Mechanism-Based Inactivation of Cytochromes P450 2E1 and 2E1

Chem. Res. Toxicol. 2002, 15, 1561-1571

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Mechanism-Based Inactivation of Cytochromes P450 2E1 and 2E1 T303A by tert-Butyl Acetylenes: Characterization of Reactive Intermediate Adducts to the Heme and Apoprotein Anna L. Blobaum,† Ute M. Kent,† William L. Alworth,‡ and Paul F. Hollenberg*,† Department of Pharmacology, The University of Michigan, Ann Arbor, Michigan 48109, and Department of Chemistry, Tulane University, New Orleans, Louisiana 70118 Received June 17, 2002

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 KI values for the inactivation of P450s 2E1 and 2E1 T303A by tBA were 1.0 and 2.0 mM, the kinact values were 0.20 and 0.38 min-1, and the t1/2 values were 3.5 and 1.8 min, respectively. The KI values for the tBMP-inactivated P450s were 0.1 and 1.0 mM, the kinact values were 0.12 and 0.07 min-1, and the t1/2 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 paranitrophenol 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.

Introduction The cytochromes P450 form a superfamily of hemecontaining monooxygenases that are involved in the oxidative, peroxidative, and reductive metabolism of a wide variety of endogenous and exogenous compounds (1, 2). Individual P450s1 exhibit unique substrate specificity and regio- and stereoselectivity profiles that reflect different tertiary structures of the proteins (3). Crystal structures of various soluble bacterial P450 enzymes such as P450s 101, 102, and 108 have been determined (4-6) and have allowed researchers to predict regions of the membrane-bound mammalian P450 active sites that may be important for catalysis. Recently, the first three* To whom correspondence should be addressed. Phone: (734) 7648166. Fax: (734) 763-4450. E-mail: [email protected]. † The University of Michigan. ‡ Tulane University. 1 Abbreviations: tBA, tert-butyl acetylene; tBMP, tert-butyl 1-methyl-2-propynyl ether; P450, cytochrome P450; reductase, NADPH-cytochrome P450 reductase; tBITC, tert-butyl isothiocyanate; BITC, benzyl isothiocyanate; PEITC, phenethyl isothiocyanate; 2EN, 2-ethynylnaphthalene; BSA, bovine serum albumin; p-NP, para-nitrophenol; GSH, glutathione; DTT, dithiothreitol; DLPC, dilauroyl-L-R-phosphatidylcholine; 7-EC, 7-ethoxycoumarin; 7-EFC, 7-ethoxy-4-(trifluoromethyl)coumarin; 7-HFC, 7-hydroxy-4-(trifluoromethyl)coumarin; TFA, trifluoroacetic acid; HPLC, high-performance liquid chromatography; ESI-LC-MS, electrospray ionization liquid chromatography-mass spectrometry.

dimensional structure of modified microsomal P450 2C9 has been elucidated (7); however, our knowledge concerning active site structure and the relevance of critical amino acids in P450 catalytic processes, particularly for P450 2E1, remains limited. Cytochrome P450 2E1 is a constitutively expressed and inducible P450 found predominantly in the endoplasmic reticulum of mammalian hepatocytes. Lower levels of P450 2E1 are also found in the kidney, lung, intestine, nasal mucosa, lymphocytes, and brain. P450 2E1 is involved in the metabolism of ethanol and other small molecules, including halogenated alkanes, acetaminophen, nitrosamines, benzene, and styrene (8). Evidence from previous studies in which a critical threonine 303 residue in the I helix of P450 2E1 was mutated to an alanine points to a role for this residue in substrate interactions and orientation and as a possible proton donor in acid-base reactions (9-11). Several naturally occurring isothiocyanates have been shown to be mechanism-based inactivators of P450 2E1 and the mutant 2E1 T303A (12) and studies have shown the binding of a reactive metabolite of tert-butyl isothiocyanate (tBITC) to the P450 2E1 apoprotein (13). Previously, Moreno et al. found that benzyl- and phenethyl isothiocyanates inactivated P450 2E1 in a mechanism-based manner (12).

10.1021/tx020052x CCC: $22.00 © 2002 American Chemical Society Published on Web 11/14/2002

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T303 residue and substrate structure in the metabolic reactions of P450 2E1.

Experimental Procedures

Figure 1. Structures of two tert-butyl acetylenic compounds: tBA and tBMP.

Similar studies with the mutant P450 2E1 T303A produced only competitive inhibition by BITC, while PEITC inactivated the enzyme in a manner comparable to the wild-type. The differential affects of these compounds on the wild-type and mutant enzymes may suggest a requirement for the T303 residue in the metabolism of isothiocyanates. The inactivation of cytochrome P450 enzymes by acetylenic compounds has been shown to occur through binding of a reactive species produced from the catalytic oxidation of the acetylenic group to either the heme group or apoprotein (14-17). Evidence suggests that insertion of an oxygen atom at the internal or terminal acetylenic carbon results in heme or protein modification, respectively (18). Previously, the inactivation of P450 2B1 by substituted phenylacetylenes was characterized and shown to primarily result in heme alkylation (19). Another acetylenic compound, 2-ethynylnaphthalene (2EN), was found to inactivate P450s 2B1 and 2B4 in a mechanism-based manner. Inactivation was the result of covalent modification of the apoproteins by a reactive ketene intermediate that bound to the threonine 302containing I helix of the enzyme (20, 21). Threonine 302 in 2B1 and 2B4 corresponds to the T303 in P450 2E1. Further studies with a P450 2B4 T302 mutant determined that the rate of inactivation and covalent binding were greatly decreased in this enzyme, although the mutant was functionally able to metabolize 2EN to its ketene product (21). This evidence suggests the involvement of the conserved threonine residue in the P450 inactivation event. Since little information is known regarding the P450 2E1 active site and the identity of the particular residues thought to play a role in substrate metabolism and turnover, mechanism-based inactivators specific to P450 2E1 would be useful tools in probing this area. The current report details our findings on the mechanismbased inactivation of P450s 2E1 and 2E1 T303A by tertbutyl acetylene (tBA) and tert-butyl 1-methyl-2-propynyl ether (tBMP), two structurally similar acetylenic compounds (Figure 1). Inactivation of the wild-type and mutant P450s by tBA and tBMP was NADPH-dependent and proceeded in a time- and concentration-dependent manner. Differences in the kinetics and the reversibility of the inactivation of tBA- or tBMP-modified P450s 2E1 and 2E1 T303A suggest a unique mechanistic variation in the metabolism of these two acetylenic compounds involving three distinct pathways for inactivation. Further, we provide data on the characterization of reactive intermediate adducts to the P450 heme formed by the inactivation of cytochromes P450 2E1 and 2E1 T303A by tBA and tBMP. Inactivation of the wild-type P450 2E1 by tBA also resulted in the formation of a tBA-derived protein adduct in addition to heme modification. The differential effects of these compounds on the activities of P450s 2E1 and 2E1 T303A support roles for both the

