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Chem. Res. Toxicol. 1999, 12, 582-587
Inactivation of Cytochrome P450 2E1 by Benzyl Isothiocyanate Rosa L. Moreno, Ute M. Kent, Kimberly Hodge,† and Paul F. Hollenberg* Department of Pharmacology, The University of Michigan, Ann Arbor, Michigan 48109 Received January 12, 1999
The cytochrome P450 enzymes constitute a family of phase I enzymes that play a prominent role in the metabolism of a great variety of endogenous and xenobiotic compounds. In this study, the kinetics for the inactivation of cytochrome P450 2E1 by benzyl isothiocyanate (BITC) were elucidated. BITC is a naturally occurring compound found in cruciferous vegetables such as broccoli. BITC inhibited the 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC) O-deethylation activity of purified and reconstituted P450 2E1 in a time- and concentration-dependent manner. The concentration of inactivator needed for half-maximal inactivation (KI) was 13 µM, and the maximum rate of inactivation at saturation (kinact) was 0.09 min-1. The partition ratio for the inactivation of P450 2E1 by BITC was found to have an approximate value of 27. Inactivation of P450 2E1 by BITC was dependent on the presence of NADPH. Following incubation for 5 min with BITC, a 65% loss in enzymatic activity was observed, while approximately 74% of the spectrally detectable enzyme remained. 7-Ethoxycoumarin (7-EC), a substrate of P450 2E1, protected P450 2E1 from BITC inactivation, reducing the loss in 7-EFC O-deethylation activity from 50 to 18% when a 1:20 molar ratio of BITC:7-EC was used. Inactivation of P450 2E1 by BITC was irreversible, and no activity was regained after extensive washes to remove BITC. Addition of cytochrome b5 to the reconstituted system did not affect the rate of inactivation. Reductase activity was unaffected by BITC. The results reported here indicate that BITC is a mechanism-based inactivator of cytochrome P450 2E1 and that the inactivation was primarily due to a modification of the apoprotein by BITC.
Introduction The family of cytochrome P450 (P450)1 enzymes plays a prominent role in the metabolism of a great variety of endogenous and xenobiotic compounds (1, 2). The basic catalytic steps that are shared by all the isozymes in this family consist of a two-electron reduction of molecular oxygen to form water and a reactive oxygen species. The catalytic diversity of these enzymes has been well established by studies in which either liver microsomal preparations or purified proteins were used in the reconstituted system (3). The crystal structures of various soluble bacterial P450 enzymes such as P450 101, 102, and 108 have been elucidated (4-6). These crystal structures have helped to predict regions of the membranebound, mammalian P450 proteins that comprise the active site and that may be critical in the process of catalysis. Mammalian P450 enzymes have not been crystallized to date. Therefore, most of our knowledge about the structure of the active site and the relevance of specific amino acids in catalysis has been derived from * To whom correspondence should be addressed: Department of Pharmacology, Medical Science Research Building III, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-0632. E-mail:
[email protected]. † K.H. was from Meharry Medical College and a recipient of a Charles Ross Fellowship Award. 1 Abbreviations: P450, cytochrome P450; BITC, benzyl isothiocyanate; reductase, NADPH-cytochrome P450 reductase; DLPC, dilauroyl-L-R-phosphatidylcholine; BSA, bovine serum albumin; 7-EC, 7-ethoxycoumarin; 7-EFC, 7-ethoxy-4-(trifluoromethyl)coumarin; 7-HFC, 7-hydroxy-4-(trifluoromethyl)coumarin; p-NP, p-nitrophenol; PEITC, phenethyl isothiocyanate; NDMA, N-nitrosodimethylamine; NNN, N-nitrosonornicotine; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.
