Kinetic deuterium isotope effects for 7-alkoxycoumarin O-dealkylation reactions catalyzed by human cytochromes P450 and in liver microsomes. Rate-limiting C-H bond breaking in cytochrome P450 1A2 substrate oxidation

7-Ethoxy (OEt) coumarin has been used as a model substrate in many cytochrome P450 (P450) studies, including the use of kinetic isotope effects to probe facets of P450 kinetics. P450s 1A2 and 2E1 are known to be the major catalysts of 7-OEt coumarin O-deethylation in human liver microsomes. Human P450 1A2 also catalyzed 3-hydroxylation of 7-methoxy (OMe) coumarin at appreciable rates but P450 2E1 did not. Intramolecular kinetic isotope effects were used as estimates of the intrinsic kinetic deuterium isotope effects for both 7-OMe and 7-OEt coumarin dealkylation reactions. The apparent intrinsic isotope effect for P450 1A2 (9.4 for O-demethylation, 6.1 for O-deethylation) showed little attenuation in other competitive and noncompetitive experiments. With P450 2E1, the intrinsic isotope effect (9.6 for O-demethylation, 6.1 for O-deethylation) was attenuated in the noncompetitive intermolecular experiments. High noncompetitive intermolecular kinetic isotope effects were seen for 7-OEt coumarin O-deethylation in a baculovirus-based microsomal system and five samples of human liver microsomes (7.3-8.1 for O-deethylation), consistent with the view that P450 1A2 is the most efficient P450 catalyzing this reaction in human liver microsomes and indicating that the C-H bond-breaking step makes a major contribution to the rate of this P450 (1A2) reaction. Thus, the rate-limiting step appears to be the chemistry of the breaking of this bond by the activated iron-oxygen complex, as opposed to steps involved in the generation of the reactive complex. The conclusion about the rate-limiting step applies to all of the systems studied with this model P450 1A2 reaction including human liver microsomes, the most physiologically relevant.     

Conformational modulation of human cytochrome P450 2E1 by ethanol and other substrates: a CO flash photolysis study

The alcohol-inducible cytochrome P450 2E1 is a major human hepatic P450 which metabolizes a broad array of endogenous and exogenous compounds, including ethanol, low-molecular weight toxins, and fatty acids. Several substrates are known to stabilize this P450 and inhibit its cellular degradation. Furthermore, ethanol is a known modulator of P450 2E1 substrate metabolism. We examined the CO binding kinetics of P450 2E1 after laser flash photolysis of the heme-CO bond, to probe the effects of ethanol and other substrates on protein conformation and dynamics. Ethanol had an effect on the two kinetic parameters that describe CO binding: it decreased the rate of CO binding, suggesting a decrease in the protein's conformational flexibility, and increased the photosensitivity, which indicates a local effect in the active site region such as strengthening of the heme-CO bond. Other substrates decreased the CO binding rate to varying degrees. Of particular interest is the effect of arachidonic acid, which abolished photodissociation in the absence of ethanol but had no effect in the presence of ethanol. These results are consistent with a model of P450 2E1 whereby arachidonic acid binds along a long hydrophobic binding pocket and blocks exit of CO from the heme region.     

Assimilatory nitrate reductase: lysine 741 participates in pyridine nucleotide binding via charge complementarity

