Quenching of tryptophanyl fluorescence of bovine adrenal P-450C-21 and inhibition of substrate binding by acrylamide

Quenching of the tryptophanyl fluorescence of cytochrome P-450C-21 by acrylamide and its relationship to substrate binding are investigated by using steady-state and time-resolved data. The average collisional quenching constant was 0.4 M whereas the quenching constant for the total fluorescence was 10.8 +/- 0.9 M. This indicates that the quenching is essentially static. The quencher inhibited the binding of the substrate apparently competitively. The inhibition constant was 0.092 M, giving rise to an association constant of 10.9 M which is remarkably similar to the static quenching constant. It is suggested that tryptophan(s) may represent a key to the substrate-binding site in P-450C-21.     

Adrenal microsomal hydroxylating system: purification and substrate binding properties of cytochrome P-450C-21

The substrate-cytochrome P-450C-21 binding reaction has been investigated in detail by using the purified cytochrome. The apparent substrate dissociation constant (KDapp) depended on the enzyme concentration, indicating that the binding reaction does not follow simple two-component mass action equilibrium. However, the binding data fit reasonably well to a model in which the P-450C-21 exists in a monomer-dimer equilibrium and the substrate does not bind to the dimer. The intrinsic dissociation constant (K1) and the dissociation constant for the dimerization reaction (K2) were calculated from the titration data by a pattern search procedure. K1 and K2 were found to be essentially independent of the enzyme concentration, indicating the appropriateness of the assumed model. In the present study, all factors that increased the dissociation of the dimer, as indicated by an increase in K2, decreased KDapp so that it approached the intrinsic constant K1. These results suggest that there is mutual interaction of the substrate binding and self-association reactions of cytochrome P-450C-21 in the purified preparation.     

Mechanism-based inactivation of bovine adrenal cytochromes P450 C-21 and P450 17 alpha by 17 beta-substituted steroids

A series of progesterone derivatives has been studied as potential inactivators of the bovine adrenocortical cytochromes P450, P450 17 alpha, and P450 C-21. Replacement of the 21-methyl group of progesterone with a difluoromethyl group resulted in a selective inactivator of P450 C-21 in a reconstituted system. The loss of 21-hydroxylase activity caused by this compound exhibits a number of characteristics of mechanism-based inactivation including NADPH dependence, pseudo-first-order kinetics, saturability, irreversibility, and protection by substrate. In addition to the difluoro compound, 21,21-dichloroprogesterone, the acetylenic compound pregn-4-en-20-yn-3-one, and the olefinic compound pregna-4,20-dien-3-one all inactivate P450 C-21. In contrast, the only compound to inactivate the rabbit adrenal progesterone 21-hydroxylase is 21,21-dichloroprogesterone. In binding studies, the 21,21-dihalo steroids produce a greater maximal type I spectral shift of P450 C-21 than the two 17 beta-unsaturated steroids. The dihalo compounds inactivate P450 C-21 by both heme destruction and protein modification as shown by significant decreases in residual 21-hydroxylase activity and spectrally detectable P450 after incubation with P450 C-21 in a reconstituted system. Liquid chromatographic and mass spectral analyses of the organic extracts from these incubations showed that 21-pregnenoic acid is a major metabolite of the dihalo compounds with a partition ratio of 5 nmol of acid produced/nmol of P450 C-21 inactivated. This supports the hypothesis that inactivation proceeds in part through an acyl halide intermediate. In contrast, the acetylenic compound pregn-4-en-20-yn-3-one inactivates P450 C-21 mainly by protein modification, producing an NADPH-dependent irreversible type I spectral shift. The stoichiometry of inactivation is approximately 1.5 nmol of compound bound/nmol of enzyme inactivated, indicating selective modification of the enzyme at or near the substrate binding site.     

Methoxyflurane acts at the substrate binding site of cytochrome P450 LM2 to induce a dependence on cytochrome b5

Rabbit cytochrome P450 isozyme 2 requires cytochrome b5 to metabolize the volatile anesthetic methoxyflurane but not the substrate benzphetamine [E. Canova-Davis and L. Waskell (1984) J. Biol. Chem. 259, 2541-2546]. To determine whether the requirement for cytochrome b5 for methoxyflurane oxidation is mediated by an allosteric effect on cytochrome P450 LM2 or cytochrome P450 reductase, we have investigated whether this anesthetic can induce a role for cytochrome b5 in benzphetamine metabolism. Using rabbit liver microsomes and antibodies raised in guinea pigs against rabbit cytochrome b5, we found that methoxyflurane did not create a cytochrome b5 requirement for benzphetamine metabolism. Methoxyflurane also failed to induce a role for cytochrome b5 in benzphetamine metabolism in the purified, reconstituted mixed function oxidase system. Studies of the reaction kinetics established that in the absence of cytochrome b5, methoxyflurane and benzphetamine are competitive inhibitors, and that in the presence of cytochrome b5, benzphetamine and methoxyflurane are two alternate substrates in competition for a single site on the same enzyme. These results all indicate that the methoxyflurane-induced cytochrome b5 dependence of the mixed function oxidase cytochrome P450 LM2 system is a direct result of the interaction between methoxyflurane and the substrate binding site of cytochrome P450 LM2 and suggest the focus of future studies of this question.     

Cytochrome P450 and steroid 21-hydroxylation in microsomes from beef adrenal cortex

Cytochrome P450 in beef adrenal cortex microsomal preparations reacted with progesterone and with 17-hydroxyprogesterone at pH 7.4 to produce Type I spectral changes. The magnitude of the spectral shift produced by addition of progesterone or 17-hydroxyprogesterone was related to the concentration of cytochrome P450 (over P450 concentration range of 0.1 to 0.3 μM). Prior saturation of cytochrome P450 with 17-hydroxyprogesterone prevented further spectral shift with the addition of progesterone. On the other hand, saturation of cytochrome P450 with progesterone decreases the expected shift with 17-hydroxyprogesterone by more than 50% but did not prevent the shift. The difference spectra were diminished by more than 50% at pH 9.0.The addition of NADPH resulted in loss of the spectral shifts and production of 21-hydroxylated products, predominantly DOC and 11-deoxycortisol. These reactions were not inhibited by their specific products. The rate of 21-hydroxylation was linearly related to microsomal protein (and microsomal P450) concentration. The 21-hydroxylation of progesterone was competitively inhibited by 17-hydroxyprogesterone; inhibition of the 21-hydroxylation of 17-hydroxyprogesterone by progesterone was not demonstrated.     

Inhibition of cytochrome P450 activities by oleanolic acid and ursolic acid in human liver microsomes

Oleanolic acid (OA) and ursolic acid (UA), triterpene acids having numerous pharmacological activities including anti-inflammatory, anti-cancer, and hepato-protective effects, were tested for their ability to modulate the activities of several cytochrome P450 (CYP) enzymes using human liver microsomes. OA competitively inhibited CYP1A2-catalyzed phenacetin O-deethylation and CYP3A4-catalyzed midazolam 1-hydroxylation, the major human drug metabolizing CYPs, with IC50 (Ki) values of 143.5 (74.2) microM and 78.9 (41.0) microM, respectively. UA competitively inhibited CYP2C19-catalyzed S-mephenytoin 4'-hydroxylation with an IC50 (Ki) value of 119.7 (80.3) microM. However, other CYPs tested showed no or weak inhibition by both OA and UA. The present study demonstrates that OA and UA have inhibitory effects on CYP isoforms using human liver microsomes. It is thus likely that consumption of herbal medicines containing OA or UA, or administration of OA or UA, can cause drug interactions in humans when used concomitantly with drugs that are metabolized primarily by CYP isoforms. In addition, it appears that the inhibitory effect of OA on CYP1A2 is, in part, related to its anti-inflammatory and anticancer activities.     

Molecular modeling and identification of substrate binding site of orphan human cytochrome P450 4F22

Cytochrome P450s are superfamily of heme proteins which generally monooxygenate hydrophobic compounds. The human cytochrome P450 4F22 (CYP4F22) was categorized into "orphan" CYPs because of its unknown function. CYP4F22 is a potential drug target for cancer therapy. However, three-dimensional structure, the active site topology and substrate specificity of CYP4F22 remain unclear. In this study, a three-dimensional model of human P450 4F22 was constructed by comparative modeling using Modeller 9v5. The resulting model was refined by energy minimization subjected to the quality assessment from both geometric and energetic aspects and was found to be of reasonable quality. Docking approach was employed to dock arachidonic acid into the active site of CYP4F22 in order to probe the ligand-binding modes. As a result, several key residues were identified to be responsible for the binding of arachidonic acid with CYP4F22. These findings provide useful information for understanding the biological roles of CYP4F22 and structure-based drug design.     

Complete amino acid sequence of 21-hydroxylase cytochrome P-450 from porcine adrenal microsomes

Adrenal microsomal 21-hydroxylase is essential for biosynthesis or metabolism of various steroid hormones. The complete amino acid sequence of this cytochrome P-450 from pig adrenal was determined by sequence analysis on several sets of proteolytic fragments. The mature protein consists of 492 amino acid residues, corresponding to a molecular weight of 55,484. Seven out of nine total cysteine residues are localized in the amino terminal half of the molecule. The carboxyl terminal half contains only two cysteines, one of which is located at the highly conserved heme-binding region proposed in all cytochromes P-450. A structural comparison between 21-hydroxylase and 17 alpha-hydroxylase reveals that there is a preponderance of sequence homology at the carboxyl terminal region. These studies indicate that a single gene product is expressed for steroid 21-hydroxylase in porcine adrenal glands.     

Defining resonance Raman spectral responses to substrate binding by cytochrome P450 from Pseudomonas putida

Resonance Raman spectra are reported for substrate-free and camphor-bound cytochrome P450cam and its isotopically labeled analogues that have been reconstituted with protoheme derivatives that bear -CD(3) groups at the 1, 3, 5, and 8-positions (d12-protoheme) or deuterated methine carbons (d4-protoheme). In agreement with previous studies of this and similar enzymes, substrate binding induces changes in the high frequency and low frequency spectral regions, with the most dramatic effect in the low frequency region being activation of a new mode near 367 cm(-1). This substrate-activated mode had been previously assigned as a second "propionate bending" mode (Chen et al., Biochemistry, 2004, 43, 1798-1808), arising in addition to the single propionate bending mode observed for the substrate-free form at 380 cm(-1). In this work, this newly activated mode is observed to shift by 8 cm(-1) to lower frequency in the d12-protoheme reconstituted enzyme (i.e., the same shift as that observed for the higher frequency "propionate bending" mode) and is therefore consistent with the suggested assignment. However, the newly acquired data for the d4-protoheme substituted analogue also support an earlier alternate suggestion (Deng et al., Biochemistry, 1999, 38, 13699-13706) that substrate binding activates several heme out-of-plane modes, one of which (gamma(6)) is accidentally degenerate with the 367 cm(-1) propionate bending mode. Finally, the study of the enzyme reconstituted with the protoheme-d4, which shifts the macrocycle nu(10) mode, has now allowed a definitive identification of the vinyl C==C stretching modes. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 1045-1053, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Inhibition of cytochrome P450 isozymes in rat liver microsomes by polysaccharides derived from Phellinus linteus

Polysaccharides (0.5, 1 and 3 mg ml^–1) from cultured broth and mycelia of Phellinus linteus inhibited cytochrome P450 (CYP) 1A1, CYP 1A2, CYP 2B1, and CYP 2E1 activities in rat liver microsomes. The polysaccharides from the broth of Phellinus linteus grown with 5% (v/v) mulberry extract had highest inhibitory potency for CYP 1A1, 1A2 and 2B1 activities. The most potent inhibitor of CYP 2E1 activity were the polysaccharides from the broth of Phellinus linteusgrown with 10% (v/v) mulberry extract.     

Access channels and methanol binding site to the CaMn4 cluster in Photosystem II based on solvent accessibility simulations, with implications for substrate water access

Given the tightly packed environment of Photosystem II (PSII), channels are expected to exist within the protein to allow the movement of small molecules to and from the oxygen evolving centre. In this report, we calculate solvent contact surfaces from the PSII crystal structures to identify such access channels for methanol and water molecules. In a previous study of the effects of methanol on the EPR split S1-, S3-, and S0-signals [Su et al. (2006) Biochemistry 45, 7617-7627], we proposed that methanol binds to one and the same Mn ion in all S-states. We find here that while channels of methanol dimensions were able to make contact with the CaMn4 cluster, only 3Mn and 4Mn were accessible to methanol. Combining this observation with spectroscopic data in the literature, we propose that 3Mn is the ion to which methanol binds. Furthermore, by calculating solvent contact surfaces for water, we found analogous and more extensive water accessible channels within PSII. On the basis of their structure, orientation, and electrostatic properties, we propose functional assignments of these channels as passages for substrate water access to the CaMn4 cluster, and for the exit of O2 and H+ that are released during water oxidation. Finally, we discuss the possible existence of a gating mechanism for the control of substrate water access to the CaMn4 cluster, based on the observation of a gap within the channel system that is formed by Ca2+ and several mechanistically very significant residues in the vicinity of the cluster.     

Inhibition of fipronil and nonane metabolism in human liver microsomes and human cytochrome P450 isoforms by chlorpyrifos

Previous studies have established that chlorpyrifos (CPS), fipronil, and nonane can all be metabolized by human liver microsomes (HLM) and a number of cytochrome P450 (CYP) isoforms. However, metabolic interactions between these three substrates have not been described. In this study the effect of either coincubation or preincubation of CPS with HLM or CYP isoforms with either fipronil or nonane as substrate was investigated. In both co- and preincubation experiments, CPS significantly inhibited the metabolism of fipronil or nonane by HLM although CPS inhibited the metabolism of fipronil more effectively than that of nonane. CPS significantly inhibited the metabolism of fipronil by CYP3A4 as well as the metabolism of nonane by CYP2B6. In both cases, preincubation with CPS caused greater inhibition than coincubation, suggesting that the inhibition is mechanism based.     

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

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 相似文献   

A predicted three-dimensional structure of human cytochrome P450: implications for substrate specificity

A three-dimensional structure for human cytochrome P450IA1 was predicted based on the crystal coordinates of cytochrome P450cam from Pseudomonas putida. As there was only 15% residue identity between the two enzymes, additional information was used to establish an accurate sequence alignment that is a prerequisite for model building. Twelve representative eukaryotic sequences were aligned and a net prediction of secondary structure was matched against the known alpha-helices and beta-sheets of P450cam. The cam secondary structure provided a fixed main-chain framework onto which loops of appropriate length from the human P450IA1 structure were added. The model-built structure of the human cytochrome conformed to the requirements for the segregation of polar and nonpolar residues between the core and the surface. The first 44 residues of human cytochrome P450 could not be built into the model and sequence analysis suggested that residues 1-26 formed a single membrane-spanning segment. Examination of the sequences of cytochrome P450s from distinct gene families suggested specific residues that could account for the differences in substrate specificity. A major substrate for P450IA1, 3-methyl-cholanthrene, was fitted into the proposed active site and this planar aromatic molecule could be accommodated into the available cavity. Residues that are likely to interact with the haem were identified. The sequence similarity between 59 eukaryotic enzymes was represented as a dendrogram that in general clustered according to gene family. Until a crystallographic structure is available, this model-building study identifies potential residues in cytochrome P450s important in the function of these enzymes and these residues are candidates for site-directed mutagenesis.     

Post-translational reduction of cytochrome P450IIE by CCl4, its substrate

The molecular mechanism of cytochrome P450IIE reduction by CCl4 was reexamined by measuring its enzyme activity, immunoreactive protein contents, and mRNA levels. Aniline hydroxylase and the amounts of immunoreactive P450IIE were rapidly decreased in a time-dependent manner after a single dose of CCl4. No changes were observed in the amounts of immunoreactive P450IIC and P450IA despite significant decreases decrease in their catalytic activities. However, the decreases in P450IIE enzyme activity and immunoreactive protein by CCl4 were not accompanied by a decline in its mRNA level. The data thus suggested a post-translational reduction of P450IIE by CCl4, probably due to specific destruction of the P450IIE protein by its own substrate rather than heme moiety.     

Structural determinants of active site binding affinity and metabolism by cytochrome P450 BM-3

The determinants of the regio- and stereoselective oxidation of fatty acids by cytochrome P450 BM-3 were examined by mutagenesis of residues postulated to anchor the fatty acid or to determine its active site substrate-accessible volume. R47, Y51, and F87 were targeted separately and in combination in order to assess their contributions to arachidonic, palmitoleic, and lauric acid binding affinities, catalytic rates, and regio- and stereoselective oxidation. For all three fatty acids, mutation of the anchoring residues decreased substrate binding affinity and catalytic rates and, for lauric acid, caused a significant increase in the enzyme's NADPH oxidase activity. These changes in catalytic efficiency were accompanied by decreases in the regioselectivity of oxygen insertion, suggesting an increased freedom of substrate movement within the active site of the mutant proteins. The formation of significant amounts of 19-hydroxy AA by the Y51A mutant and of 11,12-EET by the R47A/Y51A/F87V triple mutant, suggest that wild-type BM-3 shields these carbon atoms from the heme bound reactive oxygen by restricting the freedom of AA displacement along the substrate channