Studying tautomerism in an important pharmaceutical glibenclamide confined in the thin nanometric layers.

The uniform thin films with variable thicknesses (d = 49, 120, 220 nm) of active pharmaceutical ingredient (API) glibenclamide (GCM) was spin-coated and investigated using broadband dielectric, grazing incident FTIR spectroscopies, atomic force microscopy, and ellipsometry. Data analysis revealed that nanoconfined systems consist of a mixture of amide and imidic acid forms of this pharmaceutical, wherein the ratios of both tautomeric forms in the thin films were different with respect to the molten supercooled bulk system. Moreover, changes in the populations of glibenclamide tautomers, i.e. higher amide to imides ratio in the spatially restricted API with respect to the bulk sample, had a strong impact on the character of the proton transfer reaction. In this context, the kinetic curves constructed on the base of infrared data for the bulk system follow the sigmoidal shape, characteristic for the autocatalytic reaction, while results obtained for the confined samples provide exponential character and indicate first-order transformation. This allows hypothesizing that the autocatalytic nature of the tautomerism in the bulk sample is most likely related to the formation of the amide tautomers which further catalyze the progress of imide-amide transformation. Our results are the first studies showing that the change in the thickness of the film may affect the properties and isomerization kinetics in a pharmaceutical systems. Finally, our data open a new perspective for developing new drug delivery systems.

Comparison of high pressure and nanoscale confinement effects on crystallization of the molecular glass-forming liquid, dimethyl phthalate.

High pressure and nanoscopic confinement are two different strategies commonly employed to modify the physicochemical properties of various materials. Both strategies act mostly by changing the molecular packing. In this work, we performed a comparative study on the effect of compression and confined geometry on crystallization of a molecular liquid. Dielectric spectroscopy was employed to investigate the crystallization of the van der Waals liquid, dimethyl phthalate, in nanoporous alumina of different pore sizes as well as on increased pressure (up to 200 MPa). The analysis of the crystallization kinetics under varying thermodynamic conditions revealed that both strategies affect the crystallization behavior of the sample in very distinct ways. Compression shifts the maximum crystallization rate towards a higher temperature and broadens it. As a result, it is more challenging to avoid crystallization upon cooling the liquid at high pressure. In contrast, when the same material is incorporated into nanopores, crystallization significantly slows down and the maximum rate shifts towards a lower temperature with decreasing pore size. Finally, we show that crystallization in nanoporous alumina is accompanied by pre-crystallization effects upon which a shift of the α-relaxation peak is observed. An equilibration process prior to the initiation of crystallization was detected for the confined material both above and below the glass transition temperature of the interfacial layer, while not in the bulk.

First results of the search for neutrinoless double-beta decay with the NEMO 3 detector.

The NEMO 3 detector, which has been operating in the Fréjus underground laboratory since February 2003, is devoted to the search for neutrinoless double-beta decay (beta beta 0v). The half-lives of the two neutrino double-beta decay (beta beta 2v) have been measured for 100Mo and 82Se. After 389 effective days of data collection from February 2003 until September 2004 (phase I), no evidence for neutrinoless double-beta decay was found from approximately 7 kg of 100Mo and approximately 1 kg of 82Se. The corresponding limits are T1/2(beta beta0v) > 4.6 x 10(23) yr for 100Mo and T1/2(beta beta 0v) > 1.0 x 10(23) yr for 82Se (90% C.L.). Depending on the nuclear matrix element calculation, the limits for the effective Majorana neutrino mass are < 0.7-2.8 e/v for 100Mo and < 1.7-4.9 eV for 82Se.

Regiospecificity of human cytochrome P450 1A1-mediated oxidations: the role of steric effects.