The effects of benzyl (BITC) and phenethyl isothiocyanate (PEITC) on the activity of a P450 2E1 mutant where the conserved threonine at position 303 was replaced with an alanine residue (P450 2E1 T303A) were examined. PEITC inactivated the mutant enzyme with a K(I) of 1.6 microM. PEITC also inactivated the wild-type P450 2E1 as efficiently with a K(I) of 2.7 microM. The inactivation was entirely dependent on NADPH and followed pseudo-first-order kinetics. Previously we reported the mechanism-based inactivation of wild-type P450 2E1 by BITC with a K(I) of 13 microM. In contrast to the wild-type enzyme, the P450 2E1 T303A mutant was not inactivated by BITC but it was inhibited in a competitive manner with a K(i) of 3 microM. The binding constants determined by spectral binding studies were similar for both enzymes. The binding of BITC produced characteristic Type I spectral changes in the wild-type and mutant enzyme. A radiolabeled BITC metabolite bound to P450 2E1 and to P450 2E1 T303A when both enzymes were incubated with [(14)C]BITC and NADPH. Whole protein electrospray ion trap mass spectrometry indicated that a mass consistent with one molecule of benzylisocyanate and oxygen was adducted to the wild-type enzyme. The mass adducted to the T303A mutant was consistent with the addition of one hydroxylated BITC or of one benzylisocyanate moiety and one sulfur molecule. Analysis of the metabolites of BITC indicated that each enzyme produced similar metabolites but that the mutant enzyme generated significantly higher amounts of benzaldehyde and benzoic acid when compared to the wild-type enzyme.     

Spectral studies of tert-butyl isothiocyanate-inactivated P450 2E1

Inactivation of cytochrome P450 2E1 by tert-butyl isothiocyanate (tBITC) resulted in a loss in the spectrally detectable P450-reduced CO complex. The heme prosthetic group does not appear to become modified, since little loss of the heme was observed in the absolute spectra or the pyridine hemochrome spectra, or in the amount of heme recovered from HPLC analysis of the tBITC-inactivated samples. Prolonged incubations of the inactivated P450 2E1 with dithionite and CO resulted in a recovery of both the CO complex and the enzymatic activity. Inactivated samples that were first reduced with dithionite for 1 h prior to CO exposure recovered their CO spectrum to the same extent as samples not pretreated with dithionite, suggesting that the major defect was an inability of the inactivated sample to bind CO. Spectral binding studies with 4-methylpyrazole indicated that the inactivated P450 2E1 had an impaired ability to bind the substrate. Enzymatic activity could not be restored with iodosobenzene as the alternate oxidant. EPR analysis indicated that approximately 24% of the tBITC-inactivated P450 2E1 was EPR-silent. Of the remaining tBITC-inactivated P450 2E1, approximately 45% exhibited an unusual low-spin EPR signal that was attributed to the displacement of a water molecule at the sixth position of the heme by a tBITC modification to the apoprotein. ESI-LC-MS analysis of the inactivated P450 2E1 showed an increase in the mass of the apoprotein of 115 Da. In combination, the data suggest that tBITC inactivated P450 2E1 by binding to a critical active site amino acid residue(s). This modified amino acid(s) presumably acts as the sixth ligand to the heme, thereby interfering with oxygen binding and substrate binding.     

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.     

Kinetics of CO binding to and dissociation from microsomal cytochrome P-450 induced by phenobarbital in rat liver

The kinetics of CO binding to hepatic microsomes from phenobarbital-treated Sprague-Dawley rats, measured by stopped flow spectrophotometry, can be resolved into three components with second order velocity constants of 2.23 +/- 0.35 X 10(5) M-1 S-1, 1.59 +/- 0.18 X 10(6) M-1 S-1, and 8.7 +/- 1.7 X 10(6) M-1 S-1. The three CO-binding species were present in ratios of 1:1.25:1.39 as judged by the relative amplitude of the change in absorbance at 450 nm associated with each of the kinetic components. Similar results were obtained in a range of [CO] from 10 to 700 micron when CO recombination was followed subsequent to flash photolysis of the CO-associated microsomes. In contrast, the dissociation rate of CO from its cytochrome P-450 complex measured by the NO replacement method was biphasic. Approximately 40% of the bound CO dissociated at a rate of 0.40 +/- 0.071 s-1, whereas the remaining 60% dissociated at a rate of 0.049 +/- 0.008 s-1 at 20 degrees C.     

