Introduction

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Cytochrome P450 (P450)⁴ enzymes are microsomal heme-containing monooxygenases found in almost every biological kingdom (Nelson et al., 1996). The significance of these enzymes lies in their ability to detoxify endogenous and exogenous compounds through a two-electron reduction of molecular oxygen to form a reactive oxygen species and water (Porter and Coon, 1991). P450s can also generate potentially carcinogenic or toxic compounds in the process of catalyzing the metabolism of xenobiotics (Ortiz de Montellano, 1996). To better understand this phenomenon, extensive studies have been undertaken to identify the critical P450 active site amino acid residues involved in substrate binding and catalysis.

The P450 2B subfamily was among the first mammalian P450s to be extensively studied and characterized because of its ability to readily be induced by phenobarbital. P450 2B1 is the major phenobarbital-inducible P450 in rat liver (Gonzalez, 1989). The human homolog of P450 2B1 is P450 2B6. Although P450 2B6 constitutes only 0.2% of the total P450s in human liver microsomes, it metabolizes 3% of drugs in clinical use (Lewis et al., 1998). P450 2B1 and 2B6 are 78% homologous in terms of their cDNA sequence, and share 74% identity at the amino acid level. Differences occur in the putative active site region and may therefore have some relevance to species differences in P450 2B-mediated metabolism (Lewis et al., 1998). Currently, no high-resolution three-dimensional crystal structure of a mammalian microsomal cytochrome is available. Therefore, several methods have been used to determine the critical amino acid residues involved in P450 activity, such as comparison of naturally occurring mutants (Kedzie et al., 1991), site-directed mutagenesis (Halpert, 1995), use of mechanism-based inactivators, and homology modeling with bacterial cytochromes (Roberts et al., 1993; He et al., 1996; Strobel et al., 1999).

Mechanism-based inactivation is one method that has been successfully used to analyze the structure of mammalian P450s. A mechanism-based inactivator is a relatively inert compound that must be activated by the target enzyme to a reactive species, which then inactivates the enzyme before its release from the active site. If the mechanism-based inactivator binds to the apoprotein of the P450 rather than the heme moiety, then the radiolabeled inactivator can be used to determine part of the peptide chain, or even which amino acid residue(s) may be involved in substrate binding or catalysis.

Phencyclidine (PCP), a popular drug of abuse known for its long-term psychotic effects, has been shown to inactivate P450 2B1 in a time-, concentration-, and NADPH-dependent manner that exhibited pseudo first order kinetics (Crowley and Hollenberg, 1995). The inactivation of P450 2B1 by PCP was not due to a modification of the heme moiety, because little loss in either the absolute or CO-reduced spectrum of the inactivated protein was seen (Crowley and Hollenberg, 1995). Subsequently, Sharma et al. (1997) showed that an anti-PCP antibody recognized the PCP-inactivated P450 2B1 protein by Western blot analysis. The PCP-metabolite adducts were detected by an antibody raised against the 5-[N-(1'-phenylcyclohexyl)amino] pentanoic acid (PCHAP) hapten-BSA conjugate, which contained both the phenyl- and cyclohexyl rings of PCP (Owens et al., 1988).

The aim of a previous study using liver microsomes from phenobarbital-induced rabbits was to determine the structural basis of inactivation of the benzphetamine demethylation activity by PCP with a PCP analog lacking the readily metabolized cyclohexane ring, namely, 2-phenyl-2-(1-piperidinyl)propane (PPP; the structure is shown in Fig. 1) (Sayre et al., 1995). PPP was therefore thought to be a useful tool to study the structure of the active sites of purified P450s 2B1 and 2B6 in a reconstituted system and potentially provide further information about the metabolic pathway of PCP and PCP analogs. In this report, we investigated the interaction of PPP and cytochrome 2B enzymes with the expectation that PPP would be a more effective mechanism-based inactivator than PCP and would thus be useful in labeling and identifying amino acid residues in the active site critical for PPP/PCP binding or catalysis. PPP exhibited concentration-, time-, and NADPH-dependent inactivation of P450 2B1 and P450 2B6. PPP was also shown to inactivate both P450 isozymes primarily through modification of the apoprotein rather than alkylation of the heme moiety.

