Biomedical subjects

J R Halpert

Publications and source records attributed to J R Halpert.

At least 55 records · Page 3Linked to original sources

PubMed1997

Alanine-scanning mutagenesis of a putative substrate recognition site in human cytochrome P450 3A4. Role of residues 210 and 211 in flavonoid activation and substrate specificity.

Alanine-scanning mutagenesis was performed on amino acid residues 210-216 of cytochrome P450 3A4, the major drug-metabolizing enzyme of human liver. Mutagenesis of this region, which has been proposed to align with the C-terminal ends of F-helices from cytochromes P450BM-3, P450terp, and P450cam, served as a test of the applicability of the substrate recognition site model of Gotoh (Gotoh, O. (1992) J. Biol. Chem. 267, 83-90) to P450 3A4. The results, using two steroid substrates, indicated that substitution of Ala for Leu210 altered the responsiveness to the effector alpha-naphthoflavone and the regioselectivity of testosterone hydroxylation. Replacement of Leu211 by Ala also decreased the stimulation by alpha-naphthoflavone, whereas mutations at residues 212-216 had little effect. The diminished flavonoid responses of the 210 and 211 mutants were observed over a wide range of progesterone and alpha-naphthoflavone concentrations. Further characterization was performed with the additional effectors beta-naphthoflavone, flavone, and 4-chromanone. The finding that P450 3A4 with one altered residue, Leu210 --> Ala, can have both an altered testosterone hydroxylation profile and response to flavonoid stimulation provides evidence that the substrate binding and effector sites are at least partially overlapping.

Alanine↗

PubMed1997

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.

Animals↗

PubMed1997

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.

Amino Acid Sequence↗

PubMed1997

Inactivation of cytochrome P450s 2B1, 2B4, 2B6, and 2B11 by arylalkynes.

The time-dependent loss of the 7-ethoxy-4-trifluoromethylcoumarin (EFC) O-deethylase activity of rat P450 2B1, rabbit P450 2B4, or dog P450 2B11 by 1-ethynylnaphthalene (1EN), 2-ethynylnaphthalene (2EN), 2-(1-propynyl)naphthalene (2PN), 1-ethynylanthracene (1EA), 2-ethynylanthracene, 2-ethynylphenanthrene, 3-ethynylphenanthrene, 9-ethynylphenanthrene (9EPh), 9-(1-propynyl)phenanthrene (9PPh), 4-ethynylpyrene (4EP), and 4-(1-propynyl)biphenyl (4PbP) was investigated. The rate constants for inactivation by the arylalkynes in descending order of effectiveness for the top five compounds were 9EPh>9PPh>1EN, 2EN, 2PN for 2B1, 9EPh>2EN>4EP>1EN, 1EA for 2B4, and 9EPh>1EA>4EP, 9PPh>2EN for 2B11. The size and the shape of the aromatic ring system and the placement of the alkyne functional group were important for inactivation. The most effective inactivator with all the isozymes was 9EPh. This compound also inactivated the EFC activity in microsomes from human lymphoblastoid cells expressing human P450 2B6. The specificity of 9EPh for the inhibition or inactivation of different P450 activities in microsomes from rats treated with various inducing agents was determined by measuring lidocaine, testosterone, p-nitrophenol, or erythromycin metabolism. The greatest effect was observed with the 2B-specific products from lidocaine and testosterone, whereas no effect was seen with p-nitrophenol or erythromycin. When the covalent binding of [3H]2EN to microsomal protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, a radiolabeled protein band that corresponds to 2B1 was observed in the lanes containing microsomes from rats treated with phenobarbital and, to a lesser extent, pyridine and isosafrole after incubation with NADPH. When these microsomes were incubated with [3H]9EPh or [3H]1EP, two NADPH-dependent bands were radiolabeled. One corresponded to 2B1/2 and the other to a protein of approximately 59 kDa, which was observed in the lanes from phenobarbital-treated male and female rats and pyridine-treated male rats. No radiolabeled bands were observed with [3H,14C]4PbP with any of the microsomes.

Alkynes↗

PubMed1997

Isolation, heterologous expression and functional characterization of a novel cytochrome P450 3A enzyme from a canine liver cDNA library.

