Multi-step oxidations catalyzed by cytochrome P450 enzymes: Processive vs. distributive kinetics and the issue of carbonyl oxidation in chemical mechanisms

Catalysis of sequential oxidation reactions is not unusual in cytochrome P450 (P450) reactions, not only in steroid metabolism but also with many xenobiotics. One issue is how processive/distributive these reactions are, i.e., how much do the “intermediate” products dissociate. Our work with human P450s 2E1, 2A6, and 19A1 on this subject has revealed a mixture of systems, surprisingly with a more distributive mechanism with an endogenous substrate (P450 19A1) than for some xenobiotics (P450s 2E1, 2A6). One aspect of this research involves carbonyl intermediates, and the choice of catalytic mechanism is linked to the hydration state of the aldehyde. The non-enzymatic rates of hydration and dehydration of carbonyls are not rapid and whether P450s catalyze the reversible hydration is unknown. If carbonyl hydration and dehydration are slow, the mechanism may be set by the carbonyl hydration status.     

Regulation of three forms of cytochrome P-450 and epoxide hydrolase in rat liver microsomes. Effects of age, sex, and induction

A procedure for the preparation of monospecific antibody directed against rat liver microsomal cytochrome P-45-a is described. This antibody, together with monospecific antibodies to cytochromes P-450b and P-450c, has been used to show that these three forms of cytochrome P-450 are distinct and share no common antigenic determinants. These antibodies (a) give single immunoprecipitin bands with detergent-solubilized microsomes; (b) do not cross-react with the purified heterologous antigens in Ouchterlony double diffusion analyses; (c) have no effect on catalytic activity of the heterologous antigens but completely inhibit the enzymatic activity of the homologous antigens; and (d) remove only the homologous antigen from detergent-solubilized microsomes when covalently bound to a solid support. With radial immunodiffusion assay, we have quantitated these three forms of cytochrome P-450 in liver microsomes after treatment of rats with seven different inducers of cytochrome P-450. The levels of these cytochrome P-450 isozymes vary independently and are also regulated by the age and sex of the animal. The antibodies have also been used to assess the contribution of cytochromes P-450a, P-450b, and P-450c in the metabolism of xenobiotics by rat liver microsomes. A large proportion of benzo(a)pyrene metabolism and testosterone 16 alpha-hydroxylation in microsomes from untreated rats is not catalyzed by cytochromes P-450a, P-450b, and P-450c. Epoxide hydrolase, another microsomal enzyme involved in the metabolism of xenobiotics, was also quantitated by radial immunodiffusion after prior treatment of rats with microsomal enzyme inducers. The inductions of epoxide hydrolase varies independently of the induction of cytochromes P-450a, P-450b, and P-450c.     

Expression and catalysis of sex-specific cytochrome P450 isozymes in rat liver

Research interest in the study of cytochromes P450 has recently been shifting to the characterization of "constitutively" expressed isozymes from that of the inducible forms. Several "constitutive" cytochrome P450 isozymes have been purified from rat liver including five immunochemically related proteins designated cytochromes P450f, P450g, P450h, P450i, and P450k. These hemoproteins have been identified as distinct isozymes on the basis of spectral, electrophoretic, and catalytic properties and NH2-terminal sequence analysis. Purification and immunoquantitation studies have indicated that these isozymes are expressed in a developmental as well as sex-related manner, and are relatively refractory to induction by xenobiotics. Cytochromes P450h and P450g are male-specific proteins, cytochrome P450i is a female-specific isozyme, while cytochromes P450f and P450k are present in both male and female adult rats. In addition, the expression of cytochrome P450g was shown to segregate into two phenotypes in outbred rats. Genetic studies utilizing inbred strains have indicated that the gene responsible for inheritance of high levels of cytochrome P450g is autosomal. Although considerable progress has been made in understanding the role of gonadal hormones and growth hormone in the hepatic regulation of cytochromes P450g, P450h, and P450i in particular, the physiological significance of the "constitutive" isozymes in the liver remains largely unresolved.     

Prokaryotic cytochromes P450 (Review)

The research on the structure and role of bacterial cytochromes P450 are summarized in this review. We consider the organizational features of these enzymes, cytochrome-catalyzed reactions, the distribution of cytochromes among prokaryotes, and their functions in bacterial cells. We cite the data on cytochrome genes and the regulation of their expression in prokaryotes and classify cytochromes by components involved in the electronic transition. We consider the role of bacterial cytochromes in the biodegradation of carbohydrates and xenobiotics by microorganisms and the possible involvement of reactive oxygen species, which are generated in the catalytic cycle of these enzymes, at the initial stages of carbohydrate biodegradation.     

