenation of verapamil is observed (S/R ratio of 3.1 and 4.1, respectively). With purified hog and rat hepatic flavin-containing monooxygenase, the stereoselectivity for verapamil N-oxygenation (S/R ratio of 10.1 and 6.6, respectively) suggests a role for this enzyme in the stereoselective first-pass metabolism of verapamil.
TL;DR: The genetics, purification and properties of cytochrome P-450 isozymes of the house fly (Musca domestica) are discussed in relation to their role in resistance to xenobiotics and with reference to polyphagy.
Abstract: 1. The insect monooxygenase system is involved in the oxidative metabolism of both endogenous and exogenous substrates. 2. Monooxygenases appear to be important in insect growth and development, in adaptation to multiple food plants in phytophagous insects and in pesticide resistance. 3. The genetics, purification and properties of cytochrome P-450 isozymes of the house fly (Musca domestica) are discussed in relation to their role in resistance to xenobiotics. 4. Induction of insect cytochrome P-450 isozymes is discussed in comparison to induction in mammals and with reference to polyphagy.
TL;DR: The reaction catalyzed by 2-aminobenzoyl-coenzyme-A monooxygenase/reductase from a denitrifying Pseudomonas sp.
Abstract: A novel aerobic mechanism of 2-aminobenzoate metabolism was proposed in a denitrifying Pseudomonas species. 2-Aminobenzoic acid is activated in a coenzyme-A-ligase reaction to 2-aminobenzoyl-CoA and this intermediate is dearomatized by a unique enzyme, tentatively named 2-aminobenzoyl-CoA monooxygenase/reductase. This paper describes the purification and some molecular, kinetic and spectral properties of this flavoenzyme which catalyzes the hydroxylation and reduction of 2-aminobenzoyl-CoA to an unknown non-aromatic compound.
2-Aminobenzoyl-CoA monooxygenase/reductase was purified 25-fold to a specific activity of 25 μmol · min−1· mg−1 protein using ammonium sulfate precipitation, DEAE-cellulose anion-exchange, hydroxylapatite and Mono Q FPLC anion-exchange chromatography. Superose 6 gel filtration for estimation of molecular mass resulted in one symmetrical protein peak corresponding to a molecular mass of 170 kDa. Several experimental data suggest that the protein is probably an α2 dimer; however, it may exist in three dimeric forms, αα, αα′ and α′α′, where α′ may be a subunit with a different conformation. Approximately 2 mol noncovalently bound FAD/mol enzyme was found, which in the absence of O2 was reduced by NADH. The enzyme was specific for the substrates 2-aminobenzoyl-CoA (Km≤ 25 μM) and O2 (Km≤ 5 μM), but less specific for the reduced pyridine nucleotides NADH (Km= 42 μM) or NADPH [Km= 500 μM; Vmax (NADH)/Vmax (NADPH) = 1.7:1]. The turnover number was 4250 min−1. The enzyme also reduced N-ethylmaleimide and maleimide with NAD(P)H. The substrate, the products and the reaction stoichiometry are described in two following papers.
TL;DR: Data indicate that a significant pathway of the plant activation of 2-aminofluorene is via a cytochrome P-448-type N-hydroxylase.
Abstract: Using specific inhibitors, a plant cell/microbe coincubation assay was employed to investigate biochemical mechanisms of plant activation. The biological endpoints of mutation induction, inhibition of mutagenicity and viability of the plant-activating system as well as viability of the microbiological indicator were simultaneously assayed from the same reaction tube. We investigated six inhibitors of monooxygenases and oxidases (diethyldithiocarbamate, methimazole, metyrapone, (+)-catechin, 7,8-benzoflavone and potassium cyanide). The activation of 2-aminofluorene by TX1 cells was mediated by an enzyme system(s) that was inhibited by microM amounts of diethyldithiocarbamate or 7,8-benzoflavone. (+)-Catechin (at low concentrations) or methimazole enhanced the activation of 2-aminofluorene while higher concentrations of (+)-catechin were inhibitory. These data indicate that a significant pathway of the plant activation of 2-aminofluorene is via a cytochrome P-448-type N-hydroxylase. The presence of a FAD-dependent monooxygenase was not detected.