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)

A key step in fungal biosynthesis of lysine, enzymatic reduction of alpha-aminoadipate at C6 to the semialdehyde, requires two gene products in Saccharomyces cerevisiae, Lys2 and Lys5. Here, we show that the 31-kDa Lys5 is a specific posttranslational modification catalyst, using coenzyme A (CoASH) as a cosubstrate to phosphopantetheinylate Ser880 of the 155-kDa Lys2 and activate it for catalysis. Lys2 was subcloned from S. cerevisiae and expressed in and purified from Escherichia coli as a full-length 155-kDa enzyme, as a 105-kDa adenylation/peptidyl carrier protein (A/PCP) fragment (residues 1-924), and as a 14-kDa PCP fragment (residues 809-924). The apo-PCP fragment was covalently modified to phosphopantetheinylated holo-PCP by pure Lys5 and CoASH with a Km of 1 microM and kcat of 3 min-1 for both the PCP and CoASH substrates. The adenylation domain of the A/PCP fragment activated S-carboxymethyl-L-cysteine (kcat/Km = 840 mM-1 min-1) at 16% the efficiency of L-alpha-aminoadipate in [32P]PPi/ATP exchange assays. The holo form of the A/PCP 105-kDa fragment of Lys2 covalently aminoacylated itself with [35S]S-carboxymethyl-L-cysteine. Addition of NADPH discharged the covalent acyl-S-PCP Lys2, consistent with a reductive cleavage of the acyl-S-enzyme intermediate. These results identify the Lys5/Lys2 pair as a two-component system in which Lys5 covalently primes Lys2, allowing alpha-aminoadipate reductase activity by holo-Lys2 with catalytic cycles of autoaminoacylation and reductive cleavage. This is a novel mechanism for a fungal enzyme essential for amino acid metabolism.
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PMID:Lysine biosynthesis in Saccharomyces cerevisiae: mechanism of alpha-aminoadipate reductase (Lys2) involves posttranslational phosphopantetheinylation by Lys5. 1032 Mar 45

NADPH-d (nicotinamide-adenine dinucleotide phosphate-diaphorase) neurons are thought to migrate improperly during development in the brains of schizophrenic patients. This enzyme is a nitric oxide synthase (NOS). Nitric oxide (NO) is known to affect neurodevelopmental processes in the CNS. Therefore, we hypothesized that interference of NO generation during development may produce some aspects of schizophrenia symptomatology in a rat model. In these experiments, neonatal rats were challenged with a NOS inhibitor (L-nitroarginine 1-100 mg/kg s.c.) daily on post-natal days 3-5. L-Nitroarginine (L-NoArg) treated male rats developed a hypersensitivity to amphetamine in adulthood versus vehicle treated controls, whereas female rats did not. However, L-NoArg treated female rats developed a hypersensitivity to phencyclidine (PCP) at juvenile and adult ages versus vehicle treated controls, whereas male animals did not. L-NoArg treated male rats also had deficits in pre-pulse inhibition of startle whereas adult female rats did not. The results are discussed in terms of a new neurodevelopmental model of schizophrenia and male/female differences inherent in this disease.
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PMID:On the effect of neonatal nitric oxide synthase inhibition in rats: a potential neurodevelopmental model of schizophrenia. 1047 Oct 83

