Oxidation Kinetics of Ethanol by Human Cytochrome P450 2E1
1997; Elsevier BV; Volume: 272; Issue: 47 Linguagem: Inglês
10.1074/jbc.272.47.29643
ISSN1083-351X
AutoresL. Chastine Bell, F. Peter Guengerich,
Tópico(s)Eicosanoids and Hypertension Pharmacology
ResumoA number of cytochrome P450 (P450) 2E1 substrates are known to show kinetic deuterium isotope effects of ∼5 onK m (D K =D K m /H K m ), but not on k cat, in rat liver microsomes (e.g. N-nitrosodimethylamine, ethanol, and CH2Cl2). We observedD K m values of 3–5 for recombinant human P450 2E1-catalyzed ethanol oxidation. Replacing NADPH and O2 with the oxygen surrogate cumene hydroperoxide yielded similar results. Ferric P450 2E1 reduction was fast (k>1000 min−1) even in the absence of substrate. These results indicate that the basis for the increase inK m is in the latter portion of the catalytic cycle. The intrinsic isotope effect (D k) for ethanol oxidation was determined (competitively) to be 3.8, indicating that C–H bond cleavage is isotopically sensitive. Pre-steady-state studies showed a burst of product formation (k = 410 min−1), with the burst amplitude corresponding to the P450 concentration. Deuteration of ethanol resulted in an isotope effect of 3.2 on the rate of the burst. We conclude that product release is rate-limiting in the oxidation of ethanol to acetaldehyde by P450 2E1. The steady-state kinetics can be described by a paradigm in which thek cat approximates the rate of product release, and K m is an expression in which the denominator is dominated by the rate of C–H bond breaking. A number of cytochrome P450 (P450) 2E1 substrates are known to show kinetic deuterium isotope effects of ∼5 onK m (D K =D K m /H K m ), but not on k cat, in rat liver microsomes (e.g. N-nitrosodimethylamine, ethanol, and CH2Cl2). We observedD K m values of 3–5 for recombinant human P450 2E1-catalyzed ethanol oxidation. Replacing NADPH and O2 with the oxygen surrogate cumene hydroperoxide yielded similar results. Ferric P450 2E1 reduction was fast (k>1000 min−1) even in the absence of substrate. These results indicate that the basis for the increase inK m is in the latter portion of the catalytic cycle. The intrinsic isotope effect (D k) for ethanol oxidation was determined (competitively) to be 3.8, indicating that C–H bond cleavage is isotopically sensitive. Pre-steady-state studies showed a burst of product formation (k = 410 min−1), with the burst amplitude corresponding to the P450 concentration. Deuteration of ethanol resulted in an isotope effect of 3.2 on the rate of the burst. We conclude that product release is rate-limiting in the oxidation of ethanol to acetaldehyde by P450 2E1. The steady-state kinetics can be described by a paradigm in which thek cat approximates the rate of product release, and K m is an expression in which the denominator is dominated by the rate of C–H bond breaking. Microsomal cytochromes P450 1The abbreviations used are: P450, microsomal cytochrome P450; b 5, cytochromeb 5; DLPC,l-α-dilauroyl-sn-glycero-3-phosphocholine; PFB-, pentafluorobenzyl-; GC, gas chromatography; PIEIMS, positive ion electron ionization mass spectrometry; NICIMS, negative ion chemical ionization mass spectrometry; MMO, methane monooxygenase; HPLC, high performance liquid chromatography. 1The abbreviations used are: P450, microsomal cytochrome P450; b 5, cytochromeb 5; DLPC,l-α-dilauroyl-sn-glycero-3-phosphocholine; PFB-, pentafluorobenzyl-; GC, gas chromatography; PIEIMS, positive ion electron ionization mass spectrometry; NICIMS, negative ion chemical ionization mass spectrometry; MMO, methane monooxygenase; HPLC, high performance liquid chromatography. (also termed heme-thiolate protein P450 by the Enzyme Commission, EC 1.14.14.1) (1Palmer G. Reedijk J. J. Biol. Chem. 1992; 267: 665-677Abstract Full Text PDF PubMed Google Scholar) catalyze a variety of mixed-function monooxygenase reactions that often result in detoxication of drugs and other xenobiotics (2Brodie B.B. Gillette J.R. LaDu B.N. Annu. Rev. Biochem. 