A cDNA encoding a new member of the cytochrome P450 3A subfamily, P450 3A26, has been isolated from phenobarbital-induced canine liver. The sequence encodes a protein of 503 amino acids with 33 nucleotide differences conferring 22 amino acid substitutions when compared with the previously identified canine CYP3A12 enzyme. Nine of the amino acid differences are within the substrate recognition sites (SRSs) identified for P450 family 2, with five residue substitutions clustered within SRS-6. To facilitate heterologous expression in Escherichia coli, the N-terminus of 3A26 was modified. The expressed protein comigrated with a 3A-immunoreactive protein in dog liver microsomes with a slightly greater electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than 3A12, which suggests that 3A26 corresponds to a previously noted but never characterized 3A enzyme in dogs. Functional characterization of 3A26 was undertaken with use of progesterone, testosterone and androstenedione as substrates. Assays of expressed 3A26 and 3A12 demonstrated that 3A26 displays low steroid hydroxylase activity. Identification of an additional canine 3A enzyme should increase our understanding of xenobiotic metabolism in this important animal model. These findings also suggest that 3A26 and 3A12 may be an interesting model system for the investigation of structure-function relationships involved in steroid metabolism catalyzed by members of the cytochrome P450 3A subfamily.

Amino Acid Sequence↗

PubMed1996

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.

Androstenedione↗

PubMed1996

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.

Alkylation↗

PubMed1996

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.

Amino Acid Sequence↗

PubMed1996

Role of residue 480 in substrate specificity of cytochrome P450 2B5 and 2B11.

The role of residue 480 as a determinant of the specificities of cytochrome P450 2B5 and 2B11 toward androstenedione, progesterone, 2,2',4,4',5,5'-hexachlorobiphenyl (245-HCB), and benzyloxyresorufin has been investigated. Two reciprocal mutants at position 480, 2B5 Val-480 -> Leu and 2B11 Leu -> Val, two hybrid enzymes, H1B (amino acid residues 1-370 from P450 2B11, 371-491 from P450 2B5) and H2B (amino acid residues 1-370 from P450 2B5, 372-494 from P450 2B11) (Kedzie et al., 1993, Biochim. Biophys. Acta 1164, 124-132), and two hybrid back mutants, H1B Val-480 -> Leu and H2B Leu-480 -> Val, were constructed and expressed in Escherichia coli, and compared with the wild-type enzymes P450 2B5 and 2B11. For androstenedione metabolism, the Leu-480 -> Val mutation in P450 2B11 resulted in an increase in the 16 beta-OH:16 alpha-OH ratio from 1.2 to 3, whereas the Val-480 -> Leu mutation in hybrid H1B decreased the 16 beta-OH:16 alpha-OH ration from 4.2 to 1.1 In the case of progesterone, the Leu-480 -> Val mutant of P450 2B11 displayed 3-fold higher 16 alpha- and 21-hydroxylase activities than the wild-type 2B11. In the absence of cytochrome b5, 2B11 L480V displayed half of the benzyloxyresorufin O-dealkylase (BROD) activity of 2B11 wild-type, whereas H1B V480L showed 5,6-fold higher activity than H1B. Therefore, residue 480 seems to play an important role in steroid and benzyloxyresorufin metabolism by P450 2B11. In contrast, the mutation Leu-480 -> Val did not have a significant effect on the 245-HCB hydroxylase activity of P450 2B11, and the mutation Val-480 -> Leu in P450 2B5 had no notable effect on its progesterone hydroxylase activity. Cytochrome b5 caused marked stimulation of the BROD activity of P450 2B11 and H1B and their mutants. Furthermore, P450 2B5 exhibited different progesterone metabolite profiles in the absence or presence of cytochrome b5.

Amino Acid Sequence↗

PubMed1996

Mutagenesis study of Asp-290 in cytochrome P450 2B11 using a fusion protein with rat NADPH-cytochrome P450 reductase.

Asp-290 of the phenobarbital-inducible dog liver cytochrome P450 (P450) 2B11 was mutated to nine other amino acid residues by site-directed mutagenesis, and the functional significance of the unique negative charge in P450 2B11 at that position was studied. To facilitate the analysis of mutated P450 2B11 enzymes heterologously expressed in Escherichia coli, an enzymatically active fusion enzyme was genetically engineered between the cDNAs for P450 2B11 and rat liver NADPH-cytochrome P450 reductase using a Ser-Thr linker as previously described (Fisher et al., 1992, Proc. Natl. Acad. Sci. USA 89, 10817-10821). Sonicated whole-cell lysates of E. coli cells expressing the wild-type fusion protein were able to catalyze the 16-hydroxylation of androstenedione (AD) in the absence of added reductase, and exhibited activities and androstenedione metabolite profiles very similar to those of purified and reconstituted enzyme preparations. The substitution of Ala, Glu, Gly, Met, Asn, Arg, Ser, Thr, or Val for Asp-290 of P450 2B11 resulted in decreased AD hydroxylase activities as assessed using solubilized membranes. Replacement of Asp-290 with Glu yielded the highest activity (55% of wild type), while substituting the positively charged amino acid Arg created an enzyme with the lowest activity (< 1% of wild-type activity). Regioselectivity of AD hydroxylation was not affected although the stereoselectivity of hydroxylation at the 16 carbon position was altered in some cases. The use of the fused enzyme to study the effects of site-directed mutagenesis has resulted in the demonstration of the importance of size and charge at position 290 for enzymatic activity of P450 2B11.

Animals↗

PubMed1996

Suppression of rat hepatic microsomal cytochromes P450 by cyclophosphamide is correlated with plasma thyroid hormone levels and displays differential strain sensitivity.

Strain differences in cytochrome P450 (P450) expression were investigated in Sprague-Dawley (SDs) compared with Fischer 344s (F344s) rats after administration of cyclophosphamide (CPA). Animals received a single dose of CPA with sacrifice occurring 6 days post-treatment. At 130 mg/kg, male F344s displayed a greater sensitivity to CPA, as evidenced by a 68% loss of total hepatic microsomal P450 compared with only 35% in SDs. The most dramatic change in P450 was the loss of 2C11 (84% in F344s, 52% in SDs). In the SD, individual rat 2C11 activity was correlated (r2 = 0.76), with the level of plasma thyroxine in that animal. In male F344s administered CPA at 50 mg/kg, 43 and 44% losses in 2C11 activity (P < .05) and thyroxine (P < .01), respectively, were observed, whereas activities characteristic of P450s 2C11, 3A2, 2A2, 2C6 and 2E1/1A2 were unaffected in SDs at this dose. CPA also produced suppression of P450 in female SDs, including female-specific 2C12. Correlation was observed between the loss of P450 expression and change in body weight after treatment in both male and female animals, suggesting that CPA downregulates P450 expression secondary to decreased caloric intake. The anorectic effect of CPA is believed to result from potent central nervous system stimulation, accompanied by a state of adaptive hypothyroidism. It has been reported that CPA produces "feminization" of P450 expression in male rats. However, our findings suggest the alternative explanation that the effects of CPA on P450 expression result from decreased caloric intake.

Animals↗

PubMed1996

Escherichia coli expression and substrate specificities of canine cytochrome P450 3A12 and rabbit cytochrome P450 3A6.

High level Escherichia coli expression of cytochromes P450 3A12 and 3A6 has facilitated the characterization of proteins which exhibit limited activity as purified hepatic enzymes in reconstituted systems. Three 3A12 and two 3A6 constructs modified at the 5'-end to encode the bovine 17 alpha-sequence (Barnes et al., Proc. Natl. Acad. Sci. U.S.A. 88: 5597-5601, 1991), or related sequences, exhibited expression levels ranging from 2 to 89 nmol of cytochrome P450 liter-1. Recombinant canine 3A12 catalyzed steroid 6 beta-hydroxylation and erythromycin demethylation at rates comparable to those obtained in phenobarbital-induced canine liver microsomes. In contrast, 3A12 troleandomycin demethylase activity (2.5 nmol/min/nmol) was significantly lower than that of canine phenobarbital-induced liver microsomes (6.6 nmol/min/nmol). This difference in activity suggests that at least two 3A forms, which may differ functionally, are present within the canine liver. Purification of recombinant rabbit 3A6 revealed that homogeneous and E. coli-solubilized membrane preparations of 3A6 exhibit similar metabolic rates and identical substrate specificities; 3A activity was modulated by 25 microM alpha-naphthoflavone, which stimulated an unidentified progesterone metabolite 9-fold in 3A6 reconstituted systems in contrast to the 4-fold stimulation of 3A12. Furthermore, 25 microM alpha-naphthoflavone inhibited erythromycin demethylation 64 and 33% by purified recombinant 3A6- or 3A6-solubilized membrane fractions, respectively; 3A12-mediated erythromycin demethylation in solubilized membrane fractions was resistant to flavonoid inhibition. These results indicate that, although 3A substrate specificities are highly conserved between species, functional differences exist between canine 3A12 and rabbit 3A6, which may be utilized to better understand 3A structure-function relationships.

Amino Acid Sequence↗

PubMed1996

Participation of cytochromes P4502B and P4503A in cocaine toxicity in rat hepatocytes.

The contributions of cytochromes P4502B (P4502B) and cytochrome P4503A (P4503A) to the bioactivation of cocaine in hepatocytes isolated from Sprague-Dawley rats were assessed using a number of approaches. Hepatocytes were isolated from rats pretreated with either phenobarbital or dexamethasone. Exposure to from 50 to 500 microM of either cocaine or norcocaine resulted in toxicity in hepatocytes from phenobarbital-induced rats. Hepatocytes from dexamethasone-induced rats displayed greater resistance to toxicity mediated by either compound. Although microsomes from dexamethasone- and phenobarbital-induced rats catalyzed cocaine N-demethylation at the same rate, only inhibition of P4502B activity by chloramphenicol and not inhibition of P4503A activity by troleandomycin was associated with protection against cocaine or norcocaine-mediated toxicity. Further, inhibition of P4502B was only effective in protecting against toxicity in hepatocytes isolated from phenobarbital-induced rats. The effects of phenobarbital induction in rats, dogs, and guinea pigs, and the abilities of purified P4502B proteins from rats, dogs, and rabbits to N-demethylate cocaine were investigated. Cytochromes P4502B from different species exhibited different rates of cocaine N-demethylation; microsomes from the guinea pig were able to N-demethylate cocaine at the fastest rate, followed by the dog and the rat. Expressed human P4502B6 exhibited no ability to either N-demethylate cocaine or produce cocaine- or norcocaine-mediated toxicity in lymphoblastoid cells. These results suggest that, although P4502B and P4503A both catalyze the initial oxidation of cocaine in rats, only P4502Bs are involved in further oxidations leading to toxicity. The importance of P4502Bs toward cocaine bioactivation will depend on species-specific isoform activities.

