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PubMed1989

Sequence of a chicken phenobarbital-inducible cytochrome P450 cDNA: regulation of two P450 mRNAs transcribed from different genes.

The nucleic acid and derived amino acid sequences of a chicken phenobarbital-inducible cytochrome P450 cDNA clone, pCHP7, are presented. The amino acid sequence shares 92% identity with that of a previously characterized chicken cytochrome P450 cDNA clone, pCHP3. The two clones, pCHP7 and pCHP3, represent two distinct mRNAs of 2.2 and 3.5 kb and appear to be transcribed from separate cytochrome P450 genes. Sequence comparisons with P450s from mammalian species indicate that the chicken P450s are most closely related to the IIC subfamily. According to the cytochrome P450 nomenclature (Nebert et al., 1989), the chicken P450 gene encoding the 3.5-kb mRNA is termed P450IIF1 and the P450 gene encoding the 2.2-kb mRNA is designated P450IIF2. When chick embryos were treated with 2-allyl-2-isopropylacetamide, the levels of the 3.5- and 2.2-kb P450 mRNAs in liver were elevated maximally by about 100-fold, while phenobarbital treatment resulted in a maximal increase of about 50-fold. These increases in mRNA levels were accompanied by a less than sixfold increase in the corresponding gene transcription rates. The results indicate that the increase in the amount of these two mRNAs is due to both transcriptional activation of the P450 genes and to a marked post-transcriptional mechanism. By contrast, the drug-induced hepatic mRNA levels for 5-aminolevulinate synthase, the rate-controlling enzyme of the heme biosynthetic pathway, could be accounted for predominantly by activation of gene transcription. A close correlation was observed between the time courses of hepatic mRNA accumulation for cytochrome P450 and 5-aminolevulilnate synthase following drug treatment of chick embryos. In adult hens it was demonstrated that a tissue-specific drug induction of cytochrome P450 mRNAs occurred with levels being substantially elevated in the liver, kidney, and small intestine. The mRNA for 5-aminolevulinate synthase was also drug-induced in the same tissue-specific fashion. The results are compatible with the gene for 5-aminolevulinate synthase being activated in response to an increased cytochrome P450 heme requirement.

Amino Acid Sequence↗
PubMed1993

Importance of successive prolines in the carboxy-terminal region of P450 2C2 and P450 2C14 for the hydroxylase activities.

Proline-480 and proline-481 are highly conserved in the P450 2 family. When these successive prolines of P450 2C2 were replaced by Ala-Thr, its activities of laurate (omega-1)-hydroxylation and benzphetamine N-demethylation were lost. On the other hand, the mutated P450 which retained one of the proline residues was 60-100% active in the (omega-1)-hydroxylation and 100-120% active in the N-demethylation. Pro-Pro (480, 481) to Ala-Thr mutated P450 2C14 was also inactive in testosterone 16 alpha-hydroxylation and benzphetamine N-demethylation, the activities of the wild-type P450 2C14. In the carboxyterminal region of the P450 2C subfamily, the sequence of P450 2C2 from residues 469-473 is noticeably different from those of the other members of this subfamily. The Ser-473 to Val mutation of P450 2C2 caused a decrease in the (omega-1)-hydroxylase activity to one-fifth but the N-demethylase activity was not much affected. When the sequence of P450 2C2 from residues 469-473 was replaced by that of P450 2C14 (mutation at four residues), the 16 alpha-hydroxylase and N-demethylase activities were increased by seven- and three-fold, respectively, and the (omega-1)-hydroxylase activity was decreased to one-third. The mutated P450 2C2, in which the carboxy-terminal four residue sequence or the proline cluster adjacent to the amino-terminal membrane-anchor signal sequences was deleted, was not accumulated in yeast cells transformed with plasmids directing the synthesis of such P450s.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2006

Kinetic deuterium isotope effects for 7-alkoxycoumarin O-dealkylation reactions catalyzed by human cytochromes P450 and in liver microsomes. Rate-limiting C-H bond breaking in cytochrome P450 1A2 substrate oxidation.