Prediction of the Ligands of the CYP9e Subfamily of Ant Cytochrome P450 with the ChEBI Ontologies of Chemical and Biological Characteristics

Cytochromes P450 are involved in the metabolism of various endogenous and exogenous compounds, and their role in the detoxification of xenobiotics has been extensively studied. CYP9e, one of the subfamilies of cytochromes P450, whose functions have been poorly studied, is amplified in the black garden ant Lasius niger. We have performed molecular modeling of 23 proteins of this family belonging to L. niger and other ant species, as well as molecular docking and virtual screening of suspected ligands. The substances used as ligands have been annotated with ChEBI ontologies to predict the chemical and biological properties of molecules forming complexes with CYP9e of ants. It has been shown that, among the ligands forming energetically favorable complexes, ChEBI ontologies of mycotoxins, phytotoxins, steroids, glycosides and terpenoids are overrepresented. Nevertheless, it has been demonstrated that in carrying out a large number of inaccurate simulations, the results of function predictions can be correlated with molecular docking and the evolutionary history of a protein family.     

Heteroactivator effects on the coupling and spin state equilibrium of CYP2C9

The cytochromes P450 are capable of oxidizing a variety of xenobiotics. Binding of a small molecule heteroactivator to a P450 can alter the coupling of substrate oxidation during P450 catalysis, but the degree to which coupling or shunting via one of the three catalytic cycle branch points is linked to the heteroactivator-modified position of bound substrate is unknown. Using reconstituted CYP2C9, stoichiometric measurements were gathered with three substrates and two classes of heteroactivators to further understand the mechanisms involved in heteroactivation. Heteroactivation of P450 metabolism appeared to involve, but not require, changes in coupling and that increased uncoupling to a specific byproduct like H(2)O(2) does not necessarily correlate to the degree of coupling. In addition, spectroscopy demonstrated that every heteroactivator tested influenced the spin equilibrium of the heme iron even in the presence of saturating substrate suggesting that both substrate proximity and the ability to desolvate the heme can be involved in heteroactivation.     

Prediction of amino acid residues participated in substrate recognition by cytochrome P450 subfamilies with broad substrate specificity

Cytochromes P450 comprise a large superfamily and several of their isoforms play a crucial role in metabolism of xenobiotics, including drugs. Although these enzymes demonstrate broad and cross‐substrate specificity, different cytochrome P450 subfamilies exhibit certain selectivity for some types of substrates. Analysis of amino acid residues of the active sites of six cytochrome subfamilies (CYP1А, CYP2А, CYP2С, CYP2D, CYP2E and CYP3А) enables to define subfamily‐specific patterns that consist of four residues. These residues are located on the periphery of the active sites of these cytochromes. We suggest that they can form a primary binding site at the entrance to the active site, defining cytochrome substrate recognition. Copyright © 2013 John Wiley & Sons, Ltd.     

Involvement of steroids and cytochromes P(450) species in the triggering of immune defenses

In vertebrates the wide variety of cytochromes P(450) (P(450)) is a key for elimination of low molecular weight xenobiotics and for the production and metabolism of steroid hormones. In contrast, xenobiotics of large molecular weight are processed and eliminated after the immune response. The suppression of immune response by native P(450)-produced glucocorticoid (GC) hormones constitutes a first link between P(450) and immunity. In the last decade, mechanisms and molecules responsible for the triggering of immune response were investigated and results showed that many tissues and organs transform native 3beta-hydroxysteroids into 7-hydroxylated metabolites that trigger immunity. Present data suggest that 7-hydroxysteroids are native anti-GCs that block the GC-induced immunosuppression. Because specific P(450) are responsible for the production of 7-hydroxylated steroids resulting into increased immunity, a second link exists between P(450) and immunity. Taken together, these findings support the proposal that P(450) are keys to all of the known defense mechanisms of vertebrates against all xenobiotic forms.     