1958; 27: 427-454Crossref PubMed Scopus (245) Google Scholar, 3Wislocki P.G. Miwa G.T. Lu A.Y.H. Jakoby W.B. Enzymatic Basis of Detoxication. 1. Academic Press, New York1980: 135-182Google Scholar, 4Guengerich F.P. J. Biol. Chem. 1991; 266: 10019-10022Abstract Full Text PDF PubMed Google Scholar). Occasionally, oxidation results in the bioactivation of potentially potent carcinogens, particularly with substrates metabolized by P450 2E1 (5Guengerich F.P. Liebler D.C. Crit. Rev. Toxicol. 1985; 14: 259-307Crossref PubMed Scopus (305) Google Scholar, 6Nelson S.D. Harvison P.J. Guengerich F.P. Mammalian Cytochromes P-450. 2. CRC Press, Inc., Boca Rotan, FL1987: 19-79Google Scholar). P450 2E1 is active in the oxidation of many low molecular weight organic compounds (e.g. nitrosamines and alkenes) associated with human cancers, and the reactivity of products with DNA has been demonstrated (7Guengerich F.P. Kim D.-H. Iwasaki M. Chem. Res. Toxicol. 1991; 4: 168-179Crossref PubMed Scopus (1243) Google Scholar, 8Yamazaki H. Inui Y. Yun C.-H. Mimura M. Guengerich F.P. Shimada T. Carcinogenesis. 1992; 13: 1789-1794Crossref PubMed Scopus (385) Google Scholar). DNA alkylating ability and carcinogenicity were shown to be decreased upon deuterium substitution of N-nitrosodimethylamine, now known to be a substrate of P450 2E1 (9Keefer L.K. Lijinsky W. Garcia H. J. Natl. Cancer Inst. 1973; 51: 299-302Crossref PubMed Scopus (85) Google Scholar). When rat liver microsomes were examined, deuteration was found to increase K m for these reactions ∼5-fold, but the k cat (V max) remained unaffected by deuterium substitution (10Yang C.S. Ishizaki H. Lee M. Wade D. Fadel A. Chem. Res. Toxicol. 1991; 4: 408-413Crossref PubMed Scopus (26) Google Scholar, 11Dagani D. Archer M.C. J. Natl. Cancer Inst. 1976; 57: 955-957Crossref PubMed Scopus (41) Google Scholar, 12Wade D. Yang C.S. Metral C.J. Roman J.M. Hrabie J.A. Riggs C.W. Anjo T. Keefer L.K. Mico B.A. Cancer Res. 1987; 47: 3373-3377PubMed Google Scholar). Most P450s are considered to operate according to a general scheme (Fig. 1) (13White R.E. Coon M.J. Annu. Rev. Biochem. 1980; 49: 315-356Crossref PubMed Scopus (925) Google Scholar). Following substrate binding (step 1), ferric P450 receives 1 electron via NADPH-P450 reductase (step 2). Steps 1a and 2a represent a potential pathway by which Fe3+ is reduced to Fe2+ in the absence of substrate and suggests a possibility for later entry of the substrate into the catalytic cycle. The ferrous form of the heme binds O2 (step 3) before undergoing a second 1-electron reduction to begin O2 activation (step 4). Although this second electron originates from NADPH-P450 reductase, the accessory protein cytochrome b 5 (b 5, EC 4.4.2 group) appears to play some role in the delivery of the electron to the P450 (14Pompon D. Biochemistry. 1987; 26: 6429-6435Crossref PubMed Scopus (47) Google Scholar). Insertion of the activated oxygen into the substrate is believed to occur by way of C–H bond cleavage (step 6) followed by rapid oxygen rebound to form product (step 7) (15Groves J.T. McClusky G.A. J. Am. Chem. Soc. 1976; 98: 859-861Crossref Scopus (422) Google Scholar). Step 8 is release of the product from the enzyme active site. Within the context of this scheme, the reduction (steps 2 and 4) and chemistry (step 6) are generally considered to be rate-limiting (13White R.E. Coon M.J. Annu. Rev. Biochem. 