Cytochrome P450 1A1 oxidizes a diverse range of substrates, including the procarcinogenic xenobiotic benzo[a]pyrene (B[a]P) and endogenous fatty acid precursors of prostaglandins, such as arachidonic acid (AA) and eicosapentaenoic acid (EA). We have investigated the extent to which enzyme-substrate interactions govern regio- and stereoselectivity of oxidation of these compounds by using docking and molecular dynamics (MD) simulations to examine the likelihood of substrate oxidation at various sites. Due to structural differences between the substrates analyzed, B[a]P and its diols (planar, rigid), and the fatty acids AA and EA (long, flexible), different docking strategies were required. B[a]P, B[a]P-7,8-diols, (+) 7S,8S- and (-) 7R,8R-diols, were docked into the active site of a homology model of P450 1A1 using an automated routine, Affinity (Accelrys, San Diego, CA). AA and EA, on the other hand, required a series of restrained MD simulations to obtain a variety of productive binding modes. All complexes were evaluated by MD-based in silico site scoring to predict product profiles based on certain geometric criteria, such as angle and distance of a given substrate atom from the ferryl oxygen. For all substrates studied, the in vitro profiles were generally reflected by the in silico scores, which suggests that steric factors play a key role in determining regiospecificity in P450 1A1-mediated oxidations. We have also shown that molecular dynamics simulations may be very useful in determination of product profiles for structurally diverse substrates of P450 enzymes.

Influence of P450 3A4 SRS-2 residues on cooperativity and/or regioselectivity of aflatoxin B(1) oxidation.

The major human liver drug-metabolizing cytochrome P450 enzymes P450 3A4 and P450 3A5 share >85% amino acid sequence identity yet exhibit different regioselectivity toward aflatoxin B(1) (AFB(1)) biotransformation [Gillam et al. (1995) Arch. Biochem. Biophys. 317, 74-384]. P450 3A4 prefers AFB1 3alpha-hydroxylation, which detoxifies and subsequently eliminates the hepatotoxin, over AFB1 exo-8,9-oxidation. P450 3A5, on the other hand, is a relatively sluggish 3alpha-hydroxylase and converts AFB(1) predominantly to the genotoxic exo-8,9-epoxide. Using a combination of approaches (sequence alignment, homology modeling and site-directed mutagenesis), we have previously identified several divergent residues in four of the six putative substrate recognition sites (SRSs) of P450 3A4, which when replaced individually with the corresponding amino acid of P450 3A5, resulted in a significant switch of the characteristic P450 3A4 AFB(1) regioselectivity toward that of P450 3A5 [Wang et al. (1998) Biochemistry 37, 12536-12545]. In particular, residues N206 and L210 in SRS-2 were found to be critical for AFB(1) detoxification via 3alpha-hydroxylation, and the corresponding mutants N206S and L210F most closely mimicked P450 3A5, not only in its regioselectivity of AFB(1) metabolism but also in its overall functional capacity. We have now further explored the plausible reasons for such relative inactivity of the SRS-2 mutants by examining N206S and additional mutants (L210A, L211F, L211A, and N206E) and found that the dramatically lowered activities of the N206S mutant are accompanied by a loss of cooperativity of AFB(1) oxidation. Molecular dynamics analyses with an existing P450 3A4 homology model [Szklarz and Halpert (1997) J. Comput. Aided Mol. Des. 11, 265] suggested that N206 (helix F) interacts with E244 (helix G), creating a salt bridge that stabilizes the protein structure and/or defines the active site cavity. To examine this possibility, several E244 mutants (E244A, V, N, S) were tested, of which E244S was the most notable for its relatively greater impairment of P450 3A4-dependent AFB(1) 3alpha-hydroxylation. However, the results with these E244 mutants failed to validate the N206-E244 interaction predicted from these molecular dynamics analyses. Collectively, our findings to date have led us to reconsider our original interpretations and to reexamine them in the light of AFB(1) molecular modeling analyses with a newly refined P450 3A4 homology model. These analyses predicted that F304 in SRS-4 (I-helix) plays a pivotal role in AFB(1) binding at the active site in either orientation leading to 3alpha- or exo-8,9-oxidation. Consistent with this prediction, conversion of F304 to Ala abolished P450 3A4-dependent AFB(1) 3alpha-hydroxylation and exo-8,9-oxidation.

Molecular modeling of mammalian cytochromes P450: application to study enzyme function.