various approaches such as sequence alignments with bacterial P450 enzymes (7-9), site-directed mutagenesis (10, 11), and chemical modifications (12-14). Chemical modification of P450 enzymes can be achieved by the use of mechanism-based inactivators (15). These compounds are substrates for P450 enzymes and form reactive intermediates in the process of catalysis. These reactive species react rapidly with amino acid side chains or the P450 heme (15). Labeling of amino acids in the active site by these reactive intermediates has been useful in understanding the importance of particular residues in catalysis (16, 17). The inhibition of P450 enzymes has been implicated as a factor involved in the chemopreventive action of the isothiocyanates (18-20). Isothiocyanates are naturally occurring compounds found in cruciferous vegetables such as cabbage and broccoli and are hydrolyzed by myrosinase from glucosinolated precursors (21). These compounds have been extensively studied to determine their role as chemopreventive agents and have been shown to inhibit tumorigenesis associated with exposure to many types of nitrosamines (20, 22, 23). Isothiocyanates exhibit their chemopreventive activity when administered either prior to or during exposure to carcinogens. Their chemopreventive activity has been demonstrated in numerous tissues, including rat lung, esophagus, liver, colon, and bladder (23). Two main mechanisms for the chemopreventive action of isothiocyanates have been identified: (a) induction of phase II enzymes such as glutathione S-transferase and quinone reductase and (b) inhibition of phase I enzymes such as the P450 enzymes
10.1021/tx9900019 CCC: $18.00 © 1999 American Chemical Society Published on Web 06/09/1999
Inactivation of P450 2E1 by Benzyl Isothiocyanate
(19, 24, 25). Three main mechanisms have been identified that are responsible for the inhibition or inactivation of P450 enzymes (26, 27). Isothiocyanates inhibit or inactivate P450 enzymes by direct interaction of the parent compound with nucleophilic residues in the enzyme or by the interaction of the isocyanate metabolite with available nucleophiles in the active site. In addition, inactivation could also result from a covalent modification of the enzyme by atomic sulfur produced through oxidative desulfuration (26). This study is the first report where the kinetics for the inactivation of the ethanol-inducible enzyme P450 2E1 by benzyl isothiocyanate (BITC) in the reconstituted system were elucidated. Inactivation of P450 2E1 by BITC was NADPH-dependent and proceeded in a timeand concentration-dependent manner. The results reported here indicate that BITC is acting as a mechanismbased inactivator of P450 2E1 and that the loss in enzymatic activity is the result of an apoprotein modification by a reactive intermediate formed during BITC metabolism.
Experimental Procedures Materials. BITC and 7-ethoxycoumarin (7-EC) were obtained from Aldrich Chemical Co. (Milwaukee, WI). 7-Ethoxy-4-(trifluoromethyl)coumarin (7-EFC) was from Molecular Probes (Eugene, OR). Catalase, purified from bovine liver, NADPH, p-nitrophenol (p-NP), 4-nitrocatechol, dilauroyl-L-R-phosphatidylcholine (DLPC), Tergitol NP-10, and poly(vinylpyrolidone) were from Sigma Chemical Co. (St. Louis, MO). Topp3 Escherichia coli cells were obtained from Stratagene (La Jolla, CA). HPLC grade acetonitrile was from Fisher Scientific (Pittsburgh, PA). [14C]BITC labeled at the R carbon was provided by F. L. Chung (American Health Foundation, Valhalla, NY). Enzymes. The cDNA for rabbit P450 2E1 (provided by M. J. Coon, The University of Michigan) was expressed in E. coli cells. Expression and purification of the protein were carried out according to published methods (28). The cDNA for rat NADPHP450 reductase within the expression plasmid pOR263 (29) was expressed in E. coli Topp3 cells. Expression and purification of the protein were as described by Hanna et al. (30). Inactivation of P450 2E1 by BITC. Purified rabbit P450 2E1 (0.5 nmol) was reconstituted with reductase (0.5 nmol) and lipid (300 µg/mL) at 4 °C for 45 min. After reconstitution, catalase (17 000 units/mL) and 0.5 M potassium phosphate (pH 7.4) were added to final concentrations of 1800 units/mL and 50 mM, respectively. Primary reaction mixtures contained 1 µM P450 2E1, 1 µM reductase, and various concentrations of BITC (5, 8, 10, 20, 40, and 50 µM in a final concentration of 1% methanol). Methanol was added to the control samples instead of BITC. Reactions were carried out at 30 °C and were initiated by adding NADPH to a final concentration of 1.2 mM. The loss of activity after 0, 1, 2, and 4 min following the addition of NADPH was determined spectrofluorometrically by measuring the extent of O-deethylation of 7-EFC to HFC (31, 32) on an SLM-Aminco model SPF-500C spectrofluorometer with excitation at 410 nm (slit width of 5 nm) and emission at 510 nm (slit width of 5 nm). For this assay, 20 pmol aliquots of P450 2E1 were removed from the primary reaction mixture and added to secondary reaction mixtures containing 0.1 mM 7-EFC, and 0.2 mM NADPH in 50 mM potassium phosphate (pH 7.4) with 0.04 mg/mL BSA in a final volume of 1 mL. Reactions were allowed to proceed for 15 min and were terminated by adding 0.33 mL of ice-cold acetonitrile. Effect of BITC on P450 2E1 Activity and Content. Reconstitution and reaction conditions were as described above. The activity remaining at time zero and after incubation for 5 min with 100 µM BITC was determined using the 7-EFC
Chem. Res. Toxicol., Vol. 12, No. 7, 1999 583 O-deethylation assay. The P450 content was measured by the reduced carbon monoxide difference spectrum (33). P450 2E1 Heme Analysis. Reconstitution of P450 2E1 (1.1 nmol) and reductase (1.1 nmol) was as described previously. Primary reaction mixtures contained 2 µM P450 2E1, 2 µM reductase, and 200 µM BITC. Reactions were carried out at 30 °C, and inactivation proceeded for 20 min. The loss of activity was determined by measuring the extent of O-deethylation of 7-EFC. To determine the changes in P450 2E1 heme content, samples were analyzed by HPLC on a 4.6 mm × 250 mm Vydac C4 column with a solvent system consisting of buffer A (0.1% H2O/TFA) and buffer B (95% CH3CN/0.1% TFA). The heme content was determined by measuring the absorbance at 405 nm. The heme eluted at 4 min using a gradient of 40 to 50% B over the course of 15 min at a flow rate of 1.0 mL/min. Effect of Cytochrome b5 on the Inactivation of P450 2E1 by BITC. Cytochrome b5 was reconstituted together with P450 2E1 and reductase at a molar concentration of 1 µM each. Samples were inactivated with BITC and assayed for residual 7-EFC O-deethylation activity as described above. Partition Ratio. The partition ratio for the inactivation of P450 2E1 by BITC was determined by incubating the enzyme with increasing concentrations of BITC and allowing the inactivation to go to completion. The reconstitution was carried out as described above. Following reconstitution, catalase and potassium phosphate (pH 7.4) were added to final concentrations of 1800 units/mL and 50 mM, respectively. Primary reaction mixtures contained 1 µM P450 2E1 and 1 µM reductase. Reactions were carried out for 10 min at 30 °C, and the activity remaining after inactivation was determined by measuring the extent of 7-EFC O-deethylation activity as previously described. The percent activity remaining was plotted as a function of the molar ratio of BITC to P450 2E1. The partition ratio was obtained from the intercept between the line obtained by linear regression with the lower BITC concentrations (0-16 µM) and the straight line obtained with higher BITC concentrations (40100 µM) (34). Effect of 7-EC on the Rates of Inactivation of P450 2E1 by BITC. P450 2E1 and reductase were reconstituted as described above. Following reconstitution, catalase and potassium phosphate (pH 6.8) were added to final concentrations of 232 units/mL and 78 mM, respectively. Primary reaction mixtures containing 0.4 µM P450 2E1, 0.8 µM reductase, 10 µM BITC, and increasing concentrations of 7-EC (20-200 µM) were incubated at 30 °C for 0, 2, 4, 6, and 8 min. The activity remaining was determined electrochemically by measuring the extent of