1980; 49: 315-356Crossref PubMed Scopus (925) Google Scholar). Steps 9 and 10 reflect the potential for the Fe3+ROH complex to receive an electron from NADPH-P450 reductase, possibly leading to a second cycle of oxidation. High intramolecular kinetic hydrogen isotope effects are seen in many P450 reactions involving C–H bond cleavage and are usually interpreted as evidence for a hydrogen atom abstraction mechanism (16Groves J.T. McClusky G.A. White R.E. Coon M.J. Biochem. Biophys. Res. Commun. 1978; 81: 154-160Crossref PubMed Scopus (478) Google Scholar), with some caveats (17Guengerich F.P. Yun C.-H. Macdonald T.L. J. Biol. Chem. 1996; 271: 27321-27329Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). There are fewer reports of non-competitive intermolecular hydrogen isotope effects on P450 reactions, and these tend to be onk cat not K m (18Guengerich F.P. Peterson L.A. Böcker R.H. J. Biol. Chem. 1988; 263: 8176-8183Abstract Full Text PDF PubMed Google Scholar). Although the effects of deuterium substitution onN-nitrosodimethylamine N-demethylation were first reported in 1973, a definitive explanation for the intermolecular isotope effect on K m (and notk cat) has not been given. The observation has been repeated using several P450 2E1 substrates (9Keefer L.K. Lijinsky W. Garcia H. J. Natl. Cancer Inst. 1973; 51: 299-302Crossref PubMed Scopus (85) Google Scholar, 10Yang C.S. Ishizaki H. Lee M. Wade D. Fadel A. Chem. Res. Toxicol. 1991; 4: 408-413Crossref PubMed Scopus (26) Google Scholar, 11Dagani D. Archer M.C. J. Natl. Cancer Inst. 1976; 57: 955-957Crossref PubMed Scopus (41) Google Scholar, 19Swann P.F. Mace R. Angeles R.M. Keefer L.K. Carcinogenesis. 1983; 4: 821-825Crossref PubMed Scopus (38) Google Scholar, 20Mico B.A. Swagzdis J.E. Hu H.S.-W. Keefer L.K. Oldfield N.F. Garland W.A. Cancer Res. 1985; 45: 6280-6285PubMed Google Scholar, 21Ekström G. Norsten C. Cronholm T. Ingelman-Sundberg M. Biochemistry. 1987; 26: 7348-7354Crossref PubMed Scopus (29) Google Scholar, 22Vaz A.D.N. Coon M.J. Biochemistry. 1994; 33: 6442-6449Crossref PubMed Scopus (88) Google Scholar). Yanget al. (10Yang C.S. Ishizaki H. Lee M. Wade D. Fadel A. Chem. Res. Toxicol. 1991; 4: 408-413Crossref PubMed Scopus (26) Google Scholar) compared the competitive inhibition of alternate2H and 1H substrates of P450 2E1 and suggested that the observed deuterium effect was due, in part, to a rate difference in the breaking of the C–H bond and its relationship with other rate constants. It was also proposed (10Yang C.S. Ishizaki H. Lee M. Wade D. Fadel A. Chem. Res. Toxicol. 1991; 4: 408-413Crossref PubMed Scopus (26) Google Scholar) that the effect onV/K might be interpreted in the context of a generalized scheme (23Korzekwa K. Trager W.F. Gillette J.R. Biochemistry. 1989; 28: 9012-9018Crossref PubMed Scopus (64) Google Scholar, 24Gillette J.R. Darbyshire J.F. Sugiyama K. Biochemistry. 1994; 33: 2927-2937Crossref PubMed Scopus (26) Google Scholar) in which (i) the isotopically sensitive step (C–H bond breaking) precedes a slower step (possibly product release) and (ii) the enzyme exhibits a low commitment to catalysis. Yang et al. (10Yang C.S. Ishizaki H. Lee M. Wade D. Fadel A. Chem. Res. Toxicol. 