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Paul F. Hollenberg - One of the best experts on this subject based on the ideXlab platform.
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tert butylphenylacetylene is a potent mechanism based inactivator of cytochrome p450 2b4 inhibition of cytochrome p450 catalysis by steric hindrance
2009Co-Authors: Haoming Zhang, Djemel Hamdane, Hsia Lien Lin, Vyvyca J Walker, Paul F. HollenbergAbstract:We have demonstrated that 4-(tert-butyl)-phenylacetylene (tBPA) is a potent mechanism-based inactivator for cytochrome P450 2B4 (P450 2B4) in the reconstituted system. It inactivates P450 2B4 in a NADPH- and time-dependent manner with a KI of 0.44 μM and kinact of 0.12 min−1. The partition ratio was approximately zero, indicating that inactivation occurs without the reactive intermediate leaving the active site. Liquid chromatography-mass spectrometry analyses revealed that tBPA forms a protein adduct with a 1:1 stoichiometry. Peptide mapping of the tBPA-modified protein provides evidence that tBPA is covalently bound to Thr302. This is consistent with results of molecular modeling that show the terminal carbon of the acetylenic group is only 3.65 A away from Thr302. To characterize the effect of covalent modification of Thr302, tBPA-modified P450 2B4 was purified to homogeneity from the reconstituted system. The Soret band of tBPA-modified protein is red-shifted by 5 to 422 nm compared with unmodified protein. Benzphetamine binding to the modified P450 2B4 causes no spin shift, indicating that substrate binding and/or the heme environment has been altered by covalently bound tBPA. Cytochrome P450 reductase reduces the unmodified and tBPA-modified P450s at approximately the same rate. However, addition of Benzphetamine stimulates the rate of reduction of unmodified P450 2B4 by ∼20-fold but only marginally stimulates reduction of the tBPA-modified protein. This large discrepancy in the stimulation of the first electron transfer by Benzphetamine strongly suggests that the impairment of P450 catalysis is due to inhibition of Benzphetamine binding to the tBPA-modified P450 2B4.
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Kinetics of oxidation of Benzphetamine by compounds I of cytochrome P450 2B4 and its mutants.
2009Co-Authors: Xin Sheng, Haoming Zhang, Paul F. Hollenberg, John H. Horner, Martin NewcombAbstract:Cytochromes P450 are ubiquitous heme-containing enzymes that catalyze a wide range of reactions in nature including many oxidation reactions. The active oxidant species in P450 enzymes are widely thought to be iron(IV)-oxo porphyrin radical cations, termed Compound I species, but these intermediates have not been observed under turnover conditions. We prepared Compounds I of the mammalian hepatic P450 enzyme CYP2B4 and three mutants (E301Q, T302A, and F429H) by laser flash photolysis of the Compound II species that, in turn, were prepared by reaction of the resting enzymes with peroxynitrite. The PN treatment resulted in a small amount of nitration of the P450 as determined by mass spectrometry but no change in reactivity of the P450 in a test reaction. CYP2B4 Compound I oxidized Benzphetamine to norBenzphetamine in high yield in bulk studies. In direct kinetic studies of Benzphetamine oxidations, Compounds I displayed saturation kinetics with similar binding equilibrium constants (Kbind) for each. The fi...