formation of 4-nitrocatechol from p-NP as previously described (31, 35). Secondary reaction mixtures contained 5 mM MgCl2, 0.2 mM p-NP, 0.5 mM NADPH, and 40 µg/mL BSA in 100 mM potassium phosphate (pH 6.8). Irreversibility of P450 2E1 Inactivation by BITC. P450 2E1 and reductase were reconstituted as described previously. Following inactivation with 50 µM BITC for 5 min, the loss in activity was measured with the 7-EFC O-deethylation assay. The P450 content was determined by diluting an aliquot of the reaction mixture 1:10 in 50 mM potassium phosphate (pH 7.7) containing 40% glycerol and 0.6% Tergitol. The remainder of the primary reaction mixtures was loaded into Microcon 30 microconcentrators (Amicon, Inc., Beverly, MA) that had been pretreated overnight with 0.5% poly(vinylpyrolidone) in 100 mM acetic acid. Samples were concentrated by centrifuging at 8000g followed by three washes (200 µL each) with 50 mM potassium phosphate (pH 7.4), containing 20% glycerol. Concentrated proteins were reconstituted with 50 µg of DLPC at 4 °C for 20 min and diluted to their original volume with 50 mM potassium phosphate (pH 7.4). Activity was measured as described previously and corrected for P450 2E1 recovery. The P450 2E1 concentration was determined from the reduced carbon monoxide difference spectrum. Binding Spectra and Binding Constant. P450 2E1 (1 nmol) was reconstituted with 100 µg of lipid for 45 min at 4 °C. The reconstituted protein was diluted to 0.5 µM with 50 mM
584 Chem. Res. Toxicol., Vol. 12, No. 7, 1999
Moreno et al. Table 1. Effect of BITC on the 7-EFC O-Deethylation Activity of Purified P450 2E1 and on the Cytochrome P450 Contenta primary reaction mixture -BITC, -NADPH +BITC, -NADPH +BITC, +NADPH
% activity remaining 0 min 5 min 100 87 ( 1 83 ( 3
105 82 ( 2 35 ( 4
% P450 remaining 0 min 5 min 100
105 101 ( 2 74 ( 2
a Incubation conditions were as described in Experimental Procedures. The values shown represent the average from two different experiments.
Figure 1. Time- and concentration-dependent loss of P450 2E1 7-EFC O-deethylation activity following incubation with BITC and NADPH. Incubation conditions were as described in Experimental Procedures. The concentrations of BITC were (0) 0, (b) 5, (4) 8, ([) 10, (]) 20, (1) 40, and (O) 50 µM. The data shown represent the average and standard error from three to five separate experiments. The inset shows the double-reciprocal plot of the rates of inactivation as a function of the BITC concentration. potassium phosphate (pH 7.4). The sample was divided, and equal volumes were added to a reference and a sample cuvette. Samples were scanned from 350 to 500 nm on a DW2-UV/vis spectrophotometer (SLM Aminco, Urbana, IL) equipped with an OLIS spectroscopy operating system (On-Line Instrument Systems, Inc., Bogart, GA). Following the addition of 1 µL (2.1 nmol) aliquots of BITC dissolved in methanol to the sample cuvette and 1 µL aliquots of methanol to the reference cuvette, scans were recorded for BITC concentrations of 2.1-12.6 µM. The binding constant, Ks, was calculated from a plot of the inverse of the change in absorption versus the inverse of the BITC concentration. The Ks was obtained from the x-intercept of the linear regression line (36). Covalent Binding. Reconstitution was carried out as described above with 1.65 nmol of P450 2E1, 1.65 nmol of reductase, and 100 µg of lipid. Primary reaction mixtures containing P450 2E1 and reductase at a concentration of 2 µM each were treated either with 45 µM [14C]BITC and NADPH or with [14C]BITC alone. Samples were incubated for 20 min at 30 °C and then dialyzed against 50 mM potassium phosphate buffer (pH 7.4) containing 20% glycerol, 0.1 M EDTA, and 0.4% cholate. Following dialysis, 40 pmol aliquots of P450 2E1 from each sample were denatured in the presence or absence of β-mercaptoethanol and were loaded onto a 10% polyacrylamide gel. Proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Autoradiography was performed by exposing the membrane to Biomax MR film supplemented with a Biomax intensifying screen (Eastman Kodak Co., Rochester, NY) for 2 weeks at -80 °C.
