Mechanism-based inactivation of cytochrome P450 2B1 by 2

Luke Koenigs Lightning, Jeffrey P. Jones, Thomas Friedberg, Michael P. Pritchard, .... J. Zaluzec , Douglas A. Gage , William L. Alworth , and Paul F...
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Chem. Res. Toxicol. 1993,6, 470-479

470

Mechanism-Based Inactivation of Cytochrome P450 2B1 by 2-Ethynylnaphthalene: Identification of an Active-Site Peptide Elizabeth S. Roberts,+Nancy Eddy Hopkins,t William L. Alworth,t and Paul F. Hollenberg*$+ Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan 48201, and Department of Chemistry, Tulane University, New Orleans, Louisiana 70118 Received November 30, 1992

The 7-ethoxycoumarin 0-deethylase activity of rat liver cytochrome P450 2B1 reconstituted with NADPH-cytochrome P450 reductase and lipid was inactivated by 2-ethynylnaphthalene (2EN) in a time- and NADPH-dependent manner, and the loss of activity followed pseudofirst-order kinetics. The extrapolated KIand kinactivationwere 0.08 /IMand 0.83 min-1, respectively. The loss of 7-ethoxycoumarin 0-deethylation activity displayed a number of characteristics consistent with mechanism-based inactivation, including irreversibility, saturability, protection by an alternate substrate, and the lack of an effect of exogenous nucleophiles on the inactivation. The inactivation was not accompanied by a concomitant loss of spectrally detectable cytochrome P450. HPLC analysis showed that I3H12EN was irreversibly bound to the protein moiety of cytochrome P450 and the stoichiometry of inactivation was approximately 1.3 mol of 2EN bound per mole of cytochrome P450. Liquid chromatographic and GC-MS analyses of the organic extracts from these incubations showed that the major metabolite was 2-naphthylacetic acid, and a partition ratio of 4-5 mol of acid produced per mole of cytochrome P450 2B1 inactivated was determined. A radiolabeled peptide, approximately 6.5 kDa when analyzed by Tricinesodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was isolated by HPLC from a tryptic digest of the PHI 2EN-inactivated cytochrome P450 and NADPH-cytochrome P450 reductase. Sequence data were obtained after cyanogen bromide cleavage of this aminoterminally blocked peptide. These results, in conjunction with the results from the cleavage of the intact [3H]BEN-inactivated cytochrome P450 by cyanogen bromide and separation of the peptides either by HPLC or by Tricine-SDS-PAGE followed by transfer of the peptides to a poly(viny1idene difluoride) membrane and sequencing of the labeled peptides from both experiments, led to the identification of a 2EN-modified active-site peptide with the sequence ISLLSLFFAGTETSSTTLRYGFLLM. This corresponds to positions 290-314 in cytochrome P450 2B1. Sequence alignments of cytochrome P450 2B1 with cytochrome P450 101 predict that this region might correspond to helix I of the bacterial protein [Poulos, T. L. (1988) Pharm. Res. 5,67-751 that contains a highly conserved threonine residue involved in oxygen binding.

Introduction The cytochromes P450 (P450)I are involved in the metabolism of a wide variety of xenobiotics, including drugs and carcinogens as well as endobiotics such as steroids, retinoids, and prostaglandins, and have been the subject of many excellent reviews (1-3). The forms of P450exhibit a catalytic diversity which reflects both the structural diversity of the P450 proteins as well as the ability of individual P450 enzymes to metabolize structurally distinct compounds. The common catalytic function of these enzymes is the two-electron reduction of molecular oxygen to form water and a reactive oxygen species. Much has been learned about the catalytic and substrate recognition

* Author to whom correspondence should be addressed.

State University. t Td-ane University. 1 Abbreviations: P450, cytochrome P450; P450 2B1, the major form from liver microsomes of phenobarbital-treated rats; P450 101, the camphor monooxygenase P450 from Pseudomow putida; reductase, NADPH-cytochromeP450 reductaee; 2EN, 2-ethynylnaphthalane;DLF'C, L-a-phoephatidylcholine,dilauroyl; TFA, trifluoroacetic acid; MTBSTFA, N-methyl-N-(tert-butyldimethyleilyl)trifluoroace~ide;SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PVDF, poly(vinylidene difluoride). t Wayne

sites of P450 101 from the resolution of ita threedimensional crystal structure (4, 5). Since a crystal structure is not yet available for a mammalian P450, most of the available knowledge about substrate specificity and binding comes from studies involving chemical modification of the P4508 (61,site-directed mutagenesis (reviewed in ref 71, protein engineering with chimeric proteins (812),and sequence alignment with P450 101 (13-18). Another method which can be used to identify regions of substrate binding involves the use of mechanism-based inactivators. These are substrate molecules for the target enzymes which, in the process of catalytic conversion at the enzyme active site, are metabolized to intermediates that inactivate the enzyme. Compounds that covalently bind to the protein moiety rather than the heme prosthetic group are especiallyuseful for the identification of peptides at the active site of the enzyme. Certain compounds containing the acetylenic functional group have been established as mechanism-based inactivators of P450 (reviewed in ref 19). Many of these have been found to inactivate the protein by alkylation of the heme, which then dissociates from the protein. Recently, an acetylenic compound, 2-ethynylnaphthalene (2EN), was found to

0 1993 American Chemical Society 0093-220x/93/2706-0470~04.oo/o

Inactivation of P450 2Bl by 2-Ethynylnaphthalene

cause inactivation through modification of the protein (20). 2EN was found to inactivate the arylamine N-oxidation activity of rat P450 forms 1 A l and 1A2 and resulted in the irreversible binding of the compound to the purified P450 proteins (20).Both purified rabbit and rat P450 1A2, but not human P450 1A2, were found to be modified by I3H12EN, and in each case a singletryptic peptide was identified (21).In addition, 2EN was shown to be an effectivesuicide inhibitor of P450 2B1-dependent depentylation of 7-pentoxyphenoxazone in microsomes from phenobarbitaltreated rats (22). In this study, the BEN-dependent inactivation of the 7-ethoxycoumarin 0-deethylase activity of rat P450 2B1 reconstituted with NADPH-cytochrome P450 reductase (reductase) and lipid was characterized. The inhibition was found to be consistent with mechanism-based inactivation of P450 2B1. In addition, the labeling stoichiometry was determined, the irreversible modification of the protein moiety was confirmed, and detailed investigations involving the purification and sequence analysis of the ZEN-modified active-site peptide were carried out.