Materials. Dilauroyl-L-R-phosphatidylcholine (DLPC), NADPH, p-nitrophenol (p-NP), BSA, tert-butyl 1-methyl-2-propynyl ether (tBMP) and catalase were purchased from Sigma Chemical Co. (St. Louis, MO). 7-Ethoxy-4-(trifluoromethyl)coumarin (7-EFC) was obtained from Molecular Probes, Inc. (Eugene, OR). 7-Ethoxycoumarin (7-EC) and tert-butyl acetylene (tBA) were obtained from Aldrich Chemical Co. (Milwaukee, WI). HPLCgrade acetonitrile was from Fisher (Pittsburgh, PA) and trifluoroacetic acid (TFA) was from Pierce (Rockford, IL). Enzymes. The cDNA for rabbit P405s 2E1 and 2E1 T303A (provided by M. J. Coon, University of Michigan) were expressed in Escherichia coli cells. Expression and purification of the proteins were carried out according to published methods (22) with some modifications (23). NADPH-P450 reductase was purified after expression in E. coli Topp3 cells as previously described by Hanna et al. (24). Enzyme Activity Assays. Purified rabbit cytochrome P450 2E1 and the mutant P450 2E1 T303A were reconstituted with reductase and lipid for 45 min at 4 °C. Primary incubation mixtures contained 1 nmol of P450 2E1 or 2E1 T303A, 2 nmol of reductase, 166 µg of DLPC, 2000 units of catalase, tBA, or tBMP (in 1 µL of CH3OH/mL) and 1.2 mM NADPH in 50 mM potassium phosphate buffer (pH 7.4) containing 40 µg of BSA/ mL for a total reaction volume of 1 mL. Methanol was added to the control samples instead of tBA or tBMP. At the indicated times, 25 µL of the P450 primary reaction mixture was transferred into 975 µL of a secondary reaction mixture containing 100 µM 7-EFC, 0.2 mM NADPH, and 40 µg BSA/mL in 50 mM potassium phosphate buffer (pH 7.4). Samples were incubated for 10 min at 30 °C in a shaking water bath and enzyme activity was terminated by the addition of 334 µL of acetonitrile. Activity was assessed spectrofluorometrically by measuring the extent of O-deethylation of 7-EFC to 7-hydroxy-4-(trifluoromethyl)coumarin (7-HFC) on an SLM-Aminco model SPF-500C spectroflourometer with excitation at 410 nm and emission at 510 nm (25). Spectrophotometric Quantitation. P450s 2E1 and 2E1 T303A were reconstituted as described above for enzymatic activity. Final concentrations of 2 mM tBA and 0.5 mM tBMP (for P450 2E1) and 2 mM tBA and 1 mM tBMP (for P450 2E1 T303A) were used. At the times indicated, 100 µL aliquots of the tBA- and tBMP-inactivated P450 2E1 or 2E1 T303A primary reaction mixtures were removed and diluted with 900 µL of icecold 50 mM potassium phosphate buffer (pH 7.7), containing 40% glycerol and 0.6% Tergitol NP-40. The reduced CO spectrum was recorded from 400 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) (26). Substrate Protection. To determine whether an alternate substrate protected P450 2E1 or 2E1 T303A from inactivation by tBA or tBMP, an excess of 7-EC (3.3 mM) was added to the primary reaction mixture along with the inactivator. For wildtype P450 2E1 samples, 1 mM tBA and 0.7 mM tBMP were used; final concentrations of 2 mM tBA and 2 mM tBMP were used for the mutant P450 2E1 T303A samples. Reactions were initiated with 1.0 mM NADPH and incubated at 30 °C. At the indicated times, 25 µL of the primary reaction mixture was transferred into a secondary reaction mixture containing 10 mM MgCl2, 0.2 mM p-NP, and 0.5 mM NADPH in 100 mM potassium phosphate buffer (pH 6.8). Samples were incubated for 10 min at 30 °C in a shaking water bath. Enzymatic activity was stopped by adding 25 µL of TFA and the samples were centrifuged for 10 min at 16 000 rpm and 4 °C in a microfuge. Samples were separated by HPLC on a C18 microsorb MV column with isocratic conditions (solvent A, 25% acetonitrile and 0.1% TFA) at a flow rate of 1.0 mL/min. The catechol product

Inactivation of P450s 2E1 by tert-Butyl Acetylenes was identified by electrochemical detection as previously described (27). Irreversibility of Inactivation. P450s 2E1 and 2E1 T303A were reconstituted as described above for enzymatic activity. Final concentrations of 2 mM tBA and 0.7 mM tBMP (for P450 2E1) and 4 mM tBA and 4 mM tBMP (for P450 2E1 T303A) were used. Control samples and samples containing tBA- and tBMP-inactivated P450 2E1 or 2E1 T303A (0.5 mL) were dialyzed overnight at 4 °C against 2 × 500 mL of 50 mM potassium phosphate buffer (pH 7.4), containing 20% glycerol, to determine whether the inactivation and P450 reduced CO spectral losses were reversible. After dialysis, samples were reconstituted with lipid and in some cases fresh reductase. Samples were assayed concurrently for 7-EFC O-deethylation activity and reduced CO spectrum both prior to and after dialysis. Assayed time points were 0 and 6 min (tBA) or 10 min (tBMP). Effect of Exogenous Nucleophiles. P450s 2E1 and 2E1 T303A were reconstituted as described above for enzymatic activity. To examine the effect of exogenous nucleophiles on the rates of inactivation of the P450s, GSH (10 mM) and DTT (0.5 mM) were added to the primary reaction mixture containing the reconstituted P450 and tBA or tBMP. Reactions were carried out as described above and the 7-EFC O-deethylation activity was measured spectrofluorometrically. HPLC Analysis. P450s 2E1 and 2E1 T303A were reconstituted as described above for enzymatic activity. Final concentrations of 2 mM tBA and 0.5 mM tBMP (for P450 2E1) and 2 mM tBA and 1 mM tBMP (for P450 2E1 T303A) were used. P450s 2E1 or 2E1 T303A and reductase for control and tBA- or tBMPinactivated samples were separated by high-performance liquid chromatography (HPLC) on a reverse-phase C4 column (solvent A, 0.1% TFA and H2O; solvent B, 95% acetonitrile and 0.1% TFA). The flow rate was 1 mL/min through a linear gradient of 40% B to 100% B over 45 min. The elution of proteins and heme was monitored using diode array detection. ESI-LC-MS Analysis (Heme Adducts). P450s 2E1 and 2E1 T303A were reconstituted as described above for enzymatic activity. Primary incubation mixtures contained 0.4 nmol of P450 2E1 or 2E1 T303A, 0.8 nmol of reductase and 30 µg of DLPC. Final concentrations of 2 mM tBA and 0.5 mM tBMP (for P450 2E1) and 2 mM tBA and 3 mM tBMP (for P450 2E1 T303A) were used. Control samples incubated with tBA or tBMP in the absence of NADPH and tBA- or tBMP-inactivated P450 2E1 or P450 2E1 T303A samples were resolved on a reversephase C4 column equilibrated with 40% acetonitrile, 0.1% TFA (B) at a flow rate of 0.3 mL/min. Heme components were eluted using a linear gradient to 100% B over 25 min. The eluting peaks were subjected to mass analysis on a Thermoquest LCQ ion trap mass spectrometer. Scans were acquired with the sheath gas set at 100 (arbitrary units) and the auxiliary gas set at 30 (arbitrary units). For acquisition, the spray voltage was 4.2 kV, the capillary voltage 19 V, and the capillary temperature 220 °C. ESI-LC-MS Analysis (Protein Adducts). P450s 2E1 and 2E1 T303A were reconstituted as described above for enzymatic activity. Primary incubation mixtures contained 0.4 nmol of P450 2E1 or 2E1 T303A, 0.8 nmol of reductase, and 30 µg of DLPC. Final concentrations of 2 mM tBA and 0.5 mM tBMP (for P450 2E1) and 2 mM tBA and 1 mM tBMP (for P450 2E1 T303A) were used. Control samples incubated with tBA or tBMP in the absence of NADPH and tBA- or tBMP-inactivated P450 2E1 or P450 2E1 T303A samples were resolved on a 150 × 2.1 mm Vydac C18 reverse-phase column equilibrated with 40% acetonitrile, 0.1% TFA (B) at a flow rate of 0.3 mL/min. The proteins were eluted using a linear gradient to 90% B over 25 min. The eluting peaks were subjected to mass analysis on a Thermoquest LCQ ion trap mass spectrometer. Scans were acquired using settings and parameters that have previously been described for P450 2E1 (13). The spectra of the protein envelopes were deconvoluted to give the associated masses using Thermoquest Bioexplore 1.0 software.