Results Inactivation of P450 2E1 by BITC. The kinetics for P450 2E1 inactivation by BITC were studied by measuring the loss in 7-EFC O-deethylation activity. Purified P450 2E1 in the reconstituted system was inactivated by BITC in a time- and concentration-dependent manner (Figure 1). The inactivation followed pseudo-first-order kinetics. The kinetic constants were determined from a double-reciprocal plot of the inverse of the initial rates of inactivation as a function of the reciprocal of the BITC concentration (Figure 1, inset). The concentration of inactivator required for the half-maximal rate of inactivation (KI) was 13 µM. The maximal rate of inactivation at saturation (kinact) was 0.09 min-1, and the time
required to inactivate one-half of the enzyme molecules (t1/2) was 7.3 min. At higher BITC concentrations or longer inactivation times, the reaction kinetics became biphasic (data not shown). Therefore, the initial linear rates were used to calculate the kinetic constans. The 7-EFC O-deethylation activity remaining after inactivation of the P450 2E1 with 100 µM BITC for 5 min was compared to the cytochrome P450 content determined from the reduced CO spectrum (Table 1). No inactivation or loss of CO binding was observed in the control sample that had been incubated without BITC and NADPH. Samples that were incubated only with BITC in the primary reaction mixtures exhibited a 13% loss in activity that did not increase significantly over the course of 5 min. This loss in activity has been previously observed with other P450 inactivators and is believed to be caused by the fraction of inactivator that was transferred into the secondary reaction mixture (37, 38). Inactivation of P450 2E1 by BITC was dependent on the presence of NADPH. After incubation for 5 min with both BITC and NADPH, a 65% decrease in enzymatic activity was observed while only 26% of the spectrally detectable enzyme was lost. P450 2E1 Heme Analysis. The 7-EFC O-deethylation activity remaining after inactivation of the P450 2E1 with 200 µM BITC for 20 min was compared to the cytochrome P450 heme content determined by HPLC analysis. Samples incubated together with BITC and NADPH lost 76% of their enzymatic activity but only 9% of the heme prosthetic group as compared to control samples incubated without NADPH. Effect of Cytochrome b5 on the Inactivation of P450 2E1 by BITC. The effects of cytochrome b5 on the inactivation of P450 2E1 by BITC were studied. Cytochrome b5 has been proposed to play a role in donating the second electron required for the generation of the iron-peroxo species in P450 catalysis and in increasing the efficiency of some P450 reactions (39, 40). In this study, cytochrome b5 did not significantly affect the kinetic parameters for the inactivation of P450 2E1 by BITC (data not shown). The KI was 13 µM, the kinact 0.18 min-1, and the t1/2 4 min. Partition Ratio. The partition ratio provides an indication of the efficiency of a compound as an inactivator. It has been defined as the number of inactivator molecules leading to product per each inactivation event (34). The value for the partition ratio was estimated by plotting the percent of activity remaining following inactivation of P450 2E1 by BITC versus the ratio of the molar concentrations of BITC to P450 2E1 (Figure 2). The partition ratio was derived from the intercept between the line obtained by linear regression (lower BITC concentrations) and the straight line (higher BITC concentrations). A residual activity of approximately 20%
Inactivation of P450 2E1 by Benzyl Isothiocyanate
Figure 2. Partition ratio for the inactivation of purified P450 2E1 by BITC. Assay conditions were as described in Experimental Procedures. The percent of P450 2E1 7-EFC O-deethylation activity remaining was plotted against the ratio of BITC to P450 2E1. The data shown represent the mean and standard error from three separate experiments. For some points, the standard deviation was smaller than the size of the symbol.
