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Functional role of residues in the helix B′ region of cytochrome P450 2B1
Archives of Biochemistry and Biophysics 435 (2005) 157–165 www.elsevier.com/locate/yabbi
Functional role of residues in the helix B⬘ region of cytochrome P450 2B1 Wataru Honmaa,¤, Weihua Lib, Hong Liub, Emily E. Scotta,1, James R. Halperta a
Department of Pharmacology and Toxicology, University of Texas Medical Branch, 301 University Blvd, Galveston, TX 77555-1031, USA b Center for Drug Discovery and Design, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, PR China Received 1 November 2004, and in revised form 29 November 2004 Available online 27 December 2004
Abstract Comparison of several recently determined X-ray crystal structures of mammalian cytochrome P450 family 2 enzymes suggests considerable movement of helix B⬘ when ligands bind. To investigate the functional role of helix B⬘ in P450 2B1, residues 100–109 were substituted with alanine and phenylalanine. Kinetic properties were examined with the typical 2B substrates 7-benzyloxyresoruWn, 7-ethoxy-4-triXuoromethylcoumarin, benzphetamine, and testosterone. Several mutants showed 2- to 3-fold changes in kcat values and signiWcant diVerences in catalytic eYciencies among the substrates examined, consistent with structural information suggesting that the helix B⬘ region can adopt multiple conformations with diVerent contact residues depending on the substrate. Homology modeling of P450 2B1 was performed based on an inhibitor-bound P450 2B4 structure, and the docking analyses were consistent with experimental results. The Wndings suggest that residues in the helix B⬘ region aVect regio- and stereoselective oxidation in P450 family 2 enzymes as well as substrate entry. 2004 Elsevier Inc. All rights reserved. Keywords: Cytochrome P450; Monooxygenase; Site-directed mutagenesis
Cytochromes P450 (P450s)2 play an essential role in the oxidative biotransformation of endogenous compounds such as steroids, fatty acids, and prostaglandins, and of exogenous chemicals including drugs, natural plant products, carcinogens, and environmental pollutants. P450s form a large superfamily, and there are signiWcant substrate speciWcity diVerences among families, *
Corresponding Author. Fax: +1 409 772 9642. E-mail address: [email protected] (W. Honma). 1 Present address: Department of Medicinal Chemistry, University of Kansas, 1251 Wescoe Hall Dr., Malott 4067, Lawrence, KS 660457582, USA. 2 Abbreviations used. P450, cytochrome P450; 7-BR, 7-benzyloxyresoruWn; 7-EFC, 7-ethoxy-4-triXuoromethylcoumarin; PCR, polymerase chain reaction; Hepes, N-[2-hydroxyethyl]piperazine-N⬘-[2-ethanesulfonic acid]; NADPH, -nicotinamide adenine dinucleotide phosphate; DMZ, 4-methyl-N-methyl-N-(2-phenyl-2H-pyrazol-3-yl)benzenesulfonamide; CPI, 4-(4-chlorophenyl)imidazole. 0003-9861/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2004.12.014
subfamilies, and isoforms within a subfamily [1]. Current information indicates that selectivity of oxidation reXects initial substrate recognition, substrate access to the active site, binding orientation within the active site, as well as the chemical properties of the substrates [2,3]. An intriguing aspect of many P450 enzymes, especially those of family 2, involves the diVerential regio- and stereoselectivity among structurally related proteins [4]. The structures of several mammalian cytochromes P450 have been reported to date. As the Wrst microsomal P450 to be crystallized, the 2C5 structure (PDB: 1DT6) provided an opportunity to characterize structural features that contribute to substrate binding and enzyme conformation [5]. Subsequent structures of 2C5 with the bound substrates, diclofenac (PDB: 1NR6) or the sulfaphenazole derivative 4-methyl-N-methyl-N(2-phenyl-2H-pyrazol-3-yl)benzenesulfonamide (PDB: 1N6B), indicate that Xexible regions of the protein adapt
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for substrate binding, and that substrates may bind in multiple orientations [6,7]. Like the structures of 2C5, the structures of 2C8 and 2C9, and of 2B4 with a phenylimidazole inhibitor, generally show closed conformations without an obvious route from solvent to the buried active site [8–11]. However, the structure of inhibitor-free 2B4 (PDB: 1PO5) demonstrates a large open cleft that extends from the protein surface directly to the heme iron without perturbing the overall P450-fold [12]. This open cleft is primarily formed by repositioning of helices B⬘ to C and F through G, and is correlated with dissociation of contacts between residues in helix B⬘ and in helix G. In contrast to the bacterial cytochrome P450 enzymes, all of the reported mammalian P450 structures have an extended number of residues between helices F and G. Although there are diVerences in the length of helices F, F⬘, G, and G⬘, the overall secondary structures are maintained among the structures of mammalian P450 family 2 enzymes. To examine the functional role of the F–G region in substrate access and regioselective oxidation, selected residues in P450 2B1 were replaced and the resulting proteins were evaluated for turnover of the typical 2B substrates testosterone, 7-EFC, and 7-BR [13]. Most of the mutants demonstrated only moderate eVects on kinetic oxidation, although L209A showed 2to 6-fold increases in kcat. S221F was the only mutant that exhibited >2-fold decreases in the kcat/Km or kcat/S50 values for all three substrates. F219W was inactive, but its unaltered Ks value for benzphetamine binding suggested that substrate access was not impeded. The opposite side of the cleft in the open 2B4 structure is largely formed by helix B⬘. The region is Xanked by two GXG motifs in most P450 family 2 enzymes. In the absence of side chains, the glycine residues are relatively free to adopt a variety of conformations that contribute to the Xexibility of this region. The loops preceding and following the GXG motifs are stabilized by interactions with adjacent portions of the structure. P450 2B1 shares 77% amino-acid identity with 2B4, and six of ten residues in the helix B⬘ region (residues 100– 109) correspond to those of 2B4 (2B1: TIAVIEPIFK, 2B4: KIAVVDPIFQ). Comparison of the two structures of 2B4 suggests considerable movement of the region around helix B⬘ when ligands bind to P450 family 2 enzymes. The organization and placement of the region including helix B⬘ through the C/D loop signiWcantly alter the accessibility of the active site. The comparison between the open and closed 2B4 structures also reveals an alternate secondary conformation of helix B⬘. In the open structure of 2B4 residues I101–V104 form helix B⬘, whereas residues D105–F108 form helix B⬘ in the 2B4 structure bound with CPI [11,12]. Thus, we hypothesized that the residues in this region have an important function during substrate access and turnover. In the present study, 10 amino acid residues in the helix B⬘ region of P450 2B1 were substituted with the smaller
and larger residues alanine and phenylalanine. 2B1 was selected because of the wealth of prior mutagenesis data on other regions [4,14–17]. The mutations were generated in the background of a highly expressing form of 2B1 that is truncated at the N-terminus and modiWed by the addition of four His residues at the C-terminus [18]. Following puriWcation of the mutants from Escherichia coli, steady state kinetics were determined with the typical 2B substrate 7-BR, 7-EFC, benzphetamine, and testosterone. The results were analyzed with the help of a homology model of 2B1 based on the inhibitor-bound structure of 2B4.
Materials and methods Materials Oligonucleotide primers were obtained from Sigma Genosys (Woodlands, TX). The QuikChange SiteDirected Mutagenesis Kit was from Stratagene (La Jolla, CA). All other reagents were obtained from sources as described previously [17] or from standard suppliers. Rat NADPH-cytochrome P450 reductase and cytochrome b5 were prepared as described [19]. Mutagenesis, expression, and puriWcation The truncated version of 2B1 that served as the background for all of the mutants described in this study, 2B1dH, was generated using overlap extension PCR as described [18]. Basically, residues at positions 3–21 were deleted, several mutations were made at the new N-terminus, and a four-histidine tag was added at the C-terminus. All of the helix B⬘ mutants were generated using the QuikChange site-directed mutagenesis kit (Stratagene) with oligonucleotide primers, each complementary to opposite strands of pKK2B1dH (forward primers are shown in Table 1). Each primer contains a speciWc restriction site introduced to facilitate screening for mutants (PvuI: T100A, V103A/F, and F108A, AlwnI: I101A, I104A, SspI: A102F, I104F, E105A/F, P106A, and K109A/F, BsmI: I107A, BspEI: T100F, KpnI: I101F, DraI: P106F, and I107F). The resulting constructs were sequenced to conWrm the desired mutation and verify the absence of unintended mutations (Protein Chemistry Laboratory, University of Texas Medical Branch, Galveston, TX). 2B1dH and the mutants were expressed in Escherichia coli TOPP3 cells (Stratagene) and puriWed with a Ni–NTA aYnity column as described previously [18]. Cytochrome P450 was quantitated using the reduced-CO diVerence spectrum [20]. Enzymatic assays 7-BR O-debenzylation was measured using a standard Xuorometric assay. The 500-l Wnal reaction mixture
W. Honma et al. / Archives of Biochemistry and Biophysics 435 (2005) 157–165 Table 1 Oligonucleotides used in PCR for site-directed mutagenesis Forward primers T100A I101A V103A I104A E105A P106A I107A F108A K109A T100F I101F A102F V103F I104F E105F P106F I107F K109F
5⬘-tc tct ggt cgg gga GCG atc gct gtg a-3⬘ 5⬘-t cgg gga aca GCC gct gtg att gag-3⬘ 5⬘-atc gct GCG atc gag cca atc ttc aa-3⬘ 5⬘-aca atc gca gta GCT gag cca atc ttc-3⬘ 5⬘-atc gct gtg att GCG cca ata ttc aag-3⬘ 5⬘-gct gtg att gag GCA ata ttc aag gaa-3⬘ 5⬘-gtg att gag cca GCA ttc aag gaa ta-3⬘ 5⬘-att gag ccg atc GCC aag gaa tat ggt-3⬘ 5⬘-gag cca ata ttc GCG gaa tat ggt gtg-3⬘ 5⬘-gat ttc tcc gga cgg gga TTT atc gct gtg att ga-3⬘ 5⬘-ggt cgg ggt acc TTC gct gtg att gag-3⬘ 5⬘-cgg gga aca ata TTT gtg att gag cca-3⬘ 5⬘-g gga acg atc gct TTC att gag cca atc-3⬘ 5⬘-aca atc gct gtg TTT gag cca ata ttc a-3⬘ 5⬘-a atc gct gtg att TTC cca ata ttc aag g-3⬘ 5⬘-gct gtg att gag TTC atc ttt aaa gaa tat ggt-3⬘ 5⬘-gct gtg att gag cca TTC ttt aaa gaa ta-3⬘ 5⬘-gag cca ata ttc TTT gaa tat ggt gtg at-3⬘
The changed bases are marked in bold. Capital letters denote the codon for the altered amino acid residue. SpeciWc restriction enzyme sites (underlined) are generated to verify the desired mutation as described in Materials and methods. The reverse primers are exactly complementary to the forward primers.
