Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.16.2 (PCP)
3,761 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. Microsomal metabolism of 1-benzylpiperidine (1-BP), its cis-2,6-dimethyl (cis-2,6-DMBP), 4,4-dimethyl (4,4-DMBP), and alpha, alpha-dimethyl (alpha, alpha-DMBP) analogues, and phencyclidine (PCP) has been studied to assess the involvement of P450 oxidation of the enamine tautomers of the initial endocyclic iminium metabolites. 2. The selective prevention by cyanide of the metabolite production of 1-benzyl-3-piperidone but not 1-benzyl-3-piperidinol from 1-BP is consistent with the enamine as the source of the 3-one metabolite. 3. The parent amines and particularly the independently prepared iminium species induced a pattern of metabolism-dependent irreversible inactivation of P450 benz-phetamine demethylase activity, consistent with involvement of enamine C-3 oxidation in the inactivation process. 4. Substrate activity of the endocyclic enamines and alpha-aminoketones (presumably the enol-enamine tautomers) for horseradish peroxidase under conditions where simple aliphatic amines display no activity is consistent with metabolic one-electron oxidations of the enamines.
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PMID:Haemoprotein-mediated metabolism of enamines and the possible involvement of one-electron oxidations. 748 73

Cytochrome P4502B1, the major phenobarbital-inducible isozyme in the rat liver, is inactivated by phencyclidine (PCP). Incubation of PCP with purified P4502B1 in the reconstituted enzyme system with NADPH-cytochrome P450 reductase and phospholipid resulted in a marked loss of activity as measured using a secondary incubation mixture for 7-ethoxycoumarin O-deethylase activity. The loss of activity required NADPH and PCP, and the activity decreased in a time-dependent, pseudo-first-order process indicative of mechanism-based inactivation. The rate constants for inactivation were dependent on the PCP concentrations and displayed saturation kinetics. A KI = 3.8 microM and kinact = 0.12 min-1 were determined for the inactivation by PCP. The partition ratio calculated from a plot of the percentage activity remaining after 45 min vs. the concentration ratios of PCP to P450 was 45. Although 90% of the catalytic activity was lost after a 45-min incubation, little loss was seen in the optical spectrum at 418 nm or in the ability of the reduced enzyme to bind CO. The inactivation was not inhibited by the addition of cyanide, whereas substrates such as 7-ethoxycoumarin protected against the inactivation. The iminium ion of PCP, an oxidative metabolite, inactivated P4502B1 in the same fashion as PCP. These results demonstrate that PCP is an efficient mechanism-based inactivator of rat liver P4502B1 and does not inactivate by modification of the heme moiety.
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PMID:Mechanism-based inactivation of rat liver cytochrome P4502B1 by phencyclidine and its oxidative product, the iminium ion. 749 43

The purpose of this study was to determine whether phencyclidine (PCP) inhibits constitutive hepatic cytochrome P450 (CYP) isozymes when administered to naive adult male Sprague-Dawley rats. Animals were pretreated with PCP (25 mg/kg/day for 2 days), killed 3 and 16 hr after the last dose, and liver microsomes prepared. The washed microsomes were then assayed for benzphetamine, methamphetamine (MA), and methylenedioxymethamphetamine (MDMA) N-demethylation together with MDMA demethylenation and MA 4-hydroxylation activities. MDMA demethylenation (low substrate concentration), MA 4-hydroxylation, and metoprolol alpha-hydroxylation reactions, which are catalyzed by CYP2D isozymes, were reduced > 74% 3 hr after the last PCP dose and were only partially restored 13 hr later. Benzphetamine and (-)-MDMA N-demethylation activities were restored to control values 16 hr after the last dose. These results indicate that PCP suppresses constitutive isozymes, including CYP2C11 and members of the CYP2D subfamily. The suppression of cytochromes P450 activity by PCP in vivo is consistent with its in vitro actions found in this and other studies, and demonstrates that alteration of CYP activity is another pharmacological effect of this compound.
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PMID:Inactivation of constitutive hepatic cytochromes P450 by phencyclidine in the rat. 773 12

