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1.
J Biol Chem ; 292(27): 11230-11242, 2017 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-28533430

RESUMO

Cytochrome P450 (P450, CYP) 4A11 is a human fatty acid ω-hydroxylase that catalyzes the oxidation of arachidonic acid to the eicosanoid 20-hydroxyeicosatetraenoic acid (20-HETE), which plays important roles in regulating blood pressure regulation. Variants of P450 4A11 have been associated with high blood pressure and resistance to anti-hypertensive drugs, and 20-HETE has both pro- and antihypertensive properties relating to increased vasoconstriction and natriuresis, respectively. These physiological activities are likely influenced by the redox environment, but the mechanisms are unclear. Here, we found that reducing agents (e.g. dithiothreitol and tris(2-carboxyethyl)phosphine) strongly enhanced the catalytic activity of P450 4A11, but not of 10 other human P450s tested. Conversely, added H2O2 attenuated P450 4A11 catalytic activity. Catalytic roles of five of the potentially eight implicated Cys residues of P450 4A11 were eliminated by site-directed mutagenesis. Using an isotope-coded dimedone/iododimedone-labeling strategy and mass spectrometry of peptides, we demonstrated that the heme-thiolate cysteine (Cys-457) is selectively sulfenylated in an H2O2 concentration-dependent manner. This sulfenylation could be reversed by reducing agents, including dithiothreitol and dithionite. Of note, we observed heme ligand cysteine sulfenylation of P450 4A11 ex vivo in kidneys and livers derived from CYP4A11 transgenic mice. We also detected sulfenylation of murine P450 4a12 and 4b1 heme peptides in kidneys. To our knowledge, reversible oxidation of the heme thiolate has not previously been observed in P450s and may have relevance for 20-HETE-mediated functions.


Assuntos
Citocromo P-450 CYP4A/química , Ditiotreitol/química , Heme/química , Peróxido de Hidrogênio/química , Animais , Catálise , Citocromo P-450 CYP4A/genética , Citocromo P-450 CYP4A/metabolismo , Ditiotreitol/metabolismo , Heme/genética , Heme/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Ácidos Hidroxieicosatetraenoicos/biossíntese , Ácidos Hidroxieicosatetraenoicos/química , Ácidos Hidroxieicosatetraenoicos/genética , Rim/enzimologia , Fígado/enzimologia , Camundongos , Camundongos Transgênicos , Oxirredução , Ratos
2.
FEBS Lett ; 590(9): 1304-12, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-27059013

RESUMO

In humans, a considerable fraction of the retinoid pool in skin is derived from vitamin A2 (all-trans 3,4-dehydroretinal). Vitamin A2 may be locally generated by keratinocytes, which can convert vitamin A1 (all-trans retinol) into vitamin A2 in cell culture. We report that human cytochrome P450 (hP450) 27C1, a previously 'orphan' enzyme, can catalyze this reaction. Purified recombinant hP450 27C1 bound and desaturated all-trans retinol, retinal, and retinoic acid, as well as 11-cis-retinal. Although the physiological role of 3,4-dehydroretinoids in humans is unclear, we have identified hP450 27C1 as an enzyme capable of efficiently mediating their formation.


Assuntos
Família 27 do Citocromo P450/metabolismo , Retinoides/metabolismo , Humanos
3.
Curr Biol ; 25(23): 3048-57, 2015 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-26549260

RESUMO

Some vertebrate species have evolved means of extending their visual sensitivity beyond the range of human vision. One mechanism of enhancing sensitivity to long-wavelength light is to replace the 11-cis retinal chromophore in photopigments with 11-cis 3,4-didehydroretinal. Despite over a century of research on this topic, the enzymatic basis of this perceptual switch remains unknown. Here, we show that a cytochrome P450 family member, Cyp27c1, mediates this switch by converting vitamin A1 (the precursor of 11-cis retinal) into vitamin A2 (the precursor of 11-cis 3,4-didehydroretinal). Knockout of cyp27c1 in zebrafish abrogates production of vitamin A2, eliminating the animal's ability to red-shift its photoreceptor spectral sensitivity and reducing its ability to see and respond to near-infrared light. Thus, the expression of a single enzyme mediates dynamic spectral tuning of the entire visual system by controlling the balance of vitamin A1 and A2 in the eye.