contained 10 pmol P450, 40 pmol cytochrome P450 reductase, 20 pmol cytochrome b5, and substrate (0.01– 10 M) in 50 mM Hepes, pH 7.6, 15 mM MgCl2, and 0.1 mM EDTA. The reaction was started by adding 0.5 mM NADPH, continued for 10 min at 37 °C, and terminated with the addition of 2 ml methanol. The resoruWn formed was monitored by comparison with a standard curve using a Perkin–Elmer model 3000 spectroXuorometer with excitation at 550 and emission at 585 nm. 7-EFC O-deethylation was measured in a Wnal volume of 100 l. The reaction mixture contained 10 pmol P450, 40 pmol cytochrome P450 reductase, 20 pmol cytochrome b5, and 5–150 M 7-EFC in 50 mM Hepes, pH 7.6, 15 mM MgCl2, and 0.1 mM EDTA. The reaction was initiated by adding NADPH (1 mM), incubated for 10 min at 37 °C, and terminated with 50 l of 20% trichloroacetic acid. Metabolites were quantitated by Xuorescence using a 7-hydroxy-4-triXuoromethylcoumarin standard as described [21]. Benzphetamine N-demethylation was determined by the method of Prough and Ziegler [22]. The 300-l reaction mixture contained 30 pmol P450, 120 pmol cytochrome P450 reductase, 60 pmol cytochrome b5, and 5–500 M benzphetamine in 50 mM Hepes, pH 7.6, 15 mM MgCl2, and 0.1 mM EDTA. The reaction was started by addition of 1 mM NADPH, allowed to proceed for 10 min at 37 °C, and stopped with 300 l of 10% trichloroacetic acid. Formaldehyde formation from benzphetamine was determined by addition of the NASH reagent, heating at 60 °C for 15 min, cooling, and measuring the absorbance at 412 nm.
159
Testosterone hydroxylase activity was assayed according to the protocol described by Ciaccio and Halpert [23]. The Wnal 100-l reaction mixture contained 5 pmol P450, 20 pmol cytochrome P450 reductase, 10 pmol cytochrome b5, and 0.5–280 M [14C]testosterone (30,000 dpm/nmol) in 50 mM Hepes, pH 7.6, 15 mM MgCl2, and 0.1 mM EDTA. The reaction was initiated by the addition of 1 mM NADPH, allowed to proceed for 5 min at 37 °C, and stopped with 50 l tetrahydrofuran. Metabolites were developed on TLC plates (20 £ 20 cm, Si250F(19C), J.T. Baker, Phillipsburg, NJ) by two cycles of chromatography in (4:1 v:v) dichloromethane:acetone, identiWed by autoradiography, and quantitated by scintillation counting. Kinetic constants were assessed by GraphPad Prism (version 3) software (GraphPad Software, San Diego, CA). Spectral binding Binding spectra were recorded on a Shimadzu-2600 spectrophotometer in the diVerence mode. A 2.0-ml solution containing 1 M P450 in 10 mM potassium phosphate, 10% glycerol, and 1 mM EDTA were divided into two quartz cuvettes and a baseline was recorded between 350 and 500 nm. An aliquot of benzphetamine hydrochloride in water was added to the sample cuvette (2.5– 220 M benzphetamine), and the same amount of water was added to the reference cuvette. The diVerence spectra were obtained after the system reached equilibrium at 37 °C, and diVerences monitored in the absorbance at 387 and 420 nm. Docking of 7EFC and testosterone into the active site of homology models of P450 2B1 The initial molecular model was constructed using the Insight II software package [24]. The 2B1 model was constructed based on the crystal structure of a 2B4 complex (PDB: 1SUO). The sequence of 2B1 was obtained from SwissProt (Accession No. P00176). The coordinates of the conserved residues were assigned based on the corresponding residues of the 2B4 complex by Homology/InsightII. The heme group was copied from 2B4 into the 2B1 model. After the coordinate assignment, the initial model of 2B1 was reWned with the Gromacs force Weld using the Gromacs 3.2 software package [25,26]. The protein was placed in a periodic box large enough to contain the enzyme and 0.8 nm of solvent on all sides. The solvent was relaxed by energy minimization while restraining the protein atomic positions with a harmonic potential. The system was energy minimized without restraints for 2000 steps using a combination of steepest descent and conjugate gradient prior to MD equilibration. Equilibration was performed at 300 K with decreasing harmonic constraints over a 15-ps time interval followed by 1 ns
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of equilibration without restraints. The resulting model was energy minimized again for 1000 steps using the conjugate gradient method and was used as the model for subsequent substrate docking. For the 2B1 mutant F108A, the coordinates of the corresponding residue were changed in the 2B1 three-dimensional model by Biopolymer/InsightII, and the resulting model was minimized. The structures of 7-EFC and testosterone were constructed using the Builder module of the Insight II modeling package. Placement of 7-EFC and testosterone into the active site of the 2B1 was accomplished by using the AYnity/InsightII as described previously [17].
Results Steady-state kinetic analysis of helix B⬘ mutants All of the helix B⬘ mutants were examined in the background of a truncated form of P450 2B1. This recombinant enzyme, termed 2B1dH, was engineered for expression in E. coli as a conditionally soluble membrane protein that retained catalytic activity [18]. Eighteen mutants at 10 diVerent positions throughout the helix B⬘ region were constructed (T100A/F, I101A/F, A102F, V103A/F, I104A/F, E105A/F, P106A/F, I107A/ F, F108A, and K109A/F). These mutants expressed at 250–700 nmol P450 (L of E. coli culture)¡1 with the exception of some Phe-mutants, A102F, V103F, E105F, P106F, and K109F (100–150 nmol L¡1). For compari-
son, this same protocol yielded 700–800 nmol L¡1 of the 2B1dH holoprotein. After puriWcation, kinetic constants of the Ala/Phemutants were determined with the representative substrates of P450 2B enzymes, 7-BR, 7-EFC, benzphetamine, and testosterone (Tables 2 and 3). Most of the mutants showed Km values similar to those observed for 2B1dH for 7-BR (2B1dH: 1.9 M, Ala/Phe-mutants: 0.8– 2.6 M) and testosterone (2B1dH: 29 M for 16-OH and 27 M for 16-OH, Ala/Phe-mutants: 22–49 M for 16-OH and 20–48 M for 16-OH) and S50 values similar to those for 2B1dH for 7-EFC (2B1dH: 30 M, Ala/ Phe-mutants: 20–46 M). The few exceptions were F108A, A102F, P106A, and I107A. The substitution of F108 with Ala resulted in 2-fold reductions of S50 for 7EFC (10 M) and of Km for testosterone (17 M for 16hydroxylation). In contrast, the alteration of residue 102 from Ala to Phe resulted in 2-fold increases in the Km values for both 16- and 16-hydroxylation of testosterone (59 and 54 M, respectively). Ala-substitutions at residues P106 and I107 resulted in 2-fold increases in S50 values for 7-EFC (62 and 66 M). For benzphetamine, several Ala/Phe-mutants also showed similar Km values to that of 2B1dH (2B1dH: 37 M, Ala/Phe-mutants: 2869 M). However, a few mutants showed signiWcant increases and decreases in the Km. Ala/Phe-substitutions at residues I101–V103 resulted in 2- to 3-fold increases in Km (103–123 M) and Ala-substitution at residue F108 resulted in 2-fold decrease in the Km (16 M). In contrast to the relatively modest eVects on Km, marked changes in the kcat values and diVerent proWles