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Structures of PCP and PPP. The asterisk (*) indicates the position of the tritium label.

Experimental Procedures

Materials. Dilauroyl-L--phosphatidylcholine (DLPC), NADPH, BSA, 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) and 7-hydroxy-4-(trifluoromethyl)coumarin was purchased from Enzyme Systems Products (Livermore, CA). HPLC-grade methanol and ethyl acetate were obtained from Fisher Scientific (Pittsburgh, PA). BCA reagent and Slide-A-Lyzer cassettes were obtained from Pierce (Rockford, IL). Ultima Gold liquid scintillation cocktail was obtained from Packard (Meriden, CT). Centricon-30 micro concentrators were purchased from Amicon (Danver, MA). PCHAP antiserum was a gift from Dr. Michael Owens (University of Arkansas, Little Rock, AK); PPP and its dehydrogenated form, the PPP-iminium ion, were obtained from a previous study (Sayre et al., 1995).

Purification of P450 and NADPH-Cytochrome P450 Reductase (Reductase). P450 2B1 was purified from microsomes isolated from livers of fasted male Long Evans rats (175-190 g; Harlan Sprague-Dawley, Indianapolis, IN) and given 0.1% phenobarbital in the drinking water for 12 days according to Saito and Strobel (1981). Reductase and P450 2B6 were purified after expression in Escherichia coli as previously described (Hanna et al., 1998; Kent et al., 1999).

Microsome Preparation. Microsomal membranes were prepared from the livers of fasted male Fischer rats (175-190 g; Harlan Sprague-Dawley, Indianapolis, IN) according to Saito and Strobel (1981). P450 2E1 was induced by i.p. injection of 100 mg of pyridine in water/kg for 3 days (Kim and Novak, 1993). P450 2B1 was induced by i.p. injection of 100 mg of phenobarbital in water/kg for 3 days (Hardwick et al., 1983). P450 1A1/1A2 were induced by i.p. injection of 80 mg of -naphthoflavone in corn oil/kg for 3 days (Pasco et al., 1993). P450 3A2 was induced by i.p. injection of 50 mg of pregnenolone-16-carbonitrile in corn oil/kg for 3 days (Elshourbagy and Guzelian, 1980).

Enzyme Activity Assays and Inactivation. Microsomal P450 2B1 activity was measured with 7-benzyloxyresorufin as the substrate (Nerurkar et al., 1993). Each primary reaction mixture contained 1.5 µM P450 from microsomes isolated from phenobarbital-treated rats in 50 mM potassium phosphate buffer (pH 7.4), 0, 2, 5, 10, 25, and 50 µM PPP, and 1.2 mM NADPH. After incubating with NADPH for the indicated times at 30°C, 25-µl aliquots were transferred to 975 µl of a secondary reaction mixture containing 5 µM 7-benzloxyresorufin and 0.2 mM NADPH in 50 mM Tris-HCl buffer (pH 7.4). The reaction was quenched after 3 min with 750 µl of CH₃OH. The resorufin product was measured spectrofluorometrically with excitation at 522 nm and emission at 586 nm.

Substrate Protection. Substrate protection from PPP-dependent inactivation of P450 2B1 was assayed by including 15 µM PPP together with 7-EFC at molar ratios of 1:0, 1:1, or 1:2 of PPP/7-EFC in the primary reaction mixture. At the indicated times, duplicate 10-µl aliquots were removed and assayed for remaining activity as described above. For P450 2B6, the PPP concentration was 40 µM and the remaining activity was measured using 12-µl aliquots.

Partition Coefficient. To estimate the partition coefficient, samples were incubated in the presence of 0.01 to 1 mM PPP for 20 min to assure the inactivation had proceeded to completion. Duplicate aliquots were removed and assayed for 7-EFC activity as described above (Silverman, 1996).