Assuntos
Proteínas de Anfíbios/genética , Sistema Enzimático do Citocromo P-450/genética , Rana catesbeiana/fisiologia , Vitamina A/análogos & derivados , Vitamina A/metabolismo , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/fisiologia , Proteínas de Anfíbios/metabolismo , Animais , Sistema Enzimático do Citocromo P-450/metabolismo , Raios Infravermelhos , Células Fotorreceptoras de Vertebrados/fisiologia , Rana catesbeiana/genética , Transcriptoma , Percepção Visual , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
4.
Eur J Pharm Sci ; 73: 49-56, 2015 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-25840124

RESUMO

Aspirin (acetylsalicylic acid) is a well-known and widely-used analgesic. It is rapidly deacetylated to salicylic acid, which forms two hippuric acids-salicyluric acid and gentisuric acid-and two glucuronides. The oxidation of aspirin and salicylic acid has been reported with human liver microsomes, but data on individual cytochromes P450 involved in oxidation is lacking. In this study we monitored oxidation of these compounds by human liver microsomes and cytochrome P450 (P450) using UPLC with fluorescence detection. Microsomal oxidation of salicylic acid was much faster than aspirin. The two oxidation products were 2,5-dihydroxybenzoic acid (gentisic acid, documented by its UV and mass spectrum) and 2,3-dihydroxybenzoic acid. Formation of neither product was inhibited by desferrioxamine, suggesting a lack of contribution of oxygen radicals under these conditions. Although more liphophilic, aspirin was oxidized less efficiently, primarily to the 2,5-dihydroxy product. Recombinant human P450s 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 all catalyzed the 5-hydroxylation of salicylic acid. Inhibitor studies with human liver microsomes indicated that all six of the previously mentioned P450s could contribute to both the 5- and 3-hydroxylation of salicylic acid and that P450s 2A6 and 2B6 have contributions to 5-hydroxylation. Inhibitor studies indicated that the major human P450 involved in both 3- and 5-hydroxylation of salicylic acid is P450 2E1.


Assuntos
Aspirina/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Ácido Salicílico/metabolismo , Benzaldeídos/química , Biotransformação , Inibidores das Enzimas do Citocromo P-450/farmacologia , Sistema Enzimático do Citocromo P-450/genética , Desferroxamina/farmacologia , Inibidores Enzimáticos/farmacologia , Humanos , Hidroxilação , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/metabolismo , NADP/metabolismo , Organofosfatos/química , Oxirredução , Espécies Reativas de Oxigênio/química , Proteínas Recombinantes/metabolismo
5.
Chem Res Toxicol ; 28(4): 691-701, 2015 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-25642734

RESUMO

The carcinogenesis of urethane (ethyl carbamate), a byproduct of fermentation that is consistently found in various food products, was investigated with a combination of kinetic experiments and quantum chemical calculations. The main objective of the study was to find ΔG(⧧), the activation free energy for the rate-limiting step of the SN2 reaction among the ultimate carcinogen of urethane, vinyl carbamate epoxide (VCE), and different nucleobases of the DNA. In the experimental part, the second-order reaction rate constants for the formation of the main 7-(2-oxoethyl)guanine adduct in aqueous solutions of deoxyguanosine and in DNA were determined. A series of ab initio, density functional theory (DFT), and semiempirical molecular orbital (MO) calculations was then performed to determine the activation barriers for the reaction between VCE and nucleobases methylguanine, methyladenine, and methylcytosine. Effects of hydration were incorporated with the use of the solvent reaction field method of Tomasi and co-workers and the Langevine dipoles model of Florian and Warshel. The computational results for the main adduct were found to be in good agreement with the experiment, thus presenting strong evidence for the validity of the proposed SN2 mechanism. This allowed us to predict the activation barriers of reactions leading to side products for which kinetic experiments have not yet been performed. Our calculations have shown that the main 7-(2-oxoethyl)deoxyguanosine adduct indeed forms preferentially because the emergence of other adducts either proceeds across a significantly higher activation barrier or the geometry of the reaction requires the Watson-Crick pairs of the DNA to be broken. The computational study also considered the questions of stereoselectivity, the ease of the elimination of the leaving group, and the relative contributions of the two possible reaction paths for the formation of the 1,N(2)-ethenodeoxyguanosine adduct.


