Biomedical subjects

James R Halpert

Publications and source records attributed to James R Halpert.

At least 37 records · Page 2Linked to original sources

PubMed2004

CYP3A4 is a vitamin D-24- and 25-hydroxylase: analysis of structure function by site-directed mutagenesis.

Studies were performed to identify the microsomal enzyme that 24-hydroxylates vitamin D, whether 25-hydroxylation occurs, and structure function of the enzyme. Sixteen hepatic recombinant microsomal cytochrome P450 enzymes expressed in baculovirus-infected insect cells were screened for 24-hydroxylase activity. CYP3A4, a vitamin D-25-hydroxylase, and CYP1A1 had the highest 24-hydroxylase activity with 1 alpha-hydroxyvitamin D(2) (1 alpha OHD(2)) as substrate. The ratio of rates of 24-hydroxylation of 1 alpha-hydroxyvitamin D(3) (1 alpha OHD(3)), 1 alpha OHD(2), and vitamin D(2) by CYP3A4 was 3.6/2.8/1.0. Structures of 24-hydroxyvitamin D(2), 1,24(S)-dihydroxyvitamin D(2), and 1,24-dihydroxyvitamin D(3) were confirmed by HPLC and gas chromatography retention time and mass spectroscopy. In characterized human liver microsomes, 24-hydroxylation of 1 alpha OHD(2) by CYP3A4 correlated significantly with 6 beta-hydroxylation of testosterone, a marker of CYP3A4 activity. 24-Hydroxylase activity in recombinant CYP3A4 and pooled human liver microsomes showed dose-dependent inhibition by ketoconazole, troleandomycin, alpha-naphthoflavone, and isoniazid, known inhibitors of CYP3A4. Rates of 24- and 25-hydroxylation of 1 alpha OHD(2) and 1 alpha OHD(3) were determined in recombinant wild-type CYP3A4 and site-directed mutants and naturally occurring variants expressed in Escherichia coli. Substitution of residues showed the most prominent alterations of function at residues 119, 120, 301, 305, and 479. Thus, CYP3A4 is both a 24- and 25-hydroxylase for vitamin D(2), 1 alpha OHD(2), and 1 alpha OHD(3).

Animals↗

PubMed2004

An electrostatically driven conformational transition is involved in the mechanisms of substrate binding and cooperativity in cytochrome P450eryF.

The effect of ionic strength (I) on substrate-induced spin transitions and cooperativity in cytochrome P450eryF was studied. At a saturating concentration of 1-pyrenebutanol (1-PB) increasing ionic strength in the 0.06-1.2 M range promotes the formation of the high-spin state of P450, which fraction increases from 26% at 0.06 M to 75% at 1.2 M. This effect was associated with a considerable decrease in cooperativity as revealed in the 1-PB-induced spin shift. While P450eryF exhibits distinct positive cooperativity (S(50) = 8.3 microM, n = 2.4) with this substrate at low ionic strength (I = 0.06 M), n decreases to 1.2 (S(50) = 3.2 microM) at I = 0.66 M. Increasing ionic strength also increases the distance between the first (effector) molecule of 1-PB and the heme, as detected by the changes in the efficiency of FRET from 1-PB to the heme. The modification of Cys(154) with 7-(diethylamino)-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM) largely suppresses these effects of ionic strength and causes a prominent decrease in the cooperativity. The same effect was observed when Cys(154) was substituted with isoleucine. Importantly, Cys(154) is located at the C-terminal end of helix E and is surrounded by salt bridges formed by arginine, glutamate, and aspartate residues located in helices D, E, F, and G. Our results suggest that the binding of the first substrate molecule causes an important conformational transition in the P450eryF that facilitates the substrate-induced spin shift. This transition is apparently accompanied by dissociation or rearrangement of several salt bridges in the proximity of Cys(154) and modulates accessibility and hydration of the heme pocket.

Allosteric Site↗

PubMed2004

Structure of mammalian cytochrome P450 2B4 complexed with 4-(4-chlorophenyl)imidazole at 1.9-A resolution: insight into the range of P450 conformations and the coordination of redox partner binding.

