Chem. Res. Toxicol. 1996, 9, 223-226
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Active Site Topology of Human Cytochrome P450 2E1 Richard Mackman,† Zuyu Guo,‡,§ F. Peter Guengerich,‡ and Paul R. Ortiz de Montellano*,† Department of Pharmaceutical Chemistry and Liver Center, University of California, San Francisco, California 94143-0446, and Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146 Received July 20, 1995X
Cytochrome P450 2E1 (CYP2E1), an enzyme that is induced by ethanol, plays an important role in the metabolism of various toxic and carcinogenic substances, including dialkylnitrosamines and small halocarbons. Little information is available on the active site of the enzyme. We report here the formation of aryl-iron complexes (Fe-Ar) in the reactions of human CYP2E1 with phenyldiazene, (2-naphthyl)hydrazine, and p-biphenylhydrazine. Migration of the aryl group from the iron to the porphyrin nitrogens within the intact active site produces the four possible N-arylprotoporphyrin IX regioisomers in the following ratios (NB:NA:NC:ND, where the subscript indicates the pyrrole ring): phenyl, 03:34:02:61; 2-naphthyl, 02:18:03:77; p-biphenyl, 02:52:04:42. The results indicate that the active site of human CYP2E1 is sterically unhindered directly above the iron for a distance of 10 Å. It appears, furthermore, that the active site cavity is relatively open above pyrrole rings A and D but is closed above pyrrole rings B and C, a topology similar to that deduced for the rat enzyme.
Introduction Cytochrome P450 2E1 (CYP2E1)1 (1) catalyzes the oxidation of carcinogens, hepatotoxic xenobiotics, and drugs, including organic solvents, halocarbon anesthetics, dialkylnitrosamines, and acetaminophen (2, 3). CYP2E1 is also of interest because it oxidizes not only ethanol to acetaldehyde but also acetaldehyde to acetic acid (4). The oxidation of ethanol by CYP2E1 reportedly produces R-hydroxyethyl radicals (5). The association of CYP2E1 with ethanol is enhanced by the fact that ethanol exposure increases the levels of the enzyme and amplifies its role in the metabolism of not only ethanol but also other CYP2E1 substrates (6). Chlorzoxazone hydroxylation has been proposed as a specific test for the presence of CYP2E1, although chlorzoxazone is also oxidized, albeit less effectively, by other enzymes (9). The enzyme is inactivated by diallyl disulfide and 3-amino-1,2,4triazole (7, 8). Both the substrate specificity and mechanism-based inactivation of CYP2E1 indicate that the enzyme preferentially interacts with relatively small substrates. Little information is available on the active site of CYP2E1 beyond the inference from its specificity that the substrate binding cavity is of limited size. In an earlier study we explored the active site topology of rat CYP2E1 by examining its reaction with phenyldiazene (PhNdNH), the oxidation product of phenylhydrazine (PhNHNH2) (10). The rat enzyme was found to form a spectroscopically detectable complex with an absorption maximum at 480 nm. The 480 nm absorbance maximum, as most * Author to whom correspondence should be addressed. FAX: (415) 476-0688. † University of California, San Francisco. ‡ Vanderbilt University School of Medicine. § Present address: Department of Drug Metabolism, Rhone-PoulencRorer, 500 Arcola Road, P.O. Box 1200, Collegeville, PA 19426. X Abstract published in Advance ACS Abstracts, December 1, 1995. 1 Abbreviations: CYP2E1, cytochrome P450 2E1; heme, iron protoporphyrin IX regardless of ligation or oxidation state; NB, NA, NC, and ND refer to the N-arylprotoporphyrin regioisomers with the aryl group on the nitrogens of, respectively, pyrrole rings B, A, C, and D.
