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PubMed2006

Protein dynamics and imidazole binding in cytochrome P450 enzymes.

P450 (cytochrome P450) enzymes have major roles in the biosynthesis of endogenous factors such as steroids and eicosanoids, in the termination of the action of endogenous factors such as retinoic acid, in the metabolism of most drugs and xenobiotics and in the generation of toxic and carcinogenic products. Understanding the determinants of the substrate and inhibitor specificities of these enzymes is important for drug design. The crystallographic analysis of the deformability of two bacterial P450 active sites associated with the binding of azole (a class of inhibitors with an imidazole or triazole ring that co-ordinates to the haem iron) inhibitors described in the present study illustrates the importance of protein conformational malleability in the binding of imidazole derivatives.

Cytochrome P-450 Enzyme Inhibitors↗

PubMed1996

Structural flexibility and functional versatility of mammalian P450 enzymes.

P450 enzymes have evolved into a large superfamily that displays great diversity in substrate and product specificities by fixing the natural amino acid substitutions with high frequency. Site-directed mutagenesis has been used to correlate the substitutions with the diverse specificities in various P450s. As a result, the common residues that determine the specificities of various mammalian P450s have been identified and aligned to the corresponding residues in the substrate-heme pocket of the 3-dimensional structures of bacterial P450s. The substrate-heme pocket appears to be structurally variable so that only a minor substitution (Ala -> -> Val, for example) at the critical positions is enough to define the altered specificity. Thus, the structural variability of the P450s provides the inherent versatility in acquiring a novel activity. Recent mutational studies indicate that the side chain size is the major determining factor of specificity, outweighing other factors such as polarity. Further understanding of the paradoxical characteristics observed may provide us with the underlying principles that determine P450 activities, and may lead to the ability to predict P450 activities based on the types of key amino acid residues.

Amino Acid Sequence↗

PubMed1999

Insect P450 enzymes.

The P450 enzymes (mixed function oxidases, cytochrome P450 monooxygenases), a diverse class of enzymes found in virtually all insect tissues, fulfill many important tasks, from the synthesis and degradation of ecdysteroids and juvenile hormones to the metabolism of foreign chemicals of natural or synthetic origin. This diversity in function is achieved by a diversity in structure, as insect genomes probably carry about 100 P450 genes, sometimes arranged in clusters, and each coding for a different P450 enzyme. Both microsomal and mitochondrial P450s are present in insects and are best studied by heterologous expression of their cDNA and reconstitution of purified enzymes. P450 genes are under complex regulation, with induction playing a central role in the adaptation to plant chemicals and regulatory mutations playing a central role in insecticide resistance. Polymorphisms in induction or constitutive expression allow insects to scan their P450 gene repertoire for the appropriate response to chemical insults, and these evolutionary pressures in turn maintain P450 diversity.

Animals↗

PubMed2005

Reactivity of a new class of P450 enzyme models.

P450 enzyme models carrying a SO3(-) ligand coordinating to iron have been synthesized and characterized. These complexes show characteristics very similar to those of iron-heme cofactors of P450 enzymes. Their reactivity towards different reactions catalyzed by P450 enzymes such as epoxidation of double bonds, hydroxylation of non-activated C-H bonds, N-dealkylation of amines, and cleavage of diols (C-C-bond cleavage) has been investigated.

Computer Simulation↗

PubMed2004

The role of cytochrome P450 enzymes in the metabolism of risperidone and its clinical relevance for drug interactions.

In the recent years it has been increasingly recognized that pharmacogenetical factors play an important role in the drug treatment. These factors may influence the appearance of side-effects and drug interactions due to interindividual differences in the activity of metabolizing enzymes. Risperidone in humans is mainly metabolized to 9-hydroxyrisperidone by the polymorphic cytochrome enzyme P450 2D6 (CYP2D6). Plasma concentrations of risperidone and 9-hydroxyrisperidone show large interindividual variability, which may be partly related to the activity of the CYP2D6 enzyme. Around seven percent of Caucasians have a genetically inherited impaired activity of the CYP2D6 enzyme. Debrisoquine metabolic ratio (a marker of CYP2D6 activity) and the number of CYP2D6 active genes have been related to risperidone plasma concentrations among patients during steady-state conditions. A large number drugs have been described to be metabolized by CYP2D6, and it is therefore important to evaluate the clinical significance of the impaired metabolism and possible drug interactions on the enzyme. Since risperidone/9-hydroxyrisperidone ratio strongly correlates with CYP2D6 enzyme activity and the number of CYP2D6 active genes, thus it might be a useful tool in clinical practice to estimate the possible risk of drug interactions due to impaired CYP2D6 enzyme activity. CYP3A4 is the most abundant drug metabolizing enzyme in humans, and in vitro and in vivo results suggest also a role for the enzyme in risperidone metabolism. The consideration of the implication of cytochrome P450 enzymes in risperidone metabolism may help to individualize dose schemes in order to avoid interactions and potentially dangerous side-effects, such us QTc interval lengthening among patients with cardiac risk factors.