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Figure 2. Time- and concentration-dependent loss of P450 2E1 7-EFC O-deethylation activity following incubation with tBMP and NADPH. At the indicated time points, samples were removed from the primary reaction mixture and assayed for 7-EFC activity. The concentrations of tBMP were (9) 0, (0) 33, (b) 67, (O) 167, ([) 333, (]) 667 µM. The data shown represent the mean and standard deviation from three to five separate experiments. For some data points, the deviation was smaller than the size of the symbol. The inset shows the doublereciprocal plot of the rates of inactivation as a function of the tBMP concentration. Table 1. Kinetic Constants for the Inactivation of P450s 2E1 and 2E1 T303A by tBA and tBMPa sample

KI (mM)

kinact (min-1)

t1/2 (min)

P450 2E1 + tBMP P450 2E1 + tBA P450 2E1 T303A + tBMP P450 2E1 T303A + tBA

0.1 1.0 1.0 2.0

0.12 0.20 0.07 0.38

5.9 3.5 10.2 1.8

a Assay conditions were as described in Experimental Procedures.

Results Inactivation of P450s 2E1 and 2E1 T303A by tertButyl Acetylenes. The kinetics for the inactivation of P450s 2E1 and 2E1 T303A by tBA or tBMP were studied by measuring the loss in 7-EFC O-deethylation activity. P450s 2E1 and 2E1 T303A in the reconstituted system were inactivated by tBA or tBMP in a time- and concentration-dependent manner (Figure 2 and data not shown). A representative kinetic analysis for P450 2E1 with tBMP is shown in Figure 2. Similar results were obtained with both enzymes and both acetylenic compounds. A summary of the kinetic constants determined for the inactivation of P450s 2E1 and 2E1 T303A by tBA or tBMP is given in Table 1. The kinetic constants were determined from double reciprocal plots of the inverse of the initial rates of inactivation as a function of the reciprocal of the tBA or tBMP concentration (Figure 2, inset). As can be observed in Figure 2, the inactivation followed pseudo-first-order kinetics. In the absence of NADPH, there was no significant loss in the activity of the enzymes with either tert-butyl acetylenic compound. As expected for true mechanism-based inactivators, the presence of an exogenous nucleophile (10 mM GSH or 0.5 mM DTT) had no significant effect on the rates of inactivation of P450s 2E1 and 2E1 T303A by tert-butyl acetylenic compounds (data not shown). Spectrophotometric Quantitation. The effect of tBA and tBMP inactivation on the P450 reduced CO complex formation of P450s 2E1 and 2E1 T303A was studied. As is observed in Table 2, inactivation of wild-

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Table 2. Effect of tBA and tBMP Inactivation of P450 2E1 and P450 2E1 T303A on the 7-EFC O-Deethylation Activity, Reduced CO Spectrum, and HPLC-Detected Hemea % of control

sample

activity remaining


21 26 73 15

activity remaining (% of control)

primary reaction mixture

reduced CO HPLC spectrum heme remaining remaining 35 50 74 30

Table 3. Effects of the Alternate Substrate 7-EC on the p-NP Hydroxylation Activity of P450s 2E1 and 2E1 T303Aa

12 51 78 41

a

Assay conditions were as described in Experimental Procedures. Percent of control refers to the total amount of enzyme activity, P450 reduced CO spectrum, and HPLC-detected P450 heme that was remaining when compared to control sample values (100%). The values shown are representative of data from two to three independent experiments and reflect measurements of activity, P450 reduced CO spectrum, and HPLC-detected heme collected on the same day and from the same reconstituted system. The values from the independent experiments did not differ by more than 10%.

type P450 2E1 by tBMP or tBA resulted in significant losses in enzymatic activity (79 and 74%, respectively) that were accompanied by simultaneous decreases in the P450 CO spectrum (65 and 50%). When the mutant P450 2E1 was inactivated by tBA, an 85% loss in activity with a concurrent 70% decrease in the P450 CO spectrum was observed. However, inactivation of the mutant enzyme by tBMP resulted in only a 27% loss in activity and a 26% decrease in the P450 CO spectrum. The inability of tBITC-inactivated P450 2E1 to form a reduced CO complex was previously shown to be reversible and not due to modification or destruction of the heme (13). Therefore, the heme of the tBA- or tBMPinactivated P450 2E1 samples was measured using HPLC and diode array detection. Table 2 shows that in most instances the percentage loss in enzymatic activity correlated with the percentage decrease in the P450 CO complex and the loss in native heme detected using HPLC. Interestingly, P450 2E1 inactivated by tBA displayed a greater loss in enzymatic activity (74%) than could be accounted for by CO spectral or heme loss (50%). These observations suggest that metabolism of tBMP by P450s 2E1 and 2E1 T303A and metabolism of tBA by the mutant enzyme led to the formation of reactive intermediates that resulted in heme modification and subsequent inactivation of these enzymes. In contrast, the data for the inactivation of the wild-type P450 2E1 by tBA suggests a combination of heme and protein modification. Substrate Protection from Inactivation of P450s 2E1 and 2E1 T303A by tert-Butyl Acetylenes. The effect of the alternate substrate 7-EC on the inactivation of P450s 2E1 and 2E1 T303A by tBA or tBMP was studied. Reduced rates of inactivation were observed when P450s 2E1 and 2E1 T303A were incubated with tBA or tBMP together with an excess of 7-EC (data not shown). The percentage of activity remaining at the final time point for each inactivation assay in the presence of tBA or tBMP or tBA and tBMP together with an excess of 7-EC is shown for comparison in Table 3. A loss in enzymatic activity was seen after incubating the wildtype enzyme with tBA or tBMP and NADPH when p-NP was used as a substrate to assess residual activity. Surprisingly, the T303A mutant only showed an inability to hydroxylate p-NP when the enzyme was inactivated