Phencyclidine (PCP) inactivates the 7-ethoxy-4-trifluoromethylcoumarin O-deethylase activity of P4502B1 in a reconstituted system containing NADPH-cytochrome P450 (P450) reductase (reductase) and L-alpha-phosphatidylcholine, dilauroyl in a time-, concentration-, and NADPH-dependent manner. Catalytic activity of the enzyme could not be restored upon reconstitution with fresh reductase, indicating that the effect was on the P450 and not on the reductase. Although the kinetics suggested that PCP would be classified as a classical mechanism-based inactivator, protection against inactivation of P450 by PCP by the presence of an exogenous nucleophile, such as glutathione (GSH), indicated otherwise. There was no loss of spectrally detectable P450 associated with inactivation either in the presence or absence of GSH. When radiolabeled PCP was used to inactivate the enzyme and the reaction mixture analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, radioactivity was found to be associated with P450, reductase, and catalase that had been added to protect against oxidative damage. When GSH was included in the reaction mixtures, analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated a marked decrease in the binding to all three proteins. Correspondingly, analysis of the components of the inactivated sample by reversed-phase HPLC demonstrated that radioactivity was associated with P450, reductase, and catalase, and that there was a marked decrease in the labeling of all three proteins in the presence of GSH. The stoichiometry of binding of radiolabeled PCP to the proteins in the incubation mixture in the absence of GSH was 4:1. In the presence of GSH, no significant amount of radioactivity was incorporated into the proteins. An anti-PCP metabolite antibody was used to detect PCP metabolite adducts bound to the inactivated enzyme by Western blot analysis. The antibody recognized adducts bound to P450, reductase, and catalase. In the presence of GSH, there was a decrease in immunoreactivity, although binding of PCP to all three proteins was still detected. Because the added nucleophile protects against inactivation and protein labeling by PCP, these data suggest that the reactive intermediate may escape from the active site and attack other sites on the P450, as well as other proteins in the milieu.
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PMID:Metabolic inactivation of cytochrome P4502B1 by phencyclidine: immunochemical and radiochemical analyses of the protective effects of glutathione. 902 55

The effect of several cytochrome P450 (P450) inducers on cocaine metabolism were examined in order to characterize the metabolic events contributing to cocaine-induced hepatotoxicity. Phenobarbital (PB)-pretreatment of mice induced P450s 3A and 2B and markedly increased serum alanine aminotransferase (ALT) activity after cocaine or norcocaine administration. Although dexamethasone (Dex) induced P450s 3A and 2B at least to the same extent as PB, no increase in serum ALT activity was observed after cocaine or norcocaine administration. Phencyclidine (PCP) pretreatment did not increase either P450s 3A or 2B, yet it markedly enhanced cocaine- or norcocaine-induced serum ALT activity. In contrast to the marked induction of P450s 3A and 2B, P450 2C was increased only 2.5-fold by PB and to an even lesser extent by Dex or PCP. Cannabidiol (CBD), which inactivates P450s 3A and 2C in mice, completely protected mice against cocaine- or norcocaine-induced hepatotoxicity irrespective of whether they were induced or not with PB or PCP. Both PB and Dex pretreatment increased the in vitro hepatic microsomal formation of the first two sequential oxidative metabolites of cocaine (norcocaine and N-hydroxynorcocaine), whereas PCP pretreatment did not. Hepatic esterase activity was also determined after pretreatment with P450 inducers, since this is the major detoxification pathway in cocaine metabolism. Dex pretreatment markedly increased (> 11-fold) total hepatic esterase activity, whereas PB pretreatment increased it more modestly (less than fourfold) and PCP pretreatment had little effect. This marked effect of Dex pretreatment may decrease liver cocaine concentrations and thus protect mice against cocaine-induced hepatotoxicity, despite their increased P450 2B and 3A contents.
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PMID:Effect of cytochrome P450 inducers on cocaine-mediated hepatotoxicity. 963 Apr 65

