Biomedical subjects

A D Vaz

Publications and source records attributed to A D Vaz.

At least 19 recordsLinked to original sources

PubMed2001

Azole-antifungal binding to a novel cytochrome P450 from Mycobacterium tuberculosis: implications for treatment of tuberculosis.

Although antibiotics against Mycobacterium tuberculosis have decreased the incidence of tuberculosis infections significantly, the emergence of drug-resistant strains of this deadly pathogen renders current treatments ineffective. Therefore, it is imperative to identify biochemical pathways in M. tuberculosis that can serve as targets for new anti-mycobacterial drugs. We recently cloned, expressed, and purified MT CYP51, a soluble protein from M. tuberculosis that is similar in sequence to CYP51 (lanosterol-14alpha-demethylase) isozymes, pharmacological targets for several anti-mycotic compounds. Its striking amino acid sequence similarity to that of mammalian and fungal CYP51s led to the hypothesis that MT CYP51 plays an important role in mycobacterial biology that can be targeted for drug action. In this manuscript, we established through spectral analysis that several azole antifungals bind MT CYP51 with high affinity. The effects of several azole compounds on the growth of M. bovis and M. smegmatis, two mycobacterial species that closely resemble M. tuberculosis were examined. We established a correlation between the affinity of azole compounds to MT CYP51 and their ability to impair the growth of M. bovis and M. smegmatis. These results suggest that the metabolic functions of MT CYP51 may be comparable to those of CYP51 in yeast and fungi and may lead to the development of a new generation of anti-mycobacterial agents.

Amino Acid Sequence↗

PubMed2001

Multiple oxidants in cytochrome P450 catalyzed reactions: implications for drug metabolism.

The activation of molecular oxygen by Cytochromes P450 to the ultimate mono-oxygen oxidant species involves three distinct dioxygen species coordinated to the heme iron. These intermediates have different chemical properties, and have recently been proposed to participate in some Cytochrome P450-catalyzed oxidation reactions. This article reviews the extent of our current knowledge on the roles proposed for the heme- peroxo, hydroperoxo, and superoxo complexes in various reactions. The extent to which such species contribute to the breadth of reactions catalyzed by Cytochrome P450 has yet to be defined, and more definitive experiments are needed to establish such species in the reactions they are proposed to effect.

Animals↗

PubMed1999

Discrete species of activated oxygen yield different cytochrome P450 heme adducts from aldehydes.

Aldehydes are known to inactivate cytochrome P450 in the reconstituted enzyme system containing NADPH and NADPH-cytochrome P450 reductase under aerobic conditions in a mechanism-based reaction involving heme adduct formation [Raner, G. M., Chiang, E. W. , Vaz, A. D. N., and Coon, M. J. (1997) Biochemistry 36, 4895-4902]. In the study presented here, artificial oxidants were used to examine the mechanism of aldehyde activation by purified P450 2B4 in the absence of the usual O(2)-reducing system, and the adducts that were formed were isolated and characterized. With hydrogen peroxide as the oxidant, 3-phenylpropionaldehyde gives an adduct with a mass corresponding to that of native heme modified by a phenylethyl group, presumably arising from the reaction of a peroxy-iron species with the aldehyde to give a peroxyhemiacetal, which upon deformylation yields the alkyl radical. NMR analysis indicated that the substitution is specifically at the gamma-meso position. In contrast, with m-chloroperbenzoic acid as the oxidant, an adduct is formed from 3-phenylpropionaldehyde with a mass that is consistent with the addition of a phenylpropionyl group, apparently arising by hydrogen abstraction from the aldehyde to give the carbonyl carbon radical. m-Chloroperbenzoic acid by itself forms a heme adduct with a mass corresponding to the addition of a chlorobenzoyloxy group apparently derived from homolytic oxygen-oxygen bond cleavage. These and other results with nonanal and 2-trans-nonenal support the concept that this versatile enzyme utilizes discrete oxidizing species in heme adduct formation from aldehydes.

Aldehydes↗

PubMed1998

Epoxidation of olefins by cytochrome P450: evidence from site-specific mutagenesis for hydroperoxo-iron as an electrophilic oxidant.