Metabolism of sulphonated anthraquinones in rhubarb,maize and celery: the role of cytochromes P450 and peroxidases

Sulphonated anthraquinones are precursors of many synthetic dyes and pigments, recalcitrant to biodegradation, and thus contaminating many industrial effluents and rivers. In the development of a phytotreatment to remove sulphonated aromatic compounds, rhubarb (Rheum rhaponticum), a plant producing natural anthraquinones, as well as maize (Zea mays) and celery (Apium graveolens), plants not producing anthraquinones, were tested for their ability to metabolise these xenobiotics. Plants were cultivated under hydroponic conditions, with or without sulphonated anthraquinones, and were harvested at different times. Either microsomal or cytosolic fractions were prepared. The monooxygenase activity of cytochromes P450 towards several sulphonated anthraquinones was tested using a new method based on the fluorimetric detection of oxygen consumed during cytochromes P450-catalysed reactions. The activity of cytosolic peroxidases was measured by spectrophotometry, using guaiacol as a substrate. Results indicated that the activity of cytochromes P450 and peroxidases significantly increased in rhubarb plants cultivated in the presence of sulphonated anthraquinones. A higher activity of cytochromes P450 was also detected in maize and celery exposed to the pollutants. In these two plants, a peroxidase activity was also detected, but without a clear difference between the control plants and the plants exposed to the organic contaminants. This research demonstrated the existence in rhubarb, maize and celery of biochemical mechanisms involved in the metabolism and detoxification of sulphonated anthraquinones. Taken together, results confirmed that rhubarb might be the most appropriate plant for the phytotreatment of these organic pollutants.     

Dietary effects on cytochromes P450, xenobiotic metabolism, and toxicity.

The levels and activities of cytochrome P450 enzymes are influenced by a variety of factors, including the diet. In this article, the effects of selected non-nutritive dietary chemicals, macronutrients, micronutrients, and ethanol on cytochromes P450 and xenobiotic metabolism are reviewed in the light of our current understanding of the multiplicity and substrate specificity of cytochrome P450 enzymes. Although the mechanisms of action of several dietary chemicals on specific cytochrome P450 isozymes have been established, those for macro- and micronutrients are largely unknown. It is known, however, that specific nutrients may have varied effects on different cytochrome P450 forms and thus may affect the metabolism of various drugs differently. Nutritional deficiencies generally cause lowered rates of xenobiotic metabolism. In certain cases, such as thiamin deficiency and mild riboflavin deficiency, however, enhanced rates of metabolism of xenobiotics were observed. The effects of dietary modulation of xenobiotic metabolism on chemical toxicity and carcinogenicity are discussed.     

Molecular organization of the microsomal oxidative system: a new connotation for an old term

The central role that cytochromes P450 play in the metabolism of drugs and other xenobiotics makes these enzymes a major subject for studies of drug disposition, adverse drug effects and drug-drug interactions. Despite tremendous success in elucidating structures and mechanisms of cytochrome P450 function, the concept of the drug-metabolizing ensemble as a functionally integrated system remains undeveloped. However, eukaryotic cells typically possess a multitude of different cytochromes P450 that are co-localized in the membrane of endoplasmic reticulum (ER); they interact with each other through the formation of dynamic heteromeric complexes (mixed oligomers). There has been growing appreciation of the importance of developing an approach to study the ensemble of cytochromes P450 as an integral system inspired growing interest of researchers to the principles of molecular organization of the microsomal monooxygenase system. Academician Archakov and his colleagues made important contributions to this field during the initial period of studies. Subsequent exploration of the molecular organization of the microsomal monooxygenase system as an integral multienzyme and multifunctional system have had an essential impact on our understanding of the key factors that determine the changes in human drug metabolism and other cytochrome P450-related functions in development and aging, as well as under the influence of various pathologies and environmental factors.     

The phenobarbital-type induction of rat liver microsomal monooxygenases by perfluorodecalin

Induction of perfluorodecalin (PFD) of the liver microsomal system of metabolism of xenobiotics has been studied and compared with the inductions by phenobarbital (PB) and 3-methylcholanthrene (MC). It has been shown that PFD increases the content of cytochrome P-450, NADPH-cytochrome c reductase activity. Like PB, PFD induces the activities of benzphetamine-N-demethylase, aldrine-epoxidase, 16 beta-androstendion-hydroxylase. Using specific antibodies against cytochromes P-450b and P-450c (which are the main isoenzymes of cytochrome P-450 in the PB- and MC-microsomes respectively), an immunological identity of the cytochrome P-450 isoforms during PFD and PB induction has been found. According to the rocket immunoelectrophoresis the content of cytochrome P-450 in PFD-microsomes, which is immunologically indistinguishable from P-450b, was approximately 70% of the total cytochrome P-450. Two forms of cytochrome P-450 were isolated from the liver microsomes of PFD-induced rats and purified to homogeneity. A comparison of these forms with cytochromes P-450b and P-450e obtained from the PB-induced rat liver microsomes revealed their similarity in a number of properties, e.g., chromotographic behavior on DEAE-Sephacel column, molecular weight determined by sodium dodecyl sulphate (SDS) electrophoresis in polyacrylamide gel, immunoreactivity, peptide mapping, catalytic activity. The data presented demonstrate that PFD induced in rat liver microsomes the cytochrome P-450 forms whose immunological properties and substrate specificity correspond to those of the PB-type cytochrome P-450. These findings suggest that PFD and PB, which differ in their chemical structure, induce in the rat liver microsomes identical forms of cytochrome P-450.     