Novel macrolides epothilones, produced by cellulolytic myxobacterium Sorangium cellulosum, have the activity to promote microtubule assembly, and are considered to be a potential successor to the famous antitumor drug taxol. The biosynthetic genes leading to the epothilones are clustered into a large operon. The multi-enzyme complex is a hetero-gene cluster of polyketide synthase (PKS) and non-ribosomal peptide synthetases (NRPS) and contains several functional modules, i.e. a loading module, one NRPS module, eight PKS modules, and a P450 epoxidase. The former ten modules biosynthesize desoxyepothilone (epothilones C and D), which is then epoxidized at C12 and C13 and converted into epothilones (epothilones A and B) by the P450 epoxidase. The NRPS module is responsible for the formation of the thiazole side chain from cysteine. The biosynthesis procedure of epothilones can be divided into 5 stages, i.e. formation of holo-ACP/PCP, chain initiation and thiazole ring formation, chain elongation, termination and epoxidation, and post-modification. The analysis of the gene cluster and the biosynthetic pathway reveals that novel epothilone analogs could not only be produced by chemical synthesis/modification, tranditional microbial technologies, but also can be genetically manipulated through combinatiorial biosynthesis approaches.
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PMID:[The PKS/NRPS hetero-gene cluster of epothilones]. 1596 75

Cloning and sequencing of the morABC operon region revealed the genes encoding the three components of a cytochrome P450 monooxygenase, which is required for the degradation of the N-heterocycle morpholine by Mycobacterium sp. strain HE5. The cytochrome P450 (P450(mor)) and the Fe(3)S(4) ferredoxin (Fd(mor)), encoded by morA and morB, respectively, have been characterized previously, whereas no evidence has hitherto been obtained for a specifically morpholine-induced reductase, which would be required to support the activity of the P450(mor) system. Analysis of the mor operon has now revealed the gene morC, encoding the ferredoxin reductase of this morpholine monooxygenase. The genes morA, morB and morC were identical to the corresponding genes from Mycobacterium sp. strain RP1. Almost identical mor genes in Mycobacterium chlorophenolicum PCP-1, in addition to an inducible cytochrome P450, pointing to horizontal gene transfer, were now identified. No evidence for a circular or linear plasmid was found in Mycobacterium sp. strain HE5. Analysis of the downstream sequences of morC revealed differences in this gene region between Mycobacterium sp. strain HE5 and Mycobacterium sp. strain RP1 on the one hand, and M. chlorophenolicum on the other hand, indicating insertions or deletions after recombination. Downstream of the mor genes, the gene orf1', encoding a putative glutamine synthetase, was identified in all studied strains. The gene morC of Mycobacterium sp. strain HE5 was heterologously expressed. The purified recombinant protein FdR(mor) was characterized as a monomeric 44 kDa protein, being a strictly NADH-dependent, FAD-containing reductase. The K(m) values of FdR(mor) for the substrate NADH (37.7 +/- 4.1 microM) and the artificial electron acceptors potassium ferricyanide (14.2 +/- 1.1 microM) and cytochrome c (28.0 +/- 3.6 microM) were measured. FdR(mor) was shown to interact functionally with its natural redox partner, the Fe(3)S(4) protein Fd(mor), and with the Fe(2)S(2) protein adrenodoxin, albeit with a much lower efficiency, but not with spinach ferredoxin. In contrast, adrenodoxin reductase, the natural redox partner of adrenodoxin, could not use Fd(mor) in activity assays. These results indicated that FdR(mor) can utilize different ferredoxins, but that Fd(mor) requires the specific NADH : ferredoxin oxidoreductase FdR(mor) from the P450(mor) system for efficient catalytic function.
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PMID:Analysis of the nearly identical morpholine monooxygenase-encoding mor genes from different Mycobacterium strains and characterization of the specific NADH : ferredoxin oxidoreductase of this cytochrome P450 system. 1607 38