A 1.9-A molecular structure of the microsomal cytochrome P450 2B4 with the specific inhibitor 4-(4-chlorophenyl)imidazole (CPI) in the active site was determined by x-ray crystallography. In contrast to the previous experimentally determined 2B4 structure, this complex adopted a closed conformation similar to that observed for the mammalian 2C enzymes. The differences between the open and closed structures of 2B4 were primarily limited to the lid domain of helices F through G, helices B' and C, the N terminus of helix I, and the beta(4) region. These large-scale conformational changes were generally due to the relocation of conserved structural elements toward each other with remarkably little remodeling at the secondary structure level. For example, the F' and G' helices were maintained with a sharp turn between them but are placed to form the exterior ceiling of the active site in the CPI complex. CPI was closely surrounded by residues from substrate recognition sites 1, 4, 5, and 6 to form a small, isolated hydrophobic cavity. The switch from open to closed conformation dramatically relocated helix C to a more proximal position. As a result, heme binding interactions were altered, and the putative NADPH-cytochrome P450 reductase binding site was reformed. This suggests a structural mechanism whereby ligand-induced conformational changes may coordinate catalytic activity. Comparison of the 2B4/CPI complex with the open 2B4 structure yields insights into the dynamics involved in substrate access, tight inhibitor binding, and coordination of substrate and redox partner binding.

Amino Acid Sequence↗

PubMed2004

Mutagenesis and molecular dynamics suggest structural and functional roles for residues in the N-terminal portion of the cytochrome P450 2B1 I helix.

To investigate their potential roles in ligand access, binding, and subsequent metabolism, residues in the N-terminal portion of the cytochrome P450 2B1 I helix were mutated to alanine and phenylalanine. Of the 18 mutants from E286 to S294 only 7 yielded holoprotein in an Escherichia coli expression system. Substitutions at positions 289, 290, 292, and 294 caused >/= 2-fold changes in kcat and/or Km for two or more of the 2B1 substrates examined, testosterone, 7-ethoxy-4-trifluoromethylcoumarin, 7-benzyloxyresorufin, and benzphetamine. I290 substitutions had the largest effects on steady-state parameters for three substrates and increased benzphetamine affinity. Steered molecular dynamics simulations of testosterone egress along the I helix identified hydrophobic interactions with I290, L293, and S294 and water bridges to E286 and S294. Sensitivity of holoprotein formation to substitution and effects on substrate binding and metabolism suggest structural and functional roles for residues in the N-terminus of the cytochrome P450 2B1 I helix.

Amino Acid Substitution↗

PubMed2004

Topological changes in the CYP3A4 active site probed with phenyldiazene: effect of interaction with NADPH-cytochrome P450 reductase and cytochrome b5 and of site-directed mutagenesis.

The active site topology of heterologously expressed CYP3A4 purified from an Escherichia coli expression system was examined using phenyldiazene. Incubation of CYP3A4 with phenyldiazene and subsequent oxidation yielded all four potential N-phenylprotoporphyrin IX regioisomers derived from attack on an available nitrogen atom in pyrrole rings B, A, C, or D (N(B):N(A):N(C):N(D) = 6:73:7: 13). Further study using 28 active site mutants showed that substitution of residues closer to the heme, Ala-305, Thr-309, or Ala-370, with a larger residue caused the most drastic changes in regioisomer formation, which reflected the location of each amino acid residue replaced in a CYP3A4 homology model. Previous studies have suggested a conformational change in CYP3A4 upon binding of NADPH-cytochrome P450 reductase (CPR) or cytochrome b(5) (b(5)). Therefore, regioisomer formation was also compared in the absence of redox partners and in the presence of CPR, b(5), or both. Formation of all four regioisomers in CYP3A4 wild type, particularly the minor ones, was reduced in the presence of b(5). CPR also greatly decreased the three minor isomers but increased the major isomer significantly. The presence of b(5) and CPR restored minor isomer formation and suppressed the enhancement of N(A) formation caused by CPR alone. Interestingly, the effects of the redox partners differed among representative active site mutants. In particular, the increase in N(C) upon substitution of Ala-370 with Phe was significantly reversed in the presence of redox partners, strongly suggesting that a conformational change occurs around pyrrole ring C due to protein-protein interactions between CYP3A4 and CPR or b(5).

Animals↗

PubMed2004

Activation of the anticancer prodrugs cyclophosphamide and ifosfamide: identification of cytochrome P450 2B enzymes and site-specific mutants with improved enzyme kinetics.