Experimental Procedures Materials. 2EN and 2-ethyx1yl[ring-G-~H]naphthalene were prepared as described previously (20).L-a-Phosphatidylcholine, dilauroyl (DLPC), NADPH, and catalase were purchased from Sigma Chemical Co. (St. Louis, MO). 2-Naphthylacetic acid, benzoic acid, 7-ethoxycoumarin, and 7-hydroxycoumarin were purchased from Aldrich (Milwaukee, WI). HPLC-grade acetonitrile, ethyl acetate, and methanol were obtained from Baxter (McGraw Park, IL). Trifluoroacetic acid (TFA) and N-methylN-(tert-butyldimethylsily1)trifluoroacetamide(MTBSTFA)were purchased from Pierce (Rockford,IL). Safety Solvewas obtained from Research Products International Corp. (Mount Prospect, IL). Microconcentrators (Centricon-10,lO OOO MW cutoff)were obtained from Amicon (Danver, MA). Isolation of Enzymes. P450 2B1 andreductase were purified as described by Saito and Strobe1 (23)and Strobe1 and Dignam (24),respectively, from microsomes prepared from the livers of fastedmale Long Evans rata (150-175 g; Harlan SpragueDawley; Indianapolis, IN) given 0.1 % phenobarbital in the drinking water for 12 days. Enzyme Assays and Inactivation. In studies involving the inactivation of enzyme activity and spectrophotometric determination of P450 2B1, incubation mixtures contained 2.5 pM 2EN (in 5 pL of methanol), 0.5 pM P450 2B1,0.5 pM reductase, 30 pg/mL DLPC, 110 units of catalase, and 10 mM potassium phosphate buffer (pH 7.7) in a total volume of 0.5 mL. Solvent (5 pL of methanol) was added to the incubations in which no inactivator was present. The mixture was preincubated for 3 min at 30 OC before the addition of 1.5 mM NADPH to start the reaction or water in the reactions without NADPH. The 7-ethoxycoumarin 0-deethylase activity was measured spectrofluorometricallyas described (25)with the following modifications. The assays were performed on a SLM-Aminco Model SPF-5OOC spectrofluorometerwith the excitation at 380nm and the emission at 453 nm. At 0 and 10 min, a 100-pL aliquot of the incubation mixture was added to a secondary mixture containing 500 pM 7-ethoxycoumarin and 10 mM potassium phosphate buffer (pH 7.7) in a total volume of 1.0 mL. NADPH (0.3 mM) was also included in the secondary reaction mixtures in which no NADPH was present in the primary incubation. In addition, a 250-pL aliquot was removed at 10 min and added to a 4-fold excess of ice-cold 50 mM potassium phosphate buffer (pH 7.7) containing 40% glycerol and 0.6% Tergitol NP-10. The sample was split into two cuvettes, and the reduced carbonyl difference spectra (26) were recorded on a Uvikon Model 860 spectrophotometer. In order to follow the time-dependent loss of activity, care was taken in choosing the concentration of 2EN used in the assay. With an excess of 2EN, the loss of activity could not be measured

Chem. Res. Toxicol., Vol.6,No.4,1993 471 because it was masked by the inhibition of activity observed at 0 time. In studies involving the time- and concentration-dependent inactivation of 7-ethoxycoumarin 0-deethylase activity by 2EN, incubation mixtures contained 0.05 pM P450 2B1, 0.05 pM reductase, 30 pg/mL DLPC, 110 units of catalase, and 10 mM potassium phosphate buffer (pH 7.7). Methanol (5 pL) or 2EN in methanol was added to the incubation mixture prior to a 3-min incubation at 30 "C. The reaction was started by the addition of 0.15 mM NADPH. At the time points indicated, deethylase activity was assayed after the addition of 500 pM 7-ethoxycoumarin as described previously. In studies involving protection by 7-ethoxycoumarin against the 2EN-dependent inactivation of P450 2B1 deethylase activity, the primary incubation mixtures contained 2.8 pM 2EN, 1.0 pM P450 2B1, 0.5 pM reductase, 30 pg/mL DLPC, 110 units of catalase, 560 pM 7-ethoxycoumarin, and 10 mM potassium phosphate buffer (pH 7.7) in a total volume of 0.36 mL. The reactions were initiated by the addition of 2 mM NADPH. At various time points, the deethylase activity remaining was measured by adding aliquots to secondary reaction mixtures containing 500 pM 7-ethoxycoumarin and 10 mM potassium phosphate buffer (pH 7.7). This was compared to the activity remaining when no 7-ethoxycoumarinwas present in the primary reaction mixture. Metabolism of 2EN by P450 2B1. The incubation mixtures contained 0.5 pM P450 2B1,0.5 pM reductase, 30 pg/mL DLPC, 120 units of catalase, 12 pM [SH]2EN or unlabeled 2EN (in MezSO), and 10 mM potassium phosphate buffer (pH 7.4) in a total volume of 1.0 mL. The mixtures were preincubated at 30 O C for 3 min before initiation of the reaction with 2 mM NADPH. After a 15-minincubation, the reactions were quenched by the addition of 40 pL of 30 % phosphoric acid. The internal standard, benzoic acid, was added to the mixtures prior to extraction with four 1.0-mL aliquots of ethyl acetate. The solvent was dried under astream of compressed air and the residue dissolved in the buffer used in the initial conditions of the HPLC analysis. Quantitative analysis of the reaction products was done on a 4.6 X 250 mm Vydac Protein and Peptide CIS reverse-phase column with a HPLC system consisting of a Gilson Holochrome detector, Model 305 and 306 pumps, Model 805 manometric module, Model 811B dynamic mixer, Model 201 fraction collector, and a Hewlett-Packard HP3396 Series I1integrator. The solvent gradient consisted of buffer A, 0.1% TFA, and buffer B, 95% acetonitrile/5% HzO/O.l% TFA. Isocratic solvent conditions of 23% B at a flow rate of 1.0 mL/min were maintained for 7 min, followed by a linear gradient to 37% B over 8 min. Retention times for benzoic acid and 2-naphthylacetic acid were 8.9 and 21.0 min, respectively, and the amounts of these acids in the reaction mixtures were determined from standard curves generated with authentic compounds. For the GC-MS analysis of the reaction products, the peak representing the 2-naphthylacetic acid was manually collected from an HPLC analysis, extracted into ethyl acetate, dried under a stream of compressed air, resuspended in acetonitrile, reacted with MTBSTFA, and dried as described earlier. The gas chromatographic and electron impact mass spectral analysis of the reaction product and the standard 2-naphthylacetic acid were done on a 15-m DB1 column (0.25-mm i.d. and 0.25-pm film thickness) attached to a HP58890 gas chromatograph and a HP MSD5970 series mass-selective mass spectrometer at the MSU Mass Spectrometry Facility, Michigan State University. Stoichiometry and Specificity of Binding. A reaction mixture containing 1.0 pM P450 2B1, 1.0 pM reductase, 30 pg/ mL DLPC, 20 pM [SH]2EN, 120 units of catalase, and 50 mM potassium phosphate buffer (pH 7.7) in a total volume of 2.0 mL was incubated at 30 OC for 3 min before initiation with addition of 2 mM NADPH or water to the incubation without NADPH. The NADPH- and time-dependent loee of activity was determined at 0 and 20 min by adding an aliquot of the incubation mixture to a secondary reaction mixture containing 500 pM 7-ethoxycoumarin, 0.15 mM NADPH (in the incubation where no NADPH was present in the primary incubation), and 10 mM potassium