0893-228x/96/2709-0223$12.00/0
Figure 1. Schematic representation of the control exerted by the steric effects of protein residues on the direction of migration of the phenyl group in phenyl-iron complexes.
clearly shown by a crystal structure of the corresponding cytochrome P450cam (CYP101) complex, is due to the formation of a σ-bonded complex (Fe-Ph) between the phenyl group and the iron atom (11). Similar phenyliron complexes are formed with other cytochrome P450 enzymes (12). Oxidation of the rat CYP2E1 complex with ferricyanide resulted in migration of the phenyl group from the iron to the nitrogens of the porphyrin. The NA and ND regioisomers of N-phenylprotoporphyrin IX thus were obtained in a 1:2 ratio after removal of the iron atom from the product and separation of the regioisomers by HPLC (10). The NB and NC regioisomers were not formed in significant amounts. This regioisomer pattern provides direct information on the active site topology, since the rearrangement occurs within the intact active site and the porphyrin nitrogen(s) to which the phenyl migrates are determined by their relative steric accessibility (Figure 1). Evidence that this is so is provided by comparisons of the active site topologies deduced from the reactions with phenylhydrazine of cytochromes P450cam, P450terp (CYP108), and P450BM-3 (CYP102) with the topologies of the enzymes determined from the corresponding crystal structures (13, 14). As required © 1996 American Chemical Society
224 Chem. Res. Toxicol., Vol. 9, No. 1, 1996
for this interpretation to be valid, migration of the phenyl group in solution (i.e., in the absence of a protein environment) produces an equimolar mixture of the four possible N-phenylprotoporphyrin IX regioisomers (11). Thus, the regioisomer pattern obtained with rat CYP2E1 suggests that the active site of the protein is primarily open above pyrrole rings A and D of the heme group. We report here an examination of the active site topology of human CYP2E1 by reaction of the protein not only with phenyldiazene but also with 2-naphthyl- and p-biphenylhydrazine. As shown with other P450 enzymes (13, 15), the aryl-iron complexes obtained with the larger probes undergo the same iron-to-nitrogen shift as the phenyl complexes. The regioisomer patterns obtained with the larger aryl groups, however, are determined to a large extent by the topology of the active site at distances from the heme plane greater than those explored by the phenyl complex. The results indicate that the active site is primarily open above pyrrole rings A and D, both immediately above, and at some distance from, the heme plane. The results demonstrate, furthermore, that the active site topologies of the rat and human enzymes are very similar.
Experimental Procedures Materials. Recombinant CYP2E1 was expressed in Escherichia coli and was purified as reported elsewhere (16). Methyl phenyldiazenecarboxylate azo ester was from Research Organics Inc. (Cleveland, OH). 2-Naphthylhydrazine hydrochloride and 4-biphenylhydrazine hydrochloride were synthesized from 2-naphthylamine and 4-aminobiphenyl, respectively, by the procedure of Hunsberger (17). Determination of N-Arylprotoporphyrin IX Isomer Ratios. The N-arylprotoporphyrin IX isomer mixtures were analyzed on a Hewlett-Packard 1090 HPLC system equipped with a 4.6 × 250 mm Partisil ODS-3 5 µm column (Alltech, San Jose, CA). Isocratic analyses were carried out at a flow rate of 1 mL/min, with solvent mixtures consisting of the following percentages of solvent B in solvent A: N-phenyl, 20%; N-(2naphthyl), 30%; N-(p-biphenyl), 40%. Solvent A was 6:4:1 CH3OH/H2O/CH3COOH (v/v) and solvent B was 10:1 CH3OH/ CH3COOH (v/v). The column eluent was monitored at 416 nm (4 nm bandwidth), with the baseline reference at 600 nm. Authentic standards of the N-arylprotoporphyrin IX regioisomers were obtained as previously described (18). N-Phenylprotoporphyrin IX Isomers. P450 2E1 (2 nmol) in 500 µL of potassium phosphate buffer (100 mM, pH 7.4) was treated with 1 µL of a stock solution of methyl phenyldiazenecarboxylate azo ester prepared by dissolving the neat ester (2 µL) in aqueous 1 N KOH (200 µL). After standing at room temperature for 25 min, the solution was treated with 4 × 1 µL aliquots of K3Fe(CN)6 stock solution (62.5 mM in 100 mM potassium phosphate buffer, pH 7.4) with 5 min intervals between the aliquots. The final solution was allowed to stand for 10 min after the last addition. The reaction mixture was then added to 10 mL of a freshly prepared 95:5 CH3CN/H2SO4 solution, and the mixture was allowed to stand at 4 °C overnight. It was then concentrated in vacuo to a volume of 1-2 mL, aqueous H2SO4 (5% v/v, 2 mL) was added, and the resulting aqueous mixture was extracted with CH2Cl2 (3 × 1 mL). The combined organic extracts were washed with water (1 mL) and then concentrated in vacuo. The residue was dissolved in 100 µL of solvent A for HPLC analysis. N-(2-Naphthyl)- and N-(p-Biphenyl)protoporphyrin IX Isomers. The N-(2-naphthyl)- and N-(p-biphenyl)protoporphyrin IX isomers were obtained with the corresponding arylhydrazine hydrochlorides because the diazenes are not commercially available. A suspension of the arylhydrazine hydrochloride (25 mM nominal concentration) in potassium phosphate buffer (100 mM, pH 7.4) was sonicated for 10 min to assist dissolution.