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Mechanistic Analysis of the Inactivation of Cytochrome P450 2B6 by Phencyclidine: Effects on Substrate Binding, Electron Transfer,
2008Co-Authors: Mohamad Shebley, Ute M. Kent, David P Ballou, Paul F. HollenbergAbstract:Phencyclidine (PCP) is a mechanism-based inactivator of cyto-chrome P450 (P450) 2B6. We have analyzed several steps in the P450 catalytic cycle to determine the mechanism of inactivation of P450 2B6 by PCP. Spectral binding studies show that binding of benzphet-amine, a type I ligand, to P450 2B6 was significantly affected as a result of the inactivation, whereas binding of the inhibitor n-oc-tylamine, a type II ligand, was not compromised. Binding of these ligands to P450 2B6 occurs in two phases. Stopped-flow spectral analysis of the binding kinetics of Benzphetamine to PCP-inactivated 2B6 revealed a 15-fold decrease in the rate of binding during the second phase of the kinetics (k1 5.0 s 1, A1 30%; k2 0.02 s 1, A2 70%, where A2 indicates the fractional magnitude of the second phase) comparedwith the native enzyme (k1 8.0
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mechanistic studies with n benzyl 1 aminobenzotriazole inactivated cyp2b1 differential effects on the metabolism of 7 ethoxy 4 trifluoromethyl coumarin testosterone and Benzphetamine
2004Co-Authors: Ute M. Kent, Lise Pascual, Rebecca A Roof, David P Ballou, Paul F. HollenbergAbstract:Mechanistic studies with N-benzyl-1-aminobenzotriazole (BBT)-inactivated cytochrome P450 2B1 were conducted to determine which step(s) in the reaction cycle had been compromised. Stopped-flow studies, formation of the oxy-ferro intermediate, and analysis of products suggested that the reductive process was slower with the BBT-modified enzyme. The reduced rate of reduction alone could not account for the loss in 7-ethoxy-4-(trifluoromethyl)coumarin (EFC) O-deethylation or testosterone hydroxylation activity. Surprisingly, the ability of the BBT-modified enzyme to generate formaldehyde from Benzphetamine was much less affected. Benzphetamine metabolite analysis by electrospray ionization-mass spectrometry showed that the BBT-modified enzyme had a slightly greater propensity towards aromatic hydroxylation together with reduced levels of N-demethylation and little change in the N-debenzylation of Benzphetamine. Orientation of substrates within the active site of the BBT-inactivated enzyme may be affected such that the more flexible Benzphetamine can be metabolized, whereas metabolism of rigid, planar molecules such as EFC and testosterone is hindered.
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comparison of substrate metabolism by cytochromes p450 2b1 2b4 and 2b6 relationship of heme spin state catalysis and the effects of cytochrome b5
2003Co-Authors: James R Reed, Paul F. HollenbergAbstract:The metabolism of selected substrates by cytochromes P450 (P450) 2B1, 2B4, and 2B6 was compared, and the effects of cytochrome b(5) (b(5)) on these reactions were assessed. There did not appear to be any trends regarding the effects of b(5) when the metabolism of a given substrate by the different P450 enzymes was compared. The changes in spin states of the P450 enzymes as a result of interactions with substrates and cytochrome b(5) were also determined. Only P450 2B4 demonstrated a relationship between spin state, reaction coupling and b(5) effects. The rates of Benzphetamine and 7-ethoxy-4-trifluoromethylcoumarin metabolism by the three enzymes could be correlated with the proportions of high spin heme. Similarly, the proportion of reaction coupling during the metabolism of selected substrates was approximately equal to the proportion of high spin P450. The data are interpreted to indicate that a P450 conformational equilibrium coordinately regulates catalysis and spin state changes.
Haoming Zhang - One of the best experts on this subject based on the ideXlab platform.
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tert butylphenylacetylene is a potent mechanism based inactivator of cytochrome p450 2b4 inhibition of cytochrome p450 catalysis by steric hindrance
2009Co-Authors: Haoming Zhang, Djemel Hamdane, Hsia Lien Lin, Vyvyca J Walker, Paul F. HollenbergAbstract:We have demonstrated that 4-(tert-butyl)-phenylacetylene (tBPA) is a potent mechanism-based inactivator for cytochrome P450 2B4 (P450 2B4) in the reconstituted system. It inactivates P450 2B4 in a NADPH- and time-dependent manner with a KI of 0.44 μM and kinact of 0.12 min−1. The partition ratio was approximately zero, indicating that inactivation occurs without the reactive intermediate leaving the active site. Liquid chromatography-mass spectrometry analyses revealed that tBPA forms a protein adduct with a 1:1 stoichiometry. Peptide mapping of the tBPA-modified protein provides evidence that tBPA is covalently bound to Thr302. This is consistent with results of molecular modeling that show the terminal carbon of the acetylenic group is only 3.65 A away from Thr302. To characterize the effect of covalent modification of Thr302, tBPA-modified P450 2B4 was purified to homogeneity from the reconstituted system. The Soret band of tBPA-modified protein is red-shifted by 5 to 422 nm compared with unmodified protein. Benzphetamine binding to the modified P450 2B4 causes no spin shift, indicating that substrate binding and/or the heme environment has been altered by covalently bound tBPA. Cytochrome P450 reductase reduces the unmodified and tBPA-modified P450s at approximately the same rate. However, addition of Benzphetamine stimulates the rate of reduction of unmodified P450 2B4 by ∼20-fold but only marginally stimulates reduction of the tBPA-modified protein. This large discrepancy in the stimulation of the first electron transfer by Benzphetamine strongly suggests that the impairment of P450 catalysis is due to inhibition of Benzphetamine binding to the tBPA-modified P450 2B4.