472 Chem. Res. Toxicol., Vol. 6, No. 4, 1993 phosphate buffer (pH 7.7). Activity was assayed spectrofluorometrically over 3 min as previously described. After a 20-min incubation, the excess reagents and metabolites were removed and the samples concentrated using Amicon Centricon-10 microconcentrators. The initial centrifugation was at 5000g for 70 min at 10 "C. The protein was then resuspended in 2.0 mL of 50 mM potassiumphosphate buffer (pH 7.7) and recentrifuged. This wash procedure was repeated until less than 0.5% of the total counts were in the wash. After the final wash, the protein was brought up in a small volume of buffer and recovered by inverting the concentrator with a conical cap attached and centrifuging at l000g for 3 min. The concentrated protein samples were then analyzed by liquid scintillation counting and HPLC to determine protein recovery, binding specificity, and stoichiometry of binding. An aliquot of each sample was counted to determine the amount of 2EN in both the sample incubated with NADPH and the control (without NADPH). Another aliquot of each sample was injected onto a 4.6 X 100 mm POROS R/H perfusive-particle column from Perseptive Biosystems (Cambridge, MA). The HPLC system and solvent composition were as described previously. Initial conditions were 35% B at a flow rate of 3.0 mL/min with a linear gradient to 75% B in 13 min and then to 100% B in 2.0 min. Standard curves were generated with purified P450 2B1 and reductase. Fractions were collected every 0.5 min and monitored by liquid scintillation counting on a Beckman Model LS 3801 liquid scintillation counter. The stoichiometry of binding was then calculated as the nanomoles of 2EN, determined after subtracting the counts from the control, per nanomole of P450, determined from the HPLC standard curve. Separation and Analysis of Peptides. After concentrating and washing on an Amicon microconcentrator as previously described,the [SH]2EN-inactivated P450 2B1 and reductase were resuspended in 100 mM potassium phosphate buffer (pH 7.7). The protein solution was heated at 37 OC for 30 min in the presence of 8 M urea to denature the proteins (27).The urea concentration was then reduced to 2 M with the addition of buffer, and a 1.5-pL aliquot of a 1 pg/pL solution of ~-l-(tosylamino)-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin was added to the mixture and digestion allowed to proceed for 18h a t 37 "C. Three 1.5-pL aliquota of trypsin were added over the digestion period. After acidification with 0.1 % TFA, the peptides were analyzed on a Vydac Cl8 Protein and Peptide column. The solvent system consisted of buffer A, 0.1 % TFA, and buffer B, 95 % CHsCN/ 5%H20/0.1% TFA. The initial conditions were 5% B with a linear gradient to 70% B over 120 min, and then to 100%Bin 30 min at a flow rate of 1.0 mL/min. Cleavageat methionine residues was performed after the [3HlZEN-labeled P450 2B1 and reductase were concentrated to near drynesswith a Speed-Vacconcentrator (Savant) and resuspended in 70% TFA. After 20 pL of a cyanogen bromide solution (1.0 mg/pL in 70% TFA) was added, the reaction was allowed to proceed overnight at room temperature (27).Prior to electrophoresis or HPLC analysis, the samples were concentrated to near dryness, resuspended in 1.0 mL of H20, and again concentrated to near dryness to remove the acid and byproducts. The CNBr-generated peptides were analyzed by HPLC on a 4.6 X 250 mm Vydac C, Protein and Peptide column with a solvent system consisting of buffer A, 0.2% TFA, and buffer B, 75% CH3CN/20% isopropyl alcohol/5 % H20/0.2% TFA. After maintaining initial conditions of 5% B for 5 min, the peptides were eluted with a linear gradient to 100% B in 95 min at a flow rate of 1.0 mL/min. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out according to the method of Schagger and von Jagow (28). The composition of the separating gel was 16.5% T [T denotes the concentration of acrylamide and bis(acry1amide)l and 6% C (C denotes the concentration of the cross-linkerrelative to the totalconcentration T), the stacking gel was 4% T and 3% C, and the spacer gel was 10% T and 3% C. The 0.75-mm gel was poured and allowed to equilibrate overnight at 4 "C. Fractions containing labeled peptide were concentrated on a Speed-Vac instrument. Sample

Roberts et al. Table I. Effect of 2-Ethynylnaphthalene on Cytochrome P450 Content and 7-Ethoxycoumarin 0-Deethylase Activity of Purified Cytochrome P450 2B1* % P450 remaining % act. remaining system Omin 10min 10 min -2EN, +NADPH 100 96 100 +2EN, -NADPH 68 70 96 +2EN, +NADPH 70 2 83 a Incubation conditions were as described under Experimental Procedures. The values presented are the means of duplicate determinations that did not differ by more than 7 % . ~

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buffer containing 0.01 % Coomassie Brilliant Blue G was added to the peptide samples and to the molecular weight markers (Sigma MW-SDS-17s) before heating to 40 OC for 30 min. Electrophoresis was performed at a constant current of 20 mA per gel with water cooling until the samples had completely entered the stacking gel. The current was increased to 25 mA per gel and then maintained throughout the remainder of the run, typically 13 h. After electrophoresis, the gels were fixed, stained with 0.025% Coomassie Brilliant Blue G, and destained as described (28). After complete removal of the destaining solution, the gel was treated with ENSHANCE (Dupont NEN Research Products; Boston, MA). Autoradiography was performed by exposing the enhanced dried gel to Kodak XAR-5 film. The gel was exposed for 3 days at -70 "C. The CNBrgenerated peptides were electroblotted onto an Immobilon PQ poly(viny1idenedifluoride) (PVDF) membrane (Millipore Corp.; Milford, MA) using an LKB Model 2005 Transphor Electroblotting Unit with a buffer system of 25 mM Tris, 192mM glycine, and 20% CH30Hat 0.5 A for 2 h with stirring and water cooling. The peptides on the PVDF membrane were visualized by staining with 0.1 % Coomassie Blue R-250/45% CHaOH/10% acetic acid and destained with 45% CH30H/7% acetic acid. Labeled peptides from the HPLC analyses were collected in polypropylene tubes and concentrated using a Speed-Vac concentrator. The amino acid sequences of the HPLC-purified and PVDF membrane-boundpeptides were determined by automated Edman chemistry on a Model 470 Applied Biosystems gas-phase sequenator, with an on-lineModel 120HPLC, a Nelson Analytical Chromatography Data System, and a 900-A control/data system. CNBr cleavage on the filter was performed as described (29) to obtain the sequence of the tryptic peptide that had a blocked amino terminus.

Results Inactivation of Purified P450 2Bl. The 7-ethoxycoumarin O-deethylase activity of P450 2B1 was inactivated after incubation of the enzyme with 2EN for 10min in the presence of NADPH (Table I). In the absence of 2EN or NADPH, there was no appreciable loss of activity after 10min of incubation. Some inhibition of deethylase activity was seen without prior incubation (0time controls) and is most probably due to a competitive inhibition as seen previously with 2EN (20-22) and other inactivators (30, 31). In sharp contrast with the inactivation of deethylase activity by 2EN is the finding that there was only a 17% loss of cytochrome P450 content when the hemoprotein concentration was determined by difference spectroscopy. In other experiments, the addition of 7-ethoxycoumarin to the primary reaction mixture inhibited the 2EN-dependent inactivation of P450 2B1 deethylation: 44 % of the deethylase activity remained after a 4-min incubation when the ratio of 7-ethoxycoumarin to 2EN was 200:l in the primary reaction mixture, and only 21 % activity remained when 7-ethoxycoumarin was omitted from the primary incubation (data not shown). If an electrophilic product from 2EN was being released from the active site and then inactivating the protein, the

Inactivation of P450 2Bl by 2-Ethynylnaphthalene 1

Chem. Res. Toxicol., Vol.6,No.4, 1993 473

2.0 2.5

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-

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0.5

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-

-20 2.0 0.0

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Time (mln)

Figure 1. Time- and concentration-dependent inactivation of

7-ethoxycoumarin 0-deethylase activity by ZEN. Incubation conditions were as described under Experimental Procedures. The concentrations of ZEN were ( 0 )0, (B) 0.05 KM,(0) 0.1 pM, ( 0 )0.15 rM, and (A)0.25 pM. addition of an exogenous nucleophile such as glutathione would be expected to decrease the inhibition by 2EN by trapping the electrophile and preventing the reaction with the enzyme. The addition of 250 pM glutathione to the primary reaction mixture did not have any effect on the inactivation of P450 2B1 by 2EN. Furthermore, the initial enzyme activity was observed when fresh P450 2B1 reconstituted with lipid and reductase was added to the spent reaction mixture, suggesting that the loss of activity was not due to product inhibition or the lack of substrate or NADPH. In addition, no increase in activity was observed upon addition of 500 pM iodosobenzene to the secondary reaction mixture. Iodosobenzene is an alternate oxidant capable of producing an activated iron-oxygen species with P450 that can support the metabolism of 7-ethoxycoumarin without the presence of reductase (32). Finally, after the removal of excess2EN by repeated cycles of washing and concentrating with a microconcentrator, the P450 2B1 and reductase were reconstituted with lipid. The P450 2B1 showed the same level of inactivation as compared to pretreatment, demonstratingthat irreversible inactivation of the enzyme had occurred. The proteins were shown to withstand the washing and concentrating since a control mixture in which no NADPH was present during the inactivation retained approximately 90 % of its 0-dealkylation activity after the procedure. The rapid inactivation of the 7-ethoxycoumarin deethylase activity of P450 2B1 at various concentrations of 2EN is shown in Figure 1. In all cases, pseudo-first-order kinetics were observed for the initial phase of the inactivation. The time-dependent inactivation of P450 2B1 by 2EN is superimposed upon the competitive inhibition of deethylase activity. The extent of this inhibition is evident a t the ordinate intercept and increases with increasing 2EN concentrations. First-order inactivation constants were determined by linear regression analysis as the slopes of the lines. From the plot of the reciprocal of the initial rate of inactivation as a function of the reciprocal of the inhibitor concentration, the maximal rate of inactivation at saturating levels of 2EN (kinactivation) was determined to be 0.83min-l and the inhibitor concentration required for the half-maximal rate of inactivation (KI) was 0.08 pM (Figure 2). Metabolism of 2EN by P450 2B1. The metabolites formed by incubating radiolabeled 2EN with P450 2B1 recodstituted with reductase and lipid were analyzed by reverse-phaseHPLC with detection by UV (Figure 3, panel

-10 0 11[2EN] (pM

10

')

20

Figure 2. Double-reciprocd plot of the rate of inactivation of 7-ethoxycoumarin0-deethylaaeactivity as a functionof inhibitor

concentration.