1991; 4: 408-413Crossref PubMed Scopus (26) Google Scholar) also suggested that rate-limiting product release could explain the results if a relatively high degree of uncoupling of the activated P450-oxygen complex also occurred. MMO is a mixed-function monooxygenase that is functionally similar to P450 2E1 (25Rataj M.J. Kauth J.E. Donnelly M.I. J. Biol. Chem. 1991; 266: 18684-18690Abstract Full Text PDF PubMed Google Scholar). MMO requires a multi-enzyme complex and NADH as a co-substrate. The non-heme, two-iron catalytic center catalyzes the oxidation of a variety of low molecular weight hydrocarbons. Additionally, MMO has been shown to exhibit apparent isotope effects that are similar to those seen with P450 2E1. Chemical quench studies of the intermediates and interconversion rates in the MMO catalytic cycle revealed that product release is the rate-limiting step in this cycle (26Lee S.K. Nesheim J.C. Lipscomb J.D. J. Biol. Chem. 1993; 268: 21569-21577Abstract Full Text PDF PubMed Google Scholar, 27Nesheim J.C. Lipscomb J.D. Biochemistry. 1996; 35: 10240-10247Crossref PubMed Scopus (240) Google Scholar). Therefore, it is plausible that this might be the case for P450 2E1. To determine the basis for the kinetic hydrogen isotope effects observed with P450 2E1, we have used deuterium substitution and pre-steady-state kinetic techniques to characterize the effect of deuteration on individual steps of the catalytic cycle. The role ofb 5 and its ability to enhance the rate of product formation were also examined. We interpret the observed kinetic isotope effects terms of rate-limiting product release and isotopically sensitive C–H bond cleavage. Rabbit NADPH-P450 reductase (EC 1.6.2.4) and rabbitb 5 were purified as previously reported (28Yasukochi Y. Masters B.S.S. J. Biol. Chem. 1976; 251: 5337-5344Abstract Full Text PDF PubMed Google Scholar,29Shimada T. Misono K.S. Guengerich F.P. J. Biol. Chem. 1986; 261: 909-921Abstract Full Text PDF PubMed Google Scholar). Recombinant human P450 2E1 was expressed in Escherichia coli and purified essentially as described (30Gillam E.M.J. Guo Z. Guengerich F.P. Arch. Biochem. Biophys. 1994; 312: 59-66Crossref PubMed Scopus (149) Google Scholar). Recombinant NADPH-cytochrome P450 reductase was expressed in E. coli and purified by a modification of the method of Shen et al.(31Shen A.L. Porter T.D. Wilson T.E. Kasper C.B. J. Biol. Chem. 1989; 264: 7584-7589Abstract Full Text PDF PubMed Google Scholar). Apo-b 5 was prepared from rabbit liverb 5 by acid/acetone treatment (32Cinti D.L. Ozols J. Biochim. Biophys. Acta. 1975; 410: 32-44Crossref PubMed Scopus (23) Google Scholar). Reagent grade ethanol was obtained from McCormick Distilling Co. (Weston, MO). [1,1-2H]Ethanol andd23 -lauric acid were purchased from Cambridge Isotope Laboratories (Andover, MA). Lauric acid was purchased from Aldrich and recrystallized from CH3OH:H2O (50:50, v/v). 11-Hydroxylauric acid was synthesized from 11-dodecenoic acid (Nu-Check-Prep, Elysian, MN) as described by Brown and Geoghegan (33Brown H.C. Geoghegan Jr., P. J. Am. Chem. Soc. 1967; 89: 1522-1524Crossref Scopus (279) Google Scholar). The identity of 11-hydroxylauric acid was confirmed by NMR and fast atom bombardment-mass spectral analysis. Cumene hydroperoxide (80%, Aldrich) was purified by extraction with alkali (34Nordblom G.D. White R.E. Coon M.J. Arch. Biochem. Biophys. 1976; 175: 524-533Crossref PubMed Scopus (288) Google Scholar) and stored under argon Dinitrophenylhydrazine·HCl, purchased from Eastman Kodak Co., was recrystallized from H2O before use. 18-Crown-6-ether and 4-bromomethyl-6,7-dimethoxycoumarin were purchased from Aldrich. FloroxTM reagent (2.5 mg ofO-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine·HCl ml−1, in pyridine) was obtained from Pierce. Pesticide grade hexane was purchased from Mallinckrodt-Baker (Paris, KY), and pesticide grade CH3OH was from Burdick and Jackson (Muskegon, MI). [1-3H]Ethanol (16.3 mCi/mmol, anhydrous), [14C]ethanol (55 mCi/mmol), and [1-14C]lauric acid (55 mCi/mmol) were purchased from American Radiolabeled Chemicals (St. Louis, MO). Before