Cytochromes P450 are important heme-containing enzymes that catalyze the oxidation of a vast array of endogenous and exogenous compounds, including drugs and carcinogens. One of the more successful approaches to study P450 function involves molecular modeling. Because none of the mammalian P450s have been crystallized, a number of homology models have been constructed based on the structures of known bacterial P450s. Molecular models, generated using molecular replacement or distance geometry methods, can be used to dock substrates and/or inhibitors in the active site to explain various aspects of enzyme function. The majority of modeling research has dealt with enzyme-substrate interactions in the active site. The analysis of these interactions has helped us to better understand the mechanism of P450 catalysis and provided the structural basis for the regio- and stereospecificity of substrate oxidation as well as susceptibility to inhibition or inactivation. The models have been utilized to identify and/or confirm key residues and to rationally interpret experimental data. The alteration in activity in a mutant P450 can be related to changes in enzyme-substrate/inhibitor interactions, such as the removal or appearance of van der Waals overlaps or changes in compound mobility. Homology models can also help to analyze P450-redox partner interactions and identify critical determinants of protein stability. We can expect further development of molecular modeling methods and their increasing contribution into research on P450 function as an integral part of a combined theoretical-experimental approach.

Identification of selective mechanism-based inactivators of cytochromes P-450 2B4 and 2B5, and determination of the molecular basis for differential susceptibility.

Rabbit cytochromes P-450 (P-450) 2B4 and 2B5 differ by only 12 amino acid residues yet they exhibit unique steroid hydroxylation profiles. Previous studies have led to the identification of active site residues that are determinants of these specificities. In this study, mechanism-based inactivators were identified that discriminate between the closely related 2B4 and 2B5 enzymes. A previously characterized inhibitor, 2-ethynylnaphthalene (2EN), was found to be selective for 2B4 inactivation. As inhibitor metabolism and the partition ratio affect susceptibility, molecular dynamics simulations were performed to assess the stability of the productive binding orientation of 2EN within 2B4 and 2B5 three-dimensional models. Although 2EN was stable within the 2B4 model, it exhibited substantial movement away from the heme moiety in the 2B5 model. However, heterologously expressed 2B5 was found to catalyze the oxidation of 2EN to the stable product 2-naphthylacetic acid. Thus, the increased mobility of 2EN may result in reduced susceptibility of 2B5 by increasing the probability that the reactive ketene intermediate hydrolyzes with water instead of reacting with active site residues. Another compound, 1-adamantyl propargyl ether (1APE), selectively inactivated 2B5. The structural basis for 2EN and 1APE susceptibility was assessed using active site mutants. Interconversion of 2EN susceptibility was observed for 2B4 or 2B5 mutants containing a single alteration at residue 363. Single substitutions in 2B4 also conferred susceptibility to 1APE; however, multiple alterations were required to reduce the susceptibility of 2B5. These alterations may influence inhibitor susceptibility by affecting the stability of the productive binding orientation.

Molecular basis of P450 inhibition and activation: implications for drug development and drug therapy.

Three-dimensional homology models of cytochromes P450 (P450) 2B1 and P450 3A4 have been utilized along with site-directed mutagenesis to elucidate the molecular determinants of substrate specificity. Most of the key residues identified in 2B enzymes fall within five substrate recognition sites (SRSs) and have counterparts in bacterial P450 residues that regulate substrate binding or access. Docking of inhibitors into 2B models has provided a plausible explanation for changes in susceptibility to mechanism-based inactivation that accompany particular amino acid side-chain replacements. These studies provide a basis for predicting drug interactions due to P450 inhibition and for rational inhibitor design. In addition, the location of P450 3A4 residues capable of influencing homotropic stimulation by substrates and heterotropic stimulation by flavonoids has been identified. Steroid hydroxylation by the wild-type enzyme exhibits sigmoidal kinetics, indicative of positive cooperativity. Based on the 3A4 model and single-site mutants, a double mutant in SRS-2 has been constructed that exhibits normal Michaelis-Menten kinetics. Results of modeling and mutagenesis studies suggest that the substrate and effector bind at adjacent sites within a single large cavity in P450 3A4. A thorough understanding of the location and structural requirements of the substrate-binding and effector sites in cytochrome P450 3A4 should prove valuable in rationalizing and predicting interactions among the multitude of drugs and other compounds that bind to the enzyme.

Structure-function relationships of human liver cytochromes P450 3A: aflatoxin B1 metabolism as a probe.