Chromatography, High Pressure Liquid↗

PubMed1997

Identification of a functional water channel in cytochrome P450 enzymes.

Cytochrome P450 enzymes are monooxygenases that contain a functional heme b group linked to a conserved cysteine with a thiolate bond. In the native state, the central iron atom is hexacoordinated with a covalently bound water molecule. The exclusion of solvent molecules from the active site is essential for efficient enzymatic function. Upon substrate binding, water has to be displaced from the active site to prevent electron uncoupling that results in hydrogen peroxide or water. In contrast to typical hemoproteins, the protein surface is not directly accessible from the heme of cytochromes P450. We postulate a two-state model in which a conserved arginine, stabilizing the heme propionate in all known cytochrome P450 crystal structures, changes from the initial, stable side-chain conformation to another rotamer (metastable). In this new state, a functional water channel (aqueduct) is formed from the active site to a water cluster located on the thiolate side of the heme, close to the protein surface. This water cluster communicates with the surface in the closed state and is partly replaced by the flipping arginine side chain in the open state, allowing water molecules to exit to the surface or to reaccess the active site. This two-state model suggests the presence of an exit pathway for water between the active site and the protein surface.

Amino Acid Sequence↗

PubMed1993

Effect of immobilization on the activity of rat hepatic microsomal cytochrome P450 enzymes.

Cytochrome P450 enzyme preparations were obtained from phenobarbital-treated rats. On immobilization in Romicon PM 10 polysulfone anisotropic hollow fibers, 20-25% of the original activity remained. The immobilization procedure did not significantly alter the Km of the preparations when 7-ethoxycoumarin was the substrate. Coimmobilization of the preparations with glucose-6-phosphate dehydrogenase allowed cofactor recycling to take place but resulted in an apparent drop in the value for the Km. The Vmax of immobilized preparations with or without cofactor recycling was about 25% of that observed with the nonimmobilized preparations. The flow rate through the reactors greatly affected the time at which product was detected in the effluent from the reactor and the amount of 7-hydroxycoumarin produced. It is suggested that this reflects alternative fates of the NADPH cofactor when residence times in the reactor are lengthened. Increasing the amounts of protein immobilized increased the Km and decreased the Vmax values in comparison to the free enzyme. Immobilization increased the stability of preparations with respect to ionic strength and storage. Little effect was noted with regard to changes in temperature stability or pH optima of immobilized preparations.

Animals↗

PubMed2006

Computational models for predicting interactions with cytochrome p450 enzyme.

Cytochrome p450 (CYP) enzymes are predominantly involved in Phase 1 metabolism of xenobiotics. As only 6 isoenzymes are responsible for approximately 90 % of known oxidative drug metabolism, a number of frequently prescribed drugs share the CYP-mediated metabolic pathways. Competing for a single enzyme by the co-administered therapeutic agents can substantially alter the plasma concentration and clearance of the agents. Furthermore, many drugs are known to inhibit certain p450 enzymes which they are not substrates for. Because some drug-drug interactions could cause serious adverse events leading to a costly failure of drug development, early detection of potential drug-drug interactions is highly desirable. The ultimate goal is to be able to predict the CYP specificity and the interactions for a novel compound from its chemical structure. Current computational modeling approaches, such as two-dimensional and three-dimensional quantitative structure-activity relationship (QSAR), pharmacophore mapping and machine learning methods have resulted in statistically valid predictions. Homology models have been often combined with 3D-QSAR models to impose additional steric restrictions and/or to identify the interaction site on the proteins. This article summarizes the available models, methods, and key findings for CYP1A2, 2A6, 2C9, 2D6 and 3A4 isoenzymes.

Computational Biology↗

PubMed2006

CYP74C3 and CYP74A1, plant cytochrome P450 enzymes whose activity is regulated by detergent micelle association, and proposed new rules for the classification of CYP74 enzymes.