Assuntos
Carcinogênese , Carcinógenos/toxicidade , Uretana/toxicidade , Carcinógenos/química , DNA/química , Hidrólise , Teoria Quântica , Termodinâmica , Uretana/química
6.
J Biol Chem ; 290(6): 3248-68, 2015 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-25533464

RESUMO

Cytochrome P450 (P450) 17A enzymes play a critical role in the oxidation of the steroids progesterone (Prog) and pregnenolone (Preg) to glucocorticoids and androgens. In mammals, a single enzyme, P450 17A1, catalyzes both 17α-hydroxylation and a subsequent 17α,20-lyase reaction with both Prog and Preg. Teleost fish contain two 17A P450s; zebrafish P450 17A1 catalyzes both 17α-hydroxylation and lyase reactions with Prog and Preg, and P450 17A2 is more efficient in pregnenolone 17α-hydroxylation but does not catalyze the lyase reaction, even in the presence of cytochrome b5. P450 17A2 binds all substrates and products, although more loosely than P450 17A1. Pulse-chase and kinetic spectral experiments and modeling established that the two-step P450 17A1 Prog oxidation is more distributive than the Preg reaction, i.e. 17α-OH product dissociates more prior to the lyase step. The drug orteronel selectively blocked the lyase reaction of P450 17A1 but only in the case of Prog. X-ray crystal structures of zebrafish P450 17A1 and 17A2 were obtained with the ligand abiraterone and with Prog for P450 17A2. Comparison of the two fish P450 17A-abiraterone structures with human P450 17A1 (DeVore, N. M., and Scott, E. E. (2013) Nature 482, 116-119) showed only a few differences near the active site, despite only ∼50% identity among the three proteins. The P450 17A2 structure differed in four residues near the heme periphery. These residues may allow the proposed alternative ferric peroxide mechanism for the lyase reaction, or residues removed from the active site may allow conformations that lead to the lyase activity.


Assuntos
Domínio Catalítico , Esteroide 17-alfa-Hidroxilase/química , Proteínas de Peixe-Zebra/química , Sequência de Aminoácidos , Androstenos/farmacologia , Animais , Cinética , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Progesterona/farmacologia , Ligação Proteica , Esteroide 17-alfa-Hidroxilase/antagonistas & inibidores , Esteroide 17-alfa-Hidroxilase/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/metabolismo
7.
Biochemistry ; 53(39): 6161-72, 2014 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-25203493

RESUMO

Cytochrome P450 (P450) 4A11 is the only functionally active subfamily 4A P450 in humans. P450 4A11 catalyzes mainly ω-hydroxylation of fatty acids in liver and kidney; this process is not a major degradative pathway, but at least one product, 20-hydroxyeicosatetraenoic acid, has important signaling properties. We studied catalysis by P450 4A11 and the issue of rate-limiting steps using lauric acid ω-hydroxylation, a prototypic substrate for this enzyme. Some individual reaction steps were studied using pre-steady-state kinetic approaches. Substrate and product binding and release were much faster than overall rates of catalysis. Reduction of ferric P450 4A11 (to ferrous) was rapid and not rate-limiting. Deuterium kinetic isotope effect (KIE) experiments yielded low but reproducible values (1.2-2) for 12-hydroxylation with 12-(2)H-substituted lauric acid. However, considerable "metabolic switching" to 11-hydroxylation was observed with [12-(2)H3]lauric acid. Analysis of switching results [Jones, J. P., et al. (1986) J. Am. Chem. Soc. 108, 7074-7078] and the use of tritium KIE analysis with [12-(3)H]lauric acid [Northrop, D. B. (1987) Methods Enzymol. 87, 607-625] both indicated a high intrinsic KIE (>10). Cytochrome b5 (b5) stimulated steady-state lauric acid ω-hydroxylation ∼2-fold; the apoprotein was ineffective, indicating that electron transfer is involved in the b5 enhancement. The rate of b5 reoxidation was increased in the presence of ferrous P450 mixed with O2. Collectively, the results indicate that both the transfer of an electron to the ferrous·O2 complex and C-H bond-breaking limit the rate of P450 4A11 ω-oxidation.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Ácidos Láuricos/metabolismo , Algoritmos , Ligação Competitiva , Biocatálise , Citocromo P-450 CYP4A , Sistema Enzimático do Citocromo P-450/química , Citocromos b5/química , Citocromos b5/metabolismo , Deutério , Transporte de Elétrons , Compostos Férricos/química , Compostos Férricos/metabolismo , Compostos Ferrosos/química , Compostos Ferrosos/metabolismo , Humanos , Hidroxilação , Cinética , Ácidos Láuricos/química , Modelos Químicos , Modelos Moleculares , Oxirredução , Ligação Proteica , Estrutura Terciária de Proteína , Especificidade por Substrato , Trítio
8.
J Biol Chem ; 289(43): 29614-30, 2014 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-25160618