Table 2 Steady-state substrate kinetics for mutants of the helix B⬘ region in the 2B1dH background 7-BenzyloxyresoruWn kcata 2B1dH T100A T100F I101A I101F A102F V103A V103F I104A I104F E105A E105F P106A P106F I107A I107F F108A K109A K109F a b c
c
2.8 (0.1) 2.1 (0.1) 1.4 (0.1) 0.1 (0.0) 0.6 (0.0) 2.3 (0.1) 4.5 (0.1) 2.6 (0.1) 0.6 (0.0) 0.6 (0.0) 2.9 (0.1) 1.9 (0.1) 3.6 (0.1) 1.3 (0.1) 1.9 (0.1) 3.2 (0.1) 0.7 (0.0) 2.0 (0.0) 1.4 (0.0)
7-Ethoxy-4-triXuoromethylcoumarin
Benzphetamine
Km b
kcat /Km
kcat
n
S50
kcat/S50
kcat
1.9 (0.2) 1.8 (0.2) 1.8 (0.2) 1.7 (0.2) 1.5 (0.2) 1.5 (0.2) 2.3 (0.2) 1.3 (0.1) 1.1 (0.1) 1.3 (0.1) 1.5 (0.1) 1.5 (0.1) 1.2 (0.1) 2.6 (0.3) 1.3 (0.1) 2.1 (0.1) 1.2 (0.1) 1.1 (0.1) 0.8 (0.1)
1.46 1.18 0.77 0.05 0.42 1.55 1.94 1.95 0.57 0.51 1.93 1.30 2.93 0.48 1.47 1.53 0.60 1.89 1.71
8.1 (0.3) 6.6 (0.4) 4.9 (0.4) 0.2 (0.0) 5.2 (0.5) 6.9 (0.5) 21.7 (1.3) 11.6 (0.8) 14.7 (0.6) 3.0 (0.2) 11.0 (0.3) 7.0 (0.5) 22.9 (1.7) 5.4 (0.3) 15.3 (1.5) 14.1 (0.7) 4.2 (0.2) 13.3 (0.9) 6.6 (0.5)
1.2 (0.1) 1.3 (0.1) 1.1 (0.1) 1.2 (0.1) 1.1 (0.1) 1.3 (0.1) 1.2 (0.1) 1.4 (0.1) 1.2 (0.1) 1.3 (0.1) 1.3 (0.1) 1.2 (0.1) 1.0 (0.1) 1.0 (0.1) 1.0 (0.1) 1.1 (0.1) 0.8 (0.1) 1.1 (0.1) 1.2 (0.1)
30 (3) 32 (4) 39 (8) 41 (9) 46 (9) 40 (5) 31 (4) 41 (5) 25 (2) 38 (4) 21 (2) 32 (5) 62 (9) 40 (5) 66 (13) 32 (4) 10 (2) 34 (5) 33 (5)
0.27 0.21 0.13 0.01 0.11 0.17 0.70 0.29 0.58 0.08 0.53 0.22 0.37 0.14 0.23 0.44 0.42 0.39 0.20
36 (1) 23 (1) 23 (1) 12 (1) 37 (3) 53 (3) 81 (3) 77 (3) 35 (1) 19 (1) 39 (1) 29 (1) 66 (3) 33 (1) 75 (3) 36 (2) 26 (1) 50 (2) 36 (1)
kcat, min¡1. Km, S50, M. Standard error for Wt to the Michaelis–Menten or Hill equation shown in parentheses.
Km 37 (6) 48 (9) 69 (11) 105 (13) 103 (15) 113 (18) 123 (12) 103 (11) 28 (4) 67 (10) 35 (5) 35 (6) 54 (9) 48 (7) 41 (7) 29 (7) 16 (4) 29 (5) 58 (7)
kcat/Km 0.97 0.47 0.34 0.11 0.36 0.47 0.66 0.75 1.25 0.28 1.12 0.83 1.22 0.68 1.83 1.25 1.65 1.73 0.62
W. Honma et al. / Archives of Biochemistry and Biophysics 435 (2005) 157–165 Table 3 Steady-state kinetic constants for testosterone hydroxylation Testosterone kcata 2B1dH T100A T100F I101A I101F A102F V103A V103F I104A I104F E105A E105F P106A P106Ff I107A I107F F108A K109A K109F
Km b
kcat/Km
16
16
16
16
16
16
4.3 (0.1)d 3.6 (0.1) 2.0 (0.1) n.d.e 1.2 (0.0) 3.0 (0.1) 7.3 (0.2) 3.7 (0.1) 1.9 (0.1) n.d. 4.5 (0.1) 3.2 (0.1) 8.1 (0.2) 3.7 (0.1) 3.6 (0.1) 5.1 (0.1) 1.4 (0.0) 4.1 (0.1) 2.0 (0.1)
3.7 (0.1) 4.3 (0.1) 2.6 (0.1) n.d. 1.4 (0.0) 4.8 (0.1) 7.8 (0.2) 2.3 (0.1) 5.5 (0.2) n.d. 3.9 (0.1) 1.7 (0.0) 4.8 (0.1) 1.5 (0.0) 1.4 (0.0) 1.1 (0.0) n.d. 2.2 (0.1) 0.7 (0.0)
29 (2) 48 (4) 35 (4) n.d. 49 (5) 59 (4) 40 (3) 38 (4) 42 (5) n.d. 26 (2) 28 (3) 43 (3) 35 (3) 38 (3) 22 (2) 17 (2) 26 (2) 22 (2)
27 (3) 45 (2) 35 (3) n.d. 48 (4) 54 (4) 42 (3) 36 (5) 42 (4) n.d. 28 (2) 26 (3) 46 (4) 36 (3) 39 (4) 22 (2) n.d. 27 (3) 20 (3)
0.15 0.08 0.06 — 0.02 0.05 0.18 0.10 0.05 — 0.17 0.12 0.19 0.11 0.09 0.23 0.08 0.16 0.09
0.14 0.10 0.07 — 0.03 0.09 0.19 0.06 0.13 — 0.14 0.07 0.10 0.04 0.04 0.05 — 0.08 0.03
16/16c
1.1 0.8 0.8 — 0.8 0.6 1.0 1.6 0.3 — 1.3 1.8 1.8 2.5 2.7 4.6 — 2.0 2.6
a
kcat, min¡1. Km, S50, M. c Ratio of kcat/Km. d Standard error for Wt to Michaelis–Menten equation shown in parentheses. e n.d., not detectable (less than 0.1 min¡1). f P106F showed additional 7-hydroxylation (kcat, 1.4 min¡1; Km, 152 M; and kcat/Km, 0.01). b
were observed among substrates. The most striking result was the remarkable decrease or lack of catalytic activity with Ala-substitution at I101 and Phe-substitution at I104. These mutants showed 2- to 20-fold decreases of the kcat values for 7-BR (2B1dH: 2.8 min¡1, I101A: 0.1 min¡1, and I104F: 0.6 min¡1), for 7-EFC (2B1dH: 8.1 min¡1, I101A: 0.2 min¡1, and I104F: 3.0 min¡1), and for benzphetamine (2B1dH: 36 min¡1,
161
I101A: 12 min¡1, and I104F: 19 min¡1). These two mutants also had undetectable activities for testosterone (less than 0.1 min¡1). Besides I101A and I104F, at least 2-fold decreases were observed in the kcat values for 7BR with T100F, I101F, I104A, and F108A. While T100F, T101F, and F108A also had lower kcat values for 7-EFC, I104A had a 1.8-fold higher kcat than did 2B1dH. Ala-mutants at residues V103, P106, and I107 showed about 2-fold higher kcat values for 7-EFC (2B1dH: 8.1 min¡1, Ala-mutants: 15–23 min¡1) and for benzphetamine (2B1dH: 36 min¡1, Ala-mutants: 66–81 min¡1) but not for 7-BR. V103A and P106A also showed 2-fold higher kcat values for the 16- and 16-hydroxylation of testosterone, respectively. The greatest eVects of Ala/Phe-substitutions on testosterone hydroxylation kinetics were decreases in activity. Of special interest was the regioselectivity of the reaction. As shown in Table 3, the kcat/Km of 2B1dH for 16hydroxylation was almost identical to that of 16hydroxylation (ratio of kcat/Km, 16-OH/16-OH D 1.1). Two-fold or greater changes occurred in the ratios of 16-hydroxylation to 16-hydroxylation with several of the mutants. V104A showed the lowest relative 16hydroxylation (16-OH/16-OH: 0.3). In contrast, the substitutions at residues P106–K109 caused higher 16hydroxylation (16-OH/16-OH: 1.8–4.6). Phe-mutants showed higher ratios of 16-hydroxylation than did their corresponding Ala-mutants, and only 16-hydroxylation was observed with F108A. In addition to the diVerent proWles across the mutants for 16- and 16-hydroxylation, P106F also yielded a 7-hydroxy metabolite (kcat: 1.4 min¡1, Km: 152 M). I101A and I104F were inactive. Catalytic eYciencies (kcat/Km or kcat/S50) of the mutants are shown in Fig. 1, normalized to those of 2B1dH. For each mutant, there was at least a 2-fold decrease or increase in catalytic eYciency for one or more of the reactions monitored. In addition, at least 2-fold diVerences in relative catalytic eYciency were observed
Fig. 1. DiVerences in catalytic eYciencies among substrates. Each column indicates the catalytic eYciency (kcat/Km or kcat/S50) normalized to that of 2B1dH.