7-Ethoxy (OEt) coumarin has been used as a model substrate in many cytochrome P450 (P450) studies, including the use of kinetic isotope effects to probe facets of P450 kinetics. P450s 1A2 and 2E1 are known to be the major catalysts of 7-OEt coumarin O-deethylation in human liver microsomes. Human P450 1A2 also catalyzed 3-hydroxylation of 7-methoxy (OMe) coumarin at appreciable rates but P450 2E1 did not. Intramolecular kinetic isotope effects were used as estimates of the intrinsic kinetic deuterium isotope effects for both 7-OMe and 7-OEt coumarin dealkylation reactions. The apparent intrinsic isotope effect for P450 1A2 (9.4 for O-demethylation, 6.1 for O-deethylation) showed little attenuation in other competitive and noncompetitive experiments. With P450 2E1, the intrinsic isotope effect (9.6 for O-demethylation, 6.1 for O-deethylation) was attenuated in the noncompetitive intermolecular experiments. High noncompetitive intermolecular kinetic isotope effects were seen for 7-OEt coumarin O-deethylation in a baculovirus-based microsomal system and five samples of human liver microsomes (7.3-8.1 for O-deethylation), consistent with the view that P450 1A2 is the most efficient P450 catalyzing this reaction in human liver microsomes and indicating that the C-H bond-breaking step makes a major contribution to the rate of this P450 (1A2) reaction. Thus, the rate-limiting step appears to be the chemistry of the breaking of this bond by the activated iron-oxygen complex, as opposed to steps involved in the generation of the reactive complex. The conclusion about the rate-limiting step applies to all of the systems studied with this model P450 1A2 reaction including human liver microsomes, the most physiologically relevant.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1992

Distribution of cytochrome P450 1A1 and NADPH-cytochrome P450 reductase in lungs of rabbits treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin: ultrastructural immunolocalization and in situ hybridization.

Induction of cytochrome P450 1A1 (P450 1A1) in a variety of tissues is a well established consequence of exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. Although localization of the induced protein within the lung has been described, the precise intracellular distribution of the enzyme is not clear. Analysis of tissue sections, microsomal proteins, and mRNA from lungs of treated and untreated rabbits established that P450 1A1 had been induced by treatment with TCDD. Rabbit lungs from animals treated with TCDD were examined with immunocytochemistry and in situ hybridization, to identify the cell types that contain P450 1A1 and those that contain mRNA encoding P450 1A1. Endothelial cells of the entire vascular bed of rabbit lung reacted markedly with anti-P450 1A1. Likewise, cells lining both arteries and veins, as well as capillary endothelial cells, reacted strongly with the cDNA probe for mRNA encoding P450 1A1. Clara cells at all levels of airway labeled prominently for both P-450 1A1 and P450 1A1 mRNA. In addition, type 2 cells, alveolar macrophages, and to a lesser degree, ciliated cells reacted with the cDNA probe. P450 reductase, which is required for P450 activity, has previously been identified in Clara cells, type 2 cells, and alveolar macrophages, but not in endothelium of rabbit lung. We have now obtained similar results for the localization of mRNA encoding P-450 reductase. This finding brings into question the function of P450 1A1 in endothelium.

Animals↗
PubMed1991

N,N',N''-triethylenethiophosphoramide (thio-TEPA) oxygenation by constitutive hepatic P450 enzymes and modulation of drug metabolism and clearance in vivo by P450-inducing agents.