P450 cytochromes (P450) catalyze many types of oxidative reactions, including the conversion of olefinic substrates to epoxides by oxygen insertion. In some instances epoxidation leads to the formation of products of physiological importance from naturally occurring substrates, such as arachidonic acid, and to the toxicity, carcinogenicity, or teratogenicity of foreign compounds, including drugs. In the present mechanistic study, the rates of oxidation of model olefins were determined with N-terminal-truncated P450s 2B4 and 2E1 and their respective mutants in which the threonine believed to facilitate proton delivery to the active site was replaced by alanine. Styrene epoxidation, cyclohexene epoxidation and hydroxylation to give 1-cyclohexene-3-ol, and cis- or trans-butene epoxidation (without isomerization) and hydroxylation to give 2-butene-1-ol were all significantly decreased by the 2B4 T302A mutation. Reduced proton delivery in this mutant is believed to interfere with the activation of dioxygen to the oxenoid species, as shown earlier by decreased hydroxylation of several substrates and enhanced aldehyde deformylation via a presumed peroxo intermediate. Of particular interest, however, the T303A mutation of P450 2E1 resulted in enhanced epoxidation of all of the model olefins along with decreased allylic hydroxylation of cyclohexene and butene. These results and a comparison of the ratios of the rates of epoxidation and hydroxylation support the concept that two different species with electrophilic properties, hydroperoxo-iron (FeO2H)3+ and oxenoid-iron (FeO)3+, can effect olefin epoxidation. The ability of cytochrome P450 to use several different active oxidants generated from molecular oxygen may help account for the broad reaction specificity and variety of products formed by this versatile catalyst.

Alkenes↗

PubMed1998

Multiple activated oxygen species in P450 catalysis: contributions To specificity in drug metabolism.

A hypervalent iron-oxene species has been widely proposed as the "active oxygen" in cytochrome P450 (P450)-catalyzed reactions. We recently examined the effect of mutation of the highly conserved threonine residue in P450s 2B4 and 2E1 to alanine, a change that is believed to interfere with proton delivery to the active site, and have determined the change in rates of deformylation of aldehydes, epoxidation of olefins, and hydroxylation of various substrates. The results support the concept that three distinct oxidants are functional in P450 catalysis: nucleophilic peroxo-iron, nucleophilic or electrophilic hydroperoxo-iron, and electrophilic oxenoid-iron. The occurrence of multiple oxidizing species may contribute to the remarkable versatility of the P450 family of isozymes in the modification of drugs and other substrates. Furthermore, the relative concentrations of these oxidants in a particular P450 isozyme may contribute to substrate specificity and govern the type of reaction catalyzed.

Animals↗

PubMed1997

Significance of glycine 478 in the metabolism of N-benzyl-1-aminobenzotriazole to reactive intermediates by cytochrome P450 2B1.

The effect of mutating Gly 478 to Ala in rat cytochrome P450 2B1 on the metabolism of N-benzyl-1-aminobenzotriazole was investigated. The 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of the wild-type enzyme was completely inactivated by incubating with 1 microM BBT. The G478A mutant, however, was not inactivated by incubating with up to 10 microM BBT. Whereas metabolism of BBT by the wild-type 2B1 resulted in the formation of benzaldehyde, benzotriazole, aminobenzotriazole, and a new metabolite, the G478A mutant generated only the later. This metabolite was found by NMR, IR, and mass spectrometry to be a dimeric product formed from the reaction of two BBT molecules. Two spectral binding constants, a high-affinity constant that was the same for both enzymes (30-39 microM) and a low-affinity constant that was 5-fold lower for the mutant enzyme (0.3 mM vs 1.4 mM), were observed with BBT. The apparent Km and kcat values for the G478A mutant with BBT were 0.3 mM and 12 nmol (nmol of P450)-1 min-1, respectively. Molecular modeling studies of BBT bound in the active site of P450 2B1 suggested that a mutation of Gly 478 to Ala would result in steric hindrance and suppress oxidation of BBT at the 1-amino nitrogen. When BBT was oriented in the 2B1 active site such that oxidation at the 7-benzyl carbon could occur, no steric overlap between Ala 478 and the substrate was observed. Thus, this orientation of BBT would be preferred by the mutant leading to oxidation at the 7-benzyl carbon and subsequent dimer formation. These findings indicate that a glycine 478 to alanine substitution in P450 2B1 altered the binding of BBT such that inactivating BBT metabolites were no longer generated.