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within approximately 2/3 of the mutants among the substrates. Overall, these results strongly implicate residues 100–109 in P450 2B1 function in a substrate-dependent manner. Spectral binding studies with benzphetamine For benzphetamine, signiWcant increases and decreases in the Km were observed. To examine whether these eVects reXected altered substrate binding aYnity as opposed to turnover, spectral titrations were performed with 2B1dH and six mutants (Table 4). Since none of the other substrates examined in steady-state kinetics demonstrate signiWcant spectral changes upon binding to the enzyme, only benzphetamine was used in these studies. All of the mutants showed typical type I spectral changes, as observed for 2B1dH (data not shown). However, the KS values of the mutants (F108A: 13M, Ala/Phe-mutants at residues I101– V103: 56–158M) were diVerent from that of 2B1dH (38M). Moreover, the KS values were very close to the Km for benzphetamine with the exception of I101F and V103A, where the KS values were 2-fold lower than their Km values. In addition to changes in the KS values, 2-fold reductions of Amax were observed with I101A and F108A. Homology modeling Based on the recently reported inhibitor-bound 2B4 structure (PDB: 1SUO), a homology model of 2B1 was constructed. Using this model, we examined the orientations of 7-EFC and testosterone in the active site and the residues that interact with these ligands (Fig. 2 and Table 5). Previous analyses of 2B1 by site-directed mutagenesis and homology modeling based on P450 2C5 indicated that the active site is formed by residues V103, I114, F115, F206, L209, S294, A298, T302, V363, V367, I477, and G478 [4,16]. In addition to these residues, the present model based on the 2B4 structure suggests additional active site residues, including the helix B⬘ region. I101 and I104 are positioned less than 3 Å from 7-EFC and from testosterone docked in both the 16-OH- and 16-OHorientations. Interestingly, in the helix B⬘ region, the possiTable 4 Spectral binding constants for benzphetamine
2B1dH I101A I101F A102F V103A V103F F108A
KS [M]
Amax [unit absorbance/M P450]
38 (1) 81 (6) 56 (4) 158 (11) 69 (5) 88 (8) 13 (1)
0.040 (0.000) 0.019 (0.001) 0.031 (0.001) 0.038 (0.002) 0.030 (0.001) 0.048 (0.002) 0.017 (0.000)
Binding spectra were recorded in a diVerence mode with 2.5–220 M benzphetamine, and A was determined between 420 and 387 nm. Values in parentheses indicate standard error for Wt to Michaelis–Menten equation.
ble contact residues vary among ligands: I101, I104, and F108 for 7-EFC; T100, I101, I104, E105, and F108 for testosterone in the 16-OH-orientation; and I101, V103, and I104 for the 16-OH-orientation of testosterone. Because F108A has a unique kinetic proWle, with 2-fold higher aYnity and lower kcat values for 7-EFC and testosterone than those of 2B1dH, a model of this mutant protein was also constructed and compared with the wild-type model (data not shown). In the helix B⬘ region, there are diVerences between the 2B1 wild-type and F108A models in the distance of the nearest atoms from the ligands, especially at positions 106–109 for 7-EFC and at 103 and 108 for testosterone in the 16-OH-orientation, respectively (Table 5). The 16-OH-orientation was not observed for the F108A model, consistent with the experimental results.
Discussion In the present study, residues 100–109 in the helix B⬘ region of P450 2B1 were substituted with Ala and Phe, and the resulting mutants were expressed in E. coli and puriWed. Steady-state kinetic analysis with 7-BR, 7-EFC, testosterone, and benzphetamine revealed at least twofold changes in catalytic eYciency of each mutant with at least one substrate. In general, eVects on kcat were more prominent than those on Km. The results of the Ala and Phe substitutions were position and substrate dependent. For example, I101A and I104F exhibited very limited catalytic activities for all of the substrates examined, whereas I107F showed higher kcat values than did 2B1dH. The docked models of 2B1 also suggest diVerences in the contact residues between 7-EFC and two orientations of testosterone. These results correlate with structural information suggesting that the helix B⬘ region can adopt diVerent conformations depending on the substrate [27], which would result in diVerent substrate contact residues for diVerent substrates. Large eVects were also observed with some mutants in the binding aYnity for benzphetamine. Ala/Phe-mutants at positions I101–V103 and F108 showed 2- to 3-fold changes in the Ks values, indicating the possibility of enhanced or impeded substrate entry and/or binding. Substitution with Ala at positions I101 and F108 resulted in reductions of the Amax. Low catalytic activities were observed with these two mutants for all of the substrates examined, suggesting that these residues are likely to aVect the environment of the heme pocket. In the 2B4-CPI structure, the corresponding residue I101 interacts directly with the inhibitor (distance of the nearest atom from CPI: 4.6 Å) [11]. The nearest atoms of I101 shown in the docked models of 2B1 are located less than 3 Å from 7-EFC and testosterone. The docked model of F108A shows alternate contact residues from wild-type and does not allow the 16-OH-orientation of testosterone, consistent with the kinetic results and suggesting
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Fig. 2. Relative position of the active site residues in the helix B⬘ region. The possible active site residues in the helix B⬘ region are shown in homology models of 2B1 with 7-EFC (A) and with the 16-OH-orientation (B) and 16-OH-orientation (C) of testosterone. Heme and ligands are illustrated in red and green, respectively. Table 5 Substrate docking results with 2B1 wild-type and F108A models Residues
Shortest distance between ligand and indicated residue (Å) 7-EFC
T100 I101 A102 V103 I104 E105 P106 I107 F108 (A) K109 d Active site residues in WT model Active site residues in F108A model a b c d
a
b
6.68 (6.18) 2.53 (2.36) 7.03 (6.11) 5.62 (4.91) 2.14 (2.11) 6.31 (6.77) 8.02 (8.82) 5.81 (6.84) 3.34 (5.92) 7.50 (9.00) 101, 104, 108, 114, 115, 206, 209, 294, 297, 298, 301, 302, 362, 363, 365–368, 477 101, 103, 104, 114, 115, 209, 297, 298, 301, 302, 363, 365–368, 477
Testosterone 16-OH
Testosterone 16-OH
4.54 (5.36) 2.17 (2.13) 6.68 (6.16) 5.10 (2.97) 2.44 (2.54) 4.46 (4.46) 7.45 (6.86) 5.46 (5.86) 2.92 (4.18) 5.45 (6.26) 98, 99, 100, 101, 104, 105, 108, 114, 115, 206, 209, 294, 297–299, 301–303, 362, 363, 365, 367, 368, 477 98, 101, 103, 104, 105, 108, 114, 115, 206, 209, 297, 298, 301, 302, 362–368, 476–478
5.53 (—)c 1.92 (—) 5.85 (—) 3.10 (—) 2.11 (—) 6.10 (—) 7.71 (—) 7.25 (—) 5.13 (—) 7.84 (—) 98, 101, 103, 104, 114, 115, 206, 209, 297, 298, 301, 302, 362, 363, 365–368, 392, 477, 478
Distance between the nearest atoms of each residue and ligand in the 2B1 wild-type model. Results with F108A model. 16-OH-orientation was not observed in F108A model. Residues located within 5 Å from each ligand. Residues in helix B⬘ region are marked in bold.
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that F108 has a role in maintaining the required structure for testosterone 16-hydroxylation. The nearest atoms of I104 are also located close to 7-EFC and testosterone. Thus, both experimental and computational analyses suggest that residues 101, 104, and 108 likely contribute directly to substrate binding. However, it is also possible that I104 and F108 are involved in hydrophobic interactions with other parts of the 2B1 structure that contribute to the packing interactions in the enzyme, as for the corresponding residues in P450 2B4 [11]. Several other mutants also showed altered testosterone metabolite proWles, implicating diVerent conformations of the helix B⬘ region in testosterone binding. For example, the mutants at residues E105-K109 showed higher 16-hydroxylation, especially the Phe-mutants. Interestingly, in the case of P106F, hydroxylation is also redirected toward the 7-position. Steered molecular dynamics simulations investigating the access of testosterone to the 2B1 active site were performed in a recent report [17], which suggested that a number of residues in the helix B⬘ region (T100–I104 and I107–K109) could interact with testosterone directly or with a water bridge during movement of the substrate. Homology modeling and docking analyses of 2B1 based on the closed 2B4 structure suggest that substrate contact residues in the helix B⬘ region vary in a liganddependent manner that reXects kinetic results with mutants. However, there are some caveats with this interpretation in that X-ray structures of other cytochromes P450 demonstrate movements in the peptide backbone as well as shifts of the side chains in this region. For example, in two 2C5 structures, there is a 2.5 Å diVerence in the location of L103 between the structures bound with diclofenac and DMZ [27]. Unfortunately, the current methodology used for docking substrates into P450 models does not allow signiWcant motion of the backbone, and new rapid docking algorithms will be needed to model conformational changes in regions such as helix B⬘ that accompany ligand binding. Even more extreme movement of the helix B⬘ region is observed upon comparison of the open and closed 2B4 structures, while ligand binding also inXuences the C-terminal turn of helix F, and relocates helices F⬘, G⬘, and G [11,12]. Similar, smaller motions have been observed for the bacterial P450 BM-3 [28,29]. In addition, a ligand-free structure of P450 154C1 shows a wideopen cleft similar to that observed in the open 2B4 structure [30]. Therefore, substrate access and product egress likely occur as a consequence of dynamic changes in P450 conformations. In summary, site-directed mutagenesis strongly suggests that the helix B⬘ region of P450 2B1 plays roles in substrate access, binding, or oxidation. More than one decade of prior study of P450 2B structure–function relationships revealed 13 active site residues distributed among Wve substrate recognition sites. It is noteworthy that the helix B⬘
region is the only one where 10 contiguous residues have now been shown to have functional importance with at least one substrate. Further elucidation of ligand-induced conformational changes in helix B⬘ should greatly facilitate prediction of P450 substrate speciWcity.
Acknowledgments The authors thank Dr. Dmitri R. Davydov, You Qun He, and You Ai He for expert advice and assistance. This work was supported by NIH Grant ES03619 and Center Grant ES06676.