RESUMO

In this study, we found that the full-length CYP2C8 (WT CYP2C8) and N-terminal truncated splice variant 3 (∼ 44-kDa mass) are localized in mitochondria in addition to the endoplasmic reticulum. Analysis of human livers showed that the mitochondrial levels of these two forms varied markedly. Molecular modeling based on the x-ray crystal structure coordinates of CYP2D6 and CYP2C8 showed that despite lacking the N-terminal 102 residues variant 3 possessed nearly complete substrate binding and heme binding pockets. Stable expression of cDNAs in HepG2 cells showed that the WT protein is mostly targeted to the endoplasmic reticulum and at low levels to mitochondria, whereas variant 3 is primarily targeted to mitochondria and at low levels to the endoplasmic reticulum. Enzyme reconstitution experiments showed that both microsomal and mitochondrial WT CYP2C8 efficiently catalyzed paclitaxel 6-hydroxylation. However, mitochondrial variant 3 was unable to catalyze this reaction possibly because of its inability to stabilize the large 854-Da substrate. Conversely, mitochondrial variant 3 catalyzed the metabolism of arachidonic acid into 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids and 20-hydroxyeicosatetraenoic acid when reconstituted with adrenodoxin and adrenodoxin reductase. HepG2 cells stably expressing variant 3 generated higher levels of reactive oxygen species and showed a higher level of mitochondrial respiratory dysfunction. This study suggests that mitochondrially targeted variant 3 CYP2C8 may contribute to oxidative stress in various tissues.


Assuntos
Processamento Alternativo/genética , Hidrocarboneto de Aril Hidroxilases/genética , Citocromo P-450 CYP2C8/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Sequência de Aminoácidos , Aminoácidos/metabolismo , Animais , Ácido Araquidônico/metabolismo , Hidrocarboneto de Aril Hidroxilases/química , Hidrocarboneto de Aril Hidroxilases/metabolismo , Biocatálise , Células COS , Respiração Celular , Chlorocebus aethiops , Simulação por Computador , Citocromo P-450 CYP2C8/química , Citocromo P-450 CYP2C8/metabolismo , Heme/metabolismo , Células Hep G2 , Humanos , Isoenzimas/metabolismo , Microssomos Hepáticos/enzimologia , Modelos Moleculares , Dados de Sequência Molecular , Oxirredução , Estresse Oxidativo , Ligação Proteica , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Alinhamento de Sequência
9.
J Biol Chem ; 289(24): 16867-82, 2014 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-24759104