Mackman et al. Table 1. N-Arylprotoporphyrin IX NB:NA:NC:ND Regioisomer Ratios Obtained in the Reactions of Human Recombinant CYP2E1 with Phenyl-, 2-Naphthyl-, and p-Biphenylhydrazinesa
enzyme
phenyl
naphthyl
biphenyl
CYP2E1 (human) CYP2E1 (rat)b CYP2B1 (rat)b CYP101 (P450cam)c CYP102 (P450BM-3)d CYP108 (P450terp)d
03:34:02:61 00:33:00:67 00:39:00:61 00:05:25:70 13:67:13:07 00:00:00:100
01:18:03:77 na 00:00:00:100 00:00:100:00 05:07:51:37 00:00:00:100
02:52:04:42 na 00:87:00:13 00:14:40:46 21:37:19:22 06:44:11:39
a The abbreviations in the figure are V ) vinyl and P ) propionyl. Values listed as 00 indicate that the product was less than the limit of detection (∼2%); na indicates not available. b These data are from ref 15. c These data are from ref 18. d These data are from refs 13 and 14.
An aliquot of the arylhydrazine suspension (50 µL) was added to 2 nmol of the P450 2E1 in 500 µL of potassium phosphate buffer (100 mM, pH 7.4), and the resulting solution was allowed to stand at 25 °C for either 6 (2-naphthyl) or 2 h (p-biphenyl). The mixture was then centrifuged at 104 rpm for 1 min, and the supernatant was decanted from the residual undissolved hydrazine. The solution was then treated with 2 × 2 µL (2naphthyl) or 3 × 2 µL (p-biphenyl) aliquots of K3Fe(CN)6 solution (62.5 mM in 100 mM potassium phosphate buffer, pH 7.4) with 5 min intervals between the aliquots. The final solution was allowed to stand for 5-10 min after the last addition and was then worked up and analyzed as described for the N-phenylprotoporphyrin isomers.
Results and Discussion The reaction of CYP2E1 with phenyldiazene results in a decrease in the Soret band of the enzyme with the concomitant appearance of a new absorbance maximum at 480 nm. The complex responsible for the 480 nm chromophore is stable for a period of at least 30 min at 0 °C. Controlled addition of ferricyanide to the complex, transfer of the mixture to CH3CN and H2SO4, extraction of the porphyrin products from the acidic mixture, and HPLC analysis establish that the four possible isomers of N-phenylprotoporphyrin IX are formed in the reaction. The identities of the four peaks found in the chromatogram were established by comparing their absorption spectra and retention times with those of the authentic isomers produced in the reaction of myoglobin with phenyldiazene (18). Integration and averaging of the peaks obtained from four independent experiments indicates that the four possible regioisomers of N-phenylprotoporphyrin IX are obtained in the ratio NB:NA:NC: ND ) 03:34:02:61 (Table 1). The regioisomers are listed in the order of their elution from the HPLC column. The reactions of CYP2E1 with (2-naphthyl)hydrazine and p-biphenylhydrazine also give rise to complexes with absorption maxima at 480 nm. Exposure to ferricyanide, removal of the iron by treatment with acid, and HPLC analysis of the resulting products demonstrate that the
Active Site Topology of CYP2E1
Figure 2. Model of the CYP2E1 active site topology based on the finding that the active site is open above pyrrole ring D and to some extent above pyrrole ring A.
N-(2-naphthyl)- and N-(p-biphenyl)protoporphyrin IX derivatives are formed in the corresponding reactions (Table 1). The average ratio of the N-(2-naphthyl) regioisomers is NB:NA:NC:ND ) 01:18:03:77, and that of the N-(p-biphenyl) regioisomers is NB:NA:NC:ND ) 02:52: 04:42. The yields of these products are lower than those for the N-phenyl products, but the ratios are reproducible. Formation of a p-biphenyl-iron complex establishes that the active site of CYP2E1 is open to a height of 10 Å directly above the iron atom (15). The active site must also have significant width and depth as well as height in order to accommodate the 2-naphthyl-iron complex. The results with all three probes indicate that the active site cavity is primarily located above pyrrole rings A and D, with the region above pyrrole ring D being the most accessible (Figure 2). The differences in the regioisomer patterns obtained with the three probes are relatively small, which suggests that the topology at a distance from the heme is similar to that directly above the heme plane. Nevertheless, the small differences in the regioisomer patterns for the phenyl and (p-biphenyl) probes suggest that, if anything, the higher reaches of the cavity are slightly more open above pyrrole ring A (or slightly more closed above pyrrole ring D) than they are immediately above the heme. The N-phenylprotoporphyrin IX regioisomer patterns for rat and human CYP2E1 are essentially identical (Table 1) (10). A comparison of the two active sites with the 2-naphthyl and p-biphenyl probes is not possible because the required data are not available for the rat enzyme (10). Nevertheless, the phenyl probe provides strong evidence that the active sites of the rat and human enzymes are topologically congruent. A comparison of the data for CYP2E1 with those of other P450 enzymes indicates that the CYP2E1 active site differs from those of three bacterial enzymes, including P450BM-3, for which crystal structures are available (Table 1) (13-15). Only in the case of rat CYP2B1 is there some similarity in the N-aryl shift patterns (18). Indeed, the N-phenylprotoporphyrin IX patterns are virtually identical for CYP2B1 and CYP2E1 (Table 1). The N-(2-naphthyl)- and N-(pbiphenyl)porphyrin patterns differ, however, which suggests that the similarities in the two active sites decrease as one moves to increasing distances from the heme plane.
Chem. Res. Toxicol., Vol. 9, No. 1, 1996 225
CYP2E1 catalyzes the ω-1 hydroxylation of lauric acid (19), a reaction also catalyzed by P450BM-3. The substrate binding site of P450BM-3 appears from the crystal structure to be quite large, although the active site in the immediate vicinity of the heme iron atom appears from both the crystal structure and the aryl shift method to be highly constrained (13, 20). Lauric acid, even if flexible, is a bulky substrate that may be too large to be accommodated in the active site of CYP2E1. It is possible that only the ω-terminus of the fatty acid chain is actually bound in the active site cavity above pyrrole rings A and D. In such a binding mode, the carboxyl terminus would extend out of the crevice immediately above the heme into the substrate access channel or into medium surrounding the protein. A model for binding of the polar end of a fatty acid within the substrate access channel is provided by P450BM-3. A model for binding of the fatty acid with the polar end extending into the medium is provided by the manner in which linoleic acid must bind in the heme crevice of myoglobin to explain the regio- and stereospecificities of its H2O2-dependent conversion to a hydroperoxide (21). Independent support for an active site architecture in which the hydrocarbon terminus of a long fatty acid is bound in an environment different from that in which the carboxyl terminus is bound is provided by a recent study of the relationship between the chain lengths of linear alcohols and acids and their activities as CYP2E1 inhibitors (22).
Acknowledgment. This work was supported by National Institutes of Health Grants GM25515 (P.R.O.M.), CA 44353 (F.P.G.), and ES00267 (F.P.G). Support for the core facilities of the UCSF Liver Center was provided by Grant 5 P30 DK26743.
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