Role of the highly conserved threonine in cytochrome P450 2E1: prevention of H2O2-induced inactivation during electron transfer.

A highly conserved threonine in the I-helix of cytochrome P450s has been suggested to play an important role in dioxygen activation, a critical step for catalytic turnover. However, subsequent studies with some P450s in which this highly conserved threonine was replaced by another residue such as alanine showed that significant catalytic activities were still retained when the variants were compared with the wild type enzymes. These results make the role of this residue unclear. We provide data here that suggest a novel role for this highly conserved threonine (Thr303) in the function of P450 2E1. We found that the P450 2E1 T303A mutant undergoes rapid autoinactivation in the reconstituted system during catalytic turnover when the electrons are provided by NADPH. This inactivation was much faster than that of the wild type P450 2E1 and was prevented by catalase. Both the P450 2E1 wild type and T303A mutants produce hydrogen peroxide during the incubations. The inactivation was accompanied by heme destruction with part of the heme becoming covalently attached to protein. The heme destruction was prevented by catalase or by the presence of substrate. Interestingly, this inactivation occurred much more rapidly in the presence of both an electron transfer system and hydrogen peroxide externally added to the enzyme. This accelerated inactivation during catalytic turnover was also found with a 2B4 T302A mutant, which corresponds to 2E1 T303A. Our results suggest that the conserved threonine in these P450s prevents rapid autoinactivation during the catalytic cycle and that this residue may be highly conserved in P450s since it allows them to remain catalytically active for longer periods of time.

The inactivation of human CYP2E1 by phenethyl isothiocyanate, a naturally occurring chemopreventive agent, and its oxidative bioactivation.

Phenethylisothiocyanate (PEITC), a naturally occurring isothiocyanate and potent cancer chemopreventive agent, works by multiple mechanisms, including the inhibition of cytochrome P450 (P450) enzymes, such as CYP2E1, that are involved in the bioactivation of carcinogens. PEITC has been reported to be a mechanism-based inactivator of some P450s. We describe here the possible mechanism for the inactivation of human CYP2E1 by PEITC, as well as the putative intermediate that might be involved in the bioactivation of PEITC. PEITC inactivated recombinant CYP2E1 with a partition ratio of 12, and the inactivation was not inhibited in the presence of glutathione (GSH) and not fully recovered by dialysis. The inactivation of CYP2E1 by PEITC is due to both heme destruction and protein modification, with the latter being the major pathway for inactivation. GSH-adducts of phenethyl isocyanate (PIC) and phenethylamine were detected during the metabolism by CYP2E1, indicating formation of PIC as a reactive intermediate following P450-catalyzed desulfurization of PEITC. Surprisingly, PIC bound covalently to CYP2E1 to form protein adducts but did not inactivate the enzyme. Liquid chromatography mass spectroscopy analysis of the inactivated CYP2E1 apo-protein suggests that a reactive sulfur atom generated during desulfurization of PEITC is involved in the inactivation of CYP2E1. Our data suggest that the metabolism of PEITC by CYP2E1 that results in the inactivation of CYP2E1 may occur by a mechanism similar to that observed with other sulfur-containing compounds, such as parathion. Digestion of the inactivated enzyme and analysis by SEQUEST showed that Cys 268 may be the residue modified by PIC.

Mechanistic analysis of the inactivation of cytochrome P450 2B6 by phencyclidine: effects on substrate binding, electron transfer, and uncoupling.

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

Differential inhibition of cytochromes P450 3A4 and 3A5 by the newly synthesized coumarin derivatives 7-coumarin propargyl ether and 7-(4-trifluoromethyl)coumarin propargyl ether.