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Kinetics of oxidation of Benzphetamine by compounds I of cytochrome P450 2B4 and its mutants.
2009Co-Authors: Xin Sheng, Haoming Zhang, Paul F. Hollenberg, John H. Horner, Martin NewcombAbstract:Cytochromes P450 are ubiquitous heme-containing enzymes that catalyze a wide range of reactions in nature including many oxidation reactions. The active oxidant species in P450 enzymes are widely thought to be iron(IV)-oxo porphyrin radical cations, termed Compound I species, but these intermediates have not been observed under turnover conditions. We prepared Compounds I of the mammalian hepatic P450 enzyme CYP2B4 and three mutants (E301Q, T302A, and F429H) by laser flash photolysis of the Compound II species that, in turn, were prepared by reaction of the resting enzymes with peroxynitrite. The PN treatment resulted in a small amount of nitration of the P450 as determined by mass spectrometry but no change in reactivity of the P450 in a test reaction. CYP2B4 Compound I oxidized Benzphetamine to norBenzphetamine in high yield in bulk studies. In direct kinetic studies of Benzphetamine oxidations, Compounds I displayed saturation kinetics with similar binding equilibrium constants (Kbind) for each. The fi...
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cytochrome b5 increases the rate of product formation by cytochrome p450 2b4 and competes with cytochrome p450 reductase for a binding site on cytochrome p450 2b4
2007Co-Authors: Haoming ZhangAbstract:Abstract The kinetics of product formation by cytochrome P450 2B4 were compared in the presence of cytochrome b5 (cyt b5) and NADPH-cyt P450 reductase (CPR) under conditions in which cytochrome P450 (cyt P450) underwent a single catalytic cycle with two substrates, Benzphetamine and cyclohexane. At a cyt P450:cyt b5 molar ratio of 1:1 under single turnover conditions, cyt P450 2B4 catalyzes the oxidation of the substrates, Benzphetamine and cyclohexane, with rate constants of 18 ± 2 and 29 ± 4.5 s–1, respectively. Approximately 500 pmol of norBenzphetamine and 58 pmol of cyclohexanol were formed per nmol of cyt P450. In marked contrast, at a cyt P450:CPR molar ratio of 1:1, cyt P450 2B4 catalyzes the oxidation of Benzphetamine ≅100-fold (k = 0.15 ± 0.05 s–1) and cyclohexane ≅10-fold (k = 2.5 ± 0.35 s–1) more slowly. Four hundred picomoles of norBenzphetamine and 21 pmol of cyclohexanol were formed per nmol of cyt P450. In the presence of equimolar concentrations of cyt P450, cyt b5, and CPR, product formation is biphasic and occurs with fast and slow rate constants characteristic of catalysis by cyt b5 and CPR. Increasing the concentration of cyt b5 enhanced the amount of product formed by cyt b5 while decreasing the amount of product generated by CPR. Under steady-state conditions at all cyt b5:cyt P450 molar ratios examined, cyt b5 inhibits the rate of NADPH consumption. Nevertheless, at low cyt b5:cyt P450 molar ratios ≤1:1, the rate of metabolism of cyclohexane and Benzphetamine is enhanced, whereas at higher cyt b5:cyt P450 molar ratios, cyt b5 progressively inhibits both NADPH consumption and the rate of metabolism. It is proposed that the ability of cyt b5 to enhance substrate metabolism by cyt P450 is related to its ability to increase the rate of catalysis and that the inhibitory properties of cyt b5 are because of its ability to occupy the reductase-binding site on cyt P450 2B4, thereby preventing reduction of ferric cyt P450 and initiation of the catalytic cycle. It is proposed that cyt b5 and CPR compete for a binding site on cyt P450 2B4.