Pre-steady-state charge translocation in NaK-ATPase from eel electric organ

Time-resolved measurements of charge translocation and phosphorylation kinetics during the pre-steady state of the NaK-ATPase reaction cycle are presented. NaK-ATPase-containing microsomes prepared from the electric organ of Electrophorus electricus were adsorbed to planar lipid bilayers for investigation of charge translocation, while rapid acid quenching was used to study the concomitant enzymatic partial reactions involved in phosphoenzyme formation. To facilitate comparison of these data, conditions were standardized with respect to pH (6.2), ionic composition, and temperature (24 degrees C). The different phases of the current generated by the enzyme are analyzed under various conditions and compared with the kinetics of phosphoenzyme formation. The slowest time constant (tau 3(-1) approximately 8 s-1) is related to the influence of the capacitive coupling of the adsorbed membrane fragments on the electrical signal. The relaxation time associated with the decaying phase of the electrical signal (tau 2(-1) = 10-70 s-1) depends on ATP and caged ATP concentration. It is assigned to the ATP and caged ATP binding and exchange reaction. A kinetic model is proposed that explains the behavior of the relaxation time at different ATP and caged ATP concentrations. Control measurements with the rapid mixing technique confirm this assignment. The rising phase of the electrical signal was analyzed with a kinetic model based on a condensed Albers-Post cycle. Together with kinetic information obtained from rapid mixing studies, the analysis suggests that electroneutral ATP release, ATP and caged ATP binding, and exchange and phosphorylation are followed by a fast electrogenic E1P-->E2P transition. At 24 degrees C and pH 6.2, the rate constant for the E1P-- >E2P transition in NaK-ATPase from eel electric organ is > or = 1,000 s- 1.     

Binding and oxidation of alkyl 4-nitrophenyl ethers by rabbit cytochrome P450 1A2: evidence for two binding sites

Although most cytochrome P450 (P450) reactions demonstrate saturation kinetics that fit to the standard Michaelis-Menten equation, there are important exceptions where sigmoidal or nonhyperbolic behavior is observed and have been fit instead to kinetic models involving two binding sites. To assess these models, we demonstrate the consistency of a two binding site model to interpret both steady-state kinetics and binding events. Rates of 4-nitrophenol and formaldehyde production from the O-demethylation of 1-methoxy-4-nitrobenzene by P450 1A2 isolated from rabbit liver produced biphasic plots, when plotted against substrate concentration. Experiments confirmed the absence of the further oxidation of the products. Recombinant rabbit P450 1A2 yielded the same maximal velocity and more marked biphasicity. Overall, these steady-state data fit well to kinetic models involving two binding sites. Steady-state studies of substrates with bulkier O-ethyl or O-isopropoxy groups indicated decreased affinity for the second site. Based on binding studies, the affinity of P450 1A2 for these substrates increased 200-fold with the larger alkyl groups. To analyze the single binding site model, competition studies were conducted with 1,4-phenyldiisocyanide and the alkyl 4-nitrophenyl ethers. Although the observed dissociation constants and the competing titrant demonstrated a linear dependence, the affinity for the competing titrant depended on the presence of the other titrant, which violates the single binding site model. Alternatively, we applied a two binding site model to these data to obtain dissociation constants for the binary and ternary complexes. The agreement between the dissociation constants for the heterogeneous complexes supports the appropriateness of the two binding site model. This novel finding for P450 1A2 may be more common than originally perceived for P450s.     

Hepatic microsomes from beer fed rats contain a cytochrome P-450 metabolic intermediate complex.

Cytochrome P450 (CYP450) 2E1 (CYP2E1) is induced by pure ethanol following its chronic administration, and commercial alcoholic beverages, whose major constituent is ethanol, are generally assumed to have a similar effect on this isoform of CYP450. Recently, we serendipitously discovered that beer administered to rats for six weeks had only a minimal inductive effect on hepatic microsomal CYP2E1 activity, while rats on 10% ethanol had CYP2E1 levels five-fold greater than controls. The daily ethanol intake levels for the beer fed and 10% ethanol fed rats were equivalent. In addition, CYP450 spectral features of microsomes from beer fed and ethanol fed rats were markedly different. Spectral examination of microsomes from beer fed rats revealed that about 40% of the total CYP450 content existed in the form of a metabolic intermediate (MI) complex, while no evidence was found for MI complex formation in microsomes of ethanol fed rats. We conclude that beer contains an unidentified component(s) that apparently blocks the typical ethanol induction of CYP2E1 and form an MI complex with CYP450.     