The abilities of 7-coumarin propargyl ether (CPE) and 7-(4-trifluoromethyl)coumarin propargyl ether (TFCPE) to act as mechanism-based inactivators of P450 3A4 and 3A5 in the reconstituted system have been investigated using 7-benzyloxy-4-(trifluoromethyl)coumarin (BFC) and testosterone as probes. CPE inhibited the BFC O-debenzylation activity of P450 3A4 in a time-, concentration-, and NADPH-dependent manner characteristic of a mechanism-based inactivator with a half-maximal inactivation (K(I)) of 112 microM, a maximal rate of inactivation (k(inact)) of 0.05 min(-1), and a t(1/2) of 13.9 min. Similarly, TFCPE inhibited the BFC O-debenzylation activity of P450 3A4 in a time-, concentration-, and NADPH-dependent manner with a K(I) of 14 microM, a k(inact) of 0.04 min(-1), and a t(1/2) of 16.5 min. Parallel losses of P450 3A4 enzymatic activity and heme were observed with both compounds as measured by high-performance liquid chromatography and reduced CO spectra. Interestingly, neither compound inhibited the BFC O-debenzylation activity of P450 3A5. Reactive intermediates of CPE and TFCPE formed by P450 3A4 were trapped with glutathione, and the resulting adducts were identified using tandem mass spectral analysis. Metabolism studies using TFCPE resulted in the identification of a single metabolite that is formed by P450 3A4 but not by P450 3A5 and that may play a role in the mechanism-based inactivation.

Modification of serine 360 by a reactive intermediate of 17-alpha-ethynylestradiol results in mechanism-based inactivation of cytochrome P450s 2B1 and 2B6.

17-alpha-Ethynylestradiol (17EE) is a mechanism-based inactivator of P450 2B1 and P450 2B6 in the reconstituted monooxygenase system. The loss in enzymatic activity was due to the binding of a reactive intermediate of 17EE to the apoprotein. P450 2B1 and P450 2B6 were inactivated by 17EE and digested with trypsin. The peptides obtained following digestion with trypsin of 17EE-inactivated P450 2B1 and P450 2B6 were separated by liquid chromatography and analyzed by ESI-MS. Adducted peptides exhibiting an increase in mass consistent with the addition of the mass of the reactive intermediate of 17EE were identified for each enzyme. Analysis of these modified peptides by ESI-MS/MS and precursor ion scanning facilitated the identification of the Ser360 in both enzymes as a site that had been adducted by a reactive intermediate of 17EE. A P450 2B1 mutant where Ser360 was replaced by alanine was constructed, expressed, and purified. Activity and inactivation studies indicated that mutation of the Ser360 residue to alanine did not prevent inactivation of the mutant enzyme by 17EE. These observations suggest that Ser360 is not critical for the catalytic function of these P450s. Spectral binding studies of the 17EE-inactivated P450 2B1 and P450 2B6 indicated that modification of the enzymes by the reactive intermediate of 17EE resulted in an enzyme that was no longer capable of binding substrates. These results suggest that the inactivation by 17EE may be due to modification of an amino acid residue in the substrate access channel near the point of entry into the active site.

Mechanism-based inactivation of human cytochromes p450s: experimental characterization, reactive intermediates, and clinical implications.

The P450 type cytochromes are responsible for the metabolism of a wide variety of xenobiotics and endogenous compounds. Although P450-catalyzed reactions are generally thought to lead to detoxication of xenobiotics, the reactions can also produce reactive intermediates that can react with cellular macromolecules leading to toxicity or that can react with the P450s that form them leading to irreversible (i.e., mechanism-based) inactivation. This perspective describes the fundamentals of mechanism-based inactivation as it pertains to P450 enzymes. The experimental approaches used to characterize mechanism-based inactivators are discussed, and the criteria required for a compound to be classified as a mechanism-based inactivator are outlined. The kinetic scheme for mechanism-based inactivation and the calculation of the relevant kinetic constants that describe a particular inactivation event are presented. The structural aspects and important functional groups of several classes of molecules that have been found to impart mechanism-based inactivation upon metabolism by P450s such as acetylenes, thiol-containing compounds that include isothiocyanates, thiazolidinediones, and thiophenes, arylamines, quinones, furanocoumarins, and cyclic tertiary amines are described. Emphasis throughout this perspective is placed on more recent findings with human P450s where the site of modification, whether it be the apoprotein or the heme moiety, and, at least in part, the identity of the reactive intermediate responsible for the loss in P450 activity are known or inferred. Recent advances in trapping procedures as well as new methods for identification of reactive intermediates are presented. A variety of clinically important drugs that act as mechanism-based inactivators of P450s are discussed. The irreversible inactivation of human P450s by these drugs has the potential for causing serious drug-drug interactions that may have severe toxicological effects. The clinical significance of inactivating human P450s for improving drug efficacy as well as drug safety is discussed along with the potential for exploiting mechanism-based inactivators of P450s for therapeutic benefits.