The cancer chemotherapeutic drug N,N',N''-triethylenephosphoramide (thio-TEPA) is oxidatively desulfurated to yield the active metabolite N,N',N''-triethylenephosphoramide (TEPA) in a reaction catalyzed by the phenobarbital-inducible rat liver P450 enzyme IIB1. In the current study, the role of constitutively expressed P450 enzymes in thio-TEPA metabolism was studied using purified P450s, isolated liver microsomes, and intact rats. Metabolism of thio-TEPA (100 microM) to TEPA by uninduced adult female and male rat liver microsomes proceeded at initial rates of 0.10 and 0.28 nmol TEPA formed/min/mg microsomal protein, respectively. Although these rates are low compared to those catalyzed by phenobarbital-induced liver microsomes (3.5 nmol TEPA/min/mg), they are sufficient to contribute to the systemic metabolism of this drug. Thio-TEPA metabolism catalyzed by uninduced female liver microsomes was approximately 70% inhibitable by antibodies selectively reactive with P450 IIC6. For the uninduced male liver microsomes, which exhibit a severalfold higher rate of thio-TEPA metabolism, enzyme activity was only 15-20% inhibitable by these antibodies but was 80-85% inhibited by an anti-P450 IIC6 monoclonal antibody cross-reactive with P450 IIC11, which is expressed only in the males. Consistent with these observations, purified P450s IIC11 and IIC6 both oxidized thio-TEPA in reconstituted systems (turnover, 1.1 and 0.3 min-1 P450-1, respectively, at 100 microM substrate), while several other constitutive hepatic P450s exhibited significantly lower or undetectable activities (turnover, less than or equal to 0.15 min-1 P450-1). Metabolism of thio-TEPA by purified P450 IIC11 was associated with a time-dependent inactivation of the cytochrome analogous to that previously shown to accompany thio-TEPA metabolism catalyzed by P450 IIB1. Depletion of hepatic P450 IIC11 by cisplatin treatment of adult male rats led to a 70% reduction of TEPA formation catalyzed by the isolated liver microsomes, suggesting that cisplatin may influence thio-TEPA pharmacokinetics when these two drugs are given in combination. The extent to which hepatic P450s contribute to thio-TEPA metabolism and clearance in vivo was assessed by monitoring thio-TEPA and TEPA pharmacokinetics in rats that exhibit widely differing rates of microsomal thio-TEPA metabolism, i.e., uninduced female and male rats, and male rats treated with the P450 IIB1 inducers clofibrate and phenobarbital. In accord with the microsomal activities, conversion of thio-TEPA to TEPA was less extensive and thio-TEPA elimination slower in female than in male rats.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Bioactivation of arachidonic acid by the cytochrome P450 monooxygenases of guinea pig lung: the orthologue of cytochrome P450 2B4 is solely responsible for formation of epoxyeicosatrienoic acids.

Guinea pig lung microsomes converted arachidonic acid (AA) to two classes of cytochrome P450 (P450)-dependent metabolites, 16- through 20-hydroxyeicosatetraenoic acids [(16-20)-OH-AA] and epoxyeicosatrienoic acids (EETs). The rate of formation of (16-20)-OH-AA was approximately 3-fold higher in microsomes from beta-naphthoflavone-induced versus untreated animals. In microsomes from untreated or induced animals EETs, the major class of P450 metabolites in guinea pig lung, were formed in a regioselective manner, with 8,9-, 11,12-, and 14,15-regioisomers accounting for > or = 90% of the total EETs. With isozyme-selective inhibitors and inhibitory antibodies the role of individual pulmonary P450 isozymes in AA metabolism was examined. Metyrapone and SKF-525A (P450 2B selective) inhibited EET formation by > or = 85% with little effect on (16-20)-OH-AA formation. 1-Aminobenzotriazole (1 mM), a mechanism-based inhibitor with low isozyme selectivity, inhibited the formation of both classes of metabolites by > 95%, whereas N-alpha-methylbenzyl-1-aminobenzotriazole (1 microM), a P450 2B-selective mechanism-based inhibitor, abolished EET formation with little effect on (16-20)-OH-AA formation. Antibodies to rabbit P450 2B4 also abolished EET formation without inhibiting the formation of (16-20)-OH-AA, whereas antibodies to rabbit P450 4B1 did not inhibit the formation of either class of metabolites. alpha-Naphthoflavone (P450 1A1 selective in lung) did not inhibit the formation of either class of metabolites. These data demonstrate that the guinea pig orthologue of P450 2B4 is solely responsible for the bioactivation of AA to EETs in guinea pig lung and that a form of P450 other than a 2B, 4B, or 1A isozyme, which is inducible by beta-naphthoflavone, is responsible for (16-20)-OH-AA formation.

8,11,14-Eicosatrienoic Acid↗
PubMed1993

Purification and characterization of recombinant-expressed cytochrome P450 2C3 from Escherichia coli: 2C3 encodes the 6 beta-hydroxylase deficient form of P450 3b.