RESUMO

DNA damage incurred by a multitude of endogenous and exogenous factors constitutes an inevitable challenge for the replication machinery. Cells rely on various mechanisms to either remove lesions or bypass them in a more or less error-prone fashion. The latter pathway involves the Y-family polymerases that catalyze trans-lesion synthesis across sites of damaged DNA. 7,8-Dihydro-8-oxo-2'-deoxyguanosine (8-oxoG) is a major lesion that is a consequence of oxidative stress and is associated with cancer, aging, hepatitis, and infertility. We have used steady-state and transient-state kinetics in conjunction with mass spectrometry to analyze in vitro bypass of 8-oxoG by human DNA polymerase η (hpol η). Unlike the high fidelity polymerases that show preferential insertion of A opposite 8-oxoG, hpol η is capable of bypassing 8-oxoG in a mostly error-free fashion, thus preventing GC→AT transversion mutations. Crystal structures of ternary hpol η-DNA complexes and incoming dCTP, dATP, or dGTP opposite 8-oxoG reveal that an arginine from the finger domain assumes a key role in avoiding formation of the nascent 8-oxoG:A pair. That hpol η discriminates against dATP exclusively at the insertion stage is confirmed by structures of ternary complexes that allow visualization of the extension step. These structures with G:dCTP following either 8-oxoG:C or 8-oxoG:A pairs exhibit virtually identical active site conformations. Our combined data provide a detailed understanding of hpol η bypass of the most common oxidative DNA lesion.


Assuntos
DNA Polimerase Dirigida por DNA/química , Guanina/análogos & derivados , Simulação de Acoplamento Molecular , Sequência de Aminoácidos , Domínio Catalítico , DNA Polimerase Dirigida por DNA/metabolismo , Guanina/química , Humanos , Cinética , Dados de Sequência Molecular , Nucleotídeos/química , Nucleotídeos/metabolismo , Especificidade por Substrato
10.
Xenobiotica ; 44(4): 309-319, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24010633

RESUMO

1. Metoclopramide is a widely used clinical drug in a variety of medical settings with rare acute dystonic events reported. The aim of this study was to assess a previous report of inactivation of CYP2D6 by metoclopramide, to determine the contribution of various CYPs to metoclopramide metabolism, and to identify the mono-oxygenated products of metoclopramide metabolism. 2. Metoclopramide interacted with CYP2D6 with Type I binding and a Ks value of 9.56 ± 1.09 µM. CYP2D6 was the major metabolizer of metoclopramide and the two major products were N-deethylation of the diethyl amine and N-hydroxylation on the phenyl ring amine. CYPs 1A2, 2C9, 2C19, and 3A4 also metabolized metoclopramide. 3. While reversible inhibition of CYP2D6 was noted, CYP2D6 inactivation by metoclopramide was not observed under conditions of varying concentration or varying time using Supersomes(TM) or pooled human liver microsomes. 4. The major metabolites of metoclopramide were N-hydroxylation and N-deethylation formed most efficiently by CYP2D6 but also formed by all CYPs examined. Also, while metoclopramide is metabolized primarily by CYP2D6, it is not a mechanism-based inactivator of CYP2D6 in vitro.


Assuntos
Inibidores do Citocromo P-450 CYP2D6 , Citocromo P-450 CYP2D6/metabolismo , Metoclopramida/metabolismo , Antagonistas de Dopamina/química , Humanos , Hidroxilamina/química , Hidroxilação , Cinética , Ligantes , Fígado/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Proteínas Recombinantes/metabolismo
11.
Biochem Pharmacol ; 86(4): 548-60, 2013 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-23792120

RESUMO

7-Ketocholesterol (7-KC) is found at an elevated level in patients with cancer and chronic liver disease. The up-regulation of an efflux pump, P-glycoprotein (P-gp) leads to drug resistance. To elucidate the effect of 7-KC on P-gp, P-gp function and expression were investigated in hepatoma cell lines Huh-7 and HepG2 and in primary hepatocyte-derived HuS-E/2 cells. At a subtoxic concentration, 48-h exposure to 7-KC reduced the intracellular accumulation and cytotoxicity of P-gp substrate doxorubicin in hepatoma cells, but not in HuS-E/2 cells. In Huh-7 cells, 7-KC elevated efflux function through the activation of phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway. 7-KC activated the downstream protein synthesis initiation factor 4E-BP1 and induced P-gp expression post-transcriptionally. The stimulation of efflux was reversible and could not be prevented by N-acetyl cysteine. Total cellular ATP content remained the same, whereas the lactate production was increased and fluorescence lifetime of protein-bound NADH was shortened. These changes suggested a metabolic shift to glycolysis, but glycolytic inhibitors did not eliminate 7-KC-mediated P-gp induction. These results demonstrate that 7-KC induces P-gp through PI3K/mTOR signaling and decreased the cell-killing efficacy of doxorubicin in hepatoma cells.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Cetocolesteróis/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Acetilcisteína/farmacologia , Antibióticos Antineoplásicos/metabolismo , Antibióticos Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Carcinoma Hepatocelular , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Colesterol/farmacologia , Doxorrubicina/metabolismo , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Hepatócitos/metabolismo , Humanos , Hidroxicolesteróis/farmacologia , Cetocolesteróis/metabolismo , Ácido Láctico/biossíntese , Oligomicinas/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Transdução de Sinais , Serina-Treonina Quinases TOR/antagonistas & inibidores , Regulação para Cima
12.
Chem Res Toxicol ; 26(4): 517-28, 2013 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-23432429