Animals↗

PubMed1997

Metabolic activation of trans-4-hydroxy-2-nonenal, a toxic product of membrane lipid peroxidation and inhibitor of P450 cytochromes.

Lipid peroxidation in biological membranes is known to yield reactive aldehydes, of which trans-4-hydroxy-2-nonenal (HNE) is particularly cytotoxic. This laboratory previously reported that purified liver microsomal P450 cytochromes are directly inactivated to varying extents by HNE. We have now found a mechanism-based reaction in which P450s are inactivated by HNE in the presence of molecular oxygen, NADPH, and NADPH-cytochrome P450 reductase. The sensitivity of the various isozymes in the two pathways is different as follows: P450 2B4 and the orthologous 2B1 are inactivated to the greatest extent and 2C3, 1A2, 2E1, and 1A1 to a somewhat lesser extent by the pathway in which HNE undergoes metabolic activation. In contrast, 2B4 and 2B1 are insensitive to direct inactivation, and the reductase is unaffected by HNE by either route. Recent studies on the catalytic activities of the T302A mutant of P450 2B4 have shown that the rate of oxidation of a variety of xenobiotic aldehydes to carboxylic acids is decreased, but the rates of aldehyde deformylation and mechanism-based inactivation of the cytochrome are stimulated over those of the wild-type enzyme (Raner, G. M., Vaz, A. D. N., and Coon, M. J. (1997) Biochemistry 36, 4895-4902). Inactivation by those aldehydes apparently occurs by homolytic cleavage of a peroxyhemiacetal intermediate to yield formate and an alkyl radical that reacts with the heme. In sharp contrast, the rate of mechanism-based inactivation by HNE is decreased with the T302A mutant relative to that of the wild-type P450 2B4, and mass spectral analysis of the heme adduct formed shows that deformylation does not occur. We therefore propose that the metabolic activation of HNE involves formation of an acyl carbon radical that leads to the carboxylic acid or alternatively reacts with the heme.

Aldehydes↗

PubMed1997

1,2,3-Thiadiazole: a novel heterocyclic heme ligand for the design of cytochrome P450 inhibitors.

The 1,2,3-thiadiazole heterocycle has been explored as a heme ligand and mechanism-based inactivator for the design of cytochrome P450 inhibitors. One 4,5-fused bicyclic and three 4,5-disubstituted monocyclic 1,2,3-thiadiazoles have been examined for their spectral interactions, inhibition, mechanism-based inactivation, and oxidation products by the versatile microsomal P450s 2B4, 2E1, and 1A2. The compounds generally show heteroatom coordination to the heme iron; however, the binding mode is influenced by the architecture of the active site. For example, 4,5-diphenyl-1,2,3-thiadiazole shows type I and type II difference spectra with P450s 2B4 and 2E1, respectively, and no spectral perturbation with P450 1A2. Except for the fused bicyclic compound, the spectral dissociation constants are in the 2-50 microM range. The effectiveness as an inhibitor depends on the substituents at the 4- and 5- positions and on the P450 examined. Inhibition of the P450-catalyzed 1-phenylethanol oxidation to acetophenone by the thiadiazoles does not correlate with either the type of binding spectra or the spectral dissociation constants of the compounds. P450s 2E1 and 2B4 are inactivated by the 4,5-fused bicyclic 1,2,3-thiadiazole in a mechanism-based manner. Inactivation of the P450 correlates with loss in absorbance at 450 nm for the ferrous-CO complex. The monocyclic 1,2,3-thiadiazoles do not inactivate any of the P450s examined. The 1,2,3-thiadiazole ring is oxidized by the P450 system. Oxidation of the monocyclic compounds results in extrusion of the three heteroatoms and formation of the corresponding acetylenes, whereas oxidation of the fused bicyclic compound does not yield an acetylenic product.

Acetophenones↗

PubMed1997

Mechanism-based inactivation of cytochrome P450 2B4 by aldehydes: relationship to aldehyde deformylation via a peroxyhemiacetal intermediate.

The inactivation of cytochrome P450 2B4 by aldehydes in a reconstituted enzyme system requires molecular oxygen and NADPH and is not prevented by the addition of catalase, superoxide dismutase, epoxide hydrolase, glutathione, or ascorbic acid. A strong correlation between loss of enzymatic activity and bleaching of the heme chromophore was established, and the inactivation was shown to be irreversible upon dialysis. In general, saturated aldehydes are more inhibitory than those with alpha,beta-unsaturation, as indicated by the k(inact) values, and primary aldehydes are more potent inactivators than the structurally related secondary and tertiary aldehydes. Consistent with recent studies on catalytic specificity of the T302A mutant of this cytochrome [Vaz, A. D. N., Pernecky, S. J., Raner, G. M., & Coon, M. J. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 4644-4648], the rate of aldehyde deformylation, as determined by formation of the alcohol with one less carbon atom, is greatly stimulated over that of the wild-type enzyme. Of particular interest, the rate of oxidation of aldehydes to carboxylic acids is decreased with the mutant, whereas the rate of inactivation via heme destruction is enhanced. Furthermore, comparative deuterium isotope effects and the relative rates of inactivation and product formation suggest that the mechanism of aldehyde inactivation of P450 2B4 involves the deformylation reaction and is unrelated to carboxylic acid formation. Finally, in the reaction of P450 2B4 with 3-phenylpropionaldehyde, the formation of a heme adduct with a molecular weight corresponding to that of native heme plus 104 mass units confirms the loss of the carbonyl group from the aldehyde prior to reaction with the chromophore. We conclude that inactivation of P450 by aldehydes occurs via homolytic cleavage of a peroxyhemiacetal intermediate to give an alkyl radical that reacts with the heme.

Aldehydes↗

PubMed1997

Adrenal proteins bound by a reactive intermediate of mitotane.

PURPOSE: Mitotane (o,p'-DDD), is the only adrenolytic agent available for the treatment of adrenocortical carcinoma. Previous studies have shown that mitotane covalently binds to adrenal proteins following its metabolism in adrenocortical tissue to a reactive acyl chloride intermediate. It was the objective of this study to compare the electrophoresis separation patterns of such adducts following activation of mitotane by various adrenocortical sources. METHODS: With the use of a 125I-labeled analog of mitotane, 1-(2-chlorophenyl)-1-(4-iodophenyl)-2,2-dichloroethane, gel electrophoresis patterns were obtained for homogenates from bovine, canine and human adrenocortical preparations as well as from a human adrenal preparation. Western immunoblotting analysis was used to test the resulting patterns for adducts of cytochrome P-450scc and adrenodoxin. RESULTS: The electrophoresis separations were similar for all preparations, with bands at apparent molecular weights of 49.5 and 11.5 kDa being the most pronounced. Radiolabeling of the proteins of a human adrenal cancer cell line NCI H-295 was weak, but a band at 11.5 kDa was detected. Western immunoblotting analyses indicated that the band at 49.5 kDa corresponded in molecular weight to that of adrenal cytochrome P-450scc, but the band at 11.5 kDa did not correspond to adrenodoxin. CONCLUSIONS: The similarity of the results with canine and bovine adrenal preparations to that of human material offers useful systems for studying mitotane and its analogs. This should aid in understanding the mechanism of action of mitotane and in the design of compounds for the treatment of adrenocortical carcinoma.

Adrenal Cortex↗

PubMed1997

Evidence for a role of a perferryl-oxygen complex, FeO3+, in the N-oxygenation of amines by cytochrome P450 enzymes.

Most cytochrome P450 (P450)-catalyzed reactions are believed to involve an FeO3+ intermediate as the actual oxygenating species. However, studies on the mechanism of steroid aromatization and subsequent model work have provided evidence that a peroxo-iron form (formally FeO2) can be involved directly in some oxidations. The possible involvement of peroxoiron was considered in P450-catalyzed N-oxygenations, because there is precedent for the use of H2O2 and organic peroxides in such reactions in the literature concerning synthetic and flavin reactions. The approach used was to compare P450 reactions involving the normal NADPH/NADPH-P450 reductase/O2 system with those supported by the oxygen surrogates H2O2 (which can directly form FeO2 and subsequently FeO3+) and iodosylbenzene (which can form FeO3 but not FeO2+). Iodosylbenzene was effective in supporting rabbit P450 1A2-catalyzed N,N-dimethyl-2-aminofluorene N-oxygenation, human P450 3A4-catalyzed quinidine N-oxygenation, rat P450 2B1-catalyzed oxidation of N-benzyl-(1-phenyl) cyclobutylamine to the N-hydroxyamine and nitrone, and rat P450 2B1-catalyzed and rabbit P450 2B4-catalyzed N-oxygenation of N,N-dimethylaniline (also N-demethylation). H2O2 also supported most of these reactions. A mutant of P450 2B4 with the substitution of alanine for threonine at position 302 has been shown to have decreased ability to catalyze reactions involving the putative FeO3+ but, presumably because of decreased ability to protonate the FeO2+ complex, to have enhanced activity in oxidative deformylation reactions believed to involve FeO2+. This mutant showed both decreased N,N-dimethylaniline N-demethylation and N-oxygenation activity. Although some contribution of an FeO2+ species to these reactions cannot be ruled out, formation of product in the iodosylbenzene-supported systems cannot be readily explained by an obligatory FeO2 mechanism and the involvement of FeO3+ is concluded to be more likely.

Amines↗

PubMed1996

Peroxo-iron and oxenoid-iron species as alternative oxygenating agents in cytochrome P450-catalyzed reactions: switching by threonine-302 to alanine mutagenesis of cytochrome P450 2B4.

Among biological catalysts, cytochrome P450 is unmatched in its multiplicity of isoforms, inducers, substrates, and types of chemical reactions catalyzed. In the present study, evidence is given that this versatility extends to the nature of the active oxidant. Although mechanistic evidence from several laboratories points to a hypervalent iron-oxenoid species in P450-catalyzed oxygenation reactions, Akhtar and colleagues [Akhtar, M., Calder, M. R., Corina, D. L. & Wright, J. N. (1982) Biochem. J. 201, 569-580] proposed that in steroid deformylation effected by P450 aromatase an iron-peroxo species is involved. We have shown more recently that purified liver microsomal P450 cytochromes, including phenobarbital-induced P450 2B4, catalyze the analogous deformylation of a series of xenobiotic aldehydes with olefin formation. The investigation presented here on the effect of site-directed mutagenesis of threonine-302 to alanine on the activities of recombinant P450 2B4 with N-terminal amino acids 2-27 deleted [2B4 (delta2-27)] makes use of evidence from other laboratories that the corresponding mutation in bacterial P450s interferes with the activation of dioxygen to the oxenoid species by blocking proton delivery to the active site. The rates of NADPH oxidation, hydrogen peroxide production, and product formation from four substrates, including formaldehyde from benzphetamine N-demethylation, acetophenone from 1-phenylethanol oxidation, cyclohexanol from cyclohexane hydroxylation, and cyclohexene from cyclohexane carboxaldehyde deformylation, were determined with P450s 2B4, 2B4 (delta2-27), and 2B4 (delta2-27) T302A. Replacement of the threonine residue in the truncated cytochrome gave a 1.6- to 2.5-fold increase in peroxide formation in the presence of a substrate, but resulted in decreased product formation from benzphetamine (9-fold), cyclohexane (4-fold), and 1-phenylethanol (2-fold). In sharp contrast, the deformylation of cyclohexane carboxaldehyde by the T302A mutant was increased about 10-fold. On the basis of these findings and our previous evidence that aldehyde deformylation is supported by added H202, but not by artificial oxidants, we conclude that the iron-peroxy species is the direct oxygen donor. It remains to be established which of the many other oxidative reactions involving P450 utilize this species and the extent to which peroxo-iron and oxenoid-iron function as alternative oxygenating agents with the numerous isoforms of this versatile catalyst.

Animals↗

PubMed1996

Cytochrome P450 2: peroxidative reactions of diversozymes.