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cytochrome b5 increases the rate of product formation by cytochrome p450 2b4 and competes with cytochrome p450 reductase for a binding site on cytochrome p450 2b4
2007Co-Authors: Haoming Zhang, Lucy WaskellAbstract:The kinetics of product formation by cytochrome P450 2B4 were compared in the presence of cytochrome b(5) (cyt b(5)) and NADPH-cyt P450 reductase (CPR) under conditions in which cytochrome P450 (cyt P450) underwent a single catalytic cycle with two substrates, Benzphetamine and cyclohexane. At a cyt P450:cyt b(5) molar ratio of 1:1 under single turnover conditions, cyt P450 2B4 catalyzes the oxidation of the substrates, Benzphetamine and cyclohexane, with rate constants of 18 +/- 2 and 29 +/- 4.5 s(-1), respectively. Approximately 500 pmol of norBenzphetamine and 58 pmol of cyclohexanol were formed per nmol of cyt P450. In marked contrast, at a cyt P450:CPR molar ratio of 1:1, cyt P450 2B4 catalyzes the oxidation of Benzphetamine congruent with100-fold (k = 0.15 +/- 0.05 s(-1)) and cyclohexane congruent with10-fold (k = 2.5 +/- 0.35 s(-1)) more slowly. Four hundred picomoles of norBenzphetamine and 21 pmol of cyclohexanol were formed per nmol of cyt P450. In the presence of equimolar concentrations of cyt P450, cyt b(5), and CPR, product formation is biphasic and occurs with fast and slow rate constants characteristic of catalysis by cyt b(5) and CPR. Increasing the concentration of cyt b(5) enhanced the amount of product formed by cyt b(5) while decreasing the amount of product generated by CPR. Under steady-state conditions at all cyt b(5):cyt P450 molar ratios examined, cyt b(5) inhibits the rate of NADPH consumption. Nevertheless, at low cyt b(5):cyt P450 molar ratios
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Role of cytochrome b5 in catalysis by cytochrome P450 2B4.
2005Co-Authors: Haoming Zhang, Eugene MyshkinAbstract:Cytochrome b5 has been shown to stimulate, inhibit or have no effect on catalysis by P450 cytochromes. Its action is known to depend on the isozyme of cytochrome P450, the substrate, and experimental conditions. Cytochrome P450 2B4 (CYP 2B4) has been used in our laboratory as a model isozyme to study the role of cytochrome b5 in cytochrome P450 catalysis using two substrates, methoxyflurane and Benzphetamine. One substrate is the volatile anesthetic, methoxyflurane, whose metabolism is consistently markedly stimulated by cytochrome b5. The other is Benzphetamine, whose metabolism is minimally modified by cytochrome b5. Determination of the stoichiometry of the metabolism of both substrates showed that the amount of product formed is the net result of the simultaneous stimulatory and inhibitory actions of cytochrome b5 on catalysis. Site-directed mutagenesis studies revealed that both cytochrome b5 and cytochrome P450 reductase interact with cytochrome P450 on its proximal surface on overlapping but non-identical binding sites. Comparison of the rate of reduction of oxyferrous CYP 2B4 and the rate of substrate oxidation by cyt b5 and reductase with stopped-flow spectrophotometric and rapid chemical quench experiments has demonstrated that although cytochrome b5 and reductase reduce oxyferrous CYP 2B4 at the same rate, substrate oxidation proceeds more slowly in the presence of the reductase.
Minor J. Coon - One of the best experts on this subject based on the ideXlab platform.