Rabbit cytochrome P450 2C3 was expressed from its cDNA in Escherichia coli as a chimeric enzyme in which a portion of the N-terminal membrane anchor sequence of 2C3 was replaced with a modified sequence derived from P450 17 alpha. The nucleotide sequence encoding the N-terminus of P450 17 alpha was modified previously to achieve a high level of expression of P450 17 alpha in E. coli by altering the first eight codons of P450 17 alpha to reflect second codon preferences for high expression and to minimize the potential for the formation of a stable secondary structure of the corresponding RNA transcript. The modified P450 2C3 was expressed at > 400 nmol/liter of culture. P450 2C3 was isolated to apparent electrophoretic homogeneity and a specific content > 14 nmol P450/mg protein. When reconstituted with P450 reductase and dilauroyl-L-alpha-lecithin, the purified E. coli-expressed P450 2C3 catalyzed 16 alpha, but not 6 beta-hydroxylation of progesterone. Expression of unmodified 2C3 from its cDNA in COS-1 cells confirmed the absence of detectable 6 beta-hydroxylase activity. In addition, the enzyme expressed in E. coli is activated by the allosteric effector 5 beta-pregnane-3 beta,20 alpha-diol, with a resultant Vmax = 10 min-1 and Km = 20 microM and is not inhibited by 16 alpha-methylprogesterone. These results indicate that the 2C3 cDNA encodes an enzymatic form characteristic of IIIvo/J and B/J inbred rabbits rather than a second enzymatic form expressed in most outbred and some inbred strains that catalyzes both high efficiency 16 alpha- and 6 beta-hydroxylation of progesterone. Our results have identified the enzyme variant encoded by the 2C3 cDNA and have demonstrated the utility of E. coli for the expression of recombinant P450 enzymes.

Amino Acid Sequence↗
PubMed1996

Construction of a human cytochrome P450 1A1: rat NADPH-cytochrome P450 reductase fusion protein cDNA and expression in Escherichia coli, purification, and catalytic properties of the enzyme in bacterial cells and after purification.

A plasmid (pCW) was modified to code for a fusion protein consisting of the complete sequence of human cytochrome P450 (P450) 1A1 (with only the second amino acid changed) in the N-terminal portion connected by a Ser-Thr linker to the portion of rat NADPH-P450 reductase beginning at amino acid 57. This plasmid was used to express the fusion protein in Escherichia coli DH5alpha cells and the protein was purified from detergent-solubilized bacterial membranes using DEAE and 2',5'-ADP agarose chromatography. The purified fusion protein catalyzed benzo[a]pyrene 3-hydroxylation, 7-ethoxyresorufin O-deethylation, and zoxazolamine 6-hydroxylation. Catalytic activity was not increased in the presence of added NADPH-P450 reductase, cytochrome b5, or phospholipid. The fusion protein could also transfer electrons to cytochromes c and b5 but not P450 lA2. The same oxidation products of benzo[a]pyrene were formed with the purified fusion protein and the fusion protein functioning in bacterial cells. The catalytic activity of the human P450 1A1 fusion protein toward several substrates is markedly less than that of a similar fusion protein constructed with rat P450 1A1, in line with the reported differences in catalytic activities of the rat and human P450 1A1 enzymes. The purified fusion protein also oxidized (+)- and (-)-benzo[a]pyrene 7,8-dihydrodiols and eight aryl and heterocyclic amines to genotoxic products, in the absence of added NADPH-P450 reductase. The demonstration of catalytic activities of the human fusion protein within bacterial cells suggests the prospect of utilizing such cellular systems for production of human P450 metabolites.

Animals↗
PubMed1996

The omega-hydroxlyation of lauric acid: oxidation of 12-hydroxlauric acid to dodecanedioic acid by a purified recombinant fusion protein containing P450 4A1 and NADPH-P450 reductase.

The recombinant fusion protein rF450[mRat4Al/mRatOR]L1, containing the heme domain of P450 4A1 and the flavin domains of NADPH-P450 reductase, when incubated with dilaurylphosphatidylcholine (DLPC), Chaps, cytochrome b5, and a 20-fold excess of purified NADPH-P450 reductase, catalyzes the omega- oxidation of lauric acid at a rate of about 300 nmol/min/nmol P450. This is the first report of a mammalian P450 enzyme with such a high turnover number. The resultant 12-hydroxydodecanoic acid [12-hydroxylauric acid (12-OH LA)] is further oxidized by the P450 oxygenase reaction to dodecanedioic acid (decane-1,10-dicarboxylic acid) via 12,12-dihydroxydodecanoic acid. Spectral binding studies show that 12-OH LA inhibits the binding of lauric acid to the active site of P450 with a Ki of about 1.9 microM. The construction and expression of recombinant P450 4A1 containing a six-member polyhistidine domain at the carboxy-terminus of the protein is described. Reconstitution experiments with this purified recombinant P450 4A1, DLPC, Chaps, b5, and purified NADPH-P450 reductase show results similar to those obtained with the purified fusion protein, albeit at lower turnover rates. The requirement for normal-phase HPLC in resolving the metabolites formed during lauric acid metabolism is demonstrated.