RESUMO

A total of 68 chemicals including derivatives of naphthalene, phenanthrene, fluoranthene, pyrene, biphenyl, and flavone were examined for their abilities to interact with human P450s 2A13 and 2A6. Fifty-one of these 68 chemicals induced stronger Type I binding spectra (iron low- to high-spin state shift) with P450 2A13 than those seen with P450 2A6, i.e., the spectral binding intensities (ΔAmax/Ks ratio) determined with these chemicals were always higher for P450 2A13. In addition, benzo[c]phenanthrene, fluoranthene, 2,3-dihydroxy-2,3-dihydrofluoranthene, pyrene, 1-hydroxypyrene, 1-nitropyrene, 1-acetylpyrene, 2-acetylpyrene, 2,5,2',5'-tetrachlorobiphenyl, 7-hydroxyflavone, chrysin, and galangin were found to induce a Type I spectral change only with P450 2A13. Coumarin 7-hydroxylation, catalyzed by P450 2A13, was strongly inhibited by 2'-methoxy-5,7-dihydroxyflavone, 2-ethynylnaphthalene, 2'-methoxyflavone, 2-naphththalene propargyl ether, acenaphthene, acenaphthylene, naphthalene, 1-acetylpyrene, flavanone, chrysin, 3-ethynylphenanthrene, flavone, and 7-hydroxyflavone; these chemicals induced Type I spectral changes with low Ks values. On the basis of the intensities of the spectral changes and inhibition of P450 2A13, we classified the 68 chemicals into eight groups based on the order of affinities for these chemicals and inhibition of P450 2A13. The metabolism of chemicals by P450 2A13 during the assays explained why some of the chemicals that bound well were poor inhibitors of P450 2A13. Finally, we compared the 68 chemicals for their abilities to induce Type I spectral changes of P450 2A13 with the Reverse Type I binding spectra observed with P450 1B1: 45 chemicals interacted with both P450s 2A13 and 1B1, indicating that the two enzymes have some similarty of structural features regarding these chemicals. Molecular docking analyses suggest similarities at the active sites of these P450 enzymes. These results indicate that P450 2A13, as well as Family 1 P450 enzymes, is able to catalyze many detoxication and activation reactions with chemicals of environmental interest.


Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Poluentes Ambientais/metabolismo , Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Sítios de Ligação , Carcinógenos/metabolismo , Citocromo P-450 CYP1B1 , Citocromo P-450 CYP2A6 , Escherichia coli/genética , Escherichia coli/metabolismo , Flavonoides/metabolismo , Humanos , Simulação de Acoplamento Molecular , Hidrocarbonetos Policíclicos Aromáticos/metabolismo
13.
Biochemistry ; 51(50): 9995-10007, 2012 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-23186213