Polyclonal and monoclonal antibodies as probes of rat hepatic cytochrome P-450 isozymes

Cytochrome P-450 is the terminal oxidase of an electron transport system that is responsible for the oxidative metabolism of a large variety of endogenous and exogenous compounds. This broad substrate selectivity is caused by multiple isozymes of cytochrome P-450 and the wide substrate selectivity of many of these isozymes. We have isolated 11 isozymes of cytochrome P-450 from the livers of rats (cytochromes P-450a-P-450k). We have found both polyclonal and monoclonal antibodies increasingly useful to distinguish among these isozymes and to quantitate enzyme levels in liver microsomal preparations where as many as 15 or more cytochrome P-450 isozymes are present. Several of these isozymes show considerable immunochemical relatedness to each other, and operationally they can be grouped into families of immunochemically related isozymes that include cytochromes P-450b and P-450e in one family, cytochromes P-450c and P-450d in another, and cytochromes P-450f-P-450i, and P-450k in a third family. Immunoquantitation of some of these isozymes has revealed dramatic increases of over 50-fold in the levels of certain of these isozymes when exogenous compounds are administered to rats.     

Monoclonal antibodies distinguish among isozymes of the cytochrome P-450b subfamily

Polyclonal antibody has been shown previously to react identically with cytochromes P-450b and P-450e purified from Long Evans rats and a strain variant of cytochrome P-450b purified from Holtzman rats (P-450bH). In the present study, an array of 12 different monoclonal antibodies produced against cytochrome P-450b has been used to distinguish among these closely related phenobarbital-inducible rat hepatic cytochromes P-450. In immunoblots and enzyme-linked immunosorbent assays, 10 monoclonal antibodies bind to cytochromes P-450b, P-450e, and P-450bH; one monoclonal antibody (B50) recognizes cytochromes P-450b and P-450bH but not cytochrome P-450e; and one monoclonal antibody (B51) is specific for cytochrome P-450b. In addition, one monoclonal antibody (BEF29) reacts strongly with cytochrome P-450f, and another antibody (BEA33) reacts weakly with cytochrome P-450a. No cross-reactions with cytochromes P-450c, P-450d, and P-450g-P-450j were detected with any of the monoclonal antibodies in these assays. Six spatially distinct epitopes on cytochrome P-450b were identified, and differences in antibody reactivity provided evidence for three additional overlapping epitopes. Several monoclonal antibodies are potent inhibitors of testosterone and benzphetamine metabolism supported by cytochrome P-450b in a reconstituted system. B50 and BE52 do not inhibit metabolism of the two substrates by microsomes from untreated rats, but inhibit benzphetamine N-demethylation and testosterone metabolism to 16 alpha- and 16 beta-hydroxytestosterone as well as androstenedione formation 67-94% by microsomes from phenobarbital-treated rats. No other pathways of testosterone metabolism are inhibited by these monoclonal antibodies. The differential inhibition of microsomal metabolism of benzphetamine and testosterone by these monoclonal antibodies is a reflection of the content and inducibility of cytochromes P-450b and P-450e as well as other cytochrome P-450 isozymes.     

Cytochromes P450: a success story

Cytochrome P450 proteins, named for the absorption band at 450 nm of their carbon-monoxide-bound form, are one of the largest superfamilies of enzyme proteins. The P450 genes (also called CYP) are found in the genomes of virtually all organisms, but their number has exploded in plants. Their amino-acid sequences are extremely diverse, with levels of identity as low as 16% in some cases, but their structural fold has remained the same throughout evolution. P450s are heme-thiolate proteins; their most conserved structural features are related to heme binding and common catalytic properties, the major feature being a completely conserved cysteine serving as fifth (axial) ligand to the heme iron. Canonical P450s use electrons from NAD(P)H to catalyze activation of molecular oxygen, leading to regiospecific and stereospecific oxidative attack of a plethora of substrates. The reactions carried out by P450s, though often hydroxylation, can be extremely diverse and sometimes surprising. They contribute to vital processes such as carbon source assimilation, biosynthesis of hormones and of structural components of living organisms, and also carcinogenesis and degradation of xenobiotics. In plants, chemical defense seems to be a major reason for P450 diversification. In prokaryotes, P450s are soluble proteins. In eukaryotes, they are usually bound to the endoplasmic reticulum or inner mitochondrial membranes. The electron carrier proteins used for conveying reducing equivalents from NAD(P)H differ with subcellular localization. P450 enzymes catalyze many reactions that are important in drug metabolism or that have practical applications in industry; their economic impact is therefore considerable.     