P450 2E1 + NADPH, + tBMP + NADPH, + tBMP, + 7-EC P450 2E1 + NADPH, + tBA + NADPH, + tBA, + 7-EC P450 2E1 T303A + NADPH, + tBMP + NADPH, + tBMP, + 7-EC P450 2E1 T303A + NADPH, + tBA + NADPH, + tBA, + 7-EC

33 ( 2 80 ( 4 62 ( 3 97 ( 2 63 ( 3 88 ( 4 112 89

a Assay conditions were as described in Experimental Procedures. The values shown represent the mean and standard deviation from three to four separate experiments, except for tBAinactivated P450 2E1 T303A (*) where the numbers shown are representative for several experiments where different inactivator concentrations were used. The percentage of activity remaining refers to the enzyme activity remaining at the final time point in each activity assay when compared to control sample activity at 0 min (100%).

Table 4. Irreversibility of P450 7-EFC O-Deethylation Activity and Reduced CO Spectruma activity remaining P450 remaining (% of control) (% of control) P450 2E1 + tBMP before dialysis after dialysis after dialysis + reductase P450 2E1 + tBA before dialysis after dialysis after dialysis + reductase P450 2E1 T303A + tBMP before dialysis after dialysis after dialysis + reductase P450 2E1 T303A + tBA before dialysis after dialysis after dialysis + reductase

15 18 21

38 27

48 49 59

70 76

38 19 21

79 47

12 104 102

37 110

a Assay conditions were as described in Experimental Procedures. Enzymatic activity and reduced CO complex were measured before and after dialysis. The value obtained for the noninactivated sample in each condition was assigned 100% and the numbers shown were calculated as percent of control activity and P450 remaining, respectively. The results are the averages of values obtained from two independent experiments each done in duplicate. The values from the two independent experiments did not differ by more than 10%. Spectrophotometric quantitation was used to determine the reduced CO spectrum.

with tBMP and not with tBA. P450 2E1 T303A samples incubated with tBA in the presence of NADPH were therefore simultaneously assayed for 7-EFC or p-NP activity and showed that activity loss was again observed using 7-EFC (76% loss), but not with p-NP (1% loss). Irreversibility of P450 2E1 and 2E1 T303A Inactivation by tert-Butyl Acetylenes. Control and tBAor tBMP-inactivated P450 2E1 and 2E1 T303A samples were dialyzed extensively to determine whether the inactivation by either acetylenic compound was reversible (Table 4). Samples were also simultaneously tested before and after dialysis to determine whether the P450-reduced CO spectrum could be recovered. Neither dialysis nor the addition of fresh reductase led to a significant recovery of enzymatic activity or reduced CO complex for tBA-

Inactivation of P450s 2E1 by tert-Butyl Acetylenes


Figure 3. HPLC and LC-MS heme analyses of tBA-inactivated P450 2E1. Inactivation of wild-type P450 2E1 by tBA results in the formation and identification of two distinct heme adducts having the same molecular mass. Panel I shows an HPLC chromatogram of P450 2E1 incubated with tBA and NADPH. The unmodified heme peak (A) elutes at 21 min and the two heme adducts (B and C) elute at approximately 25 and 27 min, respectively. Panel II shows the individual diode array spectra of the unmodified heme, A, and the two heme adducts, B and C. Panel III displays the individual mass spectra of the unmodified heme (616.3 Da) and the heme adducts (661.3 Da). The mass of each adduct corresponds to an iron-depleted heme, the molecular weight of tBA, and the addition of one oxygen atom.

inactivated P450 2E1 and tBMP-inactivated P450 2E1 and 2E1 T303A. Interestingly, both the inactivation and the decrease in the reduced CO spectrum of the tBAinactivated P450 2E1 T303A mutant were reversible. HPLC and LC-MS Analysis of P450s 2E1 After Inactivation by tert-Butyl Acetylenes. P450s 2E1 and 2E1 T303A inactivated by tBA or tBMP were analyzed by HPLC to determine the amount of native heme remaining after inactivation and to determine whether heme-acetylene adducts could be detected. Figure 3 (panel I) shows the HPLC profile at 405 nm of P450 2E1 incubated with tBA and NADPH. In addition to the native heme eluting at approximately 21 min (A), two additional peaks of similar size (B and C) were observed eluting at approximately 25 and 27 min, respectively. Only peak A, corresponding to the unmodified heme, was observed in all of the control incubations

where the two enzymes were incubated with tBA or tBMP but in the absence of NADPH (data not shown). The diode array spectra of peaks B and C were similar to the spectrum of the native heme, except that a shift from 395 nm to 406-410 nm of the soret peaks of B and C was observed. This suggested that both peaks were tBA-modified heme products (Figure 3, panel II). LC-MS analysis of the tBA-inactivated P450 2E1 indicated that peak A had a mass consistent with heme (616.3 Da) and that peaks B and C had masses of 661.3 Da, presumably corresponding to the mass of an iron-depleted heme (561 Da) plus the addition of a tBA reactive intermediate (83 Da) containing one oxygen atom (16 Da). The HPLC and diode array spectra of the tBA-inactivated T303A mutant were similar to those observed with the wild-type sample (data not shown) except that peak B was formed at an approximate 5-fold higher amount compared to peak C.

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Figure 4. HPLC and LC-MS heme analyses of tBMP-inactivated P450 2E1. Inactivation of wild-type P450 2E1 by tBMP results in the formation one primary (C) and one secondary (B) heme adduct. Although equivalent masses were found at the retention times associated with each heme adduct, the secondary adduct did not show a significant spectrum at 405 nm. Panel I shows an HPLC chromatogram of P450 2E1 incubated with tBMP and NADPH. The unmodified heme peak (A) elutes at 21 min and the two heme adducts (B and C) elute at approximately 26 and 28 min, respectively. Panel II shows the individual diode array spectra of the unmodified heme, A, and the two heme adducts, B and C. Panel III displays the individual mass spectra of the unmodified heme (616.2 Da) and the heme adducts (705.4 Da). The mass of each adduct corresponds to an iron-depleted heme, the molecular weight of tBMP, and the addition of one oxygen atom.