CYP74C3 (cytochrome P450 subfamily 74C3), an HPL (hydroperoxide lyase) from Medicago truncatula (barrel medic), and CYP74A1, an AOS (allene oxide synthase) from Arabidopsis thaliana, are key membrane-associated P450 enzymes in plant oxylipin metabolism. Both recombinant detergent-free enzymes are monomeric proteins with dual specificity and very low enzyme activity that can be massively activated with detergent. This effect is a result of the formation of a complex between the protein monomer and a single detergent micelle and, in the case of CYP74A1, has a major effect on the substrate specificity of the enzyme. Association with a detergent micelle without an effect on protein oligomeric state represents a new mechanism of activation for membrane-associated P450 enzymes. This may represent a second unifying feature of CYP74 enzymes, in addition to their known differences in reaction mechanism, which separates them functionally from more classical P450 enzymes. Highly concentrated and monodispersed samples of detergent-free CYP74C3 and CYP74A1 proteins should be suitable for structural resolution. On the basis of recent evidence for incorrect assignment of CYP74 function, using the current rules for CYP74 classification based on sequence relatedness, we propose an alternative based on substrate and product specificity for debate and discussion.

Aldehyde-Lyases↗

PubMed1996

Selective localisation of P450 enzymes and NADPH-P450 oxidoreductase in rat basal ganglia using anti-peptide antisera.

Environmental or endogenous toxins may cause nigral cell death in Parkinson's disease (PD) due to altered expression of P450 enzymes. In rat brain, immunohistochemistry using anti-peptide antisera showed NADPH-P450 oxidoreductase and CYP2B1/2 in various hypothalamic nuclei and CYP1A1 in the globus pallidus, but neither enzyme was expressed in substantia nigra. No specific immunoreactivity to CYP2D1 or CYP3A1 was found in any brain region examined. In contrast, CYP2E1 was expressed in substantia nigra and in striatal blood vessels. Since CYP2E1 is associated with free radical production, it may contribute to the oxidative stress believed to underlie nigral degeneration.

Alleles↗

PubMed2005

Thermophilic cytochrome P450 enzymes.

Thermophilic cytochrome P450 enzymes are of potential interest from structural, mechanistic, and biotechnological points of view. The structures and properties of two such enzymes, CYP119 and CYP175A1, have been investigated and provide the foundation for future work on thermophilic P450 enzymes.

Archaeal Proteins↗

PubMed2005

Highly miniaturized formats for in vitro drug metabolism assays using vivid fluorescent substrates and recombinant human cytochrome P450 enzymes.

Highly miniaturized P450 screening assays designed to enable facile analysis of P450 drug interactions in a 1536-well plate format with the principal human cytochrome P450 enzymes (CYP3A4, 2D6, 2C9, 2C19, and 1A2) and Vivid fluorogenic substrates were developed. The detailed characterization of the assays included stability, homogeneity, and reproducibility of the recombinant P450 enzymes and the kinetic parameters of their reactions with Vivid fluorogenic substrates, with a focus on the specific characteristics of each component that enable screening in a low-volume 1536-well plate assay format. The screening assays were applied for the assessment of individual cytochrome P450 inhibition profiles with a panel of selected assay modifiers, including isozyme-specific substrates and inhibitors. IC(50) values obtained for the modifiers in 96- and 1536-well plate formats were similar and comparable with values obtained in assays with conventional substrates. An overall examination of the 1536-well assay statistics, such as signal-to-background ratio and Z' factor, demonstrated that these assays are a robust, successful, and reliable tool to screen for cytochrome P450 metabolism and inhibition in an ultra-high-throughput screening format.

Cytochrome P-450 Enzyme Inhibitors↗

PubMed2006

Recent progress in cytochrome P450 enzyme electrochemistry.

Cytochrome P450 (CYP) enzymes perform crucial functions in humans, including the metabolism of drugs and hormone synthesis. The catalytic reactions performed by these enzymes (typically monoxygenation) require the transfer of electrons. Thermodynamic and mechanistic detail of the electron transfer component of these catalytic processes has been obtained traditionally from potentiometric titrations. More recently, voltammetric approaches (that are inherently simpler and require less sample) have been used. This has been made possible by the creation of biocompatible electrode surfaces at which the P450 enzyme is confined and able to undergo physiologically relevant electron transfer processes. The continuing challenge has been to obtain an in vivo-like enzyme response, and to provide the basis for the creation of an artificial bioprocess in vitro. A powerful instrumental electrochemical method, employing Fourier-transformed large-amplitude ac voltammetry, offers the potential for greater insight and new opportunities to understand the nuances of the electron transfer process. This review highlights several recent advances in the electrochemistry of P450 enzymes rather than providing a comprehensive review of P450 electrochemistry.

Cytochrome P-450 Enzyme System↗

PubMed1995

Oxidation of aflatoxin B1 by bacterial recombinant human cytochrome P450 enzymes.

Human cytochromes P450 (P450) 1A2 and P450 3A4 were expressed in Escherichia coli, purified, and used in reconstituted oxidation systems. The optimal system for P450 3A4 included a mixture of phospholipids, sodium cholate, cytochrome b5, GSH, and MgCl2. Relatively high catalytic activities were obtained with such a system for aflatoxin (AF) B1 3 alpha-hydroxylation and 8,9-epoxidation. P450 3A4 was more active than P450 1A2 in forming genotoxic AFB1 oxidation products. Analysis of the AFB1 products indicated that P450 3A4 formed AFQ1 and the exo-8,9-epoxide; P450 1A2 formed AFM1, a small amount of AFQ1, and both the exo- and endo-8,9-epoxides. The endo epoxide is essentially nongenotoxic in the umu test, as found previously in bacterial mutagenicity assays [Iyer, R. S., Coles, B. F., Raney, K. D., Thier, R., Guengerich, F. P., and Harris, T. M. (1994) J. Am. Chem. Soc. 116, 1603-1609]. 7,8-Benzoflavone (alpha-naphthoflavone, alpha NF) stimulated AFB1 (exo) 8,9-epoxidation and inhibited 3 alpha-hydroxylation in human liver microsomes and a reconstituted P450 3A4 system but was a potent inhibitor of all reactions catalyzed by P450 1A2. Plots of AFB1 3 alpha-hydroxylation and 8,9-epoxidation vs AFB1 concentration were sigmoidal in both human liver microsomes and the reconstituted P450 3A4 system. The results are consistent with the view that P450 3A4 is a major human liver P450 enzyme involved in AFB1 activation, although the in vivo situation may be more complex due to the presence of the enzyme in the gastrointestinal tract.

Aflatoxin B1↗

PubMed2006

Progress towards the easier use of P450 enzymes.

The cytochrome P450 enzymes (P450s or CYPs) form a large family of heme proteins involved in drug metabolism and in the biosynthesis of steroids, lipids, vitamins and natural products. Their remarkable ability to catalyze the insertion of oxygen into non-activated C-H bonds has attracted the interest of chemists for several decades. Very few chemical methods exist that directly hydroxylate aliphatic or aromatic C-H bonds, and most of them are not selective or of limited scope. Biocatalysts such as P450s represent a promising alternative: however, their applications have been limited by substrate specificity, low activity, poor stability and the need for cofactors. This review covers the attempts to overcome these limitations using approaches such as mutagenesis, chemical modifications, conditions engineering and immobilization.

Bacterial Proteins↗

PubMed1994

Theory for the observed isotope effects on the formation of multiple products by different kinetic mechanisms of cytochrome P450 enzymes.

Cytochrome P450 systems are unusual in that many of them can convert a substrate to a number of different metabolites. Several kinetic mechanisms may be envisioned by which the metabolites may be formed. In each of the mechanisms, the substrate combines with the enzyme in different orientations to form a set of (ES) complexes that then are activated to a set of (EOS) complexes. The fate of these (EOS) complexes determines the kinetic mechanism. In the "parallel pathway" mechanism, the (EOS) complexes are so stable and rigid they cannot be converted either directly or indirectly to complexes with different orientations; the orientation of the (ES) complexes thus determines which metabolite will be formed. In the "nondissociative" mechanisms, the complexes are not rigid; instead they undergo interconversion while the substrate remains in the active site of the enzyme. In the "dissociative" mechanisms, the (EOS) complexes dissociate to (EO) and (S), but recombine to form (EOS) complexes with either the same or different orientations. Steady-state equations describing the deuterium isotope effects for these kinetics mechanisms have been derived and solved for competitive experiments, in which equal concentrations of both deuterated and nondeuterated substrates are present in incubation mixtures, and for noncompetitive experiments, in which only one of the substrates is present. The equations reveal that comparisons of the isotope effects on the formation of a metabolite by a pathway that does not involve the abstraction of a deuterium from a deuterated substrate (the non-deuterium abstraction pathway) in both experiments can differentiate between the kinetic mechanisms. A value of 1.0 for (v)H/(v)D in the competitive experiment, but < 1.0 to > 1.0 for the value of (Vmax/Km)H/(Vmax/Km)D in the noncompetitive experiment, is diagnostic for the "dissociative" mechanisms. Values of 1.0 in both kinds of experiments are diagnostic for the "parallel pathway" mechanism. Values of < 1.0 in both types of experiments are diagnostic for the "nondissociative" mechanisms. The equations also predict possible unusual substrate-inhibitor interactions when the "dissociative" mechanism is operative.