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Functional role of residues in the helix B⬘ region of cytochrome P450 2B1 Wataru Honmaa,¤, Weihua Lib, Hong Liub, Emily E. Scotta,1, James R. Halperta a
Department of Pharmacology and Toxicology, University of Texas Medical Branch, 301 University Blvd, Galveston, TX 77555-1031, USA b Center for Drug Discovery and Design, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, PR China Received 1 November 2004, and in revised form 29 November 2004 Available online 27 December 2004
Abstract Comparison of several recently determined X-ray crystal structures of mammalian cytochrome P450 family 2 enzymes suggests considerable movement of helix B⬘ when ligands bind. To investigate the functional role of helix B⬘ in P450 2B1, residues 100–109 were substituted with alanine and phenylalanine. Kinetic properties were examined with the typical 2B substrates 7-benzyloxyresoruWn, 7-ethoxy-4-triXuoromethylcoumarin, benzphetamine, and testosterone. Several mutants showed 2- to 3-fold changes in kcat values and signiWcant diVerences in catalytic eYciencies among the substrates examined, consistent with structural information suggesting that the helix B⬘ region can adopt multiple conformations with diVerent contact residues depending on the substrate. Homology modeling of P450 2B1 was performed based on an inhibitor-bound P450 2B4 structure, and the docking analyses were consistent with experimental results. The Wndings suggest that residues in the helix B⬘ region aVect regio- and stereoselective oxidation in P450 family 2 enzymes as well as substrate entry. 2004 Elsevier Inc. All rights reserved. Keywords: Cytochrome P450; Monooxygenase; Site-directed mutagenesis
Cytochromes P450 (P450s)2 play an essential role in the oxidative biotransformation of endogenous compounds such as steroids, fatty acids, and prostaglandins, and of exogenous chemicals including drugs, natural plant products, carcinogens, and environmental pollutants. P450s form a large superfamily, and there are signiWcant substrate speciWcity diVerences among families, *
Corresponding Author. Fax: +1 409 772 9642. E-mail address: [email protected] (W. Honma). 1 Present address: Department of Medicinal Chemistry, University of Kansas, 1251 Wescoe Hall Dr., Malott 4067, Lawrence, KS 660457582, USA. 2 Abbreviations used. P450, cytochrome P450; 7-BR, 7-benzyloxyresoruWn; 7-EFC, 7-ethoxy-4-triXuoromethylcoumarin; PCR, polymerase chain reaction; Hepes, N-[2-hydroxyethyl]piperazine-N⬘-[2-ethanesulfonic acid]; NADPH, -nicotinamide adenine dinucleotide phosphate; DMZ, 4-methyl-N-methyl-N-(2-phenyl-2H-pyrazol-3-yl)benzenesulfonamide; CPI, 4-(4-chlorophenyl)imidazole. 0003-9861/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2004.12.014
subfamilies, and isoforms within a subfamily [1]. Current information indicates that selectivity of oxidation reXects initial substrate recognition, substrate access to the active site, binding orientation within the active site, as well as the chemical properties of the substrates [2,3]. An intriguing aspect of many P450 enzymes, especially those of family 2, involves the diVerential regio- and stereoselectivity among structurally related proteins [4]. The structures of several mammalian cytochromes P450 have been reported to date. As the Wrst microsomal P450 to be crystallized, the 2C5 structure (PDB: 1DT6) provided an opportunity to characterize structural features that contribute to substrate binding and enzyme conformation [5]. Subsequent structures of 2C5 with the bound substrates, diclofenac (PDB: 1NR6) or the sulfaphenazole derivative 4-methyl-N-methyl-N(2-phenyl-2H-pyrazol-3-yl)benzenesulfonamide (PDB: 1N6B), indicate that Xexible regions of the protein adapt
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for substrate binding, and that substrates may bind in multiple orientations [6,7]. Like the structures of 2C5, the structures of 2C8 and 2C9, and of 2B4 with a phenylimidazole inhibitor, generally show closed conformations without an obvious route from solvent to the buried active site [8–11]. However, the structure of inhibitor-free 2B4 (PDB: 1PO5) demonstrates a large open cleft that extends from the protein surface directly to the heme iron without perturbing the overall P450-fold [12]. This open cleft is primarily formed by repositioning of helices B⬘ to C and F through G, and is correlated with dissociation of contacts between residues in helix B⬘ and in helix G. In contrast to the bacterial cytochrome P450 enzymes, all of the reported mammalian P450 structures have an extended number of residues between helices F and G. Although there are diVerences in the length of helices F, F⬘, G, and G⬘, the overall secondary structures are maintained among the structures of mammalian P450 family 2 enzymes. To examine the functional role of the F–G region in substrate access and regioselective oxidation, selected residues in P450 2B1 were replaced and the resulting proteins were evaluated for turnover of the typical 2B substrates testosterone, 7-EFC, and 7-BR [13]. Most of the mutants demonstrated only moderate eVects on kinetic oxidation, although L209A showed 2to 6-fold increases in kcat. S221F was the only mutant that exhibited >2-fold decreases in the kcat/Km or kcat/S50 values for all three substrates. F219W was inactive, but its unaltered Ks value for benzphetamine binding suggested that substrate access was not impeded. The opposite side of the cleft in the open 2B4 structure is largely formed by helix B⬘. The region is Xanked by two GXG motifs in most P450 family 2 enzymes. In the absence of side chains, the glycine residues are relatively free to adopt a variety of conformations that contribute to the Xexibility of this region. The loops preceding and following the GXG motifs are stabilized by interactions with adjacent portions of the structure. P450 2B1 shares 77% amino-acid identity with 2B4, and six of ten residues in the helix B⬘ region (residues 100– 109) correspond to those of 2B4 (2B1: TIAVIEPIFK, 2B4: KIAVVDPIFQ). Comparison of the two structures of 2B4 suggests considerable movement of the region around helix B⬘ when ligands bind to P450 family 2 enzymes. The organization and placement of the region including helix B⬘ through the C/D loop signiWcantly alter the accessibility of the active site. The comparison between the open and closed 2B4 structures also reveals an alternate secondary conformation of helix B⬘. In the open structure of 2B4 residues I101–V104 form helix B⬘, whereas residues D105–F108 form helix B⬘ in the 2B4 structure bound with CPI [11,12]. Thus, we hypothesized that the residues in this region have an important function during substrate access and turnover. In the present study, 10 amino acid residues in the helix B⬘ region of P450 2B1 were substituted with the smaller
and larger residues alanine and phenylalanine. 2B1 was selected because of the wealth of prior mutagenesis data on other regions [4,14–17]. The mutations were generated in the background of a highly expressing form of 2B1 that is truncated at the N-terminus and modiWed by the addition of four His residues at the C-terminus [18]. Following puriWcation of the mutants from Escherichia coli, steady state kinetics were determined with the typical 2B substrate 7-BR, 7-EFC, benzphetamine, and testosterone. The results were analyzed with the help of a homology model of 2B1 based on the inhibitor-bound structure of 2B4.
Materials and methods Materials Oligonucleotide primers were obtained from Sigma Genosys (Woodlands, TX). The QuikChange SiteDirected Mutagenesis Kit was from Stratagene (La Jolla, CA). All other reagents were obtained from sources as described previously [17] or from standard suppliers. Rat NADPH-cytochrome P450 reductase and cytochrome b5 were prepared as described [19]. Mutagenesis, expression, and puriWcation The truncated version of 2B1 that served as the background for all of the mutants described in this study, 2B1dH, was generated using overlap extension PCR as described [18]. Basically, residues at positions 3–21 were deleted, several mutations were made at the new N-terminus, and a four-histidine tag was added at the C-terminus. All of the helix B⬘ mutants were generated using the QuikChange site-directed mutagenesis kit (Stratagene) with oligonucleotide primers, each complementary to opposite strands of pKK2B1dH (forward primers are shown in Table 1). Each primer contains a speciWc restriction site introduced to facilitate screening for mutants (PvuI: T100A, V103A/F, and F108A, AlwnI: I101A, I104A, SspI: A102F, I104F, E105A/F, P106A, and K109A/F, BsmI: I107A, BspEI: T100F, KpnI: I101F, DraI: P106F, and I107F). The resulting constructs were sequenced to conWrm the desired mutation and verify the absence of unintended mutations (Protein Chemistry Laboratory, University of Texas Medical Branch, Galveston, TX). 2B1dH and the mutants were expressed in Escherichia coli TOPP3 cells (Stratagene) and puriWed with a Ni–NTA aYnity column as described previously [18]. Cytochrome P450 was quantitated using the reduced-CO diVerence spectrum [20]. Enzymatic assays 7-BR O-debenzylation was measured using a standard Xuorometric assay. The 500-l Wnal reaction mixture
W. Honma et al. / Archives of Biochemistry and Biophysics 435 (2005) 157–165 Table 1 Oligonucleotides used in PCR for site-directed mutagenesis Forward primers T100A I101A V103A I104A E105A P106A I107A F108A K109A T100F I101F A102F V103F I104F E105F P106F I107F K109F
5⬘-tc tct ggt cgg gga GCG atc gct gtg a-3⬘ 5⬘-t cgg gga aca GCC gct gtg att gag-3⬘ 5⬘-atc gct GCG atc gag cca atc ttc aa-3⬘ 5⬘-aca atc gca gta GCT gag cca atc ttc-3⬘ 5⬘-atc gct gtg att GCG cca ata ttc aag-3⬘ 5⬘-gct gtg att gag GCA ata ttc aag gaa-3⬘ 5⬘-gtg att gag cca GCA ttc aag gaa ta-3⬘ 5⬘-att gag ccg atc GCC aag gaa tat ggt-3⬘ 5⬘-gag cca ata ttc GCG gaa tat ggt gtg-3⬘ 5⬘-gat ttc tcc gga cgg gga TTT atc gct gtg att ga-3⬘ 5⬘-ggt cgg ggt acc TTC gct gtg att gag-3⬘ 5⬘-cgg gga aca ata TTT gtg att gag cca-3⬘ 5⬘-g gga acg atc gct TTC att gag cca atc-3⬘ 5⬘-aca atc gct gtg TTT gag cca ata ttc a-3⬘ 5⬘-a atc gct gtg att TTC cca ata ttc aag g-3⬘ 5⬘-gct gtg att gag TTC atc ttt aaa gaa tat ggt-3⬘ 5⬘-gct gtg att gag cca TTC ttt aaa gaa ta-3⬘ 5⬘-gag cca ata ttc TTT gaa tat ggt gtg at-3⬘
The changed bases are marked in bold. Capital letters denote the codon for the altered amino acid residue. SpeciWc restriction enzyme sites (underlined) are generated to verify the desired mutation as described in Materials and methods. The reverse primers are exactly complementary to the forward primers.