Temperature-jump relaxation kinetics of substrate-induced spin-state transition in cytochrome P450 (comparison of the wild-type and C334A mutant P450(CAM) and P450(2B4))

The kinetics of binding of the substrate camphor to the cytochrome P450(CAM) and the C334A mutant as well as the kinetics of binding of benzphetamine to the wild-type P450(2B4) have been studied by the temperature-jump relaxation technique in order to distinguish between the two models for substrate-induced spin-state transition. These models are the bimolecular model in which spin-state transition occurs in parallel with substrate binding, and the two-step spin-equilibrium model in which substrate binding is a separate step preceding the spin-state transition. With all three P450s, the relaxation rate versus concentration data were linear as predicted by the bimolecular model and inconsistent with the spin-equilibrium model, which predicts a curve reaching saturation. With all three P450s, the relaxation rate versus concentration data exhibited maxima. These results are considered to resolve the controversy in favor of the bimolecular model for substrate-induced spin-state transition. In addition, the results suggest that the bimolecular model may be applicable to other P450s as well.     

Cytochrome P-450-mediated oxidation of 2-hydroxyestrogens to reactive intermediates

2-Hydroxyestradiol, 2-hydroxyestrone and 2-hydroxy-17α-ethynylestradiol, oxidation products of naturally occurring estrogens and synthetic estrogens in some oral contraceptives were found to be converted by rat liver microsomes to reactive metabolites that become irreversibly bound to microsomal protein. The irreversible binding required microsomes, oxygen and NADPH. The NADPH could be replaced by a xanthine-xanthine oxidase system which is known to generate superoxide anions. The irreversible binding was substantially inhibited by superoxide dismutase, 30% in those incubations containing NADPH and 98% in those incubations containing the xanthine-xanthine oxidase system. Further studies with 2-hydroxyestradiol showed that microsomal cytochrome P-450 was rate limiting in the NADPH-dependent irreversible binding, because the binding was inhibited 62% by an antibody against NADPH-cytochrome $\text{c}$ reductase and 70% in an atmosphere of CO:O₂ (9:1) when compared to an atmosphere of N₂:O₂ (9:1). Phenobarbital, a known inducer of cytochrome P-450, had no effect on the irreversible binding of 2-hydroxyestradiol, whereas another inducer of P-450, pregnenolone-16α-carbonitrile, markedly increased the irreversible binding. In contrast, cobaltous chloride, an inhibitor of the synthesis of cytochrome P-450, decreased both P-450 and the irreversible binding. These results are consistent with a mechanism for irreversible binding of estrogens and 2-hydroxyestrogens to microsomes that requires oxidation of the catechol nucleus by cytochrome P-450-generated superoxide anion.     

Cytochrome P450 7A1 cholesterol 7alpha-hydroxylation: individual reaction steps in the catalytic cycle and rate-limiting ferric iron reduction

Cytochrome P450 (P450) 7A1 is well known as the cholesterol 7α-hydroxylase, the first enzyme involved in bile acid synthesis from cholesterol. The human enzyme has been reported to have the highest catalytic activity of any mammalian P450. Analyses of individual steps of cholesterol 7α-hydroxylation reaction revealed several characteristics of this reaction: (i) two-step binding of cholesterol to ferric P450, with an apparent K(d) of 0.51 μM, (ii) a rapid reduction rate in the presence of cholesterol (～10 s(-1) for the fast phase), (iii) rapid formation of a ferrous P450-cholesterol-O(2) complex (29 s(-1)), (iv) the lack of a non-competitive kinetic deuterium isotope effect, (v) the lack of a kinetic burst, and (vi) the lack of a deuterium isotope effect when the reaction was initiated with the ferrous P450-cholesterol complex. A minimum kinetic model was developed and is consistent with all of the observed phenomena and the rates of cholesterol 7α-hydroxylation and H(2)O and H(2)O(2) formation. The results indicate that the first electron transfer step, although rapid, becomes rate-limiting in the overall P450 7A1 reaction. This is a different phenomenon compared with other P450s that have much lower rates of catalysis, attributed to the much more efficient substrate oxidation steps in this reaction.     