use, [1-3H]ethanol and [1-14C]ethanol were purified on BakerbondTM octadecylsilane 3-ml disposable extraction columns (J. T. Baker Inc.) to remove, respectively, 3H2O and [14C]acetaldehyde, by-products of radioactive ethanol synthesis. For purification, extraction columns were first washed with 10 ml of CH3OH and then equilibrated with 10 ml of H2O. [1-3H]Ethanol (100 μl diluted with H2O to 340 mm, 16.3 mCi mmol−1) or 150 μl of [14C]ethanol (diluted with [1H]ethanol and H2O to 170 mm, 10 mCi of mmol−1) was loaded onto the column. Analytes were eluted with <3 ml of H2O, and ∼150-μl fractions were collected. A portion of each fraction was counted by liquid scintillation spectrometry. Radioactivity was plotted against fraction number, and the fractions comprising the major radioactive peak were pooled. The purity of the pooled solutions was confirmed by HPLC interfaced with radioflow detection (described under “Intrinsic Isotope Effect Estimation,” see below). The concentrations of ethanol solutions were determined by measuring NADH formation spectrophotometrically at 340 nm in a reduction assay using yeast alcohol dehydrogenase (35Sund H. Theorell H. Boyer P.D. Lardy H. Myrback K. 2nd Ed. The Enzymes. VII. Academic Press, New York1963: 25Google Scholar). The purified solutions were then counted by liquid scintillation to determine the specific activity. Unless otherwise specified, P450 2E1 (1.0 μm) was reconstituted withb 5 (2.0 μm), NADPH-P450 reductase (3.0 μm), and DLPC (30 μm) in 100 mm potassium phosphate buffer, pH 7.4. For steady-state measurements, 100-μl reactions were initiated with either an NADPH-generating system (36Guengerich F.P. Hayes A.W. Principles and Methods of Toxicology. Raven Press, Ltd., New York1989: 777-814Google Scholar) or 1.0 mm cumene hydroperoxide and incubated for 10 min at 37 °C in reaction vials sealed with Teflon-lined rubber septa. Reactions supported by cumene hydroperoxide lacked NADPH-P450 reductase and b 5. Reactions were terminated with 20 μl of a mixture of 17% ZnSO4(w/v) and 0.55 mm semicarbazide (1:1, v/v) and centrifuged following addition of saturated Ba(OH)2 (5.0 μl). The acetaldehyde product was rederivatized to form the 2,4-dinitrophenylhydrazone (37Shriner R.L. Fuson R.C. Curtin D.Y. The Systematic Identification of Organic Compounds. John Wiley & Sons, Inc., New York1965: 253-254Google Scholar) and then analyzed by HPLC using a Zorbax 6.2 × 80-mm octadecylsilane reversed-phase analytical column (3 μm, DuPont Chromatography Products, Wilmington, DE) (H2O:CH3CN, 45:55, v:v; 2.0 ml min−1), monitoring A 340) (38Brady J.F. Lee M.J. Li M. Ishizaki H. Yang C.S. Mol. Pharmacol. 1988; 33: 148-154PubMed Google Scholar). For reactions with [3H]- and [14C]ethanol, the HPLC peaks containing the radioactive product were collected and counted by liquid scintillation spectrometry, with calibration of counting efficiency using external [3H]- and [14C]toluene standards. P450 2E1 (0.5 μm) was reconstituted with NADPH-P450 reductase andb 5 as described for the ethanol oxidation assay. Reactions (400 μl) with lauric acid ord 23-lauric acid (0–200 μm, as sodium salt) were terminated after 10 min at 37 °C by adding 50 μl of 12.5% H2SO4 (v:v). The quenched reactions were extracted twice with 6 ml of (C2H5)2O, and the combined extracts were dried over Na2SO4 and then evaporated under a stream of N2. The residue was redissolved in 100 μl of 18-crown-6-ether dissolved in CH3CN (2.5 mg ml−1), to which was added 100 μl of 4-bromomethyl-6,7-dimethoxycoumarin dissolved in (CH3)2CO (10 mg ml−1) (in the presence of 2 mg of anhydrous K2CO3). The samples were incubated 60 min at 70 °C to form fluorescent derivatives. Product formation was measured by HPLC using a Zorbax 6.2 × 80 mm octadecylsilane reversed-phase analytical column (3 μm, DuPont, H2O:CH3CN, 41:59, v:v; 2.0 ml min−1). Fluorescence was monitored at λexcitation 375 nm, λemission 470 nm (39Amet Y. Berthou F. Goasduff T. Salaun J.P. Le Breton L. Menez J.F. Biochem. Biophys. Res. Commun. 