During iron starvation the Gram-negative pathogenic bacterium Pseudomonas aeruginosa makes the nonribosomal peptide siderophore pyochelin by a four protein, 11 domain assembly line, involving a cascade of acyl-S-enzyme intermediates on the PchE and PchF subunits that are elongated, heterocyclized, reduced, and N-methylated before release. Purified PchG is shown to be an NADPH-dependent reductase for the hydroxyphenylbisthiazoline-S-PchF acyl enzyme, regiospecifically converting one of the dihydroheterocyclic thiazoline rings to a thiazolidine. The K(m) for the PchG protein is 1 microM, and the k(cat) for throughput to pyochelin is 2 min(-1). The nitrogen of the newly generated thiazolidine ring can be N-methylated upon addition of SAM, to yield the mature pyochelin chain still tethered as a pyochelinyl-S-PchF at the PCP domain. A presumed methyltransferase (MT) domain embedded in the PchF subunit catalyzes this N-methylation. Mutation of a conserved G to R in the MT core motif abolishes MT activity and subsequent chain release from PchF. The thioesterase (TE) domain of PchF catalyzes hydrolytic release of the fully mature pyochelinyl chain to produce the pyochelin siderophore at a rate of 2 min(-1), at least 30-40-fold faster than in the absence of hydroxyphenylbisthiazolinyl-COOH (HPTT-COOH) chain reduction and N-methylation. A mutation in the PchF TE domain does not catalyze autodeacylation and release of the pyochelinyl-S-enzyme. Thus, full reconstitution of the nonribosomal peptide synthetase assembly line by purified protein components has been obtained for production of this tandem bisheterocyclic siderophore.
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PMID:In vitro reconstitution of the Pseudomonas aeruginosa nonribosomal peptide synthesis of pyochelin: characterization of backbone tailoring thiazoline reductase and N-methyltransferase activities. 1146 65

Pentachlorophenol 4-monooxygenase (PCP4MO) from Sphingomonas chlorophenolica is a flavoprotein that hydroxylates PCP in the presence of NADPH and oxygen. In order to investigate the structure and function of active site, recombinant PCP4MO (rePCP4MO) was produced in Escherichia coli as a glutathione S-transferase (GST) fusion protein. Moreover, a tobacco etch virus (TEV) protease cleavage site (EKLYFQG) was introduced into GST-PCP4MO and a his-tagged TEV protease was employed. Hence, a two-step purification protocol was developed which allowed obtaining 15-20 mg of rePCP4MO from 1 L culture. The rePCP4MO revealed identity with native enzyme by SDS-PAGE and N-terminal sequence analyses. Furthermore, a polyclonal PCP4MO antibody was produced with GST-PCP4MO and purified by immunoaffinity chromatography, where both the native and recombinant forms of PCP4MO showed interaction. However, rePCP4MO was identified as apoprotein with no evidence for a typical flavoprotein spectrum. The catalytic activity could be detected in the presence of FAD. The K(m) and V(max) values for PCP were 50 microM and 30 nmol/min/mg, respectively.
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PMID:Production and characterization of the recombinant Sphingomonas chlorophenolica pentachlorophenol 4-monooxygenase. 1170 94

Phencyclidine (PCP) was analyzed for its ability to inactivate human cytochrome p450 (p450) 2B6. PCP inactivated the 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of p450 2B6 in a concentration-, time-, and NADPH-dependent manner and exhibited pseudo-first order kinetics. The K(I) was 10 microM, k(inact) was 0.01 min(-1), which corresponds to a t(1/2) of 31 min. The partition ratio was approximately 45. Spectral analysis of the heme moiety demonstrated that the heme was not modified during inactivation. Extensive dialysis of the PCP-inactivated p450 2B6 did not cause a return in catalytic activity demonstrating PCP inactivation was irreversible. Including 7-ethoxycoumarin, an alternate substrate, protected 2B6 from inactivation by PCP indicating competition of the two substrates for the active site. Exogenous nucleophiles such as glutathione (GSH) and cyanide could not protect p450 2B6 from PCP inactivation demonstrating that the reactive intermediate remained within the p450 active site. High performance liquid chromatography analysis of p450 2B6 inactivated in the presence of (3)H-labeled PCP showed that PCP binding was specific for the p450 and not to other proteins in the reaction mixture. The stoichiometry of binding of PCP to p450 2B6 was demonstrated using (3)H-labeled PCP. In the absence of GSH, the stoichiometry was 5.5:1 (PCP/p450). In the presence of GSH, the stoichiometry was 1:1. This stoichiometry was further supported using electrospray ionization-liquid chromatography-mass spectrometry to analyze PCP-inactivated p450 2B1, 2B4, and 2B6.
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PMID:The mechanism-based inactivation of human cytochrome P450 2B6 by phencyclidine. 1248 52