Animals↗

PubMed1996

Dithionite-supported hydroxylation of palmitic acid by cytochrome P450BM-3.

The ability of dithionite, an inexpensive reducing agent routinely used to produce the ferrous-carbonyl form of P450, to support P450BM-3-catalyzed hydroxylation of palmitate was studied. The hydroxylation products in the presence of dithionite were 15, 14, and 13-hydroxyhexadecanoate, with relative distributions similar to those observed with NADPH. The hydroxylation reaction was carried out in two separate steps, anaerobic reduction and subsequent oxidation of P450BM-3 by oxygen bubbling. The reduction step was much slower than the oxidation step, thus limiting the overall rate of hydroxylation. Upon addition of dithionite, the reductase domain of P450BM-3 seemed to be reduced before significant reduction of the heme domain occurred. The discovery of new reducing agents for P450-catalyzed reaction raises the possibility of replacing NADPH in specialty chemical hydroxylation catalyzed by P450s, especially catalytically self-sufficient P450s, such as P450BM-3 or recombinant fusion proteins of P450 covalently linked to a reductase.

Bacterial Proteins↗

PubMed1995

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.

Amino Acid Sequence↗

PubMed1995

Escherichia coli expression of site-directed mutants of cytochrome P450 2B1 from six substrate recognition sites: substrate specificity and inhibitor selectivity studies.

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.

Androstenedione↗

PubMed1995

Structural basis of selective cytochrome P450 inhibition.

Isoform-selective cytochrome P450 inhibitors have greatly facilitated the characterization of the catalytic specificities and pharmacological and toxicological significance of individual P450 enzymes in experimental animals and humans. Recent advances in elucidating the enzymatic determinants of P450 substrate specificity now make it possible to explore how complementary properties of inhibitors and their target enzymes dictate inhibitor selectivity. A thorough understanding of the basis of specificity should lead to the rational design of a new generation of structure-based cytochrome P450 inhibitors for use as probes and modulators of P450 function in vivo.

Animals↗

PubMed1995

Inactivation of Escherichia coli-expressed rabbit cytochrome P-450 2C enzymes by 17 beta-substituted steroids.

The specific inactivation of rabbit cytochromes P-450 2C by 17 beta-substituted steroids has been investigated by using purified, Escherichia coli-expressed enzymes. The expressed P-450s provided a means to characterize accurately the effects of 21,21-dichloroprogesterone, 21,21-dichloropregnenolone, 21-chloro-21-fluoropregnenolone, pregn-5,20-diene-3 beta-ol and pregn-4,20-diene-3-one on progesterone hydroxylation by P-450 2C5, 2C4, 2C3 and 2C3v. Previous studies using rabbit liver microsomes had suggested that 21-chloro-21-fluoropregnenolone is a selective inactivator of 2C5, a progesterone 21-hydroxylase. Studies of the expressed P-450 2C forms showed little selectivity of 21,21-dichloroprogesterone, pregn-5,20-diene-3 beta-ol or pregn-4,20-diene-3-one, whereas 21,21-dichloropregnenolone and 21-chloro-21-fluoropregnenolone preferentially inactivate 2C5. The data indicate the importance of progesterone 21-hydroxylase activity in facilitating selective mechanism-based inactivation of 2C subfamily P-450s by 21,21-dihalogenated steroids. Studies of the inactivation of P-450 2C16, a progesterone 16 alpha-hydroxylase, by the three dihalogenated steroids yielded results consistent with previous findings of 16 alpha-hydroxylase inactivation in rabbit liver microsomes from the inbred B/J strain. Additionally, two mutants, 2C3v:V113A and 2C3v:V113A, T364N were created which confer progesterone 21-hydroxylation on 2C3v. The single mutant, a 6 beta- and 21-hydroxylase, is inactivated rapidly by all three of the 21,21-dihalogenated steroids, whereas the double mutant, a 16 alpha- and 21-hydroxylase, is preferentially inactivated by 21,21-dichloroprogesterone.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