A) or liquid scintillation counting (panel B). As was seen with P450 1A2 in microsomes from O-naphthoflavonetreatedrats (201,there was one major peak with aretention time of 21 min that coeluted on HPLC with 2-naphthylacetic acid. This metabolite was collected, derivatized with MTBSTFA, and analyzed by GC-MS. The analyte displayed a fragmentation pattern consistent with the 2-naphthylacetic acid standard. In addition, there were two smaller peaks that accounted for less than 12% of the total products formed. The average yield of the 2-naphthylacetic acid determined from two independent experiments was 4.0 mol of acid/mol of P450 2B1. The enzyme lost greater than 90% of its original activity under the conditions used to determine product formation. Therefore, the partition ratio, defined as the number of metabolite molecules produced per enzyme molecule inactivated, is approximately 4-5. Irreversible Labeling of P450 2Bl. A series of experiments were performed in order to determine the stoichiometry of binding and the pathway for the mechanism-based inactivation of P450 2B1. Shown in Figure 4 are the HPLC profiles of the reconstituted system after incubation with i3H12EN in the presence (panel A) or absence (panel B) of NADPH. The components of the system, catalase, reductase (both the short and full-length forms), and P450 2B1, were separated on the POROS column. In addition, under the acidic conditions used for the analysis, the heme was stripped from the P450 and eluted earlier than the protein components with aretention time of 3.0 min. The HPLC profile of the radioactivity is shown in panel C. In the presence of NADPH, there was radioactivity associated with the peak representing P450 2B1. The other radioactive peak elutes with 100% solvent B and is also seen in the control incubation in which NADPH was absent. In preparing these samples for HPLC analysis, the excess PHI 2EN was removed by concentrating the samples with microconcentrators. The proteins were then resuspended in buffer (20times volume) and concentrated again. It was difficult to remove all the counts from the sample incubated without NADPH. The HPLC chromatogram in panel C shows that the counts remaining in the control after extensive washing were not associatedwith any of the protein components. The results from the HPLC analysis were then used to determine the amount of P450 2B1 in the samples after inactivation and concentration. An aliquot of each sample was counted to determine the amount of 2EN. After subtracting the amount of 2EN in the control sample, the binding ratio was determined to be 1.3 mol of i3H12EN/mol of P450 2B1. When the HPLC separation of the proteins was monitored for heme absorbance at 410 nm, the only peak

474 Chem. Res. Toxicol., Vol. 6, No.4,1993

Roberts et a1.

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Figure 3. HPLC reverse-phase analysis of the metabolites extracted from incubationmixturescontaining [SH] 2EN and a reconstituted system including P450 2B1. The incubation and chromatographic conditions were as described under Experimental Procedures. Panel A HPLC elution profile with detection at 220 nm. Peak 1represents the internal standard benzoic acid. The compound represented by peak 2 was identified by GC-MS as 2-naphthylaceticacid. Panel B: HPLC elution profile monitored by liquid scintillation counting of a reaction mixture incubated with ( 0 )or without NADPH (+).

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Figure 4. HPLC reverse-phase analysis of the reconstituted system after incubation with 2EN in the absence or presence of NADPH. The incubation and chromatographic conditions were as described under Experimental Procedures. Panel A HPLC elution profile monitored at 214 nm of the reconstituted system incubated in the presence of NADPH. Peak 1-4 represent catalase, short reductase, reductase, and P450 2B1, respectively. Panel B: HPLC elution profile of a control mixture incubated without NADPH. Panel C: HPLC elution profile monitored by liquid scintillation counting of a reaction incubated with (a) or without NADPH (e).

seen after incubation in the presence or absence of NADPH was that of the free heme and no counts were associated with this peak. Many laboratories have used dialysis to remove excess inactivator in the determination of the binding stoichiometry. Due to the hydrophobic nature of 2EN, we encountered problems in removing the excess 2EN by dialysis and found the HPLC analysis more informative and reliable. The washed and concentrated samples were analyzed on a 7.5 % polyacrylamide gel under denaturing conditions, and essentially all of the radioactivity was found to be associated with the band corresponding to P450 2B1. Protein labeling in this system was also NADPH-dependent. In agreement with the results from the perfusiveparticle chromatography, the radioactivity is associated with the apoprotein and not with the heme moiety since the noncovalently attached heme is stripped from the

protein under the denaturing conditions used for electrophoresis. Identification of Labeled Peptides. The r3H12ENinactivated P450 2B1 and reductase were digested with trypsin, and the resulting peptides were separated by reverse-phase HPLC and analyzed by UV detection or liquid scintillation counting (Figures 5 and 6). There were two NADPH-dependent radiolabeled peaks. The peak eluting at 52 min comigrated with 2-naphthylacetic acid, and the peak at 96-98 min was further analyzed by Tricine SDS-PAGE (Figure 7). There was an NADPH-independent radiolabeled peak that eluted a t a high concentration of organic solvent (160 min) just as seen in the HPLC analyses of the proteins (Figure 4). It is important to note that this peak, the large radioactive peak eluting at 96-98 min, and almost all of the broad UV-absorbing peaks eluting after 80 min would not be present if the proteins

Chem. Res. Toxicol., Vol. 6, No. 4, 1993 475

Inactivation of P450 2B1 by 2-Ethynylnaphthalene

I

I

0

I

1

I

20

I

I

I

60

40

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100

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Figure 5. Reverse-phase HPLC analysis of a tryptic digest of reductase and [3H]2EN-inactivated P450 2B1. The inactivation of P450 2B1, digestion with trypsin, and HPLC separation were carried out as described in Experimental Procedures. Detection was at 214 nm, and only the portion of the chromatogram that remained on scale is shown.

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Time (mln)

Figure 6. HPLC analysis of the tryptic digest after incubation in the presence ( 0 )or absence of NADPH (A)with detection by liquid scintillation counting. Ten percent of each 2-mL fraction was counted. Free 2-naphthylacetic acid eluted at 52 min. The peptide eluting at 96-98 min was analyzed by Tricine-SDSPAGE (see Figure 7).

had been centrifuged to pellet insoluble material prior to the HPLC analysis. When analyzed by Tricine-SDSPAGE, the 96-98-min fraction contained one radiolabeled peptide with an approximate molecular weight of 6.5 kDa when visualized by Coomassie blue staining (Figure 7). In addition, when the 6.5-kDa band was cut out of the gel and solubilized, 85% of the counts loaded onto that lane were associated with this band. After purification by HPLC, the presence of peptide was confirmed by amino acid analysis, but no sequence data could be obtained directly from the peptide, presumably because the amino terminus was blocked. After cleavage at methionine residues with cyanogen bromide directlyon the sequencing filter, two internal sequences were determined by automated Edman analysis (Table 11). The peptide sequences were IS(L)L(SL)FFand LKYP(H)VAE,which correspond to positions 290-297 and 315-322, respectively, of rat 2B1 (33,34). In each case, the first residue follows a methionine residue. In order to more specificallylocate the labeled peptide, the intact proteins were cleaved with cyanogen bromide and the resulting peptides analyzed by Tricine-SDSPAGE or reverse-phase HPLC. As shown in Figure 8, there was a radiolabeled band at approximately 3.0 kDa

8.2

- 6.2

- 3.5 2.5

Figure 7. Tricine-SDS-PAGE analysis of the radiolabeled tryptic peptide from [3H]BEN-inactivated P450 2B1. Electrophoresiswas carried out as describedin Experimental Procedures. Panel A Lane 1, Coomassie Blue-stained gel of radiolabeled fraction (96-98 min) from an HPLC separation as described in Figures 5 and 6; Lane 2, molecular weight markers. Panel B: Autoradiography of the gel shown in panel A, lane 1.

after CNBr cleavage. On the basis of the sequence of 2B1 (33,34),all of the CNBr-generated fragments from P450 2B1 except a 5-residue peptide should remain on the gel. After transfer of the peptides to PVDF membrane, the 3.0-kDa peptide was sequenced and yielded the peptides ISLLSLFF and QLREKYGD (Table 111)which correspond to positions 290-297 and 57-64, respectively. These peptides have a predicted molecular mass of approximately 3.0 kDa on the basis of the CNBr cleavage of P450 2B1 and therefore would not be expected to separate under these conditions. The CNBr-cleaved proteins were also analyzed by reverse-phase HPLC. The radiolabeled fraction that eluted late in the gradient was sequenced, and the peptides ISLLSLFFA and PFSTGKRI (at positions 428-435) were identified (Table IV). Since the latter peptide would have a molecular weight of 7.0 kDa and no labeled peptide of this size was seen on the gel (Figure 8), it follows from the results of the three

476 Chem. Res. Toxicol., VoZ. 6, No. 4, 1993

Roberts et al.

Table 11. Amino Acid Sequence Data of the 6.5-kDa Tryptic Peptide Isolated from [3H]2EN-Inactivated P450 2B 1. amino acid

cycle

vmol

Ib

22 9.3

S (L)d L

2.0

(SI

amino acid

vmol

0.7 1.5

K Y P

E

...

1

2

amino acid

...

cycle

amino acid

S L L

S

14.4

10.6 8.2

6.2 3.5 2.5

L

-

F F A

.-

&" L

R E

K Y G D

pmol 4.1 4.0 4.9 5.6 4.9 3.9 2.4 4.9

Table IV. Amino Acid Sequence Data of Peptides Isolated from [3H]2EN-Inactivated P450 2B 1 after CNBr Cleavage and HPLC Separation.

Ib

17.0

amino acid

aThe CNBr cleavage and electrophoresis were carried out as described in Experimental Procedures. The CNBr-generated peptides were transferred after electrophoresis to an Immobilon P W PVDF membrane, stained with Coomassie Blue, and a 2.5-3.5-kDa band cut out for sequencing. b The peptide corresponds to positions 290-297 of the protein. The peptide corresponds to positions 5764. The amount of amino acid a t each cycle has been corrected for background.

B

kDa

pmol 30 5.0 5.2 2.9 0.2 3.5 1.2 2.3

2.9 1.3 1.1

A

a No sequence data were obtained directly from the 6.5-kDatryptic peptide purified as described in Figures 5-7. When an aliquot of cyanogen bromide was added to the peptide on the sequencing filter, two sequences were obtained. The amount of amino acid at each cycle has been corrected for background. The peptide corresponds to positions 290-297 of the protein. The peptide corresponds to positions 315-322 of the protein. The residues in parentheses were not identified from the sequence data but were inferred from the published sequence of 2B1 (33,34). These sequences are internal sequences that follow methionine residues as shown: HENLMIS(L)L(SL)FFAGTETSSTTLRYGFLLMLKYP (H)VAE

A

cycle

8.1 5.4 6.8 6.0

LC

(HI V

(L) F F

Table 111. Amino Acid Sequence Data of Peptides from [3H]2EN-InactivatedP450 2Bl after CNBr Cleavage and Tricine-SDS-PAGE Separation.

pmol 16.5 33 13 14.4 8.0 9.8 6.3 6.7 8.9

amino acid

pmol

P"

20.8

F

14

[Sld T G K R

I

9.2 13.1 4.3 1.5 6.9

a The CNBr-generated peptides were separated by HPLC on a Vydac Cq column as described in Experimental Procedures, and the fraction containing radioactivity and eluting late in the gradient (67 min) was sequenced. The amounts of amino acid were manually calculated from the raw data. b The peptide corresponds to positions 290-298 of the protein. The peptide corresponds to positions 428435. This residue was positively identified, but the amount was difficult to determine because of carry-over from the previous cycle.