The mass spectra obtained for all three heme related peaks in the T303A mutant were identical to the spectra for peaks A, B, and C of the tBA-inactivated wild-type enzyme (data not shown). As both the inactivation and the loss in the reduced CO spectrum of the tBAinactivated P450 2E1 T303A mutant were reversible with overnight dialysis (Table 4), the reversibility of hemeacetylene adduct formation was also analyzed by HPLC. Interestingly, overnight dialysis resulted in a decrease in the alkylated heme products (peaks B and C) and a significant recovery of the native heme (Table 5), suggesting a reversible heme alkylation. Figure 4 (panel I) shows the HPLC profile at 405 nm of P450 2E1 incubated with tBMP in the presence of NADPH. Two additional peaks (B and C) eluting at 26 and 28 min were again observed in addition to the

Table 5. Reversibility of P450 2E1 T303A Inactivation by tert-Butyl Acetylene (tBA)a % recovery sample

heme

adduct B

adduct C

inactive (b.d.) inactive (a.d.)

62 94

33 6

5 0.4

a HPLC conditions were as described in Experimental Procedures. P450 2E1 T303A samples inactivated with tBA were dialyzed for 18 h at neutral pH. For each series of experiments, the combined areas of the native heme and two heme adducts (peaks B and C) were set to 100%. Sample conditions refer to before dialysis (b.d.) and after dialysis (a.d.).

unmodified heme (peak A). Approximately 10-fold more of peak C was generated compared to B. Panel II shows the diode array spectra of peaks A through C and again



Figure 5. LC-MS protein analysis of P450 2E1 incubated with tBA. Panel I shows the deconvoluted spectrum obtained after LCMS analysis of P450 2E1 incubated with 1.2 mM tBA in the absence of NADPH. A protein with a mass of 53 799 Da was revealed, corresponding to the known mass of P450 2E1. Panel II shows the deconvoluted spectrum obtained after LC-MS analysis of P450 2E1 inactivated by incubation with 1.2 mM tBA in the presence of NADPH. Two species were observed: the unmodified protein with a mass of 53 799 Da (data not shown) and a protein adduct with a mass of 53 898 Da. The mass difference (99 Da) is consistent with modification of the protein by one molecule of tBA with the addition of an oxygen atom. The panel insets show the corresponding control and tBA-inactivated P450 2E1 associated total ion chromatograms.

indicates that B and C are modified heme products. As was observed for peaks B and C in the tBA-inactivated samples, B and C for tBMP-inactivated P450 2E1 also showed a shift of the soret peak from 395 nm to 406410 nm. The slightly longer retention times of these two products would be in agreement with a modification obtained by the larger tBMP reactive intermediate. LCMS analysis of peaks B and C (panel III) resulted in masses of 705.4 Da for both components that are consistent with an iron-depleted heme plus the additional mass of a tBMP reactive intermediate (127 Da) containing one oxygen atom. P450 2E1 T303A was incubated with tBMP and NADPH and analyzed by HPLC and LC-MS (data not shown). The HPLC profile at 405 nm and the diode array spectra were again similar to those observed with the tBMP-inactivated wild-type enzyme except that the amounts of peaks B and C that were generated by the

mutant enzyme were considerably less compared to the wild-type enzyme, reflecting the lower levels of inactivation observed with the T303A mutant. LC-MS analysis of the tBMP-inactivated P450 T303A resulted in masses of 616.3 Da for the native heme and in 705.3 Da for peak B, again indicative of a mass corresponding to a tBMPadducted, iron-depleted heme plus one oxygen atom. The mass for peak C could not be obtained due to the low abundance of this product. ESI-LC-MS Analysis of the P450 2E1 Apoproteins After Inactivation by tert-Butyl Acetylenes. Samples from tBA- or tBMP-inactivated P450s 2E1 and P450 2E1 T303A were also analyzed by ESI-LC-MS to determine if any of the acetylenic reactive intermediates had formed a covalent adduct to the P450 apoprotein. Only wild-type P450 2E1 inactivated by tBA showed a mass increase compared to the native, nonadducted P450 (Figure 5).

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Table 6. ESI-LC-MS Characterization of tBA and tBMP Reactive Intermediate Adducts to the P450 Heme or Apoproteina


mass of heme adduct (s) (Da)

mass of protein adduct

705 661 705 661

N/Ob 99 Da N/Ob N/Ob

a Assay conditions were as described under Experimental Procedures. The data shown represent samples that were incubated in the presence of tBA or tBMP and NADPH. Heme and protein components were eluted using linear gradients and the eluting peaks were subjected to mass analysis on a Thermoquest LCQ mass spectrometer. b Refers to samples in which a protein adduct was not observed.

No protein adducts were found on P450 2E1 inactivated with tBMP or on the T303A mutant inactivated with either tBA or tBMP (data not shown). In all cases, adducts of tBA or tBMP reactive intermediates were not observed on reductase. Panel I of Figure 5 shows the deconvoluted mass spectrum of P450 2E1 incubated with tBA in the absence of NADPH. The observed mass of P450 2E1 (53 799 ( 1 Da) is in agreement with the calculated mass for the expressed N-terminally modified enzyme. The inset in panel I depicts the total ion chromatogram of the reconstitution mixture containing clipped reductase (3.2 min), reductase (6.9 min), and P450 2E1 (9.0 min). Incubations of P450 2E1 with tBA in the presence of NADPH resulted in the appearance of the unmodified P450 apoprotein (inset to panel II and data not shown) and an additional P450 protein peak eluting at approximately 10.4 min (inset to panel II) with a mass of 53 898 ( 1 Da (panel II). This increase in mass of 99 Da is consistent with the masses of tBA (82 Da) plus one oxygen atom (16 Da) and with the covalent modification of P450 2E1 by a tBA reactive intermediate. A summary for each of the P450s inactivated by tBA and tBMP with the observed heme adducts and their associated masses as well as the observed protein adduct is shown in Table 6 for comparison.

Discussion Mechanism-based inactivators have been used extensively to obtain structural and mechanistic information concerning the P450 2B1 active site. Ethynyl compounds, in particular, have been able to elucidate the key events involved in heme or protein alkylation of 2B1 enzymes (15-19). Previous findings with 2-ethynylnaphthalene and 2-ethynylphenanthrene and P450 2B enzymes indicate that the mechanism-based inactivation by these acetylenes was due to a covalent modification of a residue in the I-helix peptide I290-M314 by the respective reactive intermediates of the compounds (20, 21, 28). No CO spectral losses or heme alkylation products were observed. Studies with mechanism-based inactivators, such as isothiocyanates and ethynyl compounds, aimed at identifying key amino acid residues in the P450 2E1 active site have been somewhat limited due to the lack of P450 2E1 specific inactivators. With tert-butyl isothiocyanate, an inactivator that is relatively selective for P450 2E1, a decrease in the CO spectrum concurrent with a loss in enzymatic activity was observed. However, this spectral loss was not because of a tBITC-dependent heme alkylation or destruction of the P450 heme (13, 23). The

Blobaum et al.