Anandamide metabolism by human liver and kidney microsomal cytochrome p450 enzymes to form hydroxyeicosatetraenoic and epoxyeicosatrienoic acid ethanolamides.

The endocannabinoid anandamide is an arachidonic acid derivative that is found in most tissues where it acts as an important signaling mediator in neurological, immune, cardiovascular, and other functions. Cytochromes P450 (P450s) are known to oxidize arachidonic acid to the physiologically active molecules hydroxyeicosatetraenoic acids (HETEs) and epoxyeicosatrienoic acids (EETs), which play important roles in blood pressure regulation and inflammation. To determine whether anandamide can also be oxidized by P450s, its metabolism by human liver and kidney microsomes was investigated. The kidney microsomes metabolized anandamide to a single mono-oxygenated product, which was identified as 20-HETE-ethanolamide (EA). Human liver microsomal incubations with anandamide also produced 20-HETE-EA in addition to 5,6-, 8,9-, 11-12, and 14,15-EET-EA. The EET-EAs produced by the liver microsomal P450s were converted to their corresponding dihydroxy derivatives by microsomal epoxide hydrolase. P450 4F2 was identified as the isoform that is most probably responsible for the formation of 20-HETE-EA in both human kidney and human liver, with an apparent Km of 0.7 microM. The apparent Km values of the human liver microsomes for the formation of the EET-EAs were between 4 and 5 microM, and P450 3A4 was identified as the primary P450 in the liver responsible for epoxidation of anandamide. The in vivo formation and biological relevance of the P450-derived HETE and EET ethanolamides remains to be determined.

Metabolism of efavirenz and 8-hydroxyefavirenz by P450 2B6 leads to inactivation by two distinct mechanisms.

Efavirenz is a non-nucleoside human immunodeficiency virus (HIV)-1 reverse transcriptase inhibitor used in combination therapy to treat HIV-1. Efavirenz metabolism is catalyzed primarily by the polymorphic enzyme P450 2B6. Metabolism of efavirenz by P450 2B6 and the naturally occurring P450 2B6.4 mutant led to the formation of 8-hydroxyefavirenz. Efavirenz inactivated the 7-ethoxy-4-(trifluoromethyl)coumarin activity of the wild-type P450 2B6 enzyme in a time-, concentration-, and NADPH-dependent manner. However, the P450 2B6.4 variant was not inactivated by efavirenz. The ability of efavirenz to inactivate both enzymes was investigated using cyclophosphamide and bupropion, two structurally unrelated substrates of P450 2B6, as probes. Preincubations with efavirenz decreased the ability of the wild-type enzyme to hydroxylate both substrates to similar extents but had no effect on the activities of the mutant enzyme. Interestingly, the inactivation of the wild-type enzyme was completely reversible after 24 h of dialysis as determined by heme, reduced CO spectra, and activity loss. In contrast, 8-hydroxyefavirenz, a metabolite of efavirenz, was able to inactivate both enzymes irreversibly. These data suggest that incubations of P450 2B6 and P450 2B6.4 with either the parent compound efavirenz or the metabolite 8-hydroxyefavirenz in the reconstituted system result in the formation of two different reactive intermediates that lead to losses in enzymatic activity by two different mechanisms, one reversible and one irreversible.

Metabolism of bergamottin by cytochromes P450 2B6 and 3A5.

Cytochromes P450 (P450) 2B6 and 3A5 are inactivated by bergamottin (BG). P450 2B6 metabolized BG primarily to M3 and M4 and one minor metabolite (M1). The metabolites were analyzed, and the data indicated that M1 was bergaptol, M3 was 5'-OH-BG, and M4 was a mixture of 6'- and 7'-OH-BG. Because 6'- and 7'-OH-BG were the primary metabolites, it suggested that P450 2B6 preferentially oxidized the geranyloxy chain of BG. Metabolism of BG by P450 3A5 resulted in three major metabolites: [bergaptol, M3 (5'-OH-BG), and M5 (2'-OH-BG)], and two minor metabolites [M2 (6',7'-dihydroxy-BG) and M4 (6'- and 7'-OH-BG)]. Because bergaptol was the most abundant metabolite formed, it suggested that P450 3A5 metabolized BG mainly by cleaving the geranyl-oxy chain. Molecular modeling studies confirmed that docking of BG in the P450 2B6 active site favors oxidation in the terminal region of the geranyl-oxy chain, whereas positioning the 2'-carbon of BG nearest the heme iron is preferred by P450 3A5. Glutathione (GSH)-BG conjugates were formed by both P450. Each enzyme predominantly formed conjugates with m/z values of 662. Tandem mass spectrometry analysis of the GSH conjugates indicated that the oxidation forming a reactive intermediate occurred on the furan moiety of BG, presumably through the initial formation of an epoxide at the furan double bond. The data indicate that oxidation of the geranyl-oxy chain resulted in the formation of stable metabolites of BG, whereas oxidation of the furan ring produced reactive intermediates that may be responsible for binding to and inactivating P450 2B6 and 3A4.

Identification of 17-alpha-ethynylestradiol-modified active site peptides and glutathione conjugates formed during metabolism and inactivation of P450s 2B1 and 2B6.

The oral contraceptive 17-alpha-ethynylestradiol (17EE) is a mechanism-based inactivator of cytochrome P450s (P450s) 2B1 and 2B6. Inactivation of P450s 2B1 and 2B6 in the reconstituted system by [3H]17EE resulted in labeling of the P450 apoprotein. Mass spectral analysis of 17EE-inactivated P450 2B1 showed an increase in the mass of the apoprotein by 313 Da, consistent with the mass of 17EE plus one oxygen atom. P450s 2B1 and 2B6 were inactivated with [3H]17EE and digested with CNBr. Separation of these peptides resulted in the identification of one major labeled peptide for each enzyme. N-Terminal sequencing of these peptides yielded the amino acid sequences PYTDAVIHEI (for P450 2B1) and PYTEAV (for P450 2B6) that corresponded to amino acids P347-M376 and P347-M365 in P450s 2B1 and 2B6, respectively. Electrospray ionization (ESI)-liquid chromatography-mass spectrometry (LC-MS) and matrix-assisted laser desorption ionization (MALDI)-MS analysis of the P450 2B1-derived peptide resulted in a mass of 3654 Da consistent with the mass of the P347-M376 peptide (3385 Da) plus a 268 Da 17EE adduct. Chemically reactive intermediates of 17EE that were generated during the metabolism of 17EE by P450s 2B1 and 2B6 were trapped with gluthathione (GSH). ESI-LC-MS/MS analysis of 17EE-GSH conjugates from the incubation mixtures indicated that P450s 2B1 and 2B6 generated different reactive 17EE intermediates that were responsible for the inactivation and protein modification or the formation of GSH conjugates by these two enzymes.

Synthesis of substituted phenyl diaziridines and characterization as mechanism-based inactivators of human cytochrome P450 2B6.