The ability of the carcinogenic environmental toxin pentachlorophenol (PCP, 1) to react with DNA bases has been assessed using MS and NMR. Treatment of PCP (100 microM) with horseradish peroxidase (HRP/H(2)O(2)) or myeloperoxidase (MPx/H(2)O(2), from human leukocytes) in the presence of excess deoxyguanosine (dG, 2 mM) led to the isolation and identification of the oxygen-bonded C8-dG nucleoside adduct 4. The reaction was absolutely specific for dG; no detectable adduct(s) was observed from HRP/H(2)O(2) and PCP in the presence of deoxyadenosine, deoxycytidine, or thymidine. Formation of 4 was also specific for peroxidase activation that is known to oxidize PCP into the phenoxyl radical. Treatment of PCP/dG with rat liver microsomes (RLM) failed to generate 4; instead, an adduct derived from the benzoquinone electrophile tetrachloro-1,4-benzoquinone (chloranil) was observed in the extracted ion chromatogram from the RLM/NADPH-treated PCP/dG sample. The adduct 4 is the first structurally characterized O-bonded phenolic DNA nucleoside adduct and highlights the ambident electrophilicity of phenoxyl radicals (O- vs C-) in reaction at C8 of dG, as we have previously demonstrated that the para-chlorophenolic toxin, ochratoxin A (2), reacts at C8 of dG to give the C-bonded adduct 3 via the intermediacy of the OTA phenoxyl radical. Given that PCP is known to induce DNA adduct formation in vivo and human exposure has been linked to incidences of leukemia, the adduct 4 could play a key role in PCP-mediated carcinogenesis.
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PMID:An oxygen-bonded c8-deoxyguanosine nucleoside adduct of pentachlorophenol by peroxidase activation: evidence for ambident c8 reactivity by phenoxyl radicals. 1287 Aug 83

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

The hypothesis that the psychological side effects associated with the anesthetic phencyclidine (PCP) may be caused by irreversible binding of PCP or its reactive metabolite(s) to critical macromolecules in the brain has resulted in numerous in vitro studies aimed at characterizing pathways of PCP bioactivation. The studies described herein extend the current knowledge of PCP metabolism and provide details on a previously unknown metabolic activation pathway of PCP. Following incubations with NADPH- and GSH-supplemented human and rat liver microsomes and recombinant P450 2B enzymes, two sulfhydryl conjugates with MH+ ions at 547 and 482 Da, respectively, were detected by LC/MS/MS. Shebley et al. [(2006) Drug Metab. Dispos. 34, 375-383] have also observed the GSH conjugate 1 with MH+ at 547 Da in PCP incubations with rat P450 2B1 and rabbit P450 2B4 isoforms fortified with NADPH and GSH. The molecular weight of 1 is consistent with a bioactivation pathway involving Michael addition of the sulfhydryl nucleophile to the putative 2,3-dihydropyridinium metabolite of PCP obtained via a four-electron oxidation of the piperidine ring in the parent compound. The mass spectrum of the novel GSH adduct 2 with an MH+ ion at 482 Da was suggestive of a unique PCP bioactivation pathway involving initial ortho- or para-hydroxylation of the phenyl ring in PCP followed by spontaneous decomposition to piperidine and an electrophilic quinone methide intermediate, which upon reaction with GSH yielded adduct 2. The LC retention times and mass spectral properties of enzymatically generated 2 were identical to those of a reference standard obtained via reaction of GSH with synthetic p-hydroxyPCP in phosphate buffer (pH 7.4, 37 degrees C). 1H NMR and 13C-distortionless enhancement by polarization transfer (DEPT) NMR spectral studies on synthetically generated 2 suggested that the structural integrity of the p-hydroxyphenyl and cyclohexyl rings likely was preserved and that the site of GSH addition was the benzylic carbon joining the two scaffolds. The formation of 2 in human microsomes was reduced upon addition of the dual P450 2C19/P450 2B6 inhibitor (+)- N-3-benzylnirvanol. Consistent with this finding, both recombinant P450 2B6 and P450 2C19 catalyzed PCP bioactivation to 2. In the absence of GSH, synthetic p-hydroxyPCP underwent rapid decomposition (t1/2 approximately 5.2 min) to afford p-hydroxyphenylcyclohexanol and p-hydroxyphenylcyclohexene, presumably via the quinone methide intermediate. Overall, our findings on the facile degradation of synthetic p-hydroxyPCP to yield an electrophilic quinone methide intermediate capable of reacting with nucleophiles, including GSH and water, suggest an inherent instability of the putative phenolic PCP metabolite. Thus, if formed enzymatically in vivo, p-hydroxyPCP may not require further metabolism to liberate the quinone methide, which can then react with macromolecules. To our knowledge, this is the first report of a quinone methide reactive intermediate obtained in human-liver microsomal metabolism of PCP.
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PMID:Bioactivation of phencyclidine in rat and human liver microsomes and recombinant P450 2B enzymes: evidence for the formation of a novel quinone methide intermediate. 1789 69