Animals↗
PubMed1996

Kinetic analysis of the activation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone by heterologously expressed human P450 enzymes and the effect of P450-specific chemical inhibitors on this activation in human liver microsomes.

The tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is enzymatically activated by the hydroxylation of the alpha-methyl and alpha-methylene groups, leading to the formation of reactive species which can pyridyloxobutylate and methylate DNA, respectively. The present study examined the kinetic parameters of NNK-derived keto alcohol (alpha-methyl hydroxylation), and keto aldehyde (alpha-methylene hydroxylation) formation catalyzed by human P450s heterologously expressed by either the baculovirus-insect cell expression system (P450s 2A6, 2D6, 2E1, and 3A4) or by stable expression in CHO cells (P450s 3A4 and 2D6) and human B-lymphoblastoid cells (P450 2D6). Membrane preparations of the expressed P450s catalyzed the alpha-hydroxylation of NNK, leading to the formation of keto aldehyde and keto alcohol. Human P450 2A6 showed the lowest KM (118 microM) for the formation of keto aldehyde. A similar KM was observed for keto alcohol formation by expressed P450 2A6, but the kcat was lower than the value obtained for keto aldehyde formation. The addition of exogenous b5 increased the expressed 2A6-dependent NNK hydroxylation activity 2.5-fold for both alpha-hydroxylation products. Human P450s 2E1 and 2D6 exhibited a high capacity for keto alcohol formation; however, their KM values for this reaction were in the millimolar range. Expressed human P450 3A4 oxidized NNK to keto aldehyde also with a high KM. Ten human liver microsomal samples were each shown to activate NNK to keto aldehyde and keto alcohol. A positive correlation coefficient of 0.74 was found between keto aldehyde formation and both coumarin 7-hydroxylation (P450 2A6) and 6 beta-testosterone hydroxylation (3A4) activity in characterized human liver microsomes. Keto alcohol formation showed a significant correlation with ethoxyresorufin O-dealkylation (P450 1A2) in human liver microsomes. Both coumarin and troleandomycin, specific inhibitors of P450 2A6 and 3A4, respectively, inhibited the formation of keto aldehyde, but inhibited the formation of keto alcohol only slightly in human liver microsomes. Both furafylline, a P450 1A2 inhibitor, and N-nitrosodimethylamine, a P450 2E1 substrate, inhibited the formation of keto alcohol but not keto aldehyde in human liver microsomes. Quinidine, a specific inhibitor of P450 2D6, was not an effective inhibitor of NNK metabolism. These results demonstrate that P450s 2A6 and 3A4 may be important P450s for the activation of NNK to a DNA-methylating agent and keto aldehyde via the alpha-methylene hydroxylation pathway. P450s 1A2, 2E1, and 2D6 are shown to be selective for alpha-methyl hydroxylation of NNK leading to keto alcohol and a DNA-pyridyloxobutylating agent.

Animals↗
PubMed1997

Characterization of a fusion protein between human cytochrome P450 1A1 and rat NADPH-P450 oxidoreductase in Escherichia coli.

A cDNA of fusion protein between human cytochrome P450 1A1 and rat NADPH-P450 reductase was genetically engineered and expressed in Escherichia coli DH5alpha cells under the control of an inducible tac promoter (Y. J. Chun, T. Shimada, and F. P. Guengerich, (1996) Arch. Biochem. Biophys. 330, 48-58). E. coli membranes of transformed cells showed much higher P450 1Al-dependent monooxygenase and NADPH-P450 reductase activities than pCW control vector or P450 1A1 expression vector-transformed cells. Ethoxyresorufin O-deethylase and methoxyresorufin O-demethylase were 22-fold and 11-fold higher than the control activity, respectively. alpha-Naphthoflavone and beta-naphthoflavone strongly inhibited P450 1A1 activity of the fusion protein, with alpha-naphthoflavone being more potent than beta-naphthoflavone. Divalent cations (e.g. Ca2+ and Mg2+) increased P450 1A1 activity as well as NADPH-P450 reductase activity. These results demonstrate that this fusion protein in E. coli membrane may be a useful model for elucidating details of protein-protein interactions between P450 and NADPH-P450 reductase in the endoplasmic reticulum of mammalian cells.