RESUMO

Cytochrome P450 (P450) 2E1 is the major enzyme that oxidizes N-nitrosodimethylamine [N,N-dimethylnitrosamine (DMN)], a carcinogen and also a representative of some nitrosamines formed endogenously. Oxidation of DMN by rat or human P450 2E1 to HCHO showed a high apparent intrinsic kinetic deuterium isotope effect (KIE), ≥8. The KIE was not attenuated in noncompetitive intermolecular experiments with rat liver microsomes {(D)V = 12.5; (D)(V/K) = 10.9 [nomenclature of Northrop, D. B. (1982) Methods Enzymol. 87, 607-625]} but was with purified human P450 2E1 [(D)V = 3.3; (D)(V/K) = 3.7], indicating that C-H bond breaking is partially rate-limiting with human P450 2E1. With N-nitrosodiethylamine [N,N-diethylnitrosamine (DEN)], the intrinsic KIE was slightly lower and was not expressed [e.g., (D)(V/K) = 1.2] in noncompetitive intermolecular experiments. The same general pattern of KIEs was also seen in the (D)(V/K) results with DMN and DEN for the minor products resulting from the denitrosation reactions (CH(3)NH(2), CH(3)CH(2)NH(2), and NO(2)(-)). Experiments with deuterated N-nitroso-N-methyl-N-ethylamine demonstrated that the lower KIEs associated with ethyl versus methyl oxidation could be distinguished within a single molecule. P450 2E1 oxidized DMN and DEN to aldehydes and then to the carboxylic acids. No kinetic lags were observed in acid formation; pulse-chase experiments with carrier aldehydes showed only limited equilibration with P450 2E1-bound aldehydes, indicative of processive reactions, as reported for P450 2A6 [Chowdhury, G., et al. (2010) J. Biol. Chem. 285, 8031-8044]. These same features (no lag phase for HCO(2)H formation and a lack of equilibration in pulse-chase assays) were also seen with (rat) P450 2B1, which has a lower catalytic efficiency for DMN oxidation and a larger active site. Thus, the processivity of dialkyl nitrosamine oxidation appears to be shared by a number of P450s.


Assuntos
Citocromo P-450 CYP2B1/metabolismo , Citocromo P-450 CYP2E1/metabolismo , Dietilnitrosamina/metabolismo , Dimetilnitrosamina/metabolismo , Animais , Deutério/metabolismo , Humanos , Cinética , Masculino , Microssomos Hepáticos/metabolismo , Oxirredução , Ratos , Ratos Sprague-Dawley
14.
Drug Metab Lett ; 6(1): 7-14, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22372551

RESUMO

Human cytochrome P450 2D6 (CYP2D6) is involved in metabolism of approximately 25% of pharmaceutical drugs. Inactivation of CYP2D6 can lead to adverse drug interactions. Four inactivators of CYP2D6 have previously been identified: 5-Fluoro-2-[4-[(2-phenyl-1H-imidazol-5-yl)methyl]-1-piperazinyl]pyrimidine(SCH66712), (1-[(2-ethyl- 4-methyl-1H-imidazol-5-yl)-methyl]-4-[4-(trifluoromethyl)-2-pyridinyl]piperazine(EMTPP), paroxetine, and 3,4- methylenedioxymethamphetamine (MDMA). All four contain planar, aromatic groups as well as basic nitrogens common to CYP2D6 substrates. SCH66712 and EMTPP also contain piperazine groups and substituted imidazole rings that are common in pharmaceutical agents, though neither of these compounds is clinically relevant. Paroxetine and MDMA contain methylenedioxyphenyls. SCH66712 and EMTPP are both known protein adductors while paroxetine and MDMA are probable heme modifiers. The current study shows that each inactivator displays Type I binding with Ks values that vary by 2-orders of magnitude with lower Ks values associated with greater inactivation. Comparison of KI, kinact, and partition ratio values shows SCH66712 is the most potent inactivator. Molecular modeling experiments using AutoDock identify Phe120 as a key interaction for all four inactivators with face-to-face and edge-to-face pi interactions apparent. Distance between the ligand and heme iron correlates with potency of inhibition. Ligand conformations were scored according to their binding energies as calculated by AutoDock and correlation was observed between molecular models and Ks values.