Irreversibility of PPP Inactivation. P450 2B1 (0.5 nmol) and P450 2B6 (0.5 nmol) were reconstituted as described above and inactivated with 1.4 µM PPP or 48 µM PPP, respectively, in a total volume of 138 µl. Control samples were incubated with PPP but without NADPH. After 10 min at 30°C the samples (0.13 ml) were dialyzed overnight at 4°C against 2 × 500 ml of 50 mM potassium phosphate buffer (pH 7.4) containing 20% glycerol and 0.1 mM EDTA. The dialyzed samples were reconstituted with 10 µg of lipid for 30 min on ice. Some samples also received fresh reductase. Enzymatic activity was assayed with 7-EFC as the substrate as described above.

Spectrophotometric Quantitation. At the times indicated, 200-µl aliquots of the primary reaction incubation were removed and diluted with 800 µl of ice-cold 50 mM potassium phosphate buffer (pH 7.4) containing 40% glycerol and 0.6% Tergitol NP-40. The samples were gently bubbled with CO for 90 s and the 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). Dithionite was added and the reduced carbonyl spectrum was recorded (Omura and Sato, 1964). For absolute spectral determinations, P450 2B1 and reductase were reconstituted with lipid at a 1:1 ratio. The final concentration was 1 µM P450 2B1, 1 µM reductase, 200 µg/ml DLPC, and 110 U/ml catalase in 50 mM potassium phosphate buffer (pH 7.4). The reference contained catalase and lipid in 50 mM potassium phosphate (pH 7.4). Spectra were recorded by scanning from 375 to 500 nm.

Western Blotting. P450 2B1 or P450 2B6 was reconstituted and incubated with PPP (40 and 41 µM, respectively) in the presence or absence of NADPH as described above. After 10 min (P450 2B1) or 30 min (P450 2B6), aliquots were removed to test for residual activity. The remaining samples were resolved by SDS-polyacrylamide gel electrophoresis, blotted to nitrocellulose, and probed with PCHAP antibody as previously described (Sharma et al., 1997).

Synthesis of [³H]PPP. To synthesize the radiolabeled PPP, 6 mg of the iminium ion chloride salt was suspended in 1 ml of methanol. The mixture was stirred rapidly at 4°C and NaB[³H₄] (1 mCi suspended in 1 ml of ice-cold methanol) was added. The reduction was allowed to proceed for approximately 5 min until the solid iminium salt was completely dissolved. Excess unlabeled NaBH₄ was then added and stirred for an additional 5 min. The mixture was covered and allowed to return to room temperature. The solvent was evaporated and the remaining solid was washed four times with 5 ml of ether. The washings were combined and evaporated. The product was suspended in 2 ml of water, and Na₂CO₃ was added until the pH was 11. The labeled PPP was then extracted into ethyl acetate. The organic phase was evaporated and the amine was reconstituted as the Cl salt in methanol containing a trace of HCl. The [³H]PPP was chromatographed on Silica TLC plates together with an authentic standard PPP in ethyl acetate/methanol/ammonium hydroxide (4:1:0.003, v/v/v). The amine was visualized at 254 nm. The R_f value of the PPP standard was 0.63 ± 0.01 and the R_f value of [³H]PPP was 0.64. The specific activity of the [³H]PPP was 0.23 Ci/mmol.

Stoichiometry. P450 2B1 (0.5 nmol) and 2B6 (0.5 nmol) were reconstituted with reductase (0.5 nmol) and lipid. The reconstituted mixture was incubated with 20 µM [³H]PPP in a final volume of 900 µl. For inactivation, samples received 1.2 mM NADPH and were incubated for 10 min (2B1) or 30 min (2B6) at 30°C. The reaction was stopped by placing the sample on ice and adding cholate to a final concentration of 5 mM. The samples were then dialyzed extensively against several changes of 50 mM potassium phosphate buffer (pH 7.4) containing 1 mM EDTA, 20% glycerol, and 5 mM cholate, until no more counts were detected in the buffer. The stoichiometry of binding was calculated after subtracting the counts associated with the control sample incubated without NADPH from the counts bound to the inactivated sample.