Oxidations of p-alkoxyacylanilides catalyzed by human cytochrome P450 1A2: structure-activity relationships and simulation of rate constants of individual steps in catalysis

Human cytochrome P450 (P450) 1A2 is involved in the oxidation of many important drugs and carcinogens. The prototype substrate phenacetin is oxidized to an acetol as well as the O-dealkylation product [Yun, C.-H., Miller, G. P., and Guengerich, F. P. (2000) Biochemistry 39, 11319-11329]. In an effort to improve rates of catalysis of P450 1A2 enzymes, we considered a set of p-alkoxyacylanilide analogues of phenacetin and found that variations in the O-alkyl and N-acyl substituents altered the rates of the two oxidation reactions and the ratio of acetol/phenol products. Moving one methylene group of phenacetin from the O-alkyl group to the N-acyl moiety increased rates of both oxidations approximately 5-fold and improved the coupling efficiency (oxidation products formed/NADPH consumed) from 6% to 38%. Noncompetitive kinetic deuterium isotope effects of 2-3 were measured for all O-dealkylation reactions examined with wild-type P450 1A2 and the E225I mutant, which has 6-fold higher activity. A trend of decreasing kinetic deuterium isotope effect for E225I > wild-type > mutant D320A was observed for O-demethylation of p-methoxyacetanilide, which follows the trend for k(cat). The set of O-dealkylation and acetol formation results for wild-type P450 1A2 and the E225I mutant with several of the protiated and deuterated substrates were fit to a model developed for the basic catalytic cycle and a set of microscopic rate constants in which the only variable was the rate of product formation (substrate oxygenation, including hydrogen abstraction). In this model, k(cat) is considerably less than any of the microscopic rate constants and is affected by several individual rate constants, including the rate of formation of the oxygenating species, the rate of substrate oxidation by the oxygenating species, and the rates of generation of reduced oxygen species (H(2)O(2), H(2)O). This analysis of the effects of the individual rate constants provides a framework for consideration of other P450 reactions and rate-limiting steps.     

Involvement of singlet oxygen in cytochrome P450-dependent substrate oxidations.

Cytochrome P450 (P450)-dependent p-hydroxylation of aniline and o-deethylation of 7-ethoxycoumarin were examined in rat liver microsomes in the presence of radical scavengers. The addition of beta-carotene, a quencher of singlet oxygen species ((1)O(2)), suppressed the aniline hydroxylation, while the addition of sodium azide (NaN(3)) ((1)O(2) quencher) enhanced the reaction. No other reactive oxygen scavengers or chelating agents such as superoxide dismutase, catalase, dimethylsulfoxide, or deferoxamine altered the reaction. In contrast, the microsomal o-deethylation of 7-ethoxycoumarin was suppressed by the addition of NaN(3). (1)O(2) was detectable during the reaction of microsomes and NADPH by ESR spin-trapping when 2,2,6,6-tetramethyl-4-piperidone (TMPD) was used as a spin trap, and the (1)O(2) was quenched by the additions of beta-carotene, NaN(3), aniline, and 7-ethoxycoumarin. The enhancement effect of NaN(3) in the hydroxylation of aniline appeared to be due to the conformational change of P450 protein, which in turn enhances the binding of aniline to P450 in terms of the spectral dissociation constant (K(s)). In contrast, (1)O(2) appeared to be active in the o-deethylation of 7-ethoxycoumarin. On the basis of the results, the involvement of (1)O(2) in P450-dependent substrate oxygenations is proposed.     