Figure 3. Effect of the alternate substrate 7-EC on the inactivation of P450 2E1 by BITC. Incubation and reaction conditions were as described in Experimental Procedures. Primary reaction mixtures contained increasing molar ratios of BITC:7-EC: (9) 0:0, (O) 1:20, ([) 1:10, (4) 1:2, and (b) 1:0. Control samples contained BITC but no NADPH. The data shown represent the mean and standard error from three individual experiments.
was seen even with the highest concentrations of BITC that were tested. The estimated value for the partition ratio was 27. Substrate Protection from Inactivation of P450 2E1 by BITC. The effect of the alternate P450 2E1 substrate 7-EC on the inactivation of P450 2E1 by BITC was studied (Figure 3). Incubation of purified P450 2E1 with BITC together with increasing molar ratios of 7-EC (1:0, 1:2, 1:10, and 1:20 BITC:7-EC) resulted in a decrease in the rate of inactivation of P450 2E1 as determined by measuring the level of hydroxylation of p-NP. Incubation of P450 2E1 for 8 min with BITC and 7-EC at a molar ratio of 1:20 resulted in an 18% loss in activity, while 50% of the activity was lost in the absence of 7-EC. Irreversibility of P450 2E1 Inactivation by BITC. Incubation of P450 2E1 with 50 µM BITC for 5 min resulted in a loss of 63% of the 7-EFC O-deethylation activity. NADPH and unbound BITC were removed from the samples by extensive washes as described in Experimental Procedures. No increase in the 7-EFC O-deethylation activity was observed in the washed, inactivated samples as compared to control samples incubated without NADPH. This observation suggests that under these conditions the inactivation by BITC was irreversible.
Chem. Res. Toxicol., Vol. 12, No. 7, 1999 585
Figure 4. Determination of the binding constant for P450 2E1. Assay conditions were as described in Experimental Procedures. The inverse of the change in P450 2E1 absorption (380-420 nm) vs the inverse of the BITC concentration (2.1, 4.2, 6.3, 8.4, and 10.5 µM) was graphed on a Lineweaver-Burk plot. The binding constant (Ks) was calculated from the x-intercept. The data are representative of two separate experiments.
Figure 5. Autoradiograph of samples incubated with [14C]BITC in the absence or presence of NADPH. P450 2E1 (40 pmol) was loaded in each lane: lane 1, P450 2E1 incubated with BITC and without NADPH; lane 2, P450 2E1 incubated with BITC, without NADPH, and with β-mercaptoethanol; lane 3, P450 2E1 incubated with BITC and NADPH; and lane 4, P450 2E1 incubated with BITC, NADPH, and β-mercaptoethanol.
Binding Spectra. The binding of substrates to P450 enzymes can be accompanied by changes in the absorbance spectra of these proteins (36). The binding of BITC to P450 2E1 produced spectral changes characteristic of a type I compound. Changes in the absorbance at 380 and 420 nm for P450 2E1 were followed after addition of increasing concentrations of BITC. The resulting changes in absorption were plotted as the inverse of the change in absorption versus the inverse of the BITC concentration (Figure 4). From this plot, the value for the binding constant Ks was determined. Ks is defined as the concentration of reactant that results in 50% of the theoretical maximal spectral change (36). The Ks for the binding of BITC to P450 2E1 was 4.5 µM. Covalent Binding. Incubation of the reconstituted system with [14C]BITC, followed by SDS-PAGE and autoradiography (Figure 5), showed that both P450 2E1 and reductase were radiolabeled in the absence of NADPH. The extent of labeling was not decreased by adding β-mercaptoethanol, indicating that the nonspecific as well as the increased labeling was not due to the formation of a disulfide bond of the reactive BITC intermediate with the proteins. In both instances, densitometric analysis showed a 150% increase in the extent of labeling for P450 2E1 compared to a 30% increase in the extent of labeling in reductase when the samples were incubated together with NADPH and BITC.