The metabolism of phencyclidine (PCP) has been studied previously in cytochrome P450 (P450)-containing microsomal systems. However, the reactive intermediate(s) that covalently binds to the P450 and leads to inactivation or leaves the active site to modify other proteins has not been identified. In this study two electrophilic intermediates of PCP were identified by mass spectrometry and by trapping with reduced glutathione (GSH) or N-acetyl cysteine (NAC). The tentative structures of these electrophilic intermediates were determined using mass spectrometry. P450s 2B1 and 2B4 formed a metabolite that exhibited an m/z of 240 corresponding to the mass of the 2,3-dihydropyridinium species of PCP or its conjugate base, the 1,2-dihydropyridine. Chemical reduction of the incubation mixture using NaBH4 resulted in the disappearance of the signal at m/z 240, consistent with reduction of a 2,3-dihydropyridinium species. Furthermore, the reactive metabolite trapped by GSH resulted in an adduct exhibiting an m/z of 547, consistent with the mass of the 2,3-dihydropyridinium species of PCP (m/z 240), that has reacted with a molecule of GSH (m/z 308). However, P450 2B6 formed a different reactive intermediate of PCP that was isolated as a GSH adduct exhibiting an m/z of 581 and an NAC adduct with an m/z of 437. Liquid chromatography-tandem mass spectrometry analysis of these adducts suggested that a di-oxygenated iminium metabolite of PCP could be the reactive intermediate formed by P450 2B6 but not by the other 2B isoforms. These data suggest that P450 2B6 favors oxidation pathways for PCP metabolism that are different from those of P450s 2B1 and 2B4.
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PMID:Selective pathways for the metabolism of phencyclidine by cytochrome p450 2b enzymes: identification of electrophilic metabolites, glutathione, and N-acetyl cysteine adducts. 1632 15

The mechanism behind the observed inactivation of human P450 2B6 by phencyclidine (PCP) has been evaluated over the past 2 decades. The scope of the current investigation was to contribute to the fundamental knowledge of PCP oxidation and perhaps the mechanism behind P450 inactivation. To study the chemistry of PCP oxidation, we subjected PCP to the Fenton reagent. Under Fenton chemistry conditions, oxidation on all three PCP rings was observed by liquid chromatography/tandem mass spectrometry (LC-MS/MS). When PCP was incubated with the Fenton system in the presence of glutathione (GSH), three GSH-PCP conjugates were identified. Subsequent LC-MS/MS analysis of these conjugates revealed two species that had GSH attached to the cyclohexane ring of PCP and a third conjugate in which GSH was adducted to the piperidine ring. When PCP was incubated across a panel of P450 enzymes, several enzymes, including P450s 2D6 and 3A4, were able to catalyze the formation of the PCP iminium ion, whereas P450s 2B6 and 2C19 were exclusively able to hydroxylate secondary carbons on the cyclohexane ring of PCP. Subsequent mechanistic experiments revealed that only P450s 2B6 and 2C19 demonstrated loss of catalytic activity after preincubation with 10 microM PCP. Finally, investigation of P450 2B6 inactivation using structural analogs of PCP revealed that blocking the para-carbon atom on the cyclohexane ring of PCP from oxidation protected the P450 2B6 from inactivation, which suggests that a reactive intermediate generated during the hydroxylation of the cyclohexane ring may be linked to the mechanism of inactivation of P450 2B6 by PCP.
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PMID:Mechanism of inactivation of human cytochrome P450 2B6 by phencyclidine. 1678 64

Human cytochrome P450 (P450) 2B6 plays an important role in the metabolism of many drugs used in the clinic, and it has been shown to be highly polymorphic and inducible by a variety of substrates. The metabolism of phencyclidine (PCP) by P450 2B6 results in mechanism-based inactivation of the enzyme. We investigated the effects of a naturally occurring mutation of P450 2B6 where a lysine 262 is changed to an arginine (K262R) on PCP metabolism and mechanism-based inactivation of 2B6 by PCP. The K262R mutant retained the 7-ethoxy-4-trifluoromethylcoumarin O-deethylation activity when it was incubated with PCP and NADPH in the reconstituted system, whereas the wild-type enzyme was readily inactivated by PCP. Spectral binding studies showed that PCP was reversibly bound in the active site of the K262R mutant with slightly higher affinity (156 muM) compared with the wild-type 2B6 (397 muM). In addition, all the metabolites of PCP (M1-M8) that were formed by the wild-type enzyme were also formed by the K262R mutant. Although the K262R mutant metabolized PCP to give similar metabolite profiles, the overall rate of metabolite formation was lower than the wild-type enzyme. A reactive intermediate of PCP was formed by wild-type P450 2B6 and trapped with glutathione (GSH). However, no GSH conjugates were detected from incubations with the K262R mutant. These data suggest that the lysine 262 residue plays an important role in the formation of a reactive intermediate of PCP that leads to the mechanism-based inactivation of P450 2B6.
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PMID:Mutation of a single residue (K262R) in P450 2B6 leads to loss of mechanism-based inactivation by phencyclidine. 1746 30


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