Animals↗
PubMed1997

Expression, purification, and characterization of a catalytically active human cytochrome P450 1A2:rat NADPH-cytochrome P450 reductase fusion protein.

An enzymatically active human cytochrome P450 (P450) 1A2:rat NADPH-P450 reductase fusion protein was purified and partially characterized following heterologous expression in Escherichia coli. A cDNA was engineered to include the coding sequence for human P450 1A2 at its 5' end (up to but not including the stop codon) fused in-frame to the coding sequence for a truncated (soluble) rat NADPH-P450 reductase at its 3' end via an oligonucleotide sequence encoding the hydrophilic dipeptide Ser-Thr. This fusion plasmid was expressed in E. coli and the recombinant protein was purified from the detergent-solubilized membrane fraction via sequential DEAE, ADP-agarose, and hydroxylapatite chromatographies. The purified protein has the spectral characteristics of human P450 1A2 and cytochrome c reduction activity comparable to rabbit NADPH-P450 reductase. The fusion protein catalyzed 7-ethoxyresorufin O-deethylation and phenacetin O-deethylation to appreciable levels in the presence of NADPH and phospholipid. While these activities were comparable to those of other such P450:NADPH-P450 reductase fusion proteins, they were lower than those of the system reconstituted from its individual hemoprotein and flavoprotein components. Nevertheless, the production of a functional, catalytically self-sufficient monooxygenase in E. coli enhances the prospect of using bacterial systems for production and characterization of human P450 drug metabolites as well as for biodegradation of chemicals in the environment.

Animals↗
PubMed1999

Electrospray ionization mass spectrometric analysis of intact cytochrome P450: identification of tienilic acid adducts to P450 2C9.

A general scheme for the purification of baculovirus-expressed cytochrome P450s (P450s) from the crude insect cell pastes has been designed which renders the P450s suitable for analysis by high-performance liquid chromatography (HPLC) electrospray ionization mass spectrometry (ESI-MS). An HPLC/ESI-MS procedure has been developed to analyze small amounts of intact purified P450 (P450s cam-HT, 1A1, 1A2, 2A6, 2B1, 2C9, 2C9 C175R, 3A4, 3A4-HT) and rat NADPH cytochrome P450 reductase (P450 reductase). The experimentally determined and predicted (based on the amino acid sequences) molecular masses (MMs) of the various proteins had identical rank orders. For each individual protein, the difference between the experimentally determined (+/-SD, based on experiments performed on at least 3 different days) and predicted MMs ranged from 0.002 to 0.035%. Each experimentally determined MM had a standard deviation of less than 0.09% (based on the charge state distribution). Application of this HPLC/ESI-MS technique made the detection of the covalent modification to P450 2C9 following mechanism-based inactivation by tienilic acid possible. In the absence of glutathione, three P450 2C9 species were detected that produced ESI mass spectra corresponding to native P450 2C9 and both a monoadduct and a diadduct of tienilic acid to P450 2C9. In the presence of glutathione, only native P450 2C9 and the monoadduct were detected. Based on the observed mass shifts for the P450 2C9/tienilic acid adducts, a mechanism for the inactivation of P450 2C9 by tienilic acid is proposed.

Animals↗
PubMed1997

Both cytochromes P450 2E1 and 3A are involved in the O-hydroxylation of p-nitrophenol, a catalytic activity known to be specific for P450 2E1.