	Results

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Effect of PPP on the Enzymatic Activity of P450 2B1 in Microsomes. Figure 2 shows the time- and concentration-dependent inactivation of the 7-benzyloxyresorufin activity of liver microsomes obtained from phenobarbital-treated rats by 2 to 50 µM PPP. The kinetic constants describing this inactivation in microsomes by PPP at 30°C were determined by plotting the inverse of the rates of inactivation as a function of the inverse of the inactivator concentration (Fig. 2, inset). The maximal rate of inactivation at saturating PPP (k_inact) was 0.05 min¹. The concentration required to achieve the half-maximal rate of inactivation (K_I) was 11 µM, and the time required for half of the enzyme molecules to become inactivated (t_1/2) was 13 min.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Time- and concentration-dependent inactivation of the 7-benzyloxyresorufin O-dealkylation activity of P450 2B1 by PPP in rat liver microsomes. Samples were removed from the primary reaction mixture at the indicated time points and assayed for 7-benzyloxyresorufin activity as described under Experimental Procedures. The data shown are representative of the mean and S.D. of four separate experiments. The concentrations of PPP were () 0, () 2, () 5, () 10, () 25, and () 50 µM. The 100% control activity ranged from 1.2 to 5.0 nmol/nmol P450/min. The inset shows the double-reciprocal plot of the rates of inactivation of 7-benzyloxyresorufin O-dealkylation activity as a function of inhibitor concentration.

Inactivation of P450s 2B1 and 2B6 by PPP in the Reconstituted System. The inactivation of rat P450 2B1 in the reconstituted system was time- and concentration- dependent (Fig. 3). Pseudo first order kinetics were observed between 5 and 40 µM PPP. The kinetic constants were determined from the inset of Fig. 3 as described for Fig. 2. The k_inact was 0.3 min¹, the K_I was 12 µM, and the t_1/2 was 2.5 min.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Time- and concentration-dependent inactivation of the P450 2B1 7-EFC O-deethylation activity by PPP. At the indicated time points, samples were removed from the primary reaction mixture and assayed for 7-EFC activity. The concentrations of PPP were 0 (), 5 (), 10 (), 20 (), and 40 µM (). The inset shows the double-reciprocal plot of the rate of inactivation of 7-EFC O-deethylation activity as a function of inhibitor concentration. Data points shown represent the mean and S.D. from five separate experiments.

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View larger version (22K): [in this window] [in a new window]   Fig. 4.   Time- and concentration-dependent inactivation of the P450 2B6 7-EFC O-deethylation activity by PPP. Samples were removed from the primary reaction mixture at the indicated time points and assayed for 7-EFC activity as described under Experimental Procedures. The data shown are representative of the mean and S.D. of six separate experiments. The concentrations of PPP were () 0, () 0.1, () 0.5, () 2, () 5, and () 10 µM. The inset shows the double-reciprocal plot of the rates of inactivation of 7-EFC O-deethylation activity as a function of inhibitor concentration.

Effect of PPP on the 7-EFC O-Deethylation Activity and Heme Content of P450s 2B1 and 2B6. When P450 2B1 was incubated with PPP and NADPH for 5 min, a 68% loss in enzymatic activity was observed (Table 1). The same samples retained 80% of their ability to form a CO-reduced complex. Similarly, 84% of the absolute spectrum at 418 nm was retained when compared with control samples. P450 2B1 samples incubated only with PPP showed approximately 10% loss in activity and CO spectra after 5 min compared with control samples incubated without PPP.

Substrate Protection of P450 from Inactivation by PPP. Reduced rates of inactivation were observed when P450 2B1 was incubated together with 15 µM PPP and increasing concentrations of 7-EFC (Fig. 5A). Virtually no inactivation by PPP was seen when the molar concentration of the substrate exceeded that of PPP by 4-fold. P450 2B6 was incubated together with 40 µM PPP and increasing molar concentrations of 7-ethoxycoumarin. A decrease in the rate of loss in activity was observed when P450 2B6 was incubated with a 4-fold excess of 7-ethoxycoumarin over PPP (Fig. 5B).

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Substrate protection against inactivation of P450 2B1 and 2B6 by PPP. At the indicated time points, samples were removed from the primary reaction mixture and assayed. Each point represents the mean of three separate experiments. A, P450 2B1. The molar ratios of PPP/EC in the primary reaction mixture were 0:0 (), 1:0 (), 1:1 (), 1:2 (), and 1:4 (). B, P450 2B6. The molar ratios of PPP/EFC in the primary reaction mixture were 0:0 (), 1:0 (), 1:4 (), and 1:8 ().

Partition Ratio. The partition ratio, defined as the number of molecules of inactivator metabolized per molecule of enzyme inactivated was estimated from the data shown in Fig. 6 (Silverman, 1996). P450 2B1 and 2B6 were incubated with increasing concentrations of PPP, and the reactions were allowed to proceed to completion. The percentage of activity remaining was plotted as a function of the molar ratio of PPP to P450. The turnover number (partition ratio + 1) was extrapolated from the intercept of the linear regression and the straight line. The partition ratio for P450 2B1 was 31 and the ratio for P450 2B6 was 15. 

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Loss of the P450 2B1 7-EFC O-deethylation activity as a function of the ratio of PPP to P450 2B1 or P450 2B6. Each point represents the mean of three separate experiments. The partition coefficient was estimated from the intercept of the regression line derived from the lower PPP concentrations and the straight line obtained from the higher PPP concentrations. , P450 2B1; , P450 2B6.

Irreversibility of P450 Inactivation by PPP. Control P450 2B1 and 2B6 samples incubated with PPP but without NADPH and samples that were inactivated with PPP in the presence of NADPH were dialyzed extensively to test whether the inactivation by PPP was reversible. Table 3 shows that dialysis did not lead to a recovery of enzymatic activity for either enzyme. Adding fresh reductase back to the dialyzed samples also did not restore activity to the PPP-inactivated P450 2B1 or P450 2B6 samples.

Western-Blot of PPP-Inactivated P450 2B1. Figure 7 shows nitrocellulose blots of P450 2B1 either incubated with PPP in the absence of NADPH () or inactivated with PPP and NADPH (+) in the reconstituted system. Panel A shows a Ponceau S-stained blot of all the proteins in the reconstituted system. Duplicate samples probed with the PCHAP antibody are shown in the blot depicted in panel B. The antibody recognized only the PPP-inactivated P450 2B1. Panel C shows a blot of duplicate samples probed with the anti-P450 2B1 antibody. Control and inactivated P450 2B1 were both recognized.

View larger version (51K): [in this window] [in a new window]   Fig. 7.   Western blot of P450 2B1 incubated with PPP in the absence () or presence (+) of NADPH. A, Ponceau-S stain of reductase, catalase, and P450 2B1 in the reconstituted system; B, incubation with PCHAP antibody; C, incubation with an anti-P450 2B1 antibody.

Stoichiometry of PPP Binding to P450 2B1. The stoichiometry of radiolabeled PPP metabolite bound per P450 was determined after subtracting the counts from samples incubated with labeled PPP but without NADPH. With P450 2B1 and 2B6, a stoichiometry of 1.4:1 and 0.7:1 of labeled metabolite to P450 was obtained, respectively.

Effect of PPP on the Enzymatic Activities of P450s 2C9, 2D6, 2E1, 1A1/2, and 3A2. The effect of PPP on the activity of several other P450 isozymes was examined. Table 4 shows that within 10 min, P450 2C9 lost 28 and 66% of its activity with 0.1 and 1 mM PPP, respectively. P450 2D6 samples incubated with 0.01 to 1 mM PPP also showed a significant decrease in enzymatic activity. However, a considerable inhibition of in enzymatic activity was already observed at 0 min with 0.1 and 1 mM PPP. This observation could be due to inactivation by PPP carried over into the secondary reaction or due to competitive inhibition. PPP did not exhibit any effect on the activity of P450s 1A1, 1A2, 3A2, or 2E1 at concentrations up to 1 mM (data not shown).