The metabolism of arylhydrazines by cytochromes P450 (P450s) has previously been shown to yield aryl-iron complexes that inhibit P450 enzymes as a result of heme modification. These modifications of the heme have been used to probe the topology of the active site of several P450s. Therefore, diaziridines containing one or more substitutions on the phenyl ring were synthesized and evaluated as potential mechanism-based inactivators of P450 2B enzymes that could be used to elucidate the active site topology. Five of the six trifluoroaryldiaziridines tested selectively inactivated P450 2B6 in the reconstituted system in a time-, concentration-, and NADPH-dependent manner as measured using the 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation assay. The kinetic parameters for P450 2B6 inactivation by the five compounds were calculated. Analysis of the P450 heme from P450s inactivated by the five substituted diaziridines suggested that the activity loss was not due to heme destruction as measured by the reduced-CO spectrum or high-performance liquid chromatography of the P450 heme. Dialysis experiments indicated the irreversible nature of the inactivation and the reaction between the diaziridine compounds and the P450 enzyme. Interestingly, a thiomethyl-substituted phenyl diaziridine had no effect on the activity of P450 2B6 in the reconstituted system, but competitively inhibited the O-debenzylation activity of P450 3A4 with 7-benzyloxy-4-(trifluoromethyl)coumarin as substrate. Binding spectra suggest that this compound bound reversibly to P450 2B6, and preliminary results indicate that 3-(4-methylthiophenyl)-3-(trifluoromethyl)diaziridine is metabolized by P450 2B6.

Structure-activity relationship and elucidation of the determinant factor(s) responsible for the mechanism-based inactivation of cytochrome P450 2B6 by substituted phenyl diaziridines.

It has been demonstrated previously that several 3-trifluoromethyl-3-(4-alkoxyphenyl)diaziridines inhibit the 7-ethoxy-4-(trifluoroethyl)coumarin (7-EFC) O-deethylation activity of P450 2B6 in a mechanism-based manner. In contrast, 3-trifluoromethyl-3-(4-methylthio)phenyl)diaziridine did not have any effect on the activity of P450 2B6. It is interesting that both the alkoxy and the thiophenyl compounds were metabolized by P450 2B6. In this report, the structure-activity relationships for the mechanism-based inactivation of cytochrome P450 2B6 by a series of aryl diaziridines were investigated. Three diaziridines that did not contain a 4-alkoxy-substituent on their phenyl ring, namely, 3-trifluoromethyl-3-(3-methoxyphenyl)diaziridine, 3-trifluoromethyl-3-phenyl diaziridine, and 3-trifluoromethyl-3-(4-chlorophenyl)diaziridine had no effect on the P450 2B6 7-EFC activity. Another analog that did not contain a diaziridine substructure, 3-trifluoromethyl-3-(4-methoxyphenyl)ethanone, also had no effect on the activity of P450 2B6. Glutathione ethyl ester adducts of the phenyldiaziridine reactive intermediates were isolated from reaction mixtures of the inactivated samples and analyzed by liquid chromatography-tandem mass spectrometry. The structures of the conjugates suggested that the electrophilic reactive intermediate in each case was a quinone methide (quinomethane), 4-ethylidene-cyclohexa-2,5-dienone, generated from the 4-alkoxyphenyldiaziridines by removal of both of the diaziridine and the 4-alkyl groups. In conclusion, the determinant factor for the mechanism-based inactivator activity of the aryl diaziridines seems to be the formation of the reactive quinomethane intermediate, which is generated from the 4-alkoxyphenyl diaziridines by a cytochrome P450-catalyzed metabolic reaction.

The grapefruit juice effect is not limited to cytochrome P450 (P450) 3A4: evidence for bergamottin-dependent inactivation, heme destruction, and covalent binding to protein in P450s 2B6 and 3A5.