Pentachlorophenol 4-monooxygenase (PcpB) catalyzes the hydroxylation of pentachlorophenol in the pentachlorophenol biodegradation pathway in Sphingobium chlorophenolicum. Previous studies from two different research groups proposed oppositely that the catalytic product of PcpB was tetrachlorohydroquinone (TCHQ) and tetrachlorobenzoquinone (TCBQ). We re-examined the identity of the catalytic product of PcpB, because TCHQ and TCBQ are present in a redox-equilibrium in aqueous solutions and the chemical reagents NADPH, ethyl acetate and glutathione used for the product detection in the previous studies may shift the redox-equilibrium. In this study, we investigated the effects of NADPH, ethyl acetate and glutathione on the redox-equilibrium and product distribution. Under newly designed experimental conditions, we confirmed unambiguously that the catalytic product of PcpB is TCHQ instead of TCBQ. We also propose that TCBQ may be produced non-specifically by peroxidases within the bacterial cells and that TCBQ reductase (PcpD) might act as a self-protective rather than a PCP-degradation enzyme.
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PMID:The Catalytic Product of Pentachlorophenol 4-Monooxygenase is Tetra-chlorohydroquinone rather than Tetrachlorobenzoquinone. 1908 19

Phencyclidine (PCP) is a mechanism-based inactivator of cytochrome P450 (P450) 2B6. We have analyzed several steps in the P450 catalytic cycle to determine the mechanism of inactivation of P450 2B6 by PCP. Spectral binding studies show that binding of benzphetamine, a type I ligand, to P450 2B6 was significantly affected as a result of the inactivation, whereas binding of the inhibitor n-octylamine, a type II ligand, was not compromised. Binding of these ligands to P450 2B6 occurs in two phases. Stopped-flow spectral analysis of the binding kinetics of benzphetamine to PCP-inactivated 2B6 revealed a 15-fold decrease in the rate of binding during the second phase of the kinetics (k(1) = 5.0 s(-1), A(1) = 30%; k(2) = 0.02 s(-1), A(2) = 70%, where A(2) indicates the fractional magnitude of the second phase) compared with the native enzyme (k(1) = 8.0 s(-1), A(1) = 58%; k(2) = 0.3 s(-1), A(2) = 42%). Analysis of benzphetamine metabolism by the inactivated protein using liquid chromatography/electrospray ionization/mass spectrometry showed that the rates of formation of nor-benzphetamine and hydroxylated nor-benzphetamine were decreased by 75 and 69%, respectively, whereas the rates of formation for amphetamine, hydroxybenzphetamine, and methamphetamine showed slight but statistically insignificant decreases after the inactivation. The rate of reduction of P450 2B6 by NADPH and reductase was decreased by 6-fold as a result of the modification by PCP. In addition, the extent of uncoupling of NADPH oxidation from product formation, a process leading to futile production of H(2)O(2), increased significantly during the metabolism of ethylbenzene as a result of the inactivation.
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PMID:Mechanistic analysis of the inactivation of cytochrome P450 2B6 by phencyclidine: effects on substrate binding, electron transfer, and uncoupling. 1914 70


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