defect was found to be due in part to a reduced ability to bind substrate or CO because of a tBITC adduct on the apoprotein. The present studies were designed to probe the active sites of P450 2E1 and the mutant P450 2E1 T303A with two structurally similar compounds that contained a tertbutyl moiety for P450 2E1 specificity and an ethynyl functional group for P450-dependent metabolism to a reactive intermediate capable of covalently modifying the active site. As predicted, the two acetylenes (tBA and tBMP) were mechanism-based inactivators of P450 2E1 based on the following criteria: (1) the loss in enzymatic activity followed pseudo-first order kinetics and was entirely dependent on the presence of NADPH together with the acetylenes, (2) exogenous nucleophiles, such as GSH and DTT, did not significantly reduce the rate of inactivation, and with the notable exception of the tBAinactivated T303A mutant (3) enzymatic activity was not recovered after dialysis and an alternate substrate could protect the 2E1 enzymes from inactivation. Surprisingly, the inactivation kinetics of tBA and tBMP were quite different from those seen with the tBITC. Approximately 100-fold higher concentrations were required for half-maximal inactivation of P450 2E1 or the mutant P450 2E1 T303A. Although the maximal rate of inactivation of the wild-type P450 2E1 was similar for both tBA and tBMP (0.20 and 0.12 min-1), a 10-fold higher concentration of inactivator was required for halfmaximal inactivation with tBA (1.0 mM) compared to tBMP (0.1 mM). This observation suggests that tBMP could be more readily and tightly bound in the active site of the wild-type enzyme. P450 2E1 inactivated by tBA or tBMP resulted in a 74 and 79% loss in enzymatic activity, respectively, with concurrent decreases in the P450 CO spectra of 50 and 65%. The maximal rate of inactivation of the T303A mutant by tBMP (0.07 min-1) was substantially slower when compared to inactivation by tBA (0.38 min-1). In contrast to the wild-type enzyme, the KI values for inactivation of the 2E1 T303A mutant by tBA or tBMP were similar. This suggests that the replacement of the threonine 303 by an alanine resulted in a structural change in the P450 2E1 active site that may have brought about a reduced affinity for tBMP or an altered orientation of tBMP such that metabolism of the compound either generated fewer reactive intermediates or that a critical site (amino acid or heme) involved in the inactivation was no longer accessible for modification by the tBMP reactive intermediate. Additionally, mutation of the threonine 303 resulted in only a 27% loss in enzymatic activity and a 26% loss in the P450-reduced CO spectrum when P450 2E1 T303A was inactivated by tBMP. When the mutant was inactivated by tBA, an 85% loss in enzyme activity and a 70% decrease in the reduced CO spectrum was observed. Evidence that changes in key active site residues can result in altered product profiles has been elegantly demonstrated with P450 2B1 enzymes (29, 30). Similarly, it was shown that a naturally occurring mutant of P450 2B1 was unable to generate a reactive intermediate from N-benzyl-1-aminobenzotriazole, a mechanism-based inactivator of 2B enzymes (31). Incubations with both tert-butyl acetylenes in the presence of NADPH resulted in the inactivation of P450 2E1 and the T303A mutant by three distinct mechanisms: (1) covalent alkylation of the heme prosthetic moiety (Scheme 1A), (2) a combination of heme alkylation and protein adduction (Scheme 1B), and (3) a novel



Scheme 1. Inactivation of P450 2E1 and the T303A Mutant by tert-Butyl Acetylenes Occurs by Three Distinct Mechanisms: (A) Covalent Alkylation of the Heme Prosthetic Moiety, (B) a Combination of Heme Alkylation and Protein Adduction, and (C) a Reversible Alkylation of the P450 Heme

reversible alkylation of the P450 heme (Scheme 1C). Metabolism of tBMP by P450 2E1 formed tBMP reactive intermediates that alkylated the heme moiety. Two alkylated hemes exhibiting different retention times on the HPLC but having the same mass were observed. In each case, the mass of the adducted heme was consistent with the mass of the reactive intermediate plus the masses of an iron-depleted heme and one oxygen atom. This loss of the heme iron would be expected for the modification of a pyrole nitrogen, resulting in a weakening in the interaction with the heme iron and leading to a loss of the iron under the LC-MS analysis conditions. In a previous study using 5-phenyl-1-pentyne as an inactivator for P450s 2B1 and 2E1, similar results were obtained (32). MALDI analysis of the modified P450 2E1 heme products also resulted in the formation of an irondepleted 5-phenyl-1-pentyne-modified heme in addition to an iron-containing modified heme product. Presumably, the ionization conditions used in our current LCMS studies led to a complete loss of the heme iron in the tBA- and tBMP-modified samples. The diode array spectra of the native heme and tBA- or tBMP-modified hemes were similar with the exception of a 10-15 nm shift for the modified samples, which is consistent with the high- and low-spin hemes of the native and tBA- or tBMP-modified hemes, respectively. No additional ab-

sorption bands in the 415-419 nm and 645-652 nm range, indicative of metal free heme, were observed (33). The amount of modified heme obtained in the present analysis was too low to yield enough sample for further spectral characterizations which would determine conclusively if the loss of the heme iron was a consequence of tBA- or tBMP-alkylation or the LC-MS analysis conditions. Comparison between the inactivation kinetics and the elution profiles of the tBMP-adducted hemes of P450 2E1 and the T303A mutant show that (1) the inactivation was much slower and did not proceed to the same extent as for the wild-type enzyme, and (2) there were roughly equal amounts of peaks B and C produced by the mutant where as 10-fold more peak C compared to B was generated with the wild-type enzyme. This observation suggests that the loss of the T303 residue altered the binding of tBMP such that a stable heme adduct with the reactive intermediate could not be as readily formed in the mutant. Previously, Swanson and Ortiz de Montellano characterized the phenylhydrazine modified heme moiety of myoglobin (34). Their findings suggested that under anaerobic conditions, each of the four pyrole nitrogens could be equally well modified and that the hemes could be separated by reverse-phase chromatography in the order of B-, A-, C-, and then

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D-pyrole ring adducts. However, under aerobic conditions a preference for the modification of the nitrogens on the C and D rings was observed suggesting that the hemoglobin active site conferred specificity for accessibility of the pyrole nitrogens. Previous studies with acetylenic compounds and P450 2B1 indicated that inactivation by larger acetylenes was due to the addition of the terminal ethynyl carbon to the nitrogen of the vinyl side-chaincontaining A or B rings of the heme prosthetic group and the addition of an oxygen atom to the internal carbon (35). Small molecules such as acetylene were less affected by active site architecture constraints and were able to react with at least two different pyrole nitrogens on the A and the D ring of the P450 heme. Since the inactivations of P450s 2E1 and 2E1 T303A were carried out under aerobic conditions and tBA and tBMP are relatively small acetylenic compounds, peak B likely corresponds to a modification of the A or the B ring of P450 2E1 heme, whereas peak C probably corresponds to a modification on the D ring of the heme. Current studies are underway in our laboratory to elucidate the precise ring location of the tBA and tBMP reactive intermediate adducts on the P450 heme. Inactivation of P450 2E1 by tBA generated both protein and heme adducts that were again consistent with an increase in the respective masses by the mass of the reactive tBA-intermediate plus the mass of one oxygen atom. The two alkylated iron-depleted hemes were produced in approximately equal amounts accounting for a 50% loss in enzymatic activity. The remaining activity loss was therefore due to the modification of 25% of the P450 2E1 apoprotein. Only alkylated heme products were seen when the P450 2E1 T303A mutant was incubated with tBA in the presence of NADPH. Approximately 5-fold more of peak B was generated compared to peak C, suggesting that the active site configuration in the mutant resulted either in an altered heme or in a tBA binding orientation that favored accessibility of the pyrole nitrogen that gave rise to peak B. Most importantly, the activity loss and the CO spectral loss seen with tBA and the T303A mutant was reversible with extensive dialysis. Similarly, HPLC analysis of the tBA-inactivated T303A mutant showed a decrease in the alkylated heme products and a significant recovery of the native heme after dialysis. These observations suggested either that the initial tBA modification of the heme in the T303A mutant was not covalent and represented an intermediate state or that the covalent bond was slowly decomposed. The possibility that the acidic conditions used for the HPLC and LC-MS analysis may have forced the heme alkylation reaction to go to completion was investigated by dialyzing the inactivated samples against a low pH buffer (pH 4.9). Reduced levels in the alkylated products (peaks B and C) were again observed with a concurrent increase in the native heme peak (data not shown). These observations suggest that, under nondenaturing conditions, the formation of tBA-alkylated heme adducts was reversible. Dexter and Hager made similar observations with allylbenzene inactivation of chloroperoxidase (36). Analogous to our results with the 2E1 T303A mutant and tBA, chloroperoxidase inactivation was accompanied by N-alkylation of the heme and a loss of the heme iron. Activity and native heme spectra were restored to the samples upon standing. The authors further demonstrated that the native enzyme structure was required for recovery of the native heme. The

Blobaum et al.

similarities between the chloroperoxidase results and our novel results with tBA and the P450 T303A mutant further underscore the mechanistic importance of the T303 residue in catalysis. Attempts are currently being made to elucidate the mechanism underlying this unique reversible heme alkylation process. Inactivation of the T303A mutant with tBA was different from the inactivation of the enzyme with tBMP or the inactivation of the wild-type enzyme in another respect. Although the tBA-inactivated mutant was severely compromised in its ability to metabolize 7-EFC, no loss in activity was observed when p-NP hydroxylation was monitored. Inactivation of the T303A mutant with tBMP or inactivation of the wild-type enzyme with either acetylene resulted in a loss in enzymatic activity with both 7-EFC and p-NP. Previous studies examining the residual P450 3A4 activity toward testosterone and triazolam hydroxylation found that preincubation with the mechanism-based inactivator, midazolam, reduced P450 3A4 6β-hydroxylase activity by 47% and triazolam hydroxylation by 75% (37). Further studies are required to understand the basis for these unexpected results. Clearly, these results suggest a role for threonine 303 in substrate interactions and orientation within the enzyme active site. Additionally, slight changes in inactivator structure may also be responsible for the differences we have observed between tBA- and tBMP-inactivated P450 2E1 T303A. Differences in the kinetics and the reversibility of the inactivation of P450s 2E1 and 2E1 T303A by structurally similar compounds suggest that relatively minor modifications in inactivator structure may significantly influence how enzymes carry out the metabolism of different compounds. Perhaps of more interest, is the supporting evidence for catalytic importance of the threonine 303 residue mutated in P450 2E1. Replacement of the threonine by an alanine residue either appears to significantly affect the enzyme’s ability to correctly orient the inactivator or disrupts essential enzyme-substrate interactions necessary for metabolism. Previous studies with acetylenic compounds have shown inactivation of different P450 isoforms through heme or protein alkylation by reactive species produced from the catalytic oxidation of the acetylenic group (14-19). It is evident from the results presented here that considerable differences in the loss of enzymatic activity and in the formation of adducts exist between the wild-type and T303A mutant enzymes and between tBA- and tBMP-inactivated samples. These data support the notion that distinct inactivator structure and critical amino acid residues within the enzyme active site influence metabolism and therefore the inactivation of P450s 2E1 and 2E1 T303A by tert-butyl acetylenes. In light of these findings, active site molecular modeling of P450 2E1 using the two different tert-butyl compounds should prove to be very interesting.

References (1) Gonzalez, F. J. (1998) The molecular biology of cytochrome P450s. Pharmacol. Rev. 40, 243-288. (2) Nelson, D. R., Koymans, L., Kamataki, T., Stegeman, J. J., Feyereisen, R., Waxman, D. J., Waterman, M. R., Gotoh, O., Coon, M. J., Eastabrook, R. W., Gunsalus, I. C., and Nebert, D. W. (1996) P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature. Pharmacogenetics 6, 1-42. (3) Wang, Q., and Halpert, J. R. (2002) Combined three-dimensional quantitative structure-activity relationship analysis of cyto-


(4) (5)

(6) (7)

(8) (9) (10)

(11) (12)

(13) (14)

(15) (16)

(17)

(18)

(19)

(20)

(21)