	Discussion

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Previous studies with liver microsomes from rabbits treated with phenobarbital as well as studies with purified P450 2B4 in the reconstituted system demonstrated that PCP inactivated P450 2B4 in an NADPH-dependent manner (Hoag et al., 1984; Osawa and Coon, 1989). It was first proposed that the oxidation of the  carbon of PCP led to the formation of an electrophilic PCP-iminium species that was then hydrolyzed to form the PCP amino aldehyde capable of forming a protein adduct and of inactivating the P450 (Ward et al., 1982; Hoag et al., 1987). That the PCP-iminium species is an intermediate in the PCP inactivation pathway was supported by observations that the loss in NADPH-dependent enzymatic activity and P450 content also occurred when the PCP-iminium ion was used as the starting substrate (Hoag et al., 1987; Osawa and Coon, 1989). Hoag et al. (1988) subsequently isolated and identified a major metabolite resulting from the PCP iminium species. Because this compound, the corresponding 2-en-4-one, was found to be only a weak inhibitor, these authors proposed that the precursor 2-en-4-ol derived from the PCP iminium ion may be the intermediate responsible for alkylating P450 2B4 (Hoag et al., 1988). Later studies showed that PCP also inactivated P450 2B1 in the reconstituted system (Crowley and Hollenberg, 1995; Sharma et al., 1997). Studies with P450 2B1 indicated that the inactivation was primarily due to the binding of a metabolite of PCP to the apoprotein. These observations were in contrast to those with P450 2B4 where a loss in the P450-reduced CO spectrum implied that inactivation might be due to heme modification. Taken together, these results suggested that the critical PCP structural components required for inactivation were the phenyl- and the piperidyl ring. The minimal PCP analog that comprises only these structural elements is PPP, which was synthesized along with its iminium derivative. The PPP-iminium ion (data not shown) and PPP were both able to inactivate P450 2B1. The PPP-iminium ion appeared to be slightly more effective but also resulted in slightly greater loss in the CO spectrum (data not shown). For this reason and because we could use the iminium ion as a convenient starting compound for the synthesis of the radiolabeled PPP, we decided to characterize the inactivation of P450 2B1 and 2B6 by PPP. The data presented in this report demonstrate that PPP is a mechanism-based inactivator for both P450s 2B1 and 2B6. Metabolic activation of PPP was necessary for activity loss as indicated by the absolute requirement for NADPH. A pseudo first order loss of activity was observed with both enzymes and the rates of inactivation were both time- and concentration-dependent. Previous kinetic studies with P450 2B4 and PCP or the PCP iminium ion resulted in a K_I of 20 to 23 µM and in rates of inactivation of 0.07 min¹ (Osawa and Coon, 1989). The kinetic constants for PPP and P450 2B1 were similar (12 µM) and 10-fold lower for P450 2B6 (1.2 µM), suggesting that P450 2B6 has a higher affinity for PPP than P450 2B1. Similar results were obtained with P450 2B6 and PCP (M. Jushchyshyn, U.M. Kent, and P.F. Hollenberg, unpublished results). The rate of inactivation for P450 2B6 with PPP was the same as that determined for P450 2B4 with PCP in the reconstituted system, whereas the k_inact for P450 2B1 was approximately 50-fold faster. The partition ratios for PPP with P450s 2B1 and 2B6 (31 and 15, respectively) were again similar to those reported previously for PCP or the PCP iminium ion and P450 2B4 (Osawa and Coon, 1989). Decreased rates of inactivation were observed when the P450s were incubated with PPP in the presence of an alternate substrate. Because no activity was regained by including fresh reductase, it appears that reductase activity was not affected by PPP metabolism.