Molecular Characterization of the <Emphasis Type="Italic">Camelus dromedarius</Emphasis> Putative Cytochrome P450s Genes

The expression levels of cytochrome P450s were examined in different camel tissues by western blotting and semi-quantitative polymerase chain reaction. Camelus dromedarius liver microsomes were found to express different P450s isoenzymes constitutively. The maximum expression of P450 protein was seen in the camel liver in the order of P450 2E1, 1A1, 3A and 2B1/2. Camel extrahepatic tissues, kidney, spleen and the lung showed detectable levels of P450s 1A1 but lower than that noticed in liver. Detectable level of P450 2B1/2 was also observed in camel lung (29.5 vs. 58% liver microsomes). P450scc and 21-hydroxylase were found to be differentially expressed only in camel testis. Partial sequences of these P450s genes showed high similarities with the human P450s. These results demonstrate that the multiple forms of P450s are differentially expressed in camel tissues and that the relative levels of expression are comparable with other mammals. These observations might be important in understanding the differential susceptibility of camel tissues to the toxic effects of xenobiotics and environmental pollution.     

The nature of the rate-limiting step in aniline hydroxylation involving cytochrome p-450 rat liver microsomes.

The kinetics of aniline hydroxylation was studied with: (1) rat liver microsomes involving NADPH and O2 (system 1), (2) hepatic microsomes and tert-butylhydroperoxide (system 2) and (3) microsomes and cumyl hydroperoxide (system 3) at 15--37 degrees C. The reactions were characterized by the values of the aniline oxidation rate constants, k2 = V/E0, where E0 is the initial concentration of cytochrome P-450: K 1/2 = 1.60 - 10(8) EXP (-13 400/RT) sec-1, k 2/2 = 1.66 - 10(9) exp (-14 500/RT) sec-1, k 3/2 = 6.83 - 10(9) exp (-15 300/RT) sec-1. The values of delta H0 and delta S0, were calculated and compared for the three systems. The evidence suggests that oxygen insertion into the substrate molecule is the rate-limiting step in the reaction of aniline oxidation for the mentioned systems. The nature of aniline binding to cytochrome P-450 and that of the hydroxylating agent have been discussed.     

A proteolytically sensitive region common to several rat liver cytochromes P450: effect of cleavage on substrate binding.

Limited proteolysis of rat liver microsomes was used to probe the topography and structure of cytochrome P450 bound to the endoplasmic reticulum. Three cytochromes P450 from two families were examined. Monoclonal antibodies to cytochrome P450 forms 1A1, 2B1, and 2E1 were used to immunopurify these proteolyzed cytochromes P450 from microsomes from rats treated with 3-methylcholanthrene, phenobarbital, and acetone, respectively. Electrophoretic and immunoblot analysis of tryptic fragments revealed a highly sensitive cleavage site in all three cytochromes P450. N-Terminal sequencing was performed on the fragments after transfer onto poly(vinylidene difluoride) membranes and showed that this preferential cleavage site is at amino acid position 298 of P450 1A1, position 277 of P450 2B1, and position 278 of P450 2E1. Multiple sequence alignment revealed that these positions are at the amino terminal of a highly conserved region of these cytochromes P450. The important functional role implied by primary sequence conservation along with the proteolytic sensitivity at its amino terminal suggests that this region is a protein domain. Comparison with the known structure of the bacterial cytochrome P450cam predicts that this proteolytically sensitive site is within an interhelical turn region connected to the distal helix that partially encompasses the heme-containing active site. Substrate binding to the cleaved cytochromes P450 was examined in order to determine whether the newly added conformational freedom near the cleavage site functionally altered these cytochromes P450. Cleavage of P450 2B1 abolished benzphetamine binding, which indicates that the cleavage site contains an important structural determinant for binding this substrate. However, cleavage did not affect benzo[a]pyrene binding to P450 1A1.     

Phospholipase D activity of cytochrome P450 in human liver endoplasmic reticulum.

Phospholipase D (PLD) activity in mammalian liver endoplasmic reticulum (ER) has not been characterized. Purified human liver microsomal cytochromes P450 (P450)-P450 1A2 and P450 2E1-were shown to have appreciable PLD activity, hydrolyzing phosphatidylcholine but not other phospholipids, generating PA and choline. The activity was confirmed using recombinant and mutated human P450s expressed in bacteria. In human liver microsomes, immunoinhibition of PLD activity was observed with anti-P450 1A2 > anti-P450 2C > anti-P450 2E1. Thus, P450 may act as a significant PLD in human liver ER and exert its biological effects by several mechanisms, including signaling functions and change of membrane properties.     