Assuntos
Inibidores do Citocromo P-450 CYP2D6 , Inibidores Enzimáticos/farmacologia , Modelos Moleculares , Citocromo P-450 CYP2D6/metabolismo , Inibidores Enzimáticos/química , Humanos , Imidazóis/química , Imidazóis/farmacologia , Ligantes , N-Metil-3,4-Metilenodioxianfetamina/química , N-Metil-3,4-Metilenodioxianfetamina/farmacologia , Paroxetina/química , Paroxetina/farmacologia , Piridinas/química , Piridinas/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia
15.
J Biol Chem ; 287(13): 10613-10622, 2012 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-22262854

RESUMO

Steroid 21-hydroxylase (cytochrome P450 21A2, CYP21A2) deficiency accounts for ∼95% of individuals with congenital adrenal hyperplasia, a common autosomal recessive metabolic disorder of adrenal steroidogenesis. The effects of amino acid mutations on CYP21A2 activity lead to impairment of the synthesis of cortisol and aldosterone and the excessive production of androgens. In order to understand the structural and molecular basis of this group of diseases, the bovine CYP21A2 crystal structure complexed with the substrate 17-hydroxyprogesterone (17OHP) was determined to 3.0 Šresolution. An intriguing result from this structure is that there are two molecules of 17OHP bound to the enzyme, the distal one being located at the entrance of the substrate access channel and the proximal one bound in the active site. The substrate binding features locate the key substrate recognition residues not only around the heme but also along the substrate access channel. In addition, orientation of the skeleton of the proximal molecule is toward the interior of the enzyme away from the substrate access channel. The 17OHP complex of CYP21A2 provides a good relationship between the crystal structure, clinical data, and genetic mutants documented in the literature, thereby enhancing our understanding of congenital adrenal hyperplasia. In addition, the location of certain CYP21A2 mutations provides general understanding of structure/function relationships in P450s.


Assuntos
17-alfa-Hidroxiprogesterona/química , Hiperplasia Suprarrenal Congênita/enzimologia , Mutação , Esteroide 21-Hidroxilase/química , 17-alfa-Hidroxiprogesterona/metabolismo , Hiperplasia Suprarrenal Congênita/genética , Animais , Sítios de Ligação , Bovinos , Cristalografia por Raios X , Humanos , Esteroide 21-Hidroxilase/genética , Esteroide 21-Hidroxilase/metabolismo , Relação Estrutura-Atividade
16.
Drug Metab Dispos ; 39(6): 974-83, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21422192

RESUMO

5-Fluoro-2-[4-[(2-phenyl-1H-imidazol-5-yl)methyl]-1-piperazinyl]pyrimidine (SCH 66712) is a potent mechanism-based inactivator of human cytochrome P450 2D6 that displays type I binding spectra with a K(s) of 0.39 ± 0.10 µM. The partition ratio is ~3, indicating potent inactivation that addition of exogenous nucleophiles does not prevent. Within 15 min of incubation with SCH 66712 and NADPH, ∼90% of CYP2D6 activity is lost with only ~20% loss in ability to bind CO and ~25% loss of native heme over the same time. The stoichiometry of binding to the protein was 1.2:1. SDS-polyacrylamide gel electrophoresis with Western blotting and autoradiography analyses of CYP2D6 after incubations with radiolabeled SCH 66712 further support the presence of a protein adduct. Metabolites of SCH 66712 detected by mass spectrometry indicate that the phenyl group on the imidazole ring of SCH 66712 is one site of oxidation by CYP2D6 and could lead to methylene quinone formation. Three other metabolites were also observed. For understanding the metabolic pathway that leads to CYP2D6 inactivation, metabolism studies with CYP2C9 and CYP2C19 were performed because neither of these enzymes is significantly inhibited by SCH 66712. The metabolites formed by CYP2C9 and CYP2C19 are the same as those seen with CYP2D6, although in different abundance. Modeling studies with CYP2D6 revealed potential roles of various active site residues in the oxidation of SCH 66712 and inactivation of CYP2D6 and showed that the phenyl group of SCH 66712 is positioned at 2.2 Å from the heme iron.


Assuntos
Inibidores do Citocromo P-450 CYP2D6 , Inibidores Enzimáticos/farmacologia , Imidazóis/farmacologia , Pirimidinas/farmacologia , Cromatografia Líquida de Alta Pressão , Citocromo P-450 CYP2D6/química , Citocromo P-450 CYP2D6/genética , Inibidores Enzimáticos/química , Escherichia coli/genética , Heme/química , Humanos , Imidazóis/química , Modelos Moleculares , Ligação Proteica , Pirimidinas/química , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
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