Binding of a PPP metabolite to the P450 apoprotein could be visualized using an antibody that had been generated to a PCP analog containing the phenyl- and cyclohexyl rings (PCHAP) conjugated to BSA (Owens et al., 1988). Only PPP-inactivated P450 2B1 was recognized on Western blots probed with the PCHAP antibody. The extent of antibody binding to P450 2B1 increased with time and correlated with the decrease in the 7-EFC activity (data not shown). No binding of a PPP metabolite to P450 2B1 or P450 2B6 could be visualized with the PCHAP antibody when the incubations were carried out with PPP in the absence of NADPH. However, when P450 2B6 was inactivated with PPP and NADPH and probed with the PCHAP antibody, reductase and catalase were also recognized along with the PPP-inactivated P450 2B6 (data not shown). Hoag et al. (1987) had observed non-NADPH-dependent binding of the PCP-iminium ion in rabbit microsomes. These observations would suggest that PPP required activation to form the adduct and that with P450 2B6, but not with 2B1, a reactive intermediate was able to escape from the active site. However, the presence of an exogenous nucleophile (1-5 mM GSH) during the primary incubation reactions with P450 2B6 and PPP had virtually no effect on the rates of inactivation. Similar observations were again made with P450 2B6 inactivated with PCP (M. Jushchyshyn, U.M. Kent, and P.F. Hollenberg, unpublished results). When P450 2B6 in the reconstituted system was inactivated with radiolabeled PCP or PPP and separated by HPLC, the majority of the label was with P450 2B6 (data not shown). No labeling by PPP was observed on P450 2B6 in samples that were not inactivated and separated under denaturing HPLC conditions. One explanation for these observations may be that the antibody recognized a PCP or PPP metabolite that was tightly bound to the protein and could not be dissociated by conditions for SDS-polyacrylamide electrophoresis, whereas the acidic denaturing conditions of the HPLC separation dissociated this noncovalently bound metabolite. A PPP adduct was detected on the apoprotein using radiolabeled PPP by reversed-phase HPLC of the reaction mixture (data not shown) and with anti-PCP antibodies. A stoichiometry of 0.7 to 1.4 mol of radiolabeled PPP metabolite/mol of P450 was observed. The approximate binding of 1 molecule of PPP/molecule of P450 suggests that the inactivation occurred exclusively at the active site and was not due to binding of a PPP metabolite elsewhere on the protein. The 1:1 stoichiometry was supported by liquid chromatography/mass spectrometry analysis of PPP-inactivated P450 2B1 (U.M. Kent, J. Chun, K. Regal, M. Schrag, and P.F. Hollenberg, unpublished results). Preliminary liquid chromatography/mass spectrometry analysis of PPP-inactivated P450 2B1 showed that the mass of the P450 protein increased by 241 Da. This increase is equivalent to the addition of the mass of one molecule of PPP plus two oxygen atoms. More extensive mass analysis studies are in progress to determine the mass of the PPP adduct. It has been suggested that the reactive intermediate responsible for the inactivation may result from the metabolism of the initial PCP iminium metabolite (Hoag et al., 1988; Osawa and Coon, 1989; Sayre et al., 1995) Hoag et al. (1988) suggested that the piperidine ring-derived 2-en-4-ol was the reactive intermediate of PCP, although additional work will be required to reveal the true inactivating species.

PPP also inactivated human P450 2C9. This result was not unexpected because previous observations indicated that P450 2C9 was inactivated by PCP (Laurenzana and Owens, 1997).

The findings described in this study indicate that PPP is an effective inactivator for P450 2B1 and its human homolog P450 2B6. Because the inactivation by PPP is primarily due to the binding of a metabolite of PPP to the apoprotein we are currently trying to identify the location of the PPP modification on P450 2B1 and 2B6. The similarities of PPP inactivation to those seen by PCP together with the recent finding of P450 2B enzymes in brain suggest that PCP metabolism in brain may lead to a modification of this isozyme (Tirumalai et al., 1998). In agreement with previous observations as cited above, these results also suggest that the cyclohexyl ring of PCP is not required for inactivation of P450s 2B and 2C9.