Role of cytochrome P450 in the oxidation of glycerol by reconstituted systems and microsomes.

Glycerol can be oxidized by rat liver microsomes to formaldehyde in a reaction that requires the production of reactive oxygen intermediates. Studies with inhibitors, antibodies, and reconstituted systems with purified cytochrome P4502E1 were carried out to evaluate whether P450 was required for glycerol oxidation. A purified system containing phospholipid, NADPH-cytochrome P450 reductase, P4502E1, and NADPH oxidized glycerol to formaldehyde. Formaldehyde production was dependent on NADPH, reductase, and P450, but not phospholipid. Formaldehyde production was inhibited by substrates and ligands for P4502E1, as well as by anti-pyrazole P4502E1 IgG. The oxidation of glycerol by the reconstituted system was sensitive to catalase, desferrioxamine, and EDTA but not to superoxide dismutase or mannitol, indicating a role for H2O2 plus non-heme iron, but not superoxide or hydroxyl radical in the overall glycerol oxidation pathway. The requirement for reactive oxygen intermediates for glycerol oxidation is in contrast to the oxidation of typical substrates for P450. In microsomes from pyrazole-treated, but not phenobarbital-treated rats, glycerol oxidation was inhibited by anti-pyrazole P450 IgG, anti-hamster ethanol-induced P450 IgG, and monoclonal antibody to ethanol-induced P450, although to a lesser extent than inhibition of dimethylnitrosamine oxidation. Anti-rabbit P4503a IgG did not inhibit glycerol oxidation at concentrations that inhibited oxidation of dimethylnitrosamine. Inhibition of glycerol oxidation by antibodies and by aminotriazole and miconazole was closely associated with inhibition of H2O2 production. These results indicate that P450 is required for glycerol oxidation to formaldehyde; however, glycerol is not a direct substrate for oxidation to formaldehyde by P450 but is a substrate for an oxidant derived from interaction of iron with H2O2 generated by cytochrome P450.     

P450 active site architecture and reversibility: inactivation of cytochromes P450 2B4 and 2B4 T302A by tert-butyl acetylenes

The inactivations of P450 2B4 and the T302A mutant of 2B4 by tert-butyl acetylene (tBA) and the inactivation of 2B4 T302A by tert-butyl 1-methyl-2-propynyl ether (tBMP) have been investigated. tBA and tBMP inactivated both enzymes in a mechanism-based manner with the losses in enzymatic activity corresponding closely to losses in P450 heme. HPLC and ESI-LC-MS analysis detected two different tBA- or tBMP-modified heme products with masses of 661 and 705 Da, respectively. Interestingly, the inactivations of the P450s 2B4 by tBA and tBMP were partially reversible by dialysis, and the tBA- or tBMP-modified heme products could only be observed with ESI-LC-MS/MS when the inactivated samples were acidified prior to analysis, indicating a requirement for protons in the formation of stable heme adducts in both the wild-type and mutant 2B4 enzymes. Results of studies using artificial oxidants to support enzyme inactivation suggest that the oxenoid-iron activated oxygen species is preferentially utilized during the inactivation of the P450s 2B4 by tBA. These results argue against the use of a peroxo-iron species by P450 2B4 T302A. Molecular dynamics studies of wild-type P450 2B4 reveal that contiguous hydrogen bond networks, including structural waters, link a conserved glutamate (E301) to the distal oxygen of the peroxo-heme species via threonine 302. Interestingly, models of 2B4 T302A reveal that a compensatory, ordered hydrogen bond network forms despite the removal of T302. These results indicate that while T302 may play a role in proton delivery in the formation of the oxenoid-iron complex and in the stabilization of acetylene heme adducts in 2B4, it is not essential for proton delivery given the presence of E301 in the binding site.