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Discussion The interaction between purified P450 2E1 in the reconstituted system and BITC was studied for the first time in this report. One of the main goals of this study was to evaluate BITC as a possible mechanism-based inactivator of P450 2E1 for future use in labeling critical amino acid residues in the active site. Since BITC is a naturally occurring compound in crusiferous vegetables, its potential inhibitory effect on P450 2E1 takes on additional physiological relevance as a potential chemopreventive agent. P450 2E1 has been suggested to play a major role in the activation of short-chain N-nitrosamines to carcinogenic species (41). In human liver microsomes, P450 2E1 is reported to be the isozyme mostly responsible for the activation of N-nitrosodimethylamine (NDMA) and N-nitrosonornicotine (NNN) and it also contributes to the N-demethylation of 4(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) (41). The inactivation of P450 2E1 by BITC followed pseudofirst-order kinetics, was time- and concentration-dependent, and required NADPH. The observed significant loss of 7-EFC O-deethylation activity did not correlate with the minimal decrease in the level of CO binding induced by BITC. This result indicated that inactivation occurred mainly due to a modification of the apoprotein rather than the heme. The observed minor decrease in CO binding does not necessarily imply that a heme modification has occurred. A similar phenomenon has been observed with other isozymes or inactivators where a decrease in the extent of CO binding did not correlate with heme content determined by HPLC analysis (31, 42). In those studies, HPLC analysis of the modified protein at 405 nm showed virtually no change in the peak corresponding to the heme following inactivation even though the amount of CO-reactive heme declined significantly. The 9% loss in the ability of the BITC-modified P450 2E1 to bind CO did not correlate with the 76% loss in P450 2E1 O-deethylation activity. These observations suggested that the primary site of modification by BITC occurred on the apoprotein. The modification by the reactive BITC intermediate appeared to be covalent since extensive washing did not lead to a recovery of enzymatic activity. The absolute requirement of NADPH for inactivation and the protection against inactivation offered by 7-EC confirmed the need for BITC to be metabolically activated. These results also suggested that the P450 2E1 active site was the place where the inactivating event occurred. BITC did not appear to affect the enzymatic activity of NADPHP450 reductase. Further studies were performed to rule out an effect of BITC on the reductase. Mainly, inactivation of P450 2E1 by BITC did not affect the ability of the reductase to reduce cytochrome c (data not shown). Phenethyl isothiocyanate, another naturally occurring structurally related isothiocyanate, was also shown to inhibit P450 2E1 without effecting reductase activity based on cytochrome c reduction assays (43). Also, addition of new reductase to the inactivated sample did not result in recovery of activity (data not shown). Even though labeling of reductase by BITC was observed in the absence and presence of NADPH, the above results indicate that this modification is not contributing to the loss in P450 2E1 activity. The labeling of both P450 2E1 and reductase in the absence of NADPH is not surprising on the basis of the known reactivity of isothiocyanates
Moreno et al.
with nucleophilic residues in proteins (18), resulting in the inhibition or inactivation of enzymes by isothiocyanate compounds in general. The partition ratio was estimated by adding increasing concentrations of BITC to the reaction mixture and letting the inactivation reaction go to completion. The points derived from the higher ratios of BITC to P450 2E1 deviated from the linear regression line. At the highest concentrations tested, the maximal loss in activity that could be obtained was 80%. The possibility that this was due to product inhibition is unlikely. Residual activity was tested by diluting an aliquot of the primary reaction by 50-fold into the secondary reaction. Therefore, the highest concentration of BITC carried over into the secondary reaction was 2 µM. Further, extensive washings of the BITC-inactivated sample did not restore enzymatic activity. The extent of inactivation appears to be dependent on the P450 isozyme and the type of inactivator used (13-15, 31, 37, 42). One possibility that should be considered is that certain modifications greatly compromise either substrate binding or catalysis but do not completely inactivate the 7-EFC O-deethylation activity. Studies with naturally occurring or engineered mutant enzymes have shown that single amino acid changes can influence the rate of catalysis or the nature of the metabolites formed (10, 16, 17, and references therein, 30). In preliminary HPLC studies with [14C]BITC, a stoichiometric binding of 0.4-1 labeled metabolite bound per nanomole of P450 2E1 was observed (data not shown). Several factors may have contributed to a