Bergamottin (BG), a component of grapefruit juice, is a mechanism-based inactivator of cytochromes P450 (P450) 2B6 and 3A5 in the reconstituted system. The inactivation of both P450s was NADPH-dependent and irreversible. The kinetic constants for the inactivation of the 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of P450 2B6 were: K(I), 5 microM; k(inact) 0.09 min(-1); and t1/2, 8 min. The kinetic constants obtained for the inactivation of the testosterone 6beta-hydroxylation activity of P450 3A5 were: K(I), 20 microM; k(inact) 0.045 min(-1); and t1/2, 15 min. Incubations of P450s 2B6 and 3A5 with 20 microM BG at 37 degrees C for 20 min resulted in an approximately 60% loss in the catalytic activity that was accompanied by a significant loss in intact heme and a similar decrease in the reduced CO difference spectrum. The extrapolated partition ratios for BG with P450s 2B6 and 3A5 were approximately 2 and approximately 20, respectively. Liquid chromatography-mass spectroscopy analysis of the BG-inactivated samples showed that the mass of the inactivated apoprotein had increased by approximately 388 Da for both P450 2B6 and P450 3A5. SDS-polyacrylamide gel electrophoresis analysis demonstrated that [14C]BG was irreversibly bound to the apoprotein in the BG-inactivated samples. The stoichiometry of binding was approximately 0.5 mol BG metabolite/mol of each P450 inactivated. High-pressure liquid chromatography analysis of the metabolites of BG showed that P450 2B6 generated two major metabolites, whereas P450 3A5 generated three additional metabolites. Two of metabolites were identified as 6',7'-dihydroxybergamottin and bergaptol.

The naturally occurring cytochrome P450 (P450) 2B6 K262R mutant of P450 2B6 exhibits alterations in substrate metabolism and inactivation.

The polymorphic human cytochrome P450 (P450) 2B6 is primarily responsible for the metabolism of several clinically relevant drugs including bupropion, cyclophosphamide, propofol, and efavirenz. Although a number of single nucleotide polymorphisms have been found in the P450 2B6 gene, the influence of these variants on the metabolism of substrates and on the response to known inactivators of P450 2B6 has not been examined. We have compared the metabolism of different substrates of P450 2B6 (P450 Delta2B6) and the effects of mechanism-based inactivators with that observed with the polymorphic P450 Delta2B6 K262R in a reconstituted monooxygenase system (reconstituted system). Metabolism of bupropion by P450 Delta2B6 K262R resulted in increased production of hydroxybupropion compared with P450 Delta2B6. However, production of formaldehyde from the metabolism of benzphetamine by the P450 Delta2B6 K262R mutant was significantly less than that of the wild-type isozyme. P450 Delta2B6 K262R formed fewer benzphetamine metabolites compared with the wild type. N,N',N''-Triethylenethiophosphoramide (tTEPA) and bergamottin decreased the ability of both enzymes to hydroxylate bupropion and to O-deethylate 7-hydroxy-4-(trifluoromethyl)coumarin (7-EFC). Incubation with 17-alpha-ethynylestradiol decreased bupropion hydroxylation and 7-EFC O-deethylation with the wild-type enzyme but had no effect on the mutant. The kinetics for inactivation of the variant by tTEPA and bergamottin were determined using 7-EFC. The KI values for inactivation of the variant were significantly greater than those determined for the wild-type enzyme. These data demonstrate a functional difference between P450 Delta2B6 and the allelic variant P450 Delta2B6 K262R.

The mechanism-based inactivation of human cytochrome P450 2B6 by phencyclidine.

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

Mechanism-based inactivation of cytochrome P450 3A4 by 17 alpha-ethynylestradiol: evidence for heme destruction and covalent binding to protein.

17 alpha-Ethynylestradiol (EE), a major constituent of many oral contraceptives, inactivated the testosterone 6 beta-hydroxylation activity of purified P450 3A4 reconstituted with phospholipid and NADPH-cytochrome P450 reductase in a mechanism-based manner. The inactivation of P450 3A4 followed pseudo first order kinetics and was dependent on NADPH. The values for the K(I) and k(inact) were 18 microM and 0.04 min(-1), respectively, and the t(1/2) was 16 min. Incubation of 50 microM EE with P450 3A4 at 37 degrees C for 30 min resulted in a 67% loss of testosterone 6 beta-hydroxylation activity accompanied by a 35% loss of the spectral absorbance of the native protein at 415 nm and a 70% loss of the spectrally detectable P450-CO complex. The inactivation of P450 3A4 by EE was irreversible. Testosterone, an alternate substrate, was able to protect P450 3A4 from EE-dependent inactivation. The partition ratio was approximately 50. The stoichiometry of binding was approximately 1.3 nmol of an EE metabolite bound per nmol of P450 3A4 inactivated. SDS-polyacrylamide gel electrophoresis analysis demonstrated that [(3)H]EE was irreversibly bound to the P450 3A4 apoprotein. After extensive dialysis of the [(3)H]EE inactivated samples, high-pressure liquid chromatography (HPLC) analysis demonstrated that the inactivation resulting from EE metabolism led to the destruction of approximately half the heme with the concomitant generation of modified heme and EE-labeled heme fragments and produced covalently radiolabeled P450 3A4 apoprotein. Electrospray mass spectrometry demonstrated that the fraction corresponding to the major radiolabeled product of EE metabolism has a mass (M - H)(-) of 479 Da. HPLC and gas chromatography-mass spectometry analyses revealed that EE metabolism by P450 3A4 generated one major metabolite, 2-hydroxyethynylestradiol, and at least three additional metabolites. In conclusion, our results demonstrate that EE is an effective mechanism-based inactivator of P450 3A4 and that the mechanism of inactivation involves not only heme destruction, but also the irreversible modification of the apoprotein at the active site.

Mechanism-based inactivation of cytochromes P450 2E1 and 2E1 T303A by tert-butyl acetylenes: characterization of reactive intermediate adducts to the heme and apoprotein.

The kinetics for the inactivation of cytochrome P450 2E1 and the mutant P450 2E1 T303A by tert-butyl acetylene (tBA) and tert-butyl 1-methyl-2-propynyl ether (tBMP) were investigated. The two acetylenes inactivated the 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC) O-deethylation activity of purified rabbit P450s 2E1 and 2E1 T303A in a reconstituted system in a time-, concentration-, and NADPH-dependent manner. The K(I) values for the inactivation of P450s 2E1 and 2E1 T303A by tBA were 1.0 and 2.0 mM, the k(inact) values were 0.20 and 0.38 min(-)(1), and the t(1/2) values were 3.5 and 1.8 min, respectively. The K(I) values for the tBMP-inactivated P450s were 0.1 and 1.0 mM, the k(inact) values were 0.12 and 0.07 min(-)(1), and the t(1/)(2) values were 5.9 and 10.2 min, respectively. Losses in enzyme activity occurred with concurrent losses in the P450 CO spectrum and P450 heme, which were accompanied by the appearance of two different tBA- or tBMP-modified heme products in each inactivated sample. LC-MS analysis of the adducts showed masses of 661 or 705 Da, consistent with the mass of an iron-depleted heme plus the masses of a tBA or tBMP reactive intermediate and one oxygen atom, respectively. Only the tBA-inactivated P450 2E1 revealed a tBA-adducted apoprotein with an increase in mass of 99 Da, corresponding to the mass of tBA plus one oxygen atom. Surprisingly, the inactivation, CO spectral and heme loss, and heme adduct formation of the tBA-inactivated T303A mutant were completely reversible after dialysis. In addition, metabolism of para-nitrophenol was not compromised by the tBA-inactivated T303A mutant. Therefore, our studies on the inactivation of P450s 2E1 and 2E1 T303A by tBA and tBMP suggest the existence of three distinct mechanisms for inactivation, among which includes a novel, reversible heme alkylation that has not been previously described with P450 enzymes.

Mutation of tyrosine 190 to alanine eliminates the inactivation of cytochrome P450 2B1 by peroxynitrite.