chrome P450 2B6 substrates and protein homology modeling. ASPET 30, 86-95. Poulos, T. L., Finzel, B. C., and Howard, A. J. (1987) Highresolution crystal structure of cytochrome P450cam. J. Mol. Biol. 195, 687-700. Ravichandran, K. G., Boddupalli, S. S., Hasemann, C. A., Peterson, J. A., and Deisenhofer, J. (1993) Crystal structure of hemoprotein domain of P450 BM-3, a prototype for microsomal P450s. Science 261, 731-736. Hasemann, C. A., Ravichandran, K. G., Peterson, J. A., and Deisenhofer, J. (1994) Crystal structure and refinement of cytochrome P450 terp at 2.3 Å resolution. J. Mol. Biol. 236, 1169-1185. Williams, P. A., Cosme, J., Sridhar, V., Johnson, E. F., and McRee, D. E. (2000) Mammalian microsomal cytochrome P450 monooxygenase: structural adaptations for membrane binding and functional diversity. Mol. Cell 5, 121-131. Perrot, N., Nalpas, B., Yang, C. S., and Beaune, P. H. (1989) Modulation of cytochrome P450 isozymes in human liver, by ethanol and drug intake. Eur. J. Clin. Invest. 19, 549-555. Raag, R., Martinis, S. A., Sligar, S. G., and Poulos, T. L. (1991) Crystal structure of the cytochrome P-450CAM active site mutant Thr252Ala. Biochemistry 30, 11420-11429. Imai, M., Shimada, H., Watanabe, Y., Matsushima-Hibiya, Y., Makino, R., Koga, H., Horiuchi, T., and Ishimura, Y. (1989) Uncoupling of the cytochrome P-450cam monooxygenase reaction by a single mutation, threonine-252 to alanine or valine: possible role of the hydroxy amino acid in oxygen activation. Proc. Natl. Acad. Sci. U.S.A. 86, 7823-7827. Tan, Y., White, S. P., Paranawithana, S. R., and Yang, C. S. (1997) A hypothetical model for the active site of human cytochrome P4502E1. Xenobiotica 27, 287-299. Moreno, R. L., Goosen, T., Kent, U. M., Chung, F., and Hollenberg, P. F. (2001) Differential effects of naturally occurring isothiocyanates on the activities of cytochrome P450 2E1 and the mutant P450 2E1 T303A. Arch. Biochem. Biophys. 391, 99-110. Kent, U. M., Roberts-Kirchhoff, E. S., Moon, N., Dunham, W. R., and Hollenberg, P. F. (2001) Spectral studies of tert-butyl isothiocyanate-inactivated P450 2E1. Biochemistry 40, 7253-7261. Osawa, Y., and Pohl, L. R. (1989) Covalent bonding of the prosthetic heme to protein: a potential mechanism for the suicide inactivation or activation of hemoproteins. Chem. Res. Toxicol. 2, 131-141. Ortiz de Montellano, P. R. (1985) Alkenes and alkynes. In Bioactivation of Foreign Compounds (Anders, M. W., Ed.) pp 121155, Academic Press, New York. Ortiz de Montellano, P. R. (1986) Oxygen activation and transfer. In Cytochrome P450: Structure, Mechanism, and Biochemistry (Ortiz de Montellano, P. R., Ed.) pp 217-271, Plenum Press, New York. Ortiz de Montellano, P. R., and Reich, N. O. (1986) Inhibition of cytochrome P450 enzymes. In Cytochrome P450: Structure, Mechanism, and Biochemistry (Ortiz de Montellano, P. R., Ed.) pp 273-314, Plenum Press, New York. Ortiz de Montellano, P. R., and Komives, E. A. (1985) Branchpoint for heme alkylation and metabolite formation in the oxidation of arylacetylenes by cytochrome P-450. J. Biol. Chem. 260, 33303336. Komives, E. A., and Ortiz de Montellano, P. R. (1987) Mechanism of oxidation of π bonds by cytochrome P450. Electronic requirements of the transition state in the turnover of phenylacetylenes. J. Biol. Chem. 262, 9793-9802. Roberts, E. S., Pernecky, S. J., Alworth, W. L., and Hollenberg, P. F. (1996) A role for threonine 302 in the mechanism-based inactivation of P450 2B4 by 2-ethynylnaphthalene. Arch. Biochem. Biophys. 331, 170-176. Roberts, E. S., Hopkins, N. E., Zaluzec, E. J., Gage, D. A., Alworth, W. L., and Hollenberg, P. F. (1994) Identification of active-site


(22)

(23) (24)

(25) (26) (27)

(28)

(29)

(30)

(31)

(32) (33)

(34)

(35)

(36) (37)

peptides from 3H-labeled 2-ethynylnaphthalene-inactivated P450 2B1 and 2B4 using amino acid sequencing and mass spectrometry. Biochemistry 33, 3766-3771. Larson, J. R., Coon, M. J., and Porter, T. D. (1991) Alcoholinducible cytochrome P-450IIE1 lacking the hydrophobic NH2terminal segment retains catalytic activity and is membranebound when expressed in Escherichia coli. J. Biol. Chem. 266, 7321-7324. Kent, U. M., Roberts, E. S., Chun, J., Hodge, K., Juncaj, J., and Hollenberg, P. F. (1998) Inactivation of cytochrome P450 2E1 by tert-butylisothiocyanate. Chem. Res. Toxicol. 11, 1154-1161. Hanna, I. H., Teiber, J. F., Kokones, K. L., and Hollenberg, P. F. (1998) Role of the alanine at position 363 of cytochrome P450 2B2 in influencing the NADPH- and hydroperoxide-supported activities. Arch. Biochem. Biophys. 350, 324-332. Buters, J. T., Schiller, C. D., and Chou, R. C. (1993) A highly sensitive tool for the assay of cytochrome P450 enzyme activity in rat, dog and man. Biochem. Pharmacol. 46, 1577-1584. Omura, T., and Sato, R. (1964) The carbon monoxide-binding pigment of liver microsomes. J. Biol. Chem. 239, 2370-2378. Mishin, V. M., Koivisto, T., and Lieber, C. S. (1996) The determination of cytochrome P450 2E1-dependent p-nitrophenol hydroxylation by high-performance liquid chromatography with electrochemical detection. Anal. Biochem. 233, 212-215. Roberts, E. S., Hopkins, N. E., Alworth, W. L., and Hollenberg, P. F. (1993) Mechanism-based inactivation of cytochrome P450 2B1 by 2-ethynylnaphthalene: identification of an active site peptide. Chem. Res. Toxicol. 6, 470-479. Spatzenegger, M., Wang, Q., He, Y. Q., Wester, M. R., Johnson, E. F., and Halpert, J. R. (2001) Amino acid residues critical for differential inhibition of CYP2B4, CYP2B5, and CYP2B1 by phenylimidazoles. Mol. Pharmacol. 59, 475-485. Strobel, S. M., Szklarz, G. D., He, Y. Q., Foroozesh, M., Alworth, W. L., Roberts, E. S., Hollenberg, P. F., and Halpert, J. R. (1999) Identification of selective mechanism-based inactivators of cytochrome P450 2B4 and 2B5, and determination of the molecular basis for differential susceptibility. J. Pharmacol. Exp. Ther. 290, 445-451. Kent, U. M., Hanna, I. H., Szklarz, G. D., Vaz, A. D., Halpert, J. R., Bend, J. R., and Hollenberg, P. F. (1997) Significance of glycine 478 in the metabolism of N-benzyl-1-aminobenzotriazole to reactive intermediates by cytochrome P450 2B1. Biochemistry 36, 11707-11716. Roberts, E. S., Alworth, W. L., and Hollenberg, P. F. (1998) Mechanism-based inactivation of cytochromes P450 2E1 and 2B1 by 5-phenyl-1-pentyne. Arch. Biochem. Biophys. 354, 295-302. Ortiz de Montellano, P. R., Beilan, H. S., and Mathews, J. M. (1982) Alkylation of the prosthetic heme in cytochrome P-450 during oxidative metabolism of the sedative-hypnotic ethchlorvynol. J. Med. Chem. 25, 1174-1179. Swanson, B. A., and Ortiz de Montellano, P. R. (1991) Structure and absolute stereochemistry of the four N-phenylprotoporphyrin IX regioisomers isolated from phenylhydrazine-treated myoglobin. J. Am. Chem. Soc. 113, 8146-8153. Kunze, K. L., Mangold, B. L. K., Wheeler, C., Beilan, H. S., and Ortiz de Montellano, P. R. (1983) The cytochrome P-450 active site. Regiospecificity of prosthetic heme alkylation by olefins and acetylenes. J. Biol. Chem. 258, 4202-4207. Dexter, A. F., and Hager, L. P. (1995) Transient heme Nalkylation of chloroperoxidase by terminal alkenes and alkynes. J. Am. Chem. Soc. 117, 817-818. Schrag, M. L., and Wienkers, L. C. (2001) Covalent alteration of the CYP3A4 active site: evidence for multiple substrate binding domains. Arch. Biochem. Biophys. 391, 49-55.

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Mechanism-Based Inactivation of Cytochromes P450 2E1 and 2E1

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