Cytochromes P450 3A4 and 3A5, the dominant drug-metabolizing enzymes in the human liver, share >85% primary amino acid sequence identity yet exhibit different regioselectivity toward aflatoxin B1 (AFB1) biotransformation [Gillam et al., (1995) Arch. Biochem. Biophys. 317, 374-384]. P450 3A4 apparently prefers AFB1 3alpha-hydroxylation, which results in detoxification and subsequent elimination of the hepatotoxin, over AFB1 exo-8,9-oxidation. In contrast, P450 3A5 is incapable of appreciable AFB1 3alpha-hydroxylation and converts it predominantly to the exo-8,9-oxide which is genotoxic. To elucidate the structural features that govern the regioselectivity of the human liver 3A enzymes in AFB1 metabolism and bioactivation, a combination of approaches including sequence alignment, homology modeling, and site-directed mutagenesis was employed. Specifically, the switch in AFB1 regioselectivity was examined after individual substitution of the divergent amino acids in each of the six putative substrate recognition sites (SRSs) of P450 3A4 with the corresponding amino acid of P450 3A5. Of the P450 3A4 mutants examined, P107S, F108L, N206S, L210F, V376T, S478D, and L479T mutations resulted in a significant switch of P450 3A4 regioselectivity toward that of P450 3A5. The results confirmed the importance of some of these residues in substrate contact in the active site, with residue N206 (SRS-2) being critical for AFB1 detoxification via 3alpha-hydroxylation. Moreover, the P450 3A4 mutant N206S most closely mimicked P450 3A5, not only in its regioselectivity of AFB1 metabolism but also in its overall functional capacity. Furthermore, the other SRS-2 mutant, L210F, also resembled P450 3A5 in its overall AFB1 metabolism and regioselectivity. These findings reveal that a single P450 3A5 SRS domain (SRS-2) is capable of conferring the P450 3A5 phenotype on P450 3A4. In addition, some of these P450 3A4 mutations that affected AFB1 regioselectivity had little influence on testosterone 6beta-hydroxylation, thereby confirming that each substrate-P450 active site fit is indeed unique.

Probing the active site of cytochrome P450 2B1: metabolism of 7-alkoxycoumarins by the wild type and five site-directed mutants.

A series of 7-alkoxycoumarins (chain length of 1-7 carbon atoms) was utilized as active site probes of purified Escherichia coli-expressed cytochrome P450 2B1 wild type and five site-directed mutants (I114V, F206L, V363A, V363L, and G478S). The production of 7-hydroxycoumarin, the O-dealkylation product, by the wild-type enzyme exhibited a rank order of C2 > C4 > C3 > C1 > C5 > C6 = C7. The pattern observed for the P450 I114V mutant was similar to that of the wild-type enzyme, whereas with F206L and G478S mutants, the rate of O-dealkylation was low with all the compounds. In contrast, with V363A, the highest rate of product formation was observed with 7-butoxycoumarin. The V363L mutant preferentially catalyzed the O-dealkylation of 7-methoxy- and 7-ethoxycoumarin, and a further increase in the length of the alkyl chain led to a marked decrease in product formation. The stoichiometry of 7-butoxycoumarin oxidation by V363L suggested that products other than 7-hydroxycoumarin were also formed. HPLC and GC-EIMS analyses revealed that P450 2B1 V363L produced 7-(3-hydroxybutoxy)coumarin and 7-(4-hydroxybutoxy)coumarin as major oxidation products, while the V363A mutant mainly catalyzed the O-dealkylation of 7-butoxycoumarin. Docking of alkoxycoumarins into the active site of a P450 2B1 homology model confirmed the importance of the studied residues in substrate dealkylation and explained the formation of novel 7-butoxycoumarin products by the V363L mutant.

Structural determinants of progesterone hydroxylation by cytochrome P450 2B5: the role of nonsubstrate recognition site residues.

The highly related rabbit cytochromes P450 2B4 and 2B5 differ in only 12 amino acid positions, but only 2B5 has activity toward progesterone. Previously, simultaneous site-directed mutagenesis of four key substrate recognition site (SRS) residues (114, 294, 363, and 367) was shown to result in interconversion of the androstenedione hydroxylase specificities of cytochrome P450 2B4 and 2B5. However, the progesterone metabolite profiles of the 2B4 quadruple mutant or of a quintuple mutant in which residue 370 was also mutated to the 2B5 residue were not identical to that of P450 2B5. Therefore, single mutants of P450 2B5 at the remaining seven positions were constructed, expressed in Escherichia coli, and studied with progesterone as the substrate. The single mutants at positions 120 and 221, which are outside any known SRS, exhibited a significant alteration in progesterone hydroxylation. Based on these results, Ile-114, Arg-120, Ser-221, Ser-294, Ile-363, and Val-367 in cytochrome P450 2B4 were replaced simultaneously with Phe, His, Pro, Thr, Val, and Ala, respectively, from 2B5. This yielded a mutant with a very similar progesterone metabolite profile to that of 2B5, although the total activity was lower. An additional substitution at residue 370 produced a multiple mutant P450 2B4 I114F-R120H-S221P-S294T-I363V-V367A- T370M with very similar or identical substrate specificity, regio- and stereospecificity and kinetic properties to that of P450 2B5 wild type.