contained 10 pmol P450, 40 pmol cytochrome P450 reductase, 20 pmol cytochrome b5, and substrate (0.01– 10 M) in 50 mM Hepes, pH 7.6, 15 mM MgCl2, and 0.1 mM EDTA. The reaction was started by adding 0.5 mM NADPH, continued for 10 min at 37 °C, and terminated with the addition of 2 ml methanol. The resoruWn formed was monitored by comparison with a standard curve using a Perkin–Elmer model 3000 spectroXuorometer with excitation at 550 and emission at 585 nm. 7-EFC O-deethylation was measured in a Wnal volume of 100 l. The reaction mixture contained 10 pmol P450, 40 pmol cytochrome P450 reductase, 20 pmol cytochrome b5, and 5–150 M 7-EFC in 50 mM Hepes, pH 7.6, 15 mM MgCl2, and 0.1 mM EDTA. The reaction was initiated by adding NADPH (1 mM), incubated for 10 min at 37 °C, and terminated with 50 l of 20% trichloroacetic acid. Metabolites were quantitated by Xuorescence using a 7-hydroxy-4-triXuoromethylcoumarin standard as described [21]. Benzphetamine N-demethylation was determined by the method of Prough and Ziegler [22]. The 300-l reaction mixture contained 30 pmol P450, 120 pmol cytochrome P450 reductase, 60 pmol cytochrome b5, and 5–500 M benzphetamine in 50 mM Hepes, pH 7.6, 15 mM MgCl2, and 0.1 mM EDTA. The reaction was started by addition of 1 mM NADPH, allowed to proceed for 10 min at 37 °C, and stopped with 300 l of 10% trichloroacetic acid. Formaldehyde formation from benzphetamine was determined by addition of the NASH reagent, heating at 60 °C for 15 min, cooling, and measuring the absorbance at 412 nm.
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Testosterone hydroxylase activity was assayed according to the protocol described by Ciaccio and Halpert [23]. The Wnal 100-l reaction mixture contained 5 pmol P450, 20 pmol cytochrome P450 reductase, 10 pmol cytochrome b5, and 0.5–280 M [14C]testosterone (30,000 dpm/nmol) in 50 mM Hepes, pH 7.6, 15 mM MgCl2, and 0.1 mM EDTA. The reaction was initiated by the addition of 1 mM NADPH, allowed to proceed for 5 min at 37 °C, and stopped with 50 l tetrahydrofuran. Metabolites were developed on TLC plates (20 £ 20 cm, Si250F(19C), J.T. Baker, Phillipsburg, NJ) by two cycles of chromatography in (4:1 v:v) dichloromethane:acetone, identiWed by autoradiography, and quantitated by scintillation counting. Kinetic constants were assessed by GraphPad Prism (version 3) software (GraphPad Software, San Diego, CA). Spectral binding Binding spectra were recorded on a Shimadzu-2600 spectrophotometer in the diVerence mode. A 2.0-ml solution containing 1 M P450 in 10 mM potassium phosphate, 10% glycerol, and 1 mM EDTA were divided into two quartz cuvettes and a baseline was recorded between 350 and 500 nm. An aliquot of benzphetamine hydrochloride in water was added to the sample cuvette (2.5– 220 M benzphetamine), and the same amount of water was added to the reference cuvette. The diVerence spectra were obtained after the system reached equilibrium at 37 °C, and diVerences monitored in the absorbance at 387 and 420 nm. Docking of 7EFC and testosterone into the active site of homology models of P450 2B1 The initial molecular model was constructed using the Insight II software package [24]. The 2B1 model was constructed based on the crystal structure of a 2B4 complex (PDB: 1SUO). The sequence of 2B1 was obtained from SwissProt (Accession No. P00176). The coordinates of the conserved residues were assigned based on the corresponding residues of the 2B4 complex by Homology/InsightII. The heme group was copied from 2B4 into the 2B1 model. After the coordinate assignment, the initial model of 2B1 was reWned with the Gromacs force Weld using the Gromacs 3.2 software package [25,26]. The protein was placed in a periodic box large enough to contain the enzyme and 0.8 nm of solvent on all sides. The solvent was relaxed by energy minimization while restraining the protein atomic positions with a harmonic potential. The system was energy minimized without restraints for 2000 steps using a combination of steepest descent and conjugate gradient prior to MD equilibration. Equilibration was performed at 300 K with decreasing harmonic constraints over a 15-ps time interval followed by 1 ns
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of equilibration without restraints. The resulting model was energy minimized again for 1000 steps using the conjugate gradient method and was used as the model for subsequent substrate docking. For the 2B1 mutant F108A, the coordinates of the corresponding residue were changed in the 2B1 three-dimensional model by Biopolymer/InsightII, and the resulting model was minimized. The structures of 7-EFC and testosterone were constructed using the Builder module of the Insight II modeling package. Placement of 7-EFC and testosterone into the active site of the 2B1 was accomplished by using the AYnity/InsightII as described previously [17].
Results Steady-state kinetic analysis of helix B⬘ mutants All of the helix B⬘ mutants were examined in the background of a truncated form of P450 2B1. This recombinant enzyme, termed 2B1dH, was engineered for expression in E. coli as a conditionally soluble membrane protein that retained catalytic activity [18]. Eighteen mutants at 10 diVerent positions throughout the helix B⬘ region were constructed (T100A/F, I101A/F, A102F, V103A/F, I104A/F, E105A/F, P106A/F, I107A/ F, F108A, and K109A/F). These mutants expressed at 250–700 nmol P450 (L of E. coli culture)¡1 with the exception of some Phe-mutants, A102F, V103F, E105F, P106F, and K109F (100–150 nmol L¡1). For compari-
son, this same protocol yielded 700–800 nmol L¡1 of the 2B1dH holoprotein. After puriWcation, kinetic constants of the Ala/Phemutants were determined with the representative substrates of P450 2B enzymes, 7-BR, 7-EFC, benzphetamine, and testosterone (Tables 2 and 3). Most of the mutants showed Km values similar to those observed for 2B1dH for 7-BR (2B1dH: 1.9 M, Ala/Phe-mutants: 0.8– 2.6 M) and testosterone (2B1dH: 29 M for 16-OH and 27 M for 16-OH, Ala/Phe-mutants: 22–49 M for 16-OH and 20–48 M for 16-OH) and S50 values similar to those for 2B1dH for 7-EFC (2B1dH: 30 M, Ala/ Phe-mutants: 20–46 M). The few exceptions were F108A, A102F, P106A, and I107A. The substitution of F108 with Ala resulted in 2-fold reductions of S50 for 7EFC (10 M) and of Km for testosterone (17 M for 16hydroxylation). In contrast, the alteration of residue 102 from Ala to Phe resulted in 2-fold increases in the Km values for both 16- and 16-hydroxylation of testosterone (59 and 54 M, respectively). Ala-substitutions at residues P106 and I107 resulted in 2-fold increases in S50 values for 7-EFC (62 and 66 M). For benzphetamine, several Ala/Phe-mutants also showed similar Km values to that of 2B1dH (2B1dH: 37 M, Ala/Phe-mutants: 2869 M). However, a few mutants showed signiWcant increases and decreases in the Km. Ala/Phe-substitutions at residues I101–V103 resulted in 2- to 3-fold increases in Km (103–123 M) and Ala-substitution at residue F108 resulted in 2-fold decrease in the Km (16 M). In contrast to the relatively modest eVects on Km, marked changes in the kcat values and diVerent proWles
Table 2 Steady-state substrate kinetics for mutants of the helix B⬘ region in the 2B1dH background 7-BenzyloxyresoruWn kcata 2B1dH T100A T100F I101A I101F A102F V103A V103F I104A I104F E105A E105F P106A P106F I107A I107F F108A K109A K109F a b c
c
2.8 (0.1) 2.1 (0.1) 1.4 (0.1) 0.1 (0.0) 0.6 (0.0) 2.3 (0.1) 4.5 (0.1) 2.6 (0.1) 0.6 (0.0) 0.6 (0.0) 2.9 (0.1) 1.9 (0.1) 3.6 (0.1) 1.3 (0.1) 1.9 (0.1) 3.2 (0.1) 0.7 (0.0) 2.0 (0.0) 1.4 (0.0)
7-Ethoxy-4-triXuoromethylcoumarin
Benzphetamine
Km b
kcat /Km
kcat
n
S50
kcat/S50
kcat
1.9 (0.2) 1.8 (0.2) 1.8 (0.2) 1.7 (0.2) 1.5 (0.2) 1.5 (0.2) 2.3 (0.2) 1.3 (0.1) 1.1 (0.1) 1.3 (0.1) 1.5 (0.1) 1.5 (0.1) 1.2 (0.1) 2.6 (0.3) 1.3 (0.1) 2.1 (0.1) 1.2 (0.1) 1.1 (0.1) 0.8 (0.1)
1.46 1.18 0.77 0.05 0.42 1.55 1.94 1.95 0.57 0.51 1.93 1.30 2.93 0.48 1.47 1.53 0.60 1.89 1.71
8.1 (0.3) 6.6 (0.4) 4.9 (0.4) 0.2 (0.0) 5.2 (0.5) 6.9 (0.5) 21.7 (1.3) 11.6 (0.8) 14.7 (0.6) 3.0 (0.2) 11.0 (0.3) 7.0 (0.5) 22.9 (1.7) 5.4 (0.3) 15.3 (1.5) 14.1 (0.7) 4.2 (0.2) 13.3 (0.9) 6.6 (0.5)
1.2 (0.1) 1.3 (0.1) 1.1 (0.1) 1.2 (0.1) 1.1 (0.1) 1.3 (0.1) 1.2 (0.1) 1.4 (0.1) 1.2 (0.1) 1.3 (0.1) 1.3 (0.1) 1.2 (0.1) 1.0 (0.1) 1.0 (0.1) 1.0 (0.1) 1.1 (0.1) 0.8 (0.1) 1.1 (0.1) 1.2 (0.1)
30 (3) 32 (4) 39 (8) 41 (9) 46 (9) 40 (5) 31 (4) 41 (5) 25 (2) 38 (4) 21 (2) 32 (5) 62 (9) 40 (5) 66 (13) 32 (4) 10 (2) 34 (5) 33 (5)
0.27 0.21 0.13 0.01 0.11 0.17 0.70 0.29 0.58 0.08 0.53 0.22 0.37 0.14 0.23 0.44 0.42 0.39 0.20
36 (1) 23 (1) 23 (1) 12 (1) 37 (3) 53 (3) 81 (3) 77 (3) 35 (1) 19 (1) 39 (1) 29 (1) 66 (3) 33 (1) 75 (3) 36 (2) 26 (1) 50 (2) 36 (1)
kcat, min¡1. Km, S50, M. Standard error for Wt to the Michaelis–Menten or Hill equation shown in parentheses.
Km 37 (6) 48 (9) 69 (11) 105 (13) 103 (15) 113 (18) 123 (12) 103 (11) 28 (4) 67 (10) 35 (5) 35 (6) 54 (9) 48 (7) 41 (7) 29 (7) 16 (4) 29 (5) 58 (7)
kcat/Km 0.97 0.47 0.34 0.11 0.36 0.47 0.66 0.75 1.25 0.28 1.12 0.83 1.22 0.68 1.83 1.25 1.65 1.73 0.62
W. Honma et al. / Archives of Biochemistry and Biophysics 435 (2005) 157–165 Table 3 Steady-state kinetic constants for testosterone hydroxylation Testosterone kcata 2B1dH T100A T100F I101A I101F A102F V103A V103F I104A I104F E105A E105F P106A P106Ff I107A I107F F108A K109A K109F
Km b
kcat/Km
16
16
16
16
16
16
4.3 (0.1)d 3.6 (0.1) 2.0 (0.1) n.d.e 1.2 (0.0) 3.0 (0.1) 7.3 (0.2) 3.7 (0.1) 1.9 (0.1) n.d. 4.5 (0.1) 3.2 (0.1) 8.1 (0.2) 3.7 (0.1) 3.6 (0.1) 5.1 (0.1) 1.4 (0.0) 4.1 (0.1) 2.0 (0.1)
3.7 (0.1) 4.3 (0.1) 2.6 (0.1) n.d. 1.4 (0.0) 4.8 (0.1) 7.8 (0.2) 2.3 (0.1) 5.5 (0.2) n.d. 3.9 (0.1) 1.7 (0.0) 4.8 (0.1) 1.5 (0.0) 1.4 (0.0) 1.1 (0.0) n.d. 2.2 (0.1) 0.7 (0.0)
29 (2) 48 (4) 35 (4) n.d. 49 (5) 59 (4) 40 (3) 38 (4) 42 (5) n.d. 26 (2) 28 (3) 43 (3) 35 (3) 38 (3) 22 (2) 17 (2) 26 (2) 22 (2)
27 (3) 45 (2) 35 (3) n.d. 48 (4) 54 (4) 42 (3) 36 (5) 42 (4) n.d. 28 (2) 26 (3) 46 (4) 36 (3) 39 (4) 22 (2) n.d. 27 (3) 20 (3)
0.15 0.08 0.06 — 0.02 0.05 0.18 0.10 0.05 — 0.17 0.12 0.19 0.11 0.09 0.23 0.08 0.16 0.09
0.14 0.10 0.07 — 0.03 0.09 0.19 0.06 0.13 — 0.14 0.07 0.10 0.04 0.04 0.05 — 0.08 0.03
16/16c
1.1 0.8 0.8 — 0.8 0.6 1.0 1.6 0.3 — 1.3 1.8 1.8 2.5 2.7 4.6 — 2.0 2.6
a
kcat, min¡1. Km, S50, M. c Ratio of kcat/Km. d Standard error for Wt to Michaelis–Menten equation shown in parentheses. e n.d., not detectable (less than 0.1 min¡1). f P106F showed additional 7-hydroxylation (kcat, 1.4 min¡1; Km, 152 M; and kcat/Km, 0.01). b
were observed among substrates. The most striking result was the remarkable decrease or lack of catalytic activity with Ala-substitution at I101 and Phe-substitution at I104. These mutants showed 2- to 20-fold decreases of the kcat values for 7-BR (2B1dH: 2.8 min¡1, I101A: 0.1 min¡1, and I104F: 0.6 min¡1), for 7-EFC (2B1dH: 8.1 min¡1, I101A: 0.2 min¡1, and I104F: 3.0 min¡1), and for benzphetamine (2B1dH: 36 min¡1,
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I101A: 12 min¡1, and I104F: 19 min¡1). These two mutants also had undetectable activities for testosterone (less than 0.1 min¡1). Besides I101A and I104F, at least 2-fold decreases were observed in the kcat values for 7BR with T100F, I101F, I104A, and F108A. While T100F, T101F, and F108A also had lower kcat values for 7-EFC, I104A had a 1.8-fold higher kcat than did 2B1dH. Ala-mutants at residues V103, P106, and I107 showed about 2-fold higher kcat values for 7-EFC (2B1dH: 8.1 min¡1, Ala-mutants: 15–23 min¡1) and for benzphetamine (2B1dH: 36 min¡1, Ala-mutants: 66–81 min¡1) but not for 7-BR. V103A and P106A also showed 2-fold higher kcat values for the 16- and 16-hydroxylation of testosterone, respectively. The greatest eVects of Ala/Phe-substitutions on testosterone hydroxylation kinetics were decreases in activity. Of special interest was the regioselectivity of the reaction. As shown in Table 3, the kcat/Km of 2B1dH for 16hydroxylation was almost identical to that of 16hydroxylation (ratio of kcat/Km, 16-OH/16-OH D 1.1). Two-fold or greater changes occurred in the ratios of 16-hydroxylation to 16-hydroxylation with several of the mutants. V104A showed the lowest relative 16hydroxylation (16-OH/16-OH: 0.3). In contrast, the substitutions at residues P106–K109 caused higher 16hydroxylation (16-OH/16-OH: 1.8–4.6). Phe-mutants showed higher ratios of 16-hydroxylation than did their corresponding Ala-mutants, and only 16-hydroxylation was observed with F108A. In addition to the diVerent proWles across the mutants for 16- and 16-hydroxylation, P106F also yielded a 7-hydroxy metabolite (kcat: 1.4 min¡1, Km: 152 M). I101A and I104F were inactive. Catalytic eYciencies (kcat/Km or kcat/S50) of the mutants are shown in Fig. 1, normalized to those of 2B1dH. For each mutant, there was at least a 2-fold decrease or increase in catalytic eYciency for one or more of the reactions monitored. In addition, at least 2-fold diVerences in relative catalytic eYciency were observed
Fig. 1. DiVerences in catalytic eYciencies among substrates. Each column indicates the catalytic eYciency (kcat/Km or kcat/S50) normalized to that of 2B1dH.
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within approximately 2/3 of the mutants among the substrates. Overall, these results strongly implicate residues 100–109 in P450 2B1 function in a substrate-dependent manner. Spectral binding studies with benzphetamine For benzphetamine, signiWcant increases and decreases in the Km were observed. To examine whether these eVects reXected altered substrate binding aYnity as opposed to turnover, spectral titrations were performed with 2B1dH and six mutants (Table 4). Since none of the other substrates examined in steady-state kinetics demonstrate signiWcant spectral changes upon binding to the enzyme, only benzphetamine was used in these studies. All of the mutants showed typical type I spectral changes, as observed for 2B1dH (data not shown). However, the KS values of the mutants (F108A: 13M, Ala/Phe-mutants at residues I101– V103: 56–158M) were diVerent from that of 2B1dH (38M). Moreover, the KS values were very close to the Km for benzphetamine with the exception of I101F and V103A, where the KS values were 2-fold lower than their Km values. In addition to changes in the KS values, 2-fold reductions of Amax were observed with I101A and F108A. Homology modeling Based on the recently reported inhibitor-bound 2B4 structure (PDB: 1SUO), a homology model of 2B1 was constructed. Using this model, we examined the orientations of 7-EFC and testosterone in the active site and the residues that interact with these ligands (Fig. 2 and Table 5). Previous analyses of 2B1 by site-directed mutagenesis and homology modeling based on P450 2C5 indicated that the active site is formed by residues V103, I114, F115, F206, L209, S294, A298, T302, V363, V367, I477, and G478 [4,16]. In addition to these residues, the present model based on the 2B4 structure suggests additional active site residues, including the helix B⬘ region. I101 and I104 are positioned less than 3 Å from 7-EFC and from testosterone docked in both the 16-OH- and 16-OHorientations. Interestingly, in the helix B⬘ region, the possiTable 4 Spectral binding constants for benzphetamine
2B1dH I101A I101F A102F V103A V103F F108A
KS [M]
Amax [unit absorbance/M P450]
38 (1) 81 (6) 56 (4) 158 (11) 69 (5) 88 (8) 13 (1)
0.040 (0.000) 0.019 (0.001) 0.031 (0.001) 0.038 (0.002) 0.030 (0.001) 0.048 (0.002) 0.017 (0.000)
Binding spectra were recorded in a diVerence mode with 2.5–220 M benzphetamine, and A was determined between 420 and 387 nm. Values in parentheses indicate standard error for Wt to Michaelis–Menten equation.
ble contact residues vary among ligands: I101, I104, and F108 for 7-EFC; T100, I101, I104, E105, and F108 for testosterone in the 16-OH-orientation; and I101, V103, and I104 for the 16-OH-orientation of testosterone. Because F108A has a unique kinetic proWle, with 2-fold higher aYnity and lower kcat values for 7-EFC and testosterone than those of 2B1dH, a model of this mutant protein was also constructed and compared with the wild-type model (data not shown). In the helix B⬘ region, there are diVerences between the 2B1 wild-type and F108A models in the distance of the nearest atoms from the ligands, especially at positions 106–109 for 7-EFC and at 103 and 108 for testosterone in the 16-OH-orientation, respectively (Table 5). The 16-OH-orientation was not observed for the F108A model, consistent with the experimental results.
Discussion In the present study, residues 100–109 in the helix B⬘ region of P450 2B1 were substituted with Ala and Phe, and the resulting mutants were expressed in E. coli and puriWed. Steady-state kinetic analysis with 7-BR, 7-EFC, testosterone, and benzphetamine revealed at least twofold changes in catalytic eYciency of each mutant with at least one substrate. In general, eVects on kcat were more prominent than those on Km. The results of the Ala and Phe substitutions were position and substrate dependent. For example, I101A and I104F exhibited very limited catalytic activities for all of the substrates examined, whereas I107F showed higher kcat values than did 2B1dH. The docked models of 2B1 also suggest diVerences in the contact residues between 7-EFC and two orientations of testosterone. These results correlate with structural information suggesting that the helix B⬘ region can adopt diVerent conformations depending on the substrate [27], which would result in diVerent substrate contact residues for diVerent substrates. Large eVects were also observed with some mutants in the binding aYnity for benzphetamine. Ala/Phe-mutants at positions I101–V103 and F108 showed 2- to 3-fold changes in the Ks values, indicating the possibility of enhanced or impeded substrate entry and/or binding. Substitution with Ala at positions I101 and F108 resulted in reductions of the Amax. Low catalytic activities were observed with these two mutants for all of the substrates examined, suggesting that these residues are likely to aVect the environment of the heme pocket. In the 2B4-CPI structure, the corresponding residue I101 interacts directly with the inhibitor (distance of the nearest atom from CPI: 4.6 Å) [11]. The nearest atoms of I101 shown in the docked models of 2B1 are located less than 3 Å from 7-EFC and testosterone. The docked model of F108A shows alternate contact residues from wild-type and does not allow the 16-OH-orientation of testosterone, consistent with the kinetic results and suggesting
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Fig. 2. Relative position of the active site residues in the helix B⬘ region. The possible active site residues in the helix B⬘ region are shown in homology models of 2B1 with 7-EFC (A) and with the 16-OH-orientation (B) and 16-OH-orientation (C) of testosterone. Heme and ligands are illustrated in red and green, respectively. Table 5 Substrate docking results with 2B1 wild-type and F108A models Residues
Shortest distance between ligand and indicated residue (Å) 7-EFC
T100 I101 A102 V103 I104 E105 P106 I107 F108 (A) K109 d Active site residues in WT model Active site residues in F108A model a b c d
a
b
6.68 (6.18) 2.53 (2.36) 7.03 (6.11) 5.62 (4.91) 2.14 (2.11) 6.31 (6.77) 8.02 (8.82) 5.81 (6.84) 3.34 (5.92) 7.50 (9.00) 101, 104, 108, 114, 115, 206, 209, 294, 297, 298, 301, 302, 362, 363, 365–368, 477 101, 103, 104, 114, 115, 209, 297, 298, 301, 302, 363, 365–368, 477
Testosterone 16-OH
Testosterone 16-OH
4.54 (5.36) 2.17 (2.13) 6.68 (6.16) 5.10 (2.97) 2.44 (2.54) 4.46 (4.46) 7.45 (6.86) 5.46 (5.86) 2.92 (4.18) 5.45 (6.26) 98, 99, 100, 101, 104, 105, 108, 114, 115, 206, 209, 294, 297–299, 301–303, 362, 363, 365, 367, 368, 477 98, 101, 103, 104, 105, 108, 114, 115, 206, 209, 297, 298, 301, 302, 362–368, 476–478
5.53 (—)c 1.92 (—) 5.85 (—) 3.10 (—) 2.11 (—) 6.10 (—) 7.71 (—) 7.25 (—) 5.13 (—) 7.84 (—) 98, 101, 103, 104, 114, 115, 206, 209, 297, 298, 301, 302, 362, 363, 365–368, 392, 477, 478
Distance between the nearest atoms of each residue and ligand in the 2B1 wild-type model. Results with F108A model. 16-OH-orientation was not observed in F108A model. Residues located within 5 Å from each ligand. Residues in helix B⬘ region are marked in bold.
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that F108 has a role in maintaining the required structure for testosterone 16-hydroxylation. The nearest atoms of I104 are also located close to 7-EFC and testosterone. Thus, both experimental and computational analyses suggest that residues 101, 104, and 108 likely contribute directly to substrate binding. However, it is also possible that I104 and F108 are involved in hydrophobic interactions with other parts of the 2B1 structure that contribute to the packing interactions in the enzyme, as for the corresponding residues in P450 2B4 [11]. Several other mutants also showed altered testosterone metabolite proWles, implicating diVerent conformations of the helix B⬘ region in testosterone binding. For example, the mutants at residues E105-K109 showed higher 16-hydroxylation, especially the Phe-mutants. Interestingly, in the case of P106F, hydroxylation is also redirected toward the 7-position. Steered molecular dynamics simulations investigating the access of testosterone to the 2B1 active site were performed in a recent report [17], which suggested that a number of residues in the helix B⬘ region (T100–I104 and I107–K109) could interact with testosterone directly or with a water bridge during movement of the substrate. Homology modeling and docking analyses of 2B1 based on the closed 2B4 structure suggest that substrate contact residues in the helix B⬘ region vary in a liganddependent manner that reXects kinetic results with mutants. However, there are some caveats with this interpretation in that X-ray structures of other cytochromes P450 demonstrate movements in the peptide backbone as well as shifts of the side chains in this region. For example, in two 2C5 structures, there is a 2.5 Å diVerence in the location of L103 between the structures bound with diclofenac and DMZ [27]. Unfortunately, the current methodology used for docking substrates into P450 models does not allow signiWcant motion of the backbone, and new rapid docking algorithms will be needed to model conformational changes in regions such as helix B⬘ that accompany ligand binding. Even more extreme movement of the helix B⬘ region is observed upon comparison of the open and closed 2B4 structures, while ligand binding also inXuences the C-terminal turn of helix F, and relocates helices F⬘, G⬘, and G [11,12]. Similar, smaller motions have been observed for the bacterial P450 BM-3 [28,29]. In addition, a ligand-free structure of P450 154C1 shows a wideopen cleft similar to that observed in the open 2B4 structure [30]. Therefore, substrate access and product egress likely occur as a consequence of dynamic changes in P450 conformations. In summary, site-directed mutagenesis strongly suggests that the helix B⬘ region of P450 2B1 plays roles in substrate access, binding, or oxidation. More than one decade of prior study of P450 2B structure–function relationships revealed 13 active site residues distributed among Wve substrate recognition sites. It is noteworthy that the helix B⬘
region is the only one where 10 contiguous residues have now been shown to have functional importance with at least one substrate. Further elucidation of ligand-induced conformational changes in helix B⬘ should greatly facilitate prediction of P450 substrate speciWcity.
Acknowledgments The authors thank Dr. Dmitri R. Davydov, You Qun He, and You Ai He for expert advice and assistance. This work was supported by NIH Grant ES03619 and Center Grant ES06676.
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