Cytochrome P450, the most versatile biological catalyst known, was originally named as a pigment having a carbon monoxide difference spectrum at about 450 nm and no known function. Recent progress in many laboratories has revealed that the P450 superfamily has immense diversity in its functions, with hundreds of isoforms in many species catalyzing many types of chemical reactions. We believe it is safe to predict that each mammalian species may be found to have up to a hundred P450 isoforms that respond in toto to a thousand or more inducers and that, along with P450s from other sources, metabolize a million or more potential substrates. Accordingly, the name DIVERSOZYMES is proposed for this remarkable family of hemoproteins. This paper reviews the peroxidative reactions of Diversozymes, including peroxides as oxygen donors in hydroxylation reactions, as substrates for reductive beta-scission, and as peroxyhemiacetal intermediates in the cleavage of aldehydes to formate and alkenes. Lipid hydroperoxides undergo reductive beta-cleavage to give hydrocarbons and aldehydic acids. One of these products, trans-4-hydroxynonenal, inactivates P450, particularly alcohol-inducible 2E1, in what may be a negative regulatory process. Although a P450 iron-oxene species is believed to be the oxygen donor in most hydroxylation reactions, an iron-peroxy species is apparently involved in the deformylation of many aldehydes with desaturation of the remaining structure, as in aromatization reactions.

Aldehydes↗

PubMed1996

Metabolism of all-trans, 9-cis, and 13-cis isomers of retinal by purified isozymes of microsomal cytochrome P450 and mechanism-based inhibition of retinoid oxidation by citral.

The involvement of a series of microsomal cytochrome P450 (P450) isozymes in all-trans-retinoid metabolism, including the conversion of all-trans-retinal to all-trans-retinoic acid, was previously described. In the current study, we examined the role of seven liver microsomal P450 isozymes in the oxidation of three isomers of retinal. P450 1A1, which was not tested previously, is by far the most active in the conversion of all-trans-, 9-cis-, and 13-cis-retinal to the corresponding acids, as well as in the 4-hydroxylation of all-trans- and 13-cis retinal. In contrast, P450s 2B4 and 2C3 are the most active in the 4-hydroxylation of 9-cis-retinal, with turnover numbers approximately 7 times as great as that of P450 1A1. The inclusion of cytochrome b5 in the reconstituted enzyme system is without effect or inhibitory in most cases but stimulates the 4-hydroxylation of 9-cis-retinal by P450 2B4, giving a turnover of 3.7 nmol of product/min/nmol of this isozyme, the highest for any of the retinoid conversions we have studied. Evidence was obtained for two additional catalytic reactions not previously attributed to P450 oxygenases: the oxidation of all-trans- and 9-cis-retinal to the corresponding 4-oxo derivatives by isoform 1A2, and the oxidative cleavage of the acetyl ester of vitamin A (retinyl acetate) to all-trans-retinal, also by isoform 1A2. The physiological significance of the latter reaction, with a Km for the ester of 32 microM and a Vmax of 18 pmol/min/nmol of P450, remains to be established. We also examined the effect on P450 of citral, a terpenoid alpha, beta-unsaturated aldehyde and a known inhibitor of cytosolic retinoid dehydrogenases. Evidence was obtained that citral is an effective mechanism-based inactivator of isozyme 2B4, with a KI of 44 microM as determined by the oxidation of 1-phenylethanol to acetophenone, and by isozyme 1A2 in the oxidation of all-trans-retinal to the corresponding acid and by isozyme 2B4 in the 4-hydroxylation of all-trans-retinol and retinoic acid. Thus, citral is not suitable for use in attempts to distinguish between retinoid conversions catalyzed by dehydrogenases in the cytoplasm and by P450 cytochromes in the endoplasmic reticulum.

Acyclic Monoterpenes↗

PubMed1996

Metabolic activation of 2,6-dichlorobenzonitrile, an olfactory-specific toxicant, by rat, rabbit, and human cytochromes P450.