We have previously reported that cytochrome P450 2B1 was inactivated by peroxynitrite and that the decrease in the catalytic activity correlated with an increase in the nitration of tyrosine. Digestion of the peroxynitrite-treated P450 2B1 with Lys C followed by amino acid sequencing of the major nitrotyrosine-containing peptide demonstrated that it spanned residues 160-225. This peptide contains two tyrosine residues at positions 190 and 203. In this study, we mutated Tyr 190 to Ala (Y190A) and Tyr 203 to Ala (Y203A) in wild-type recombinant P450 2B1 (WT) in order to identify the specific residue(s) that is nitrated and to determine whether nitrotyrosine formation is reponsible for the peroxynitrite-mediated inactivation of P450 2B1. All three P450s were expressed in Escherichia coli, purified to homogeneity, and characterized. The catalytic activities for four different substrates of P450 2B1 increased approximately 2-fold for the Y203A mutant, but decreased by about 60% for the Y190A mutant when compared to WT. The addition of peroxynitrite to the P450s resulted in concentration-dependent decreases in the catalytic activities of WT and Y203A, but no loss of the catalytic activities of Y190A. The extent of tyrosine nitration of Y190A by peroxynitrite decreased by approximately 75% as compared with WT or the Y203A protein. Following digestion of the peroxynitrite-modified proteins with Lys C, a major nitrotyrosine-containing peptide was detected from WT and Y203A, but not from Y190A. Collectively, these results indicate that Tyr 190 is the target residue for peroxynitrite-mediated nitration and that nitration of this tyrosine is a responsible for the inactivation of P450 2B1. Modeling studies suggest that Tyr 190 may play a structural role in maintaining the integrity of the protein for maximal activity through hydrogen bonding with Glu 149.

Formation of a novel reversible cytochrome P450 spectral intermediate: role of threonine 303 in P450 2E1 inactivation.

tert-Butyl acetylene (tBA) is a mechanism-based inactivator of cytochromes P450 2E1 and 2E1 T303A; however, the inactivation of the T303A mutant could be reversed by overnight dialysis. The inactivation of P450 2E1 T303A, but not the wild-type 2E1 enzyme, by tBA resulted in the formation of a novel reversible acetylene-iron spectral intermediate with an absorption maximum at 485 nm. The formation of this intermediate required oxygen and could be monitored spectrally with time. Although the alternate oxidants tert-butyl hydroperoxide (tBHP) and cumene hydroperoxide (CHP) supported the inactivation of wild-type P450 2E1 by tBA in a reductase- and NADPH-free system, only tBHP supported the inactivation of the 2E1 T303A mutant. The losses in enzymatic activity occurred concomitantly with losses in the native P450 heme, which were accompanied by the formation of tBA-adducted heme products. The inactivations supported by tBHP and CHP were completely irreversible with overnight dialysis. Spectral binding constants (K(s)) for the binding of tBA to the 2E1 P450s together with models of the enzymes with the acetylenic inactivator bound in the active site suggest that the T303A mutation results in increased hydrophobic interactions between tBA and nearby P450 residues, leading to a higher binding affinity for the acetylene compound in the mutant enzyme. Together, these data support a role for the highly conserved T303 residue in proton delivery to the active site of P450 2E1 and in the inactivation of the 2E1 P450s by small acetylenic compounds.

Mechanistic studies with N-benzyl-1-aminobenzotriazole-inactivated CYP2B1: differential effects on the metabolism of 7-ethoxy-4-(trifluoromethyl)coumarin, testosterone, and benzphetamine.

Mechanistic studies with N-benzyl-1-aminobenzotriazole (BBT)-inactivated cytochrome P450 2B1 were conducted to determine which step(s) in the reaction cycle had been compromised. Stopped-flow studies, formation of the oxy-ferro intermediate, and analysis of products suggested that the reductive process was slower with the BBT-modified enzyme. The reduced rate of reduction alone could not account for the loss in 7-ethoxy-4-(trifluoromethyl)coumarin (EFC) O-deethylation or testosterone hydroxylation activity. Surprisingly, the ability of the BBT-modified enzyme to generate formaldehyde from benzphetamine was much less affected. Benzphetamine metabolite analysis by electrospray ionization-mass spectrometry showed that the BBT-modified enzyme had a slightly greater propensity towards aromatic hydroxylation together with reduced levels of N-demethylation and little change in the N-debenzylation of benzphetamine. Orientation of substrates within the active site of the BBT-inactivated enzyme may be affected such that the more flexible benzphetamine can be metabolized, whereas metabolism of rigid, planar molecules such as EFC and testosterone is hindered.