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SOLUBILIZED FORM OF CYTOCHROME P-450 FROM LIVER MICROSOMES: LIPID REQUIREMENT FOR DRUG DEMETHYLATION*
2016Co-Authors: Henry W. Strobel, Minor J. CoonAbstract:Summary: A solubilized hepatic microsomal enzyme system previously shown to catalyze the w-hydroxylation of fatty acids also catalyzes the hydroxylation of drugs. Benzphetamine, aminopyrine, ethylmorphine, hexobarbital, norcodeine, and p-nitroanisole undergo aerobic demethylation in the presence of NADPH and the resolved enzyme system. The required submicrosomal components for Benzphetamine demethylation, as determined either by formaldehyde liberation or by NADPH oxidation, are cytochrome P-450, NADPH-cytochrome P-450 reductase, and a heat-stable lipid fraction. Similar re-quirements were shown for the oxidation of aminopyrine, ethylmorphine, and hexobarbital. Laurate and Benzphetamine were found to be mutually inhibitory, as would be expected if a common "methyl hydroxylase " were involved. The solubilized cytochrome P-450 preparation exhibits a difference spectrum in the presence of Benzphetamine with a peak at 392 ml and a trough at 427 rnp and difference spectra with aniline and hexobarbital typical of those obtained with the microsomal bound.form of this hemoprotein. Cytochrome P-450, the carbon monoxide-binding pigment of microsomes (l), serves as a mixed function oxidase effect-ing the hydroxylation of steroids and the oxidative demethyla-tion and hydroxylation of drugs in animal tissues (2-4). Attempts to solubilize this pigment by a variety of tech-niques resulted in the extensive formation of P-420 (l), an altered form possessing no catalytic activity. Recent studies in our laboratory on the w-hydroxylation of fatty acids in liver microsomes led to "solubilization " of the enzyme system by the criterion that enzymatic activity remained i
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peroxo iron and oxenoid iron species as alternative oxygenating agents in cytochrome p450 catalyzed reactions switching by threonine 302 to alanine mutagenesis of cytochrome p450 2b4
1996Co-Authors: Alfin D N Vaz, Steven J. Pernecky, Gregory M Raner, Minor J. CoonAbstract:Among biological catalysts, cytochrome P450 is unmatched in its multiplicity of isoforms, inducers, substrates, and types of chemical reactions catalyzed. In the present study, evidence is given that this versatility extends to the nature of the active oxidant. Although mechanistic evidence from several laboratories points to a hypervalent iron-oxenoid species in P450-catalyzed oxygenation reactions, Akhtar and colleagues [Akhtar, M., Calder, M. R., Corina, D. L. & Wright, J. N. (1982) Biochem. J. 201, 569-580] proposed that in steroid deformylation effected by P450 aromatase an iron-peroxo species is involved. We have shown more recently that purified liver microsomal P450 cytochromes, including phenobarbital-induced P450 2B4, catalyze the analogous deformylation of a series of xenobiotic aldehydes with olefin formation. The investigation presented here on the effect of site-directed mutagenesis of threonine-302 to alanine on the activities of recombinant P450 2B4 with N-terminal amino acids 2-27 deleted [2B4 (delta2-27)] makes use of evidence from other laboratories that the corresponding mutation in bacterial P450s interferes with the activation of dioxygen to the oxenoid species by blocking proton delivery to the active site. The rates of NADPH oxidation, hydrogen peroxide production, and product formation from four substrates, including formaldehyde from Benzphetamine N-demethylation, acetophenone from 1-phenylethanol oxidation, cyclohexanol from cyclohexane hydroxylation, and cyclohexene from cyclohexane carboxaldehyde deformylation, were determined with P450s 2B4, 2B4 (delta2-27), and 2B4 (delta2-27) T302A. Replacement of the threonine residue in the truncated cytochrome gave a 1.6- to 2.5-fold increase in peroxide formation in the presence of a substrate, but resulted in decreased product formation from Benzphetamine (9-fold), cyclohexane (4-fold), and 1-phenylethanol (2-fold). In sharp contrast, the deformylation of cyclohexane carboxaldehyde by the T302A mutant was increased about 10-fold. On the basis of these findings and our previous evidence that aldehyde deformylation is supported by added H202, but not by artificial oxidants, we conclude that the iron-peroxy species is the direct oxygen donor. It remains to be established which of the many other oxidative reactions involving P450 utilize this species and the extent to which peroxo-iron and oxenoid-iron function as alternative oxygenating agents with the numerous isoforms of this versatile catalyst.
Lucy Waskell - One of the best experts on this subject based on the ideXlab platform.
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cytochrome b5 increases the rate of product formation by cytochrome p450 2b4 and competes with cytochrome p450 reductase for a binding site on cytochrome p450 2b4
2007Co-Authors: Haoming Zhang, Lucy WaskellAbstract:The kinetics of product formation by cytochrome P450 2B4 were compared in the presence of cytochrome b(5) (cyt b(5)) and NADPH-cyt P450 reductase (CPR) under conditions in which cytochrome P450 (cyt P450) underwent a single catalytic cycle with two substrates, Benzphetamine and cyclohexane. At a cyt P450:cyt b(5) molar ratio of 1:1 under single turnover conditions, cyt P450 2B4 catalyzes the oxidation of the substrates, Benzphetamine and cyclohexane, with rate constants of 18 +/- 2 and 29 +/- 4.5 s(-1), respectively. Approximately 500 pmol of norBenzphetamine and 58 pmol of cyclohexanol were formed per nmol of cyt P450. In marked contrast, at a cyt P450:CPR molar ratio of 1:1, cyt P450 2B4 catalyzes the oxidation of Benzphetamine congruent with100-fold (k = 0.15 +/- 0.05 s(-1)) and cyclohexane congruent with10-fold (k = 2.5 +/- 0.35 s(-1)) more slowly. Four hundred picomoles of norBenzphetamine and 21 pmol of cyclohexanol were formed per nmol of cyt P450. In the presence of equimolar concentrations of cyt P450, cyt b(5), and CPR, product formation is biphasic and occurs with fast and slow rate constants characteristic of catalysis by cyt b(5) and CPR. Increasing the concentration of cyt b(5) enhanced the amount of product formed by cyt b(5) while decreasing the amount of product generated by CPR. Under steady-state conditions at all cyt b(5):cyt P450 molar ratios examined, cyt b(5) inhibits the rate of NADPH consumption. Nevertheless, at low cyt b(5):cyt P450 molar ratios
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the stoichiometry of the cytochrome p 450 catalyzed metabolism of methoxyflurane and Benzphetamine in the presence and absence of cytochrome b5
1995Co-Authors: Larry D Gruenke, Krystyna Konopka, Marie Cadieu, Lucy WaskellAbstract:The complete stoichiometry of the metabolism of the cytochrome b5 (cyt b5)-requiring substrate, methoxyflurane, by purified cytochrome P-450 2B4 was compared to that of another substrate, Benzphetamine, which does not require cyt b5 for its metabolism. Cyt b5 invariably improved the efficiency of product formation. That is, in the presence of cyt b5 a greater percentage of the reducing equivalents from NADPH were utilized to generate substrate metabolites, primarily at the expense of the side product, superoxide. With methoxyflurane, cyt b5 addition always resulted in an increased rate of product formation, while with Benzphetamine the rate of product formation remained unchanged, increased or decreased. The apparently contradictory observations of increased reaction efficiency but decrease in total product formation for Benzphetamine can be explained by a second effect of cyt b5. Under some experimental conditions cyt b5 inhibits total NADPH consumption. Whether stimulation, inhibition, or no change in product formation is observed in the presence of cyt b5 depends on the net effect of the stimulatory and inhibitory effects of cyt b5. When total NADPH consumption is inhibited by cyt b5, the rapidly metabolized, highly coupled (approximately equal to 50%) substrate, Benzphetamine, undergoes a net decrease in metabolism not counterbalanced by the increase in the efficiency (2-20%) of the reaction. In contrast, in the presence of the slowly metabolized, poorly coupled (approximately equal to 0.5-3%) substrate, methoxyflurane, inhibition of total NADPH consumption by cyt b5 was never sufficient to overcome the stimulation of product formation due to an increase in efficiency of the reaction.
James R. Halpert - One of the best experts on this subject based on the ideXlab platform.
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rational engineering of human cytochrome p450 2b6 for enhanced expression and stability importance of a leu264 phe substitution
2007Co-Authors: Santosh Kumar, James R. Halpert, Yonghong Zhao, Surendra S Negi, Bilikallahalli K MuralidharaAbstract:Despite the emerging importance of human P450 2B6 in xenobiotic metabolism, thorough biochemical and biophysical characterization has been impeded as a result of low expression in Escherichia coli . Comparison with similar N-terminal truncated and C-terminal His-tagged constructs (rat P450 2B1dH, rabbit 2B4dH, and dog 2B11dH) revealed that P450 2B6dH showed the lowest thermal stability, catalytic tolerance to temperature, and chemical stability against guanidinium chloride-induced denaturation. Eleven P450 2B6dH mutants were rationally engineered based on sequence comparison with the three other P450 2B enzymes and the solvent accessibility of residues in the ligand-free crystal structure of P450 2B4dH. L198M, L264F, and L390P showed ∼3-fold higher expression than P450 2B6dH. L264F alone showed enhanced stability against thermal and chemical denaturation compared with P450 2B6dH and was characterized further functionally. L264F showed similar preferential inhibition by pyridine over imidazole derivatives as P450 2B6dH. The Leu 264 →Phe substitution did not alter the K s for inhibitors or the substrate Benzphetamine, the K m for 7-ethoxy-4-(trifluoromethyl)coumarin, or the Benzphetamine metabolite profiles. The enhanced stability and monodisperse nature of L264F made it suitable for isothermal titration calorimetry studies. Interaction of 1-benzylimidazole with L264F yielded a clear binding isotherm with a distinctly different thermodynamic signature from P450 2B4dH. The inhibitor docked differently in the binding pocket of a P450 2B6 homology model than in 2B4, highlighting the different chemistry of the active site of these two enzymes. Thus, L264F is a good candidate to further explore the unique structure-function relationships of P450 2B6 using X-ray crystallography and solution thermodynamics.
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Mutagenesis and molecular dynamics suggest structural and functional roles for residues in the N-terminal portion of the cytochrome P450 2B1 I helix.
2004Co-Authors: Emily E. Scott, Hong Liu, James R. HalpertAbstract: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.
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Escherichia coli expression of site-directed mutants of cytochrome P450 2B1 from six substrate recognition sites: substrate specificity and inhibitor selectivity studies.
1995Co-Authors: James R. HalpertAbstract:Cytochrome P450 2B1 wild-type and eight site-directed mutations at positions 114, 206, 236, 302, 363, 367, and 478 have been expressed in an Escherichia coli system. Solubilized membrane preparations yielded 100-180 nmol of P450/L of culture. The metabolism of a number of substrates including androstenedione, progesterone, (benzyloxy)resorufin, pentoxyresorufin, and Benzphetamine was analyzed. The E. coli-expressed enzymes displayed the same androstenedione metabolite profiles previously observed with a COS cell expression system. Several of the mutants exhibited an increased rate of progesterone hydroxylation, possibly as the result of an enlarged substrate binding pocket and increased D-ring alpha-face binding. (Benzyloxy)resorufin and pentoxyresorufin O-dealkylation by the P450 2B1 mutants exhibited activities ranging from 10% to 99% and 3% to 71% of wild-type, respectively. Interestingly, the Val-363-->Leu mutant showed markedly suppressed pentoxyresorufin but unaltered (benzyloxy)resorufin dealkylase activity. Benzphetamine N-demethylase activities ranged from 28% to 110% of wild-type. Mechanism-based inactivation of the P450 2B1 mutants showed that susceptibility to inactivation by chloramphenicol and D-erythro- and L-threo-chloramphenicol was abolished in the Val-367-->Ala mutant. The Val-363-->Leu mutant was refractory to L-threo-chloramphenicol. Studies of chloramphenicol covalent binding and metabolism by the Val-367-->Ala mutant showed that its resistance to inactivation is largely attributable to an inability to bioactivate the inhibitor. The expression of P450 2B1 wild-type and mutants in E. coli provides an excellent opportunity to study structure/function relationships by site-directed mutagenesis.