Cyclophosphamide (CPA) and ifosfamide (IFA) are oxazaphosphorine anticancer prodrugs metabolized by two alternative cytochrome P450 (P450) pathways, drug activation by 4-hydroxylation and drug inactivation by N-dechloroethylation, which generates the neurotoxic and nephrotoxic byproduct chloroacetaldehyde. CPA and IFA metabolism catalyzed by P450s 2B1, 2B4, 2B5, and seven site-specific 2B1 mutants was studied in a reconstituted Escherichia coli expression system to identify residues that contribute to the unique activities and substrate specificities of these enzymes. The catalytic efficiency of CPA 4-hydroxylation by rat P450 2B1 was 10- to 35-fold higher than that of rabbit P450 2B4 or 2B5. With IFA, approximately 50% of metabolism proceeded via N-dechloroethylation for 2B1 and 2B4, whereas CPA N-dechloroethylation corresponded to only approximately 3% of total metabolism (2B1) or was absent (2B4, 2B5). Improved catalytic efficiency of CPA and IFA 4-hydroxylation was obtained upon substitution of 2B1 Ile-114 by Val, and replacement of Val-363 by Leu or Ile selectively suppressed CPA N-dechloroethylation >or=90%. P450 2B1-V367A, containing the Ala replacement found in 2B5, exhibited only approximately 10% of wild-type 2B1 activity for both substrates. Canine P450 2B11, which has Val-114, Leu-363, and Val-367, was therefore predicted to be a regioselective CPA 4-hydroxylase with high catalytic efficiency. Indeed, P450 2B11 was 7- to 8-fold more active as a CPA and IFA 4-hydroxylase than 2B1, exhibited a highly desirable low K(m) (80-160 microM), and catalyzed no CPA N-dechloroethylation. These findings provide insight into the role of specific P450 2B residues in oxazaphosphorine metabolism and pave the way for gene therapeutic applications using P450 enzymes with improved catalytic activity toward these anticancer prodrug substrates.

Animals↗

PubMed2003

Conformational heterogeneity of cytochrome P450 3A4 revealed by high pressure spectroscopy.

We applied hydrostatic pressure perturbation to study substrate-induced transitions in human cytochrome P450 3A4 (CYP3A4) with bromocriptine (BCT) as a substrate. The barotropic behavior of the purified enzyme in solution was compared with that observed in recombinant microsomes of Saccharomyces cerevisiae coexpressing CYP3A4, cytochrome b(5), (b(5)) and NADPH-cytochrome P450 reductase (CPR). Important barotropic heterogeneity of CYP3A4 was detected in both cases. Only about 70% of CYP3A4 in solution and about 50% of the microsomal enzyme were susceptible to a pressure-induced P450-->P420 transition. The results suggest that both in solution and in the membrane CYP3A4 is represented by two conformers with different positions of spin equilibrium and different barotropic properties. No interconversion between these conformers was observed within the time frame of the experiment. Importantly, a pressure-induced spin shift, which is characteristic of all cytochromes P450 studied to date, was detected in CYP3A4 in solution only; the P450-->P420 transition was the sole pressure-induced process detected in microsomes. This fact suggests unusual stabilization of the high-spin state of CYP3A4, which is assumed to reflect decreased water accessibility of the heme moiety due to specific interactions of the hemoprotein with the protein partners (b(5) and CPR) and/or membrane lipids.

Bromocriptine↗

PubMed2003

An open conformation of mammalian cytochrome P450 2B4 at 1.6-A resolution.

The xenobiotic metabolizing cytochromes P450 (P450s) are among the most versatile biological catalysts known, but knowledge of the structural basis for their broad substrate specificity has been limited. P450 2B4 has been frequently used as an experimental model for biochemical and biophysical studies of these membrane proteins. A 1.6-A crystal structure of P450 2B4 reveals a large open cleft that extends from the protein surface directly to the heme iron between the alpha-helical and beta-sheet domains without perturbing the overall P450 fold. This cleft is primarily formed by helices B' to C and F to G. The conformation of these regions is dramatically different from that of the other structurally defined mammalian P450, 2C5/3LVdH, in which the F to G and B' to C regions encapsulate one side of the active site to produce a closed form of the enzyme. The open conformation of 2B4 is trapped by reversible formation of a homodimer in which the residues between helices F and G of one molecule partially fill the open cleft of a symmetry-related molecule, and an intermolecular coordinate bond occurs between H226 and the heme iron. This dimer is observed both in solution and in the crystal. Differences between the structures of 2C5 and 2B4 suggest that defined regions of xenobiotic metabolizing P450s may adopt a substantial range of energetically accessible conformations without perturbing the overall fold. This conformational flexibility is likely to facilitate substrate access, metabolic versatility, and product egress.

Animals↗

PubMed2003

A rational approach to Re-engineer cytochrome P450 2B1 regioselectivity based on the crystal structure of cytochrome P450 2C5.

The regioselectivity for progesterone hydroxylation by cytochrome P450 2B1 was re-engineered based on the x-ray crystal structure of cytochrome P450 2C5. 2B1 is a high K(m) progesterone 16alpha-hydroxylase, whereas 2C5 is a low K(m) progesterone 21-hydroxylase. Initially, nine individual 2B1 active-site residues were changed to the corresponding 2C5 residues, and the mutants were purified from an Escherichia coli expression system and assayed for progesterone hydroxylation. At 150 microm progesterone, I114A, F297G, and V363L showed 5-15% of the 21-hydroxylase activity of 2C5, whereas F206V showed high activity for an unknown product and a 13-fold decrease in K(m). Therefore, a quadruple mutant, I114A/F206V/F297G/V363L (Q), was constructed that showed 60% of 2C5 progesterone 21-hydroxylase activity and 57% regioselectivity. Based on their 2C5-like testosterone hydroxylation profiles, S294D and I477F alone and in combination were added to the quadruple mutant. All three mutants showed enhanced regioselectivity (70%) for progesterone 21-hydroxylation, whereas only Q/I477F had a higher k(cat). Finally, the remaining three single mutants, V103I, V367L, and G478V, were added to Q/I477F and Q/S294D/I477F, yielding seven additional multiple mutants. Among these, Q/V103I/S294D/I477F showed the highest k(cat) (3-fold higher than that of 2C5) and 80% regioselectivity for progesterone 21-hydroxylation. Docking of progesterone into a three-dimensional model of this mutant indicated that 21-hydroxylation is favored. In conclusion, a systematic approach to convert P450 regioselectivity across subfamilies suggests that active-site residues are mainly responsible for regioselectivity differences between 2B1 and 2C5 and validates the reliability of 2B1 models based on the crystal structure of 2C5.

Animals↗

PubMed2003

Analysis of homotropic and heterotropic cooperativity of diazepam oxidation by CYP3A4 using site-directed mutagenesis and kinetic modeling.

The structural basis for the cooperativity of diazepam oxidation catalyzed by human cytochrome P450 3A4 (CYP3A4) and 40 mutants has been investigated. An ordered two-site model in which substrates bind first to a catalytic/effector site and then to the catalytic site was used to explain sigmoidal kinetics for temazepam formation but hyperbolic kinetics for nordiazepam formation. In this model diazepam binds to the enzyme-substrate complex with a greater affinity (K(S2)=140 microM) than to free enzyme (K(S1)=960 microM). Residues 107, 119, 211, 301, 304, 309, 369, 370, and 373 play an important role in determining regioselectivity of diazepam oxidation. Interestingly, S119F and A370F displayed sigmoidal kinetics for nordiazepam formation, whereas I301F exhibited hyperbolic kinetics for both products. In the presence of increasing concentrations of testosterone, K(S1) for diazepam decreased, whereas K(S2) increased. The data suggest that three sites exist within the active pocket.

Binding Sites↗

PubMed2003

Homotropic versus heterotopic cooperativity of cytochrome P450eryF: a substrate oxidation and spectral titration study.

P450eryF is the only bacterial P450 to show cooperativity of substrate binding and oxidation. However, the studies reported so far have provided evidence only for homotropic cooperativity of P450eryF but not for heterotropic cooperativity. Therefore, oxidation of 7-benzyloxyquinoline (7-BQ) and 1-pyrenebutanol (1-PB) by P450eryF A245T and spectral binding of 9-aminophenanthrene (9-AP) to wild-type P450eryF were investigated in the presence of various effectors. The addition of steroids and flavones caused no stimulation but rather moderate inhibition of 7-BQ or 1-PB oxidation by P450eryF A245T. However, the binding affinity of 9-AP was significantly increased in the presence of androstenedione or alpha-naphthoflavone (ANF). A comparative study with CYP3A4 revealed a similar increase in the binding affinity of 9-AP for the enzyme at low ANF concentrations but some competition at higher ANF concentrations. These studies, to our knowledge, provide the first report of heterotropic cooperativity in P450eryF as well as spectroscopic evidence for simultaneous presence of two ligand molecules in the CYP3A4 active site.

Alanine↗

PubMed2003

Analysis of differential substrate selectivities of CYP2B6 and CYP2E1 by site-directed mutagenesis and molecular modeling.

Human CYP2B6 and CYP2E1 were used to investigate the extent to which differential substrate selectivities between cytochrome P450 subfamilies reflect differences in active-site residues as opposed to distinct arrangement of the backbone of the enzymes. Reciprocal CYP2B6 and CYP2E1 mutants at active-site positions 103, 209, 294, 363, 367, and 477 (numbering according to CYP2B6) were characterized using the CYP2B6-selective substrate 7-ethoxy-4-trifluoromethylcoumarin, the CYP2E1-selective substrate p-nitrophenol, and the common substrates 7-ethoxycoumarin, 7-butoxycoumarin, and arachidonic acid. This report is the first to study the active site of CYP2E1 by systematic site-directed mutagenesis. One of the most intriguing findings was that substitution of CYP2E1 Phe-477 with valine from CYP2B6 resulted in significant 7-ethoxy-4-trifluoromethylcoumarin deethylation. Use of three-dimensional models of CYP2B6 and CYP2E1 based on the crystal structure of CYP2C5 suggested that deethylation of 7-ethoxy-4-trifluoromethylcoumarin by CYP2E1 is impeded by van der Waals overlaps with the side chain of Phe-477. Interestingly, none of the CYP2B6 mutants acquired enhanced ability to hydroxylate p-nitrophenol. Substitution of residue 363 in CYP2E1 and CYP2B6 resulted in significant alterations of the metabolite profile for the side chain hydroxylation of 7-butoxycoumarin. Probing of CYP2E1 mutants with arachidonic acid indicated that residues Leu-209 and Phe-477 are critical for substrate orientation in the active site. Overall, the study revealed that differences in the side chains of active-site residues are partially responsible for differential substrate selectivities across cytochrome P450 subfamilies. However, the relative importance of active-site residues appears to be dependent on the structural similarity of the compound to other substrates of the enzyme.

Amino Acid Sequence↗

PubMed2002

Allosteric mechanisms in P450eryF probed with 1-pyrenebutanol, a novel fluorescent substrate.

1-Pyrenebutanol (1-PB) has been used as a new fluorescent substrate for P450eryF to explore the molecular mechanisms of cooperativity. Hydroxylation of 1-PB by P450eryF was detected by both fluorometric and chromatographic assays. Binding was monitored by a substrate-induced low-to-high spin shift, as well as by fluorescence resonance energy transfer (FRET) from 1-PB to the heme. Spectrophotometric titration showed that P450eryF has high affinity for 1-PB with distinct positive cooperativity (S(50) = 12.4 +/- 2.2 microM, n = 2.3 +/- 0.6), as also revealed in activity measurements. FRET analysis showed a different binding process obeying a simple bimolecular mechanism with a K(D) = 2.15 +/- 0.8 microM that suggests the presence of the higher affinity binding site. 1-PB binding at this site appears not to modulate the spin state directly but rather to facilitate the spin shift caused by the interactions of P450eryF with the other substrate molecule.

Allosteric Site↗

PubMed2002

7-Benzyloxyquinoline oxidation by P450eryF A245T: finding of a new fluorescent substrate probe.

The main objective of the present study was to find a fluorescent substrate probe for cytochrome P450eryF (P450eryF). P450eryF is a bacterial P450 that catalyzes the hydroxylation of 6-deoxyerythronolide B at the 6S position, a necessary step in the biosynthesis of erythromycin. The lack of a conserved threonine residue in the I-helix, in contrast to other P450s, makes P450eryF unable to oxidize other substrates. A recent study [Xiang et al. (2000) J. Biol. Chem. 275, 35999-36006] has shown that the substitution of Ala-245 by threonine confers on P450eryF significant testosterone hydroxylase activity. Therefore, we investigated various known fluorescent P450 substrates with P450eryF wild-type as well as two mutants, A245S and A245T. Among the various fluorescent compounds tested, 7-benzyloxyquinoline (7-BQ) was found to be the most suitable probe for P450eryF A245T, with rates of oxidation being lower for A245S and wild-type enzyme. The steady-state kinetics of 7-BQ oxidation by A245T are sigmoidal (V(max) = 0.71 nmol/min/nmol, n = 2.18, and S(50) = 132 microM). alpha-Naphthoflavone (alpha-NF), a well-known activator of CYP3A4, did not stimulate 7-BQ oxidation by A245T, although the S(50) value for alpha-NF binding to wild-type P450eryF was similar to P450 3A4. Interestingly, spectral binding studies of wild-type P450eryF and A245T with ketoconazole and miconazole showed differential binding behaviors. Titration of wild-type with ketoconazole and miconazole and of A245T with miconazole showed the expected type-II binding. However, titration of A245T with ketoconazole produced a spectrum similar to type-I. Inhibition studies showed that both ketoconazole and miconazole are able to inhibit 7-BQ oxidation by A245T, although miconazole showed a slightly higher potency. In brief, the present study reports the discovery of 7-BQ as the first fluorescent and only the second unnatural substrate, and of miconazole as an effective P450eryF inhibitor.

Androstenes↗

PubMed2002

Substrate routes to the buried active site may vary among cytochromes P450: mutagenesis of the F-G region in P450 2B1.

Until recently, all known structures of bacterial cytochromes P450 suggested that substrate access to the buried active site occurred via the F-G region, a surface loop distal to the heme cavity. However, the structure of P450 51 indicates a large opening from the protein surface along the I helix N-terminus, at right angles to the F-G channel. The single available microsomal P450 structure (2C5) does not obviously favor one potential access route over the other. To determine whether the F-G region forms part of the substrate access channel in the microsomal cytochrome P450 2B1, 11 residues between positions 208 and 230 were substituted with smaller and larger side chains in a highly expressed truncated form of the enzyme. Steady-state kinetic parameters were determined with the substrates testosterone, 7-ethoxy-4-trifluoromethylcoumarin (7-EFC), and 7-benzyoxyresorufin (7-BR). The largest changes, 2-6-fold increases in k(cat) with testosterone and 7-EFC, were observed for L209A, which also exhibits an altered testosterone metabolite profile and probably forms part of the active site roof. F219W demonstrated little or no activity with any of the three substrates examined, although the K(s) value for benzphetamine binding was unaltered. S221F showed little activity with 7-BR. No significant changes were observed in K(m)(testosterone) or S(50)(7-EFC) values for any of the mutants, in stark contrast to the 10-fold and 100-fold changes in K(m) observed for mutants in this region of other cytochromes P450. The minimal changes in 2B1 do not support access via the F-G region of 2B1 and suggest the alternate access route identified in P450 51.

Base Sequence↗

PubMed2002

Site-directed mutagenesis of cytochrome P450eryF: implications for substrate oxidation, cooperativity, and topology of the active site.

The role of five active-site residues (Phe-78, Gly-91, Ser-171, Ile-174, and Leu-175) has been investigated in P450eryF, the only bacterial P450 known to show cooperativity. The residues were selected based on two-ligand-bound P450eryF structures and previous mutagenesis studies of other cytochromes P450. To better understand the role of these residues in substrate catalysis and cooperativity, each mutant was generated in the wild-type and A245T background, a substitution that enables P450eryF to oxidize testosterone and 7-benzyloxyquinoline (7-BQ). Replacement of Phe-78 with tryptophan decreased cooperativity of 9-aminophenanthrene binding, with little effect on testosterone binding or oxidation. Interestingly, substitution of Gly-91 with alanine or phenylalanine abolished the type-I spectral change elicited by testosterone and significantly decreased testosterone hydroxylation. However, G91A/A245T showed a 4-fold higher k(cat) value with 7-BQ compared with A245T. Replacement of Ser-171 with alanine or phenylalanine did not alter cooperativity of testosterone binding but significantly decreased binding affinity and oxidation of testosterone and 7-BQ. The only mutant that exhibited an increased testosterone binding affinity and increased rates of testosterone and 7-BQ oxidation was I174F. Substitution of Ile-175 with phenylalanine decreased testosterone and 7-BQ oxidation. Reaction with phenyldiazene showed that P450eryF may be much more open above pyrrole ring B than other cytochromes P450 and indicated significant changes in active-site topology in some of the mutants. The study suggests a crucial role of residues Ser-171, Ile-174, and Leu-175, which are part of a distal ligand site, in addition to the proximal Gly-91 in determining the oxidative properties of P450eryF.

Bacterial Proteins↗

PubMed2002

Combined three-dimensional quantitative structure-activity relationship analysis of cytochrome P450 2B6 substrates and protein homology modeling.

Understanding the basis of the substrate specificity of cytochrome P450 2B6 (CYP2B6) is important for determining the role of this enzyme in drug metabolism and for predicting new substrates. Pharmacophores were generated for 16 structurally diverse CYP2B6 substrates with Catalyst after overlapping the reaction sites. Two pharmacophores were determined for the CYP2B6 binding site. Both include two hydrophobes and one hydrogen bond acceptor. The three-dimensional structure of CYP2B6 was then modeled based on the crystal structure of CYP2C5. Benzyloxyresorufin and 7-ethoxy-4-trifluoromethylcoumarin, the two lowest K(m) substrates in the training set, were then docked in the active site of CYP2B6. The pharmacophores were combined with the CYP2B6 model by comparing the docking results and the mapping of the two substrates with the pharmacophores. The results indicated that the active site of CYP2B6 complements the pharmacophores. The pharmacophores and the CYP2B6 model were used in conjunction to predict the K(m) values of substrates in a test set of five compounds and yielded satisfactory predictions for benzphetamine, cinnarizine, bupropion, and verapamil but not lidocaine. The CYP2B6 model, the pharmacophores, and the combination of the model with these pharmacophores provide insight into the interactions of CYP2B6 with substrates. The pharmacophores may be used as queries to search a database to predict new substrates for CYP2B6 when the reaction site is known (N- or O-dealkylation). For C-hydroxylation, the CYP2B6 model is helpful in evaluating the possible reaction sites in order for the pharmacophores to predict corresponding K(m) values.

Amino Acid Sequence↗

PubMed2002

Differential oxidation of mifepristone by cytochromes P450 3A4 and 3A5: selective inactivation of P450 3A4.

The principal enzyme involved in the oxidation of mifepristone is cytochrome P450 3A4 (CYP3A4), which undergoes mechanism-based inactivation by the drug. However, no information is available on the interaction with CYP3A5, the second most abundant CYP3A enzyme in adult human liver. Oxidation of mifepristone by recombinant CYP3A4 produced mono- and didemethylated products and one C-hydroxylated metabolite, as reported previously. However, CYP3A5 produced only the demethylated metabolites. The apparent V(max) and K(M) values for formation of the monodemethylated product by CYP3A4 and CYP3A5 were 46 and 30 nmol/min/nmol P450, and 36 and 16 microM, respectively. Unlike CYP3A4, CYP3A5 was not inactivated by mifepristone. The basis of this differential susceptibility was explored using site-directed mutants in which a CYP3A4 residue was converted to its 3A5 counterpart. Surprisingly, none of these replacements caused a significant decrease in CYP3A4 inactivation by mifepristone. Examination of selected CYP3A4 mutants at 20 other positions indicated that the relative formation rate of the C-hydroxylated product could not account for the differential susceptibility of CYP3A4 and 3A5. Together these results indicate that mifepristone fails to orient itself in the CYP3A5 active site in such a way that its propylenic group is accessible for oxidation, thus rendering CYP3A5 unable to produce the C-hydroxylated product or putative ketene that leads to enzyme inactivation. Identification of mifepristone as a selective mechanism-based inactivation of CYP3A4 may be very useful in distinguishing between the two major CYP3A enzymes collectively responsible for the oxidative metabolism of over half of the drugs currently in use.

Cytochrome P-450 CYP3A↗