1994; 203: 1168-1174Crossref PubMed Scopus (71) Google Scholar). External standards of 11-hydroxylauric acid were used for quantitation. Experiments using [1-14C]lauric acid were quenched with H2SO4 as described above, and reactions (40 μl) were extracted twice with 3 ml of (C2H5)2O, and the combined extracts were dried under N2. The residue was redissolved in 100 μl of CH3OH, and 50-μl aliquots were analyzed by HPLC interfaced with radioflow counting (ZorbaxTM 4.6 × 250-mm octadecylsilane reversed-phase analytical column, 5 μm, DuPont) (H2O:CH3CN, 50:50, v:v; 2.0 ml min−1). The [1-14C]11-hydroxylauric acid peak was collected and re-counted by liquid scintillation spectrometry. Deuterium isotope effects were determined by two methods (40Northrop D.B. Methods Enzymol. 1982; 87: 607-625Crossref PubMed Scopus (108) Google Scholar). In a non-competitive method, P450 2E1 was incubated with d0 -ethanol ord2 -[1,1-2H]ethanol (0–100 mm) or d0 -lauric acid ord23 -lauric acid (0–150 μm), and the products were analyzed as described. K m andk cat were calculated using ak cat nonlinear regression program (Bio-Metallics, Princeton, NJ). In a competitive method, P450 2E1 was incubated with a 1:1 mixture (v/v) of d0 -ethanol andd2 -ethanol (20 mm). The 2,4-dinitrophenylhydrazones were extracted into CH2Cl2 and analyzed by capillary column GC-PIEIMS (17Guengerich F.P. Yun C.-H. Macdonald T.L. J. Biol. Chem. 1996; 271: 27321-27329Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 21Ekström G. Norsten C. Cronholm T. Ingelman-Sundberg M. Biochemistry. 1987; 26: 7348-7354Crossref PubMed Scopus (29) Google Scholar). D(V/K) was determined as the ratio of 1H to 2H product formed in the competitive reaction. The approach was that of Miwa et al. (41Miwa G.T. Walsh J.S. Lu A.Y.H. J. Biol. Chem. 1984; 259: 3000-3004Abstract Full Text PDF PubMed Google Scholar) and Northrop (42Northrop D.B. Biochemistry. 1975; 14: 2644-2651Crossref PubMed Scopus (359) Google Scholar), only modified in regard to the analysis of isotopes in the products. The tritium isotope effect T(V/K) on ethanol oxidation was determined by reaction of P450 2E1 with [1-3H]ethanol (20 mm). Previous work by others (21Ekström G. Norsten C. Cronholm T. Ingelman-Sundberg M. Biochemistry. 1987; 26: 7348-7354Crossref PubMed Scopus (29) Google Scholar), with rabbit P450s, indicated no stereoselectivity in the removal of hydrogen from ethanol.3H2O formation during ethanol oxidation was measured by injecting one-half of the total reaction supernatant onto two 4.6 × 250-mm octadecylsilane reversed-phase analytical columns (ZorbaxTM column from DuPont and EconosphereTM column from Alltech Associates, Deerfield, IL, both 5 μm) connected in tandem (mobile phase 100% H2O; 1.0 ml min−1) interfaced with a radioflow counter (IN/US Systems, Inc., Tampa, FL). Retention times and separation of analytes are shown in Figs.2 and 3, respectively. The 3H2O peak was collected and re-counted by liquid scintillation spectrometry.T(V/K) and D kwere calculated according to the method of Northrop (42Northrop D.B. Biochemistry. 