-

-

3

Figure8. Tricine-SDS-PAGE analysis of the cyanogen bromide-

2EN-inactivated generated peptides from reductase and [3H] P450 2B1. The CNBr cleavage was carried out as described in Experimental Procedures. Panel A Lane 1, Coomassie Bluestained gel of molecular weight markers; lane 2, CNBr-generated peptides from P450 and reductase. Panel B: Autoradiography of gel shown in panel A, lane 2.

sequencing experiments that the [3H]2EN is bound to the peptide ISLLSLFFAGTETSSTTLRYGFLLM.

Discussion The 2EN-dependent inactivation of P450 2B1 exhibits a number of characteristics consistent with mechanismbased inactivation. The inactivationof 7-ethoxycoumarin 0-deethylation activity requires NADPH, is irreversible, is decreased in the presence of an alternate substrate, and is not affected by the presence of an exogenousnucleophile. Kinetically the loss of activity is pseudo-first-order and saturable. 2EN is an effective suicide inhibitor, with KI and kinadivation values of 0.08 pM and 0.83 min-l, respectively. These results correlate well with those of Hopkins et al. in which 2EN was found to be an effective inactivator of P450 2B1-dependent depentylation of 7-pentoxyphenoxazone (7-pentoxyresorufin)in liver microsomes from

phenobarbital-treated rats with values of 0.14 p M and 0.23 min-l for KI and kinactivation, respectively (22). Three pathways for the mechanism-based inactivation of cytochrome P450 have been characterized (35). One pathway involves the modification of the heme moiety to products that dissociate from the protein as exhibited by several allenic and acetyleniccompounds that form green pigments resultingfrom N-alkylation of the iron porphyrin (36). The second pathway involves the covalent modification of the apoprotein as exhibited by chloramphenicol (37-39). A third mechanism involvesthe covalent binding of the heme to the protein, and a number of compounds cause inactivation through this pathway, including CC4 (40). In the present study, the evidence that there is no appreciable loss of the P450 chromophore and that radiolabeled 2EN remains associated with P450 2B1 on denaturing PAGE discounts the first mechanism in which the heme is alkylated and dissociates from the protein. In addition, under acidic conditions on reverse-phase HPLC the radiolabel remains associated with the P450 moiety. No radioactivity is associated with the heme peak, and there is no increase in absorbance at 410 nm in the area where the proteins elute. Thus, the conclusion can be drawn that the mechanism of inactivation of P450 2B1 by 2EN is through irreversiblemodificationof the apoprotein. After digestion with trypsin or CNBr, the 2EN was found by analysis on Tricine-SDS-PAGE to be irreversibly attached to peptide fragments. In these types of analyses

.

Chem. Res. Toxicol., Vol. 6, No. 4, 1993 477

Inactivation of P450 2Bl by 2-Ethynylnaphthalene

0 CH3:C-O

C y

-C

NIS-P~X)

Figure 9. Proposed scheme for the formation of 2-naphthylacetic acid and labeling of P450 2B1 by 2EN.

care should be taken in showing that the radiolabeled compound is in fact attached to the peptide and not associated in a hydrophobic manner or simply eluting with the peptides in the HPLC gradient. After chemical or enzymatic digestion and under acidic conditions,we believe that some of the radiolabel is hydrolyzed and appears as free 2-naphthylacetic acid. In the reverse-phase HPLC analysis of tryptic peptides from 2B1 (Figure 6) there is a peak that comigrates with 2-naphthylacetic acid. This peak increases under acidic conditions including cleavage with CNBr. The chemistry of inactivation of P450 2B1 by 2EN is thought to proceed through the formation of a ketene intermediate within the active site of the enzyme (Figure 9) as proposed by Ortiz de Montellano et al. (41-43). This could explain the products of the reaction including the formation of radiolabeled protein from nucleophilic attack on the ketene by an amino acid at the active site of the enzyme and the formation of 2-naphthylacetic acid from hydrolysis of the ketene intermediate. The quantitation of 2-naphthylacetic acid was used to calculate a partition coefficient of approximately 4-5, which indicates that the inactivation occurs very efficiently. This is consistent with our data in Table I, which show essentially complete inactivation in incubations where the molar ratio of 2EN to P450 was 5:l. The calculated partition coefficient for 2EN can be compared to the partition ratios calculated for P450 2B1 with substituted phenylacetylenes that range from 38 for @-methylpheny1)acetylene to 4 for @nitropheny1)acetylene (43). In contrast with these inactivators that result in primarily heme alkylation, 2EN seems to be the most efficient acetylenic mechanism-based inactivator of P450 2B1 that results in predominantly protein modification. This could be explained by specific binding of 2EN in the active site of P450 2B1, with the ketene intermediate oriented in a position that facilitates nucleophilic attack by an amino acid at the active site. Hopkins et al. (22)found that both the size of the aromatic ring system and the placement of the ethynyl functional group on a polycyclic aromatic acetylene influences the effectiveness of the inhibition and the ability to produce a mechanism-based inactivation of 7-pentoxyphenoxazone depentylation catalyzed by P450 2B1 in microsomes. They found that all of the aryl acetylenes were reversible inhibitors, but only relatively small, compact compounds produced a mechanism-based inactivation (22). In this study, the sequencing of the radiolabeled peptides from P450 2B1 leads to the identification of a 2ENmodified peptide with the sequence ISLLSLFFAGTETSSTTLRYGFLLM corresponding to residues 290-314 of P450 2B1. The nucleophilic residues in this peptide include Ser, Thr, Glu, Arg, Tyr, and Met. The adducts formed by reaction of a ketene with Glu or Met would probably be too unstable to isolate. The slight acid lability