4-Nitrophenol 2-hydroxylation activity was previously shown to be mainly catalyzed by P450 2E1 in animal species and humans. As this chemical compound is widely used as an in vitro probe for P450 2E1, this study was carried out to test its catalytic specificity. First, experiments were carried out on liver microsomes and hepatocyte cultures of rat treated with different inducers. Liver microsomes from pyrazole- and dexamethasone-treated rats hydroxylated p-nitrophenol with a metabolic rate increased by 2.5- and 2.7-fold vs control. Dexamethasone treatment increased the hepatic content of P450 3A but not that of P450 2E1. Two specific inhibitors of P450 3A catalytic activities, namely, ketoconazole and troleandomycin (TAO), inhibited up to 50% of 4-nitrophenol hydroxylation in dexamethasone-treated rats but not in controls. Hepatocyte cultures from dexamethasone-treated rats transformed p-nitrophenol into 4-nitrocatechol 7.8 times more than controls. This catalytic activity was inhibited by TAO. Similarly, hepatocyte cultures from pyrazole-treated rats hydroxylated p-nitrophenol with a metabolic ratio increased by about 8-fold vs control. This reaction was inhibited by diethyl dithiocarbamate and dimethyl sulfoxide, both inhibitors of P450 2E1. Second, the capability of human P450s other than P450 2E1 to catalyze the formation of 4-nitrocatechol was examined in a panel of 13 human liver microsomes. Diethyl dithiocarbamate and ketoconazole reduced 4-nitrophenol hydroxylase activity by 77% (+/- 11) and 13% (+/- 16), respectively. Furthermore, the residual activity following diethyl dithiocarbamate inhibition was significantly correlated with seven P450 3A4 catalytic activities. Finally, the use of human cell lines genetically engineered for expression of human P450s demonstrated that P450 2E1 and 3A4 hydroxylated 4-nitrophenol with turnovers of 19.5 and 1.65 min-1, respectively. In conclusion, P450 3A may make a significant contribution to 4-nitrophenol hydroxylase activity in man and rat.

Animals↗
PubMed2000

Mechanism-based inactivation of cytochrome P450 2B1 by 7-ethynylcoumarin: verification of apo-P450 adduction by electrospray ion trap mass spectrometry.

7-Ethynylcoumarin was synthesized as a potential mechanism-based inhibitor, and it was found to be an effective inactivator of 7-ethoxy-4-(trifluoromethyl)coumarin (7EFC) O-deethylation catalyzed by purified, reconstituted P450 2B1. In contrast, 7-ethynylcoumarin demonstrated minimal inactivation of P450 2A6-mediated 7-hydroxycoumarin formation. The inactivation of P450 2B1 demonstrated pseudo-first-order kinetics and was NADPH- and inhibitor-dependent. The maximal rate constant for the inactivation of 2B1 was 0.39 min(-)(1) at 30 degrees C, and thus, the time required to inactivate 50% of the P450 2B1 that was present (t(1/2)) was 1.8 min. The estimated concentration which led to half-maximal inactivation (K(I)) was 25 microM. No protection from inactivation was seen in the presence of nucleophiles (glutathione and sodium cyanide), an iron chelator (deferroxamine), or superoxide dismutase and catalase. Addition of the substrate (7EFC) protected P450 2B1 from inactivation, in a concentration-dependent manner. The partition ratio for P450 2B1 was 25; i.e., the number of metabolic events was 25-fold higher than the number of inactivating events. Incubations of 7-ethynylcoumarin with P450 2B1 for 10 min resulted in an 80% loss in enzymatic activity, while 90% of the ability to form a reduced-CO complex remained. This activity loss was not recovered following dialysis, indicative of irreversible inactivation. Covalent attachment of the entire inhibitor and oxygen to apo-P450 2B1, in a 1:1 ratio, was shown via electrospray ion trap mass spectrometry. This method also verified the absence of modification to the heme or the cytochrome P450 reductase. Taken together, the characterization of the inhibition seen with P450 2B1 and 7-ethynylcoumarin was consistent with all of the criteria required to distinguish a mechanism-based inactivator. In addition, electrospray ion trap mass spectrometry has the potential to be applied to protein adducts above and beyond those associated with the mechanism-based inactivation of cytochrome P450s.

Animals↗
PubMed1995

Identification by in vitro mutagenesis of the interaction of two segments of C2MstC1, a chimera of cytochromes P450 2C2 and P450 2C1.

A hybrid cytochrome P450, C2MstC1, with 306 N-terminal amino acids derived from cytochrome P450 2C2 sequence and 184 C-terminal amino acids from cytochrome P450 2C1 acquires a novel progesterone 21-hydroxylase activity which is absent in the parent enzymes. Extension of the cytochrome P450 2C2 sequence to residue 382 reduced progesterone hydroxylase activity to 5% of that of C2MstC1, while further extension to residue 411 or 462 increased activity back to about 30 or 40%, respectively. In the chimera with cytochrome P450 2C2 sequence to residue 382, substitution of cytochrome P450 2C1 amino acids at positions 368, 369, and 374 increased progesterone hydroxylase activity to a level equivalent to that of C2MstC1. In the chimera with cytochrome P450 2C2 sequence extending to residue 411, substitutions of P450 2C1 amino acids at positions 386 and 388, in addition those at 368, 369, and 374, were required to obtain activities equivalent to that of C2MstC1, which suggests an interaction between these two regions. The lauric acid hydroxylase activities of all chimeras and mutant cytochromes P450 differed by 2-fold or less, demonstrating that the changes in progesterone hydroxylase activity reflected altered interactions with the substrate. Alignment of cytochrome P450 2C1 sequence with cytochromes P450cam, P450BM-3, and P450terp predicts that residues 368/369 and 386/388 are in adjacent antiparallel strands of the same beta-sheet, in agreement with the experimental data suggesting an interaction between these two regions.