1975; 14: 2644-2651Crossref PubMed Scopus (359) Google Scholar, 43.Northrop, D. B., Proceedings of the Sixth Annual Harry Steenbock Symposium, Cleland, W. W., O'Leary, M. H., Northrop, D. B., 1977, 122, 152, University Park Press, Baltimore.Google Scholar) (see Equation 1), T(V/K)=[log(1−f)]/log[1−f(SAp/SAo)]Equation 1 where f is the fractional conversion of substrate to product, determined as the ratio of 3H2O to the initial amount of [3H]ethanol; SA pis the specific activity of the product, expressed as a ratio of3H2O formed/total acetaldehyde formed, andSA o is the specific activity of the substrate. Finally, D k was determined (Equation 2) from the Swain relationship (44Swain C.G. Stivers E.C. Reuwer Jr., J.F. Schaad L.J. J. Am. Chem. Soc. 1958; 80: 5885-5893Crossref Scopus (386) Google Scholar) (Dk−1/Dk1.442−1)=[D(V/K)−1]/[T(V/K)−1]Equation 2 Figure 3HPLC separation of reaction products from P450 2E1-catalyzed [3H]ethanol oxidation. 3H was measured by radioflow detection.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Pre-steady-state ethanol oxidation reactions were done in a quench-flow apparatus (model RQF-3, KinTek Corp., State College, PA). P450 2E1 (40 or 200 pmol) was incubated in the presence of either 200 mm[1H]ethanol, 20 mm [3H]- or [14C]ethanol, or 100 μm[14C]lauric acid in a 40-μl reaction volume for a period ranging from 2 ms to 10 min (as indicated) at 37 °C. Reactions with radioactive ethanol were quenched with a ZnSO4/semicarbazide mixture and analyzed as described for steady-state reactions. Reactions using [1H]ethanol were quenched by the addition of 30% pesticide grade CH3OH and derivatized with PFB-hydroxylamine by reacting the 250-μl quenched reaction volume with 20 μl of FloroxTM reagent for 20 min at 60 °C. The oxime derivative was extracted into 1.0 ml of pesticide grade hexane, and the residual reagent was back-extracted following addition of 3 drops of concentrated H2SO4. The hexane layer (0.7 ml) was dried under N2 and then redissolved in 20 μl of hexane. The oxime derivative was analyzed by GC-NICIMS (45Koshy K.T. Kaiser D.G. VanDerSlik A.L. J. Chromatogr. Sci. 1975; 13: 97-103Crossref PubMed Scopus (57) Google Scholar, 46Knapp D.R. Handbook of Analytical Derivatization Reactions. John Wiley & Sons, Inc., New York1979: 485-486Google Scholar, 47Luo X.P. Yazdanpanah M. Bhooi N. Lehotay D.C. Anal. Biochem. 1995; 228: 294-298Crossref PubMed Scopus (177) Google Scholar). Reactions with [14C]lauric acid were analyzed as described previously. P450 2E1 was reconstituted with NADPH-P450 reductase with or without b 5 as described for steady-state ethanol oxidation experiments. Reconstituted enzyme (196 μl) was preincubated for 1 min at 37 °C in the presence or absence of substrate (20 mm ethanol or 0.15 mmchlorzoxazone). Reactions were initiated with the addition of 4 μl of 10 mm NADPH, and the decrease in absorbance at 340 nm was monitored spectrophotometrically. UV-visible spectra were recorded using a modified Cary 14/OLIS spectrophotometer (On-Line Instrument Systems, Bogart, GA). Rates of NADPH oxidation were calculated using ε340 = 6.22 mm−1cm−1 for NADPH. Reaction systems were prepared exactly as described above, except that reaction volumes were 0.5 ml. Reactions were initiated by adding the NADPH-generating system and were terminated by adding 0.8 ml of cold CF3CO2H (3%, w:v) after 10 min at 37 °C. H2O2 was determined spectrophotometrically by reaction with ferroammonium sulfate and potassium thiocyanate as described (48Kostrubsky V.E. Szakacs J.G. Jeffery E.H. Woods S.G. Bement W.J. Wrighton S.A. Sinclair P.R. Sinclair J.F. Ann. Clin. Lab. Sci. 1997; 27: 57-62PubMed Google Scholar). Enzyme mixtures and NADPH (600 μm) solutions were prepared separately as described for steady-state reactions. Some enzyme mixtures contained 20 mm ethanol (or 0.15 mm chlorzoxazone). The solutions were made anaerobic by alternating applications of vacuum and argon to a closed system as described previously (49Yamazaki H. Johnson W.W. Ueng Y.