Binding, Competitive↗

PubMed1996

A convenient method to discriminate between cytochrome P450 enzymes and flavin-containing monooxygenases in human liver microsomes.

Liver microsomes are a frequently used probe to investigate the phase I metabolism of xenobiotics in vitro. Structures containing nucleophilic hetero-atoms are possible substrates for cytochrome P450 enzymes (P450) and flavin-containing monooxygenases (FMO). Both enzymes are located in the endoplasmatic reticulum of hepatocytes and both need oxygen and NADPH as cofactors. The common method to distinguish between the two enzyme systems is to use the thermal inactivation of FMO and to inhibit P450 completely with carbon monoxide, N-octylamine or N-benzylimidazole. In the literature no indication could be found that the heat inactivation of FMO does not affect any of the human P450 enzymes or that the overall P450 inhibitors inhibit the different human P450 enzymes sufficiently and do not affect the FMO. The effect of N-benzylimidazole and heat inactivation was tested on specific activities of seven P450 enzymes in human liver microsomes, 1A2, 2A6, 2C9, 2C19, 2D6, 3A4/5, and 2E1, using methoxyresorufin O-demethylation, coumarin 7-hydroxylation, (S)-warfarin 4-hydroxylation, (S)-(+)-mephenytoin 4-hydroxylation, dextrometorphan O-demethylation, oxidation of denitronifedipine, and chlorzoxazone 6-hydroxylation respectively. The sulfoxidation of methimazole (MMI) was used as a specific probe for the determination of FMO activity. Methimazole sulfoxidation was compared with the well known assay for FMO metabolism, the formation of N,N-dimethylaniline (DMA) N-oxide, to be confirmed as an exclusively FMO mediated reaction. The participation of P450 and FMO in the sulfoxidation of four sulfur containing peptides, ametryne; terbutryne, prometryne and methiocarb was investigated using human liver microsomes. All four reactions were demonstrated to be catalysed predominantly by cytochrome P450.

Aniline Compounds↗

PubMed2003

Comparison of the formation of N-alkylprotoporphyrin IX after interaction of porphyrinogenic xenobiotics with single cDNA-expressed human P450 enzymes in microsomes prepared from baculovirus-infected insect cells and human lymphoblastoid cell lines.

In a previous study using microsomes from human lymphoblastoid cell lines (HLCL) containing single cDNA-expressed human cytochrome P450 (P450) enzymes, human P450 enzymes were identified that are susceptible to mechanism-based inactivation by the porphyrinogenic xenobiotics, 3-[(arylthio)ethyl]sydnone (TTMS), 3,5-diethoxycarbonyl-1,4-dihydro-2,6-dimethyl-4-ethylpyridine (4-ethylDDC) and allylisopropylacetamide (AIA). In this study, we tested the hypothesis that N-alkylprotoporphyrin IX (N-alkylPP) formation following interaction of porphyrinogenic xenobiotics with single cDNA-expressed human P450 enzymes in microsomes from HLCL would occur only with P450 enzymes that had undergone mechanism-based inactivation. In a previous study, when 4-ethylDDC and NADPH interacted with human liver microsomes possessing elevated levels of CYP1A2 and 2C9, N-ethylprotoporphyrin IX (N-ethylPP) was not formed despite the fact that it was formed in microsomes from baculovirus-infected insect cell lines (BIICL) containing either CYP1A2 or 2C9. In this study, we tested the hypothesis that 4-ethylDDC underwent biotransformation by CYP3A4 present in human liver microsomes, diverting the xenobiotic from CYP1A2 and 2C9. Fluorometry was used to measure N-alkylPP formation following interaction of porphyrinogenic xenobiotics and NADPH with cDNA-expressed human P450 enzymes in microsomes from HLCL or BIICL. With TTMS and 4-ethylDDC but not with AIA, N-alkylPP formation was observed only with human P450 enzymes CYP2D6, 1A2, 3A4, or 2C9 in microsomes from HLCL, which had undergone mechanism-based inactivation. Microsomes from BIICL containing CYP3A4 were added to a mixture of NADPH, 4-ethylDDC, and microsomes from BIICL containing CYP1A2 and 2C9. The addition of CYP3A4 to CYP1A2 and 2C9 did not decrease N-ethylPP formation, providing no support for the hypothesis.

Animals↗