Amino Acid Sequence↗
PubMed2001

Stimulation of cytochrome P450 reactions by apo-cytochrome b5: evidence against transfer of heme from cytochrome P450 3A4 to apo-cytochrome b5 or heme oxygenase.

Many cytochrome P450 (P450)-dependent reactions have been shown to be stimulated by another microsomal protein, cytochrome b(5) (b(5)). Two major explanations are (i) direct electron transfer from b(5) and (ii) a conformational effect in the absence of electron transfer. Some P450s (e.g. 3A4, 2C9, 17A, and 4A7) are stimulated by either b(5) or b(5) devoid of heme (apo-b(5)), indicating a lack of electron transfer, whereas other P450s (e.g. 2E1) are stimulated by b(5) but not by apo-b(5). Recently, a proposal has been made by Guryev et al. (Biochemistry 40, 5018-5031, 2001) that the stimulation by apo-b(5) can be explained only by transfer of heme from P450 preparations to apo-b(5), enabling electron transfer. We have repeated earlier findings of stimulation of catalytic activity of testosterone 6beta-hydroxylation activities with four P450 preparations, in which nearly all of the heme was accounted for as P450. Spectral analysis of mixtures indicated that only approximately 5% of the heme can be transferred to apo-b(5), which cannot account for the observed stimulation. The presence of the heme scavenger apomyoglobin did not inhibit the stimulation of P450 3A4-dependent testosterone or nifedipine oxidation activity. Further evidence against the presence of loosely bound P450 3A4 heme was provided in experiments with apo-heme oxygenase, in which only 3% of the P450 heme was converted to biliverdin. Finally, b(5) supported NADH-b(5) reductase/P450 3A4-dependent testosterone 6beta-hydroxylation, but apo-b(5) did not. Thus, apo-b(5) can stimulate P450 3A4 reactions as well as b(5) in the absence of electron transfer, and heme transfer from P450 3A4 to apo-b(5) cannot be used to explain the catalytic stimulation.

Apoproteins↗
PubMed1994

Different mechanisms of regioselection of fatty acid hydroxylation by laurate (omega-1)-hydroxylating P450s, P450 2C2 and P450 2E1.

P450 2C2 as well as P450 2E1 [Fukuda, T. et al. (1993) J. Biochem. 113, 7-12] catalyzed the hydroxylation of medium chain fatty acids, although the regioselectivity of substrates of the former contrasted with that of the latter. Whereas P450 2E1 hydroxylated C9-C18 fatty acids at the omega-1 position and to a much lesser extent at the omega and omega-2 positions, P450 2C2 hydroxylated C9-C13 fatty acids at different positions dependent on the chain length of fatty acids. Among the fatty acids used as the substrate, undecanoate was hydroxylated at the omega-1 position almost exclusively by P450 2C2. The proportion of omega-hydroxylated products produced by P450 2C2 was markedly increased with decreasing chain length of fatty acids, while the hydroxylation positions were enlarged to the omega-3 position with tridecanoate. When the conserved Thr at the putative distal helix was replaced with Ser, the substrate regioselectivity of the two P450s was affected in different manners. The mutation of P450 2C2 did not change the hydroxylation positions of C9-C12 fatty acids, but caused a significant decrease in the proportion of the omega-1 hydroxy analog in the total products. In sharp contrast to P450 2C2, the mutated P450 2E1 gave additional products to those with the wild-type P450, and the number of different products increased with increasing chain length of the fatty acids. Thus, the products of palmitate hydroxylation were identified as omega-1, omega-2, omega-3, omega-4, omega-5, omega-6, and omega-7 monohydroxy isomers using gas chromatography-electron impact mass spectrometry.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