-F. Shimada T. Guengerich F.P. J. Biol. Chem. 1996; 271: 27438-27444Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). Reduction kinetics were monitored in a stopped-flow apparatus (Applied Photophysics SX-17MV instrument, Applied Photophysics, Leatherhead, UK) at 37 °C under a CO atmosphere. Rapid reduction of P450 2E1 to a Fe2+·CO complex was observed at 450 nm upon mixing of reconstituted enzymes with NADPH. Data were collected using the Applied Photophysics software system and fitted to exponential equations using a Marquardt-Levenberg algorithm for nonlinear regression analysis. Results are reported as three to eight individually monitored reactions averaged using the manufacturer's software. The hydrazone derivatives were analyzed by GC-PIEIMS. Analytes were separated on a 15-m SPBTM-1 fused silica capillary column (Supelco, Bellefonte, PA) interfaced to a Finnigan INCOS 50 mass spectrometer (Finnigan, San Jose, CA). GC conditions were as follows: carrier gas (2He) at constant pressure of 10 p.s.i.; injection port 230 °C; transfer line 260 °C. The initial column temperature was 150 °C and was increased to 300 °C at a rate of 20 °C min−1. The [1H]acetaldehyde hydrazone was detected by selected ion monitoring at m/z 224, and the [2H]acetaldehyde hydrazone was monitored atm/z 225 (Fig. 4). GC-NICIMS of the PFB-oxime derivatives was performed using a 30-m SPBTM-5 fused silica capillary column (Supelco) coupled to a Hewlett-Packard 5989A mass spectrometer (Hewlett-Packard, Wilmington, DE). GC conditions were as follows: carrier gas (2He) at constant pressure of 0.8 p.s.i.; injection port 265 °C; transfer line 270 °C. The column was initially held at 65 °C for 10 min, increased by 5 °C min−1 to 100 °C, and then raised at 20 °C min−1 to 250 °C (Fig.5). Acetaldehyde-PFB oxime was quantitated by selected ion monitoring at m/z 181, using propionaldehyde-PFB oxime as an internal standard. The retention times of the analytes were verified by monitoring ions corresponding to M+ − 20 (M+-HF) for each of the oximes (Fig.6).Figure 5GC-MS of aldehyde-PFB oximes. Peaks 1 and 2 are identified as acetaldehyde-PFB oxime stereoisomers (E and Z), and peaks 4and 5 are identified as propionaldehyde-PFB oxime stereoisomers (used as internal standard). Peak 3 is a contaminant attributed to an acetone-PFB derivative.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6Mass spectra of acetaldehyde-PFB oxime (A) and propionaldehyde-PFB oxime (B).Samples were analyzed by GC-NICIMS.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In rat liver microsomes, deuterium substitution of P450 2E1 substrates usually results in a 3–5-fold increase in K m without any effect onk cat (9Keefer L.K. Lijinsky W. Garcia H. J. Natl. Cancer Inst. 1973; 51: 299-302Crossref PubMed Scopus (85) Google Scholar, 11Dagani D. Archer M.C. J. Natl. Cancer Inst. 1976; 57: 955-957Crossref PubMed Scopus (41) Google Scholar, 12Wade D. Yang C.S. Metral C.J. Roman J.M. Hrabie J.A. Riggs C.W. Anjo T. Keefer L.K. Mico B.A. Cancer Res. 1987; 47: 3373-3377PubMed Google Scholar, 19Swann P.F. Mace R. Angeles R.M. Keefer L.K. Carcinogenesis. 1983; 4: 821-825Crossref PubMed Scopus (38) Google Scholar, 20Mico B.A. Swagzdis J.E. Hu H.S.-W. Keefer L.K. Oldfield N.F. Garland W.A. Cancer Res. 1985; 45: 6280-6285PubMed Google Scholar, 50Andersen M.E. Clewell III, H.J. Mahle D.A. Gearhart J.M. Toxicol. Appl. Pharmacol. 1994; 128: 158-165Crossref PubMed Scopus (37) Google Scholar). Recombinant human cytochrome P450 2E1 was reconstituted with NADPH-P450 reductase andb 5 and incubated with a variety of substrates and their
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