of the adduct as evidenced by the release of 2-naphthylacetic acid from the protein (Figure 6) could be explained by an ester linkage to Tyr, Ser, or Thr, the latter two being very abundant in this segment. The inability of trypsin to cleave at Arg could be due to modification at this residue or due to the inability of trypsin to cleave in this very hydrophobic segment. To explain the size of the tryptic peptide that was isolated (6.5 kDa), we have to assume that trypsin did not cut at other Arg or Lys residues in this region. On the basis of hydropathy profiles ( l a ) ,the most polar section of P450 2B1 occurs just before the identified peptide, and the Lys276 to Ser277 peptide bond has been identified as the site of rapid proteolysis when 2B1 is cleaved in liver microsomes (44). A strongly hydrophobic segment that includes the isolated tryptic peptide follows this site of rapid cleavage. We encountered difficulty in the subdigestion of this segment with other proteases including V8 protease even in the presence of SDS, and the hydrophobicity of the peptides may be the reason for the difficulty in proteolytic digestion. It is interesting to note that the peptides identified by Yun et al. (21) after inactivation of rat and rabbit 1A2 are different from the one identified in this study. Once again, this could reflect the orientation of the ketene intermediate in the active site of each enzyme and the different amino acids that are available for nucleophilic attack. For example, the segment identified in P450 2B1 contains 5 additional nucleophilic amino acids as compared to the same region in P450 1A2. In addition, the work by Halpert et al. led to the identification of a lysine residue of P450 2B1 that was covalently modified after suicide inactivation by chloramphenicol (38). They found that chloramphenicol-inactivated P450 was able to catalyze the iodosobenzenesupported but not the reductase-supported metabolism of 7-ethoxycoumarin. This was attributed to the binding of chloramphenicol to amino acid residues of P450 2B1 close to the heme moiety which blocks electron transport from reductase (32). Thus the mechanism of inactivation by 2EN appears to be different than seen with chloramphenicol since the 7-ethoxycoumarin activity of 2ENinactivated P450 2B1 was not supported by iodosobenzene. Since there is no crystal structure available for a eukaryotic P450, many alignments with P450 101 have been used to aid in the development of a hypothetical three-dimensional model of an eukaryotic P450 (13-18). If the labeled peptide was aligned with the sequence of P450 101, this segment would correspond to helix I of the bacterial protein (17). The central region of helix I, which in P450 101 forms part of the oxygen binding pocket, is one region that exhibits a high degree of homology among the proteins. The same functional requirements for 02 binding and activation in all P450s suggest a similar structure for the oxygen binding pocket. In P450 101, helix I is located at the distal side of the heme surface and

478 Chem. Res. Toxicol., Vol. 6,No. 4, 1993

centers around Thr252. This helix undergoes a local distortion and kinking in the vicinity of Thr252. The hydrogen bonding capabilities of Thr252 disrupt the local helical hydrogen bonding pattern, forcing a widening of the helix and providing a part of the 02 binding pocket (17). The important role that this segment plays in substrate binding in eukaryotic P450s has been well documented with the use of site-directed mutagenesis (4550). Moreover, antibodies to an area in helix I would be expected to give only a weak response since it is presumed to be buried in the interior of the globular protein. This was observed in microsomes for a site-specific antibody to the predicted center of helix I in P450 2B1, while antibodies for the segment extending from the end of helix I to the end of helix J gave a strong response (51). After further analysis of the radiolabeled peptide to determine the residue that is modified, site-directed mutagenesis can be carried out to investigate the role this amino acid plays in the inactivation. In addition, it will be interesting to examine the inactivation of other similar P450s and to compare whether the inactivation and binding to this segment can be predicted by analyzing the sequences of the different enzymes.

Acknowledgment. We are grateful to David A. Putt for purifying cytochrome P450 2B1 and reductase and to June Snow of the Macromolecular Core Facility at Wayne State University for performing the peptide sequencing and for many helpful discussions regarding peptide isolation and purification. This work was supported by Grant CA 16954 from the National Cancer Institute, USPHS. E.S.R.was a postdoctoral trainee on Cancer Biology Training Grant T32-CA09531 from the National Cancer Institute. References Gonzalez, F. J. (1988) The molecular biology of cytochrome P450s. Pharmacol. Rev. 40, 243-288. Porter, T. D., and Coon,M. J. (1991)CytochromeP450. Multiplicity of ieoforms, substrates, and catalytic and regulatory mechanisms. J. Biol. Chem. 266,13469-13472. Nebert, D. W., Nelson, D. R., Coon, M. J., Estabrook, R. W., Feyereisen, R., Fujii-Kuriyama, Y., Gonzalez, F. J., Guengerich, F. P., Gunealua, I. C., Johnson, E. F., Loper, J. C., Sato, R., Waterman, M. R., and Waxman, D. J. (1991) The P450 superfamily: update on new sequences, gene mapping, and recommended nomenclature. DNA Cell Biol. 10, 1-14. Poulos, T. L., Finzel, B. C., Gunsalus, I. C., Wagner, G. C., and Kraut, J. (1985)The 2.6-8, crystal structure of Pseudomonas putida cytochrome P-450. J. Biol Chem. 260, 16122-16130. Poulos,T. L., Finzel, B. C., and Howard, A. J. (1987)High-resolution crystal structure of cytochrome P450cam. J. Mol. Biol. 196, 687700. Onoda, M., Haniu, M., Yanagibashi, K., Sweet, F., Shively, J. E., and Hall, P. F. (1987) Affinity alkylation of the active site of Czl steroid side-chain cleavagecytochrome P-450 from neonatal porcine testis: a unique cysteine residue alkylated by 17-(bromoacetoxy)progesterone. Biochemistry 26,657-662. Johnson, E. F., Kronbach, T., and Hsu, M. (1992) Analysis of the catalytic specificity of cytochrome P450 enzymes through sitedirected mutagenesis. FASEB J. 6, 700-705. Sakaki, T., Shibata, M., Yabueaki, Y., Murakami, H., and Ohkawa, H. (1989) Expression of bovine cytochrome P460c17 cDNA in Saccharomyces cerevisia. DNA 8, 409-418. Imai, Y. (1988) Characterization of rabbit liver cytochrome P-450 (laurate 0-1hydroxylase) synthesized in transformed yeast cells. J. Biochem. 109, 143-148. Pompon, D., and Nicolas, A. (1989) Protein engineering by cDNA recombination in yeast: shuffling of mammalian cytochrome P-450 functions. Gene 83, 15-24. Hanioka, N., Korzekwa, K., and Gonzalez, F. J. (1990) Sequence requirements for cytochromes P450IIA1 and P450IIA2 catalytic activiw. evidencefor both specificand non-specificsubstrate binding interactions through uee of chimeric cDNAs and cDNA expression. Protein Eng. 3, 571-575.

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