The herbicide 2,6-dichlorobenzonitrile (DCBN) is known to cause tissue-specific toxicity at very low doses in the olfactory mucosa of rodents. The toxicity of DCBN is reportedly cytochrome P450 (P450) dependent, but the isoforms involved have not been identified, and the effects of this agent on humans are not known. In the present study, DCBN metabolism was examined with microsomes and with purified P450s in a reconstituted system. Rat and rabbit olfactory microsomes act on DCBN to form DCBN-protein adducts as well as two metabolite peaks, designated M1 and M2, identified through high performance liquid chromatography with radiometric detection. The activity of rat olfactory microsomes in DCBN metabolism is much higher than that of liver or lung microsomes. Of seven purified rabbit P450s known to be expressed in the olfactory mucosa, including 1A2, 2A10/11, 2B4, 2E1, 2G1, and 3A6, the 2A10/11 preparation is the most active, producing M2 as well as DCBN-protein adducts; P450 2E1 is the only other active isoform. The addition of purified epoxide hydrolase (EC 4.2.1.63) to the reconstituted enzyme system leads to the formation of M1 and decreased formation of M2. It seems that M1 and M2 are derived from an epoxide intermediate that also forms covalent protein adducts. Gas chromatography- and liquid chromatography-mass spectrometry analyses of nasal microsomal DCBN metabolites and DCBN-glutathione conjugates indicated that the major reactive intermediate may be 2,3-oxo-DCBN and that M1 may be 2,3-dihydroxy-6-chlorobenzonitrile, whereas M2 may correspond to a monohydroxy-DCBN. Interestingly, heterologously expressed human P450s 2A6 and 2E1, but not 1A2, are active in the metabolism of DCBN, forming protein adducts as well as M2. Thus, the preferential expression of P450s of the 2A subfamily in olfactory tissue suggests a molecular basis for the tissue-specific toxicity of the herbicide and may have important implications for risk assessment in humans.

Animals↗

PubMed1995

Inactivation of ethanol-inducible cytochrome P450 and other microsomal P450 isozymes by trans-4-hydroxy-2-nonenal, a major product of membrane lipid peroxidation.

Of the microsomal P450 cytochromes, the ethanol-inducible isoform, P450 2E1, is believed to be predominant in leading to oxidative damage, including the generation of radical species that contribute to lipid peroxidation, and in the reductive beta-scission of lipid hydroperoxides to give hydrocarbons and aldehydes. In the present study, the sensitivity of a series of P450s to trans-4-hydroxy-2-nonenal (HNE), a known toxic product of membrane lipid peroxidation, was determined. After incubation of a purified cytochrome with HNE, the other components of the reconstituted system (NADPH-cytochrome P450 reductase, phosphatidylcholine, and NADPH) were added, and the rate of oxygenation of 1-phenylethanol to yield acetophenone was assayed. Inactivation occurs in a time-dependent and HNE concentration-dependent manner, with P450s 2E1 and 1A1 being the most sensitive, followed by isoforms 1A2, 3A6, and 2B4. At an HNE concentration of 0.24 microM, which was close to the micromolar concentration of the enzyme, four of the isoforms were significantly inhibited, but not P450 2B4. In other experiments, the reductase was shown to be only relatively weakly inactivated by HNE. P450s 2E1 and 2B4 in microsomal membranes from animals induced with acetone or phenobarbital, respectively, are as readily inhibited as the purified forms. Evidence was obtained that the P450 heme is apparently not altered and the sulfur ligand is not displaced, that substrate protects against HNE, and that the inactivation is reversed upon dialysis. Higher levels of reductase or substrate do not restore the activity of inhibited P450 in the catalytic assay. Our results suggest that the observed inhibition of the various P450s is of sufficient magnitude to cause significant changes in the metabolism of foreign compounds such as drugs and chemical carcinogens by the P450 oxygenase system at HNE concentrations that occur in biological membranes. In view of the known activities of P450 2E1 in generating lipid hydroperoxides and in their beta-scission, its inhibition by this product of membrane peroxidation may provide a negative regulatory function.

Aldehydes↗

PubMed1995

Oxidative cleavage of esters and amides to carbonyl products by cytochrome P450.