Regulation of xenobiotic and bile acid metabolism by the nuclear pregnane X receptor

The nuclear pregnane X receptor (PXR; NR1I2) is an integral component of the body's defense mechanism against chemical insult (chemoprotection). PXR is activated by a diverse array of lipophilic chemicals, including xenobiotics and endogenous substances, and regulates the expression of cytochromes P450, conjugating enzymes, and transporters involved in the metabolism and elimination of these potentially harmful chemicals from the body. Among the chemicals that bind and activate PXR is the toxic bile acid lithocholic acid; activation of PXR, in turn, protects against the severe liver damage caused by this bile acid.Thus, PXR serves as a physiological sensor of lithocholic acid and perhaps other bile acids and coordinately regulates genes involved in their detoxification. Interestingly, both the antibiotic rifampicin and the herbal antidepressant St. John's wort activate PXR and have anticholestatic properties, which suggests that more potent, selective PXR agonists may be useful in the treatment of biliary cholestasis or other diseases characterized by the accumulation of bile acids or other toxins in the liver.     

A novel type of allosteric regulation: functional cooperativity in monomeric proteins

Cooperative functional properties and allosteric regulation in cytochromes P450 play an important role in xenobiotic metabolism and define one of the main mechanisms of drug-drug interactions. Recent experimental results suggest that ability to bind simultaneously two or more small organic molecules can be the essential feature of cytochrome P450 fold, and often results in rich and complex pattern of allosteric behavior. Manifestations of non-Michaelis kinetics include homotropic and heterotropic activation and inhibition effects depending on the stoichiometric ratios of substrate and effector, changes in the regio- and stereospecificity of catalytic transformations, and often give rise to the clinically important drug-drug interactions. In addition, functional response of P450 systems is modulated by the presence of specific and non-specific effector molecules, metal ions, membrane incorporation, formation of homo- and hetero-oligomers, and interactions with the protein redox partners. In this article we briefly overview the main factors contributing to the allosteric effects in cytochromes P450 with the main focus on the sources of cooperative behavior in xenobiotic metabolizing monomeric heme enzymes with their conformational flexibility and extremely broad substrate specificity. The novel mechanism of functional cooperativity in P450 enzymes does not require substantial binding cooperativity, rather it implies the presence of one or more binding sites with higher affinity than the single catalytically active site in the vicinity of the heme iron.     

Flavin-containing monooxygenase isoform 2: developmental expression in fetal and neonatal rabbit lung

Mammalian flavin-containing monooxygenase functions in the oxygenation of numerous xenobiotics containing a soft nucleophile, usually a nitrogen or sulfur. A total of five distinct flavin monooxygenase (FMO) isoforms are expressed in mammals. Individual isoforms are expressed in a sex-, age-, and tissue-specific fashion. In this study, we document the early developmental appearance of the major isoform in rabbit lung, FMO2. FMO2 catalytic activity as well as protein and mRNA are not only present in fetal and neonatal lung but, in some instances, approach levels found in the adult. The expression pattern of FMO2 is similar to that of the two major constitutive cytochromes P450 found in rabbit lung, 2B4 and 4B1. The early developmental appearance of these monooxygenases indicate an important role in the protection of the fetus and neonate against toxic insult from foreign chemicals.     

Ventilator associated pneumonia and infection control

Rifampicin, an important drug in the treatment of tuberculosis, is used extensively despite its broad effects on drug-drug interactions, creating serious problems. The clinical importance of such interactions includes autoinduction leading to suboptimal or failed treatment. The concomitantly administered effects of rifampicin on other drugs can result in their altered metabolism or transportation that are metabolised by cytochromes P450 or transported by p-glycoprotein in the gastrointestinal tract and liver. This review paper summarises recent findings with emphases on the molecular mechanisms used to explain these broad drug-drug interactions. In general, rifampicin can act on a pattern: rifampicin activates the nuclear pregnane X receptor that in turn affects cytochromes P450, glucuronosyltransferases and p-glycoprotein activities. This pattern of action may explain many of the rifampicin inducing drug-drug interactions. However, effects through other mechanisms have also been reported and these make any explanation of such drug-drug interactions more complex.