Significance of glycine 478 in the metabolism of N-benzyl-1-aminobenzotriazole to reactive intermediates by cytochrome P450 2B1.

The effect of mutating Gly 478 to Ala in rat cytochrome P450 2B1 on the metabolism of N-benzyl-1-aminobenzotriazole was investigated. The 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of the wild-type enzyme was completely inactivated by incubating with 1 microM BBT. The G478A mutant, however, was not inactivated by incubating with up to 10 microM BBT. Whereas metabolism of BBT by the wild-type 2B1 resulted in the formation of benzaldehyde, benzotriazole, aminobenzotriazole, and a new metabolite, the G478A mutant generated only the later. This metabolite was found by NMR, IR, and mass spectrometry to be a dimeric product formed from the reaction of two BBT molecules. Two spectral binding constants, a high-affinity constant that was the same for both enzymes (30-39 microM) and a low-affinity constant that was 5-fold lower for the mutant enzyme (0.3 mM vs 1.4 mM), were observed with BBT. The apparent Km and kcat values for the G478A mutant with BBT were 0.3 mM and 12 nmol (nmol of P450)-1 min-1, respectively. Molecular modeling studies of BBT bound in the active site of P450 2B1 suggested that a mutation of Gly 478 to Ala would result in steric hindrance and suppress oxidation of BBT at the 1-amino nitrogen. When BBT was oriented in the 2B1 active site such that oxidation at the 7-benzyl carbon could occur, no steric overlap between Ala 478 and the substrate was observed. Thus, this orientation of BBT would be preferred by the mutant leading to oxidation at the 7-benzyl carbon and subsequent dimer formation. These findings indicate that a glycine 478 to alanine substitution in P450 2B1 altered the binding of BBT such that inactivating BBT metabolites were no longer generated.

Identification of three key residues in substrate recognition site 5 of human cytochrome P450 3A4 by cassette and site-directed mutagenesis.

Cassette mutagenesis and site-directed mutagenesis were used to investigate the importance of individual amino acid residues at positions 364-377 of cytochrome P450 3A4 in determining steroid hydroxylation or stimulation by alpha-naphthoflavone. The mutants were expressed in an Escherichia coli system, and solubilized membranes were prepared. All mutants except R365G and R365K exhibited anti-3A immunoreactivity on Western blotting, although R372S and R375K were not detected as the Fe2+-CO complex. Replacement of Arg-372 by Lys yielded a typical P450 spectrum. The results indicate that the highly conserved Arg residues at positions 365 and 375 may play a role in stabilizing the tertiary structure or in heme binding. Catalytic activities of 12 mutants were examined using progesterone and testosterone as substrates, and residues 369, 370, and 373 were found to play an important role in determining substrate specificity. Although the three mutants hydroxylated progesterone and testosterone primarily at the 6beta-position like the wild-type, replacement of Ile-369 by Val suppressed progesterone 16alpha-hydroxylase activity, whereas substitution of Ala-370 with Val enhanced progesterone 16alpha-hydroxylation. Interestingly, substitution of Leu-373 with His resulted in production of a new metabolite from both steroids. Moreover, the mutants at positions 369 and 373 were more and less responsive, respectively, than the wild-type to alpha-naphthoflavone stimulation. Alterations in activities or expression of several mutants were interpreted using a three-dimensional model of P450 3A4. The results suggest that analogy with mammalian family 2 and bacterial cytochromes P450 can be used to predict P450 3A residues that contribute to regiospecific steroid hydroxylation.

Molecular modeling of cytochrome P450 3A4.