A series of esters and several amides were shown to undergo oxidative cleavage with the formation of carbonyl products in the presence of purified isoforms of liver microsomal cytochrome P450 (P450) in a reconstituted enzyme system. The reaction also requires NADPH and NADPH-cytochrome P450 reductase and is stimulated by phosphatidylcholine. Kinetic constants were determined in experiments in which the predicted aldehyde product was identified and quantitated by gas chromatography. A relationship was seen with P450 2E1 between the structures of the esters and the Vmax values, with the rates decreasing in the series of methyl formate to methyl valerate, and similarly in the series of methyl, ethyl, propyl, butyl, and amyl acetates. Furthermore, a clear correlation exists between the Km values of the ethyl esters examined and the log of the octanol/water partition coefficients of these substrates. With P450 2E1, the Km decreases significantly between one and four carbon atoms in the chain length of the acyl component of the ester but is unaffected by a further increase in length. However, no correlation was found between the Km value and the chain length of the alcohol moiety of the esters. Similarly, with P450 2B4 a large decrease in Km occurs between one and five carbons in the acyl component of the ethyl esters but is unaffected by a further increase in chain length. The observed correlation is presumed to arise from hydrophobic interactions between the access channel to the active site of P450 and the acyl side chain of the esters. P450 1A2 is also active in ester cleavage, and the three cytochromes examined with esters are active in the conversion of N-alkyl amides to aldehydes, as are P450s 2C3, 1A1, and 3A6. Studies on 2-butyl acetate oxidation by P450 2B4 in the presence of 18O2 showed 88% 18O incorporation into the product, 2-butanone. This is consistent with a mechanism that involves hydroxylation at the alpha-carbon of the alcohol component of the ester to yield an unstable geminal hydroxy ester, as proposed earlier by F. P. Guengerich et al. (1988, J. Biol. Chem. 263, 8176-8183) for several dihydropyridine carboxylic esters. Our results further indicate that such an intermediate decomposes by a nonhydrolytic mechanism and also rule out the possibility of transient ester hydrolysis with subsequent oxidation of the alcohol formed. In addition, they establish that oxidative cleavage is a widespread reaction among P450 cytochromes and commonly used esters and amides.

Amides↗

PubMed1995

Old Yellow enzyme: aromatization of cyclic enones and the mechanism of a novel dismutation reaction.

The origin of charge transfer bands that develop on reaction of Old Yellow Enzyme with alpha,beta-unsaturated cyclic ketones such as 3-oxodecalin-4-ene (ODE, numbered according to the convention for steroids), 3-oxodecalin-4-ene-10-carboxaldehyde (ODEC), and 2-cyclohexenone is shown to be due to the aromatization of ODE and ODEC to 3-hydroxy-6,7,8,9-tetrahydronaphthalene (HTN) and of 2-cyclohexenone to phenol. The aromatization of ODEC to HTN is stereospecific and involves the trans dehydrogenation of the 1 beta, 2 alpha hydrogens. The aromatization occurs under aerobic as well as anaerobic conditions. With the exception of ODEC under aerobic conditions, the aromatization of these substrates is accompanied by a dismutation reaction in which the olefinic bond of a second molecule of each substrate is reduced to give the saturated cyclic ketone. Molecular oxygen may serve as the electron acceptor with ODEC and some other substrates under aerobic reaction conditions. The dismutation reaction involves an overall sequence of hydride transfer from one substrate molecule to the beta-carbon of a second substrate molecule along with a solvent proton uptake by the alpha-carbon. 19-Nortestosterone is aromatized to beta-estradiol; however, other 3-oxo-delta 4-steroids such as progesterone, testosterone, and androstene-3,17-dione bind tightly to the enzyme but are not aromatized. The NADPH-dependent reduction of the olefinic bond of alpha,beta-unsaturated carbonyl compounds is limited to aldehydes and ketones. alpha,beta-Unsaturated acids, esters, amides, and nitriles are not reduced. The reduction of the olefinic bond of ODE or cinnamaldehyde by NADPH occurs by an overall sequence of hydride transfer from the reduced pyridine nucleotide to the beta-carbon of the alpha,beta-unsaturated carbonyl compound and a solvent proton uptake by the alpha-carbon. The 4-pro-R hydride of NADPH is transferred in the reduction reaction. Structure-function relationships in the NADPH-dependent reduction of alpha,beta-unsaturated aldehydes or ketones indicate that increasing alkyl substitution at the beta-carbon results in marked decrease in the rate of reduction of the olefinic bond, consistent with a steric hindrance to hydride transfer at the beta-carbon.

Acrolein↗