The three-dimensional structure of human cytochrome P450 3A4 was modeled based on crystallographic coordinates of four bacterial P450s; P450 BM-3, P450cam, P450terp, and P450eryF. The P450 3A4 sequence was aligned to those of the known proteins using a structure-based alignment of P450 BM-3, P450cam, P450terp, and P450eryF. The coordinates of the model were then calculated using a consensus strategy, and the final structure was optimized in the presence of water. The P450 3A4 model resembles P450 BM-3 the most, but the B' helix is similar to that of P450eryF, which leads to an enlarged active site when compared with P450 BM-3, P450cam, and P450terp. The 3A4 residues equivalent to known substrate contact residues of the bacterial proteins and key residues of rat P450 2B1 are located in the active site or the substrate access channel. Docking of progesterone into the P450 3A4 model demonstrated that the substrate bound in a 6 beta-orientation can interact with a number of active site residues, such as 114, 119, 301, 304, 305, 309, 370, 373, and 479, through hydrophobic interactions. The active site of the enzyme can also accommodate erythromycin, which, in addition to the residues listed for progesterone, also contacts residues 101, 104, 105, 214, 215, 217, 218, 374, and 478. The majority of 3A4 residues which interact with progesterone and/or erythromycin possess their equivalents in key residues of P450 2B enzymes, except for residues 297, 480 and 482, which do not contact either substrate in P450 3A4. The results from docking of progesterone and erythromycin into the enzyme model make it possible to pinpoint residues which may be important for 3A4 function and to target them for site-directed mutagenesis.

Use of homology modeling in conjunction with site-directed mutagenesis for analysis of structure-function relationships of mammalian cytochromes P450.

In recent years, homology modeling has become an important tool to study cytochrome P450 function, especially in conjunction with experimental approaches such as site-directed mutagenesis. Molecular models of mammalian P450s can be constructed based on crystal structures of four bacterial enzymes, P450cam, P450 BM-3, P450terp and P450eryF, using molecular replacement or consensus methods. In a model built by molecular replacement, the coordinates are copied from those of a given template protein, while consensus methods utilize more then one protein as a template and are based on distance geometry calculations. The models can be used to identify or confirm key residues, evaluate enzyme-substrate interactions and explain changes in protein stability and/or regio- and stereospecificity of substrate oxidation upon residue substitution by site-directed mutagenesis. P450 models have also been utilized to analyze binding of inhibitors or activators, as well as alterations in inhibition and activation due to residue replacement.

Interconversion of the androstenedione hydroxylase specificities of cytochromes P450 2B4 and 2B5 upon simultaneous site-directed mutagenesis of four key substrate recognition residues.

Based on recent studies of single reciprocal mutants of cytochrome P450 2B4 and the highly related P450 2B5 at positions 114, 294, 363, and 367 [G. D. Szklarz, Y. Q. He, K. M. Kedzie, J. R. Halpert, and V. L. Burnett (1996) Arch. Biochem. Biophys. 327,308-318], a number of multiple mutants were constructed, expressed in Escherichia coli, and assayed with androstenedione, progesterone, and benzyloxyresorufin. Simultaneous substitutions of Ile-114, Ser-294, Ile-363, and Val-367 in cytochrome P450 2B4 with Phe, Thr, Val, and Ala, respectively from 2B5, resulted in a marked increase in androstenedione 15alpha- and 16alpha-hydroxylation compared with the wild-type enzyme and yielded a metabolite profile indistinguishable from that of cytochrome P450 2B5. Likewise, the reciprocal P450 2B5 quadruple mutant exhibited the specificity for 16beta-hydroxylation characteristic of the 2B4 wild type. The two reciprocal quadruple mutants of P450 2B4 and 2B5 also displayed benzyloxyresorufin dealkylase activities similar to those of the wild-type P450 2B5 and 2B4, respectively. However, the progesterone metabolite profile of P450 2B5 was not identical to that of the 2B4 quadruple mutant or of a quintuple mutant in which residue 370 was also mutated to the 2B5 residue. Therefore, the 17beta-acetyl group on progesterone as opposed to the oxo group on androstenedione may lead to interaction with additional amino acid residues.

Secobarbital-mediated inactivation of cytochrome P450 2B1 and its active site mutants. Partitioning between heme and protein alkylation and epoxidation.

Secobarbital (SB) is a relatively selective mechanism-based inactivator of cytochrome P450 2B1, that partitions between epoxidation and heme and protein modification during its enzyme inactivation. The SB-2B1 heme adduct formed in situ in a functionally reconstituted system has been spectrally documented and structurally characterized as N-(5-(2-hydroxypropyl)-5-(1-methylbutyl)barbituric acid)protoporphyrin IX. The SB-protein modification has been localized to 2B1 peptide 277-323 corresponding to the active site helix I of cytochrome P450 101. The targeting of heme and this active site peptide suggests that the 2B1 active site topology could influence the course of its inactivation. To explore this possibility, the individual SB epoxidation, heme and protein modification, and corresponding molar partition ratios of the wild type and seven structural 2B1 mutants, site-directed at specific substrate recognition sites, and known to influence 2B1 catalysis were examined after Escherichia coli expression. These studies reveal that Thr-302 is critical for SB-mediated heme N-alkylation, whereas Val-367 is a critical determinant of 2B1 protein modification, and Val-363 is important for SB epoxidation. SB docking into a refined 2B1 homology model coupled with molecular dynamics analyses provide a logical rationale for these findings.

Elucidation of amino acid residues critical for unique activities of rabbit cytochrome P450 2B5 using hybrid enzymes and reciprocal site-directed mutagenesis with rabbit cytochrome P450 2B4.

The molecular basis for the unique activities of rabbit cytochrome P450 2B5, compared with the highly related rabbit 2B4, was investigated using hybrid enzymes and site-directed mutagenesis. Alterations in androstenedione hydroxylase profiles observed with 2B4-2B5 hybrids expressed in COS cells showed that key amino acids are present in both the N-terminal ApaI fragment (codons 1-122) and an internal SstI fragment (codons 220-393). Based on these results, data obtained with other cytochromes P450 2B, and correlation to the six substrate recognition sites proposed by Gotoh (1992, J. Biol. Chem. 267, 83-90), reciprocal 2B4-2B5 mutants were constructed at positions 114, 294, 363, and 367. Wild-type and mutant enzymes were expressed in Escherichia coli, and the oxidation of a number of substrates was analyzed. All residues studied were found to be important for regio- and stereospecificity of androstenedione hydroxylation. Mutations at these positions also caused alterations in the oxidation of progesterone, benzyloxyresorufin, pentoxyresorufin, ethoxycoumarin, and benzphetamine, with the magnitude and direction of the changes dependent upon the enzyme, residue, and substrate. Major changes in activity were consistently observed upon mutation of residues 114 and 294 in both enzymes, and some of these alterations were interpreted with the help of a 3-D model of P450 2B4. For example, in the 2B4 Ile-114--> Phe mutant, Phe prevents androstenedione from assuming a 16 beta-binding orientation and also hinders binding of benzyloxyresorufin, leading to a loss of activity. Conversely, the presence of Phe-114 stabilizes a 16 alpha-binding orientation of androstenedione, resulting in an increase in this activity.

Site-directed mutagenesis as a tool for molecular modeling of cytochrome P450 2B1.

Prompted by our previous homology model of cytochrome P450 2B1 based on the 3-D structure of P450cam [Szklarz, G. D., Ornstein, R. L., & Halpert, J. R. (1994) J. Biomol. Struct. Dyn. 12, 61-78], we constructed 11 new site-directed mutants at positions 100, 111, 205, 209, 291, 477, and 480 and expressed the enzymes in Escherichia coli. The mutations at positions 209, 477, and 480 affected androstenedione and progesterone hydroxylation as predicted by the model. For example, the Ile-477-->Ala and Ile-480-->Ala mutants retained < or = 5% activity with androstenedione and progesterone but were active with benzphetamine, whereas the Leu-209-->Ala mutant catalyzed 21-hydroxylation of progesterone. Mutations at the other positions, i.e., 100, 111, 205, and 291, did not change enzyme activity, contrary to predictions. Therefore, an improved molecular model of cytochrome P450 2B1 was constructed. An alignment of the P450 2B1 sequence with P450 BM-3, P450cam, and P450terp was optimized using data from site-directed mutagenesis at 27 positions in various cytochromes P450 2B and docking of androstenedione into the active site of the known crystal structures. Because all three structures were found to be suitable templates for P450 2B1, the new model was formulated on the basis of the crystallographic coordinates of the three proteins using a consensus strategy, a modeling method based on distance geometry calculations. The new model provides a means to explain alterations in regio- and stereospecificity of steroid hydroxylation upon residue substitution at key amino acid positions, including positions 114, 206, 209, 290, 302, 363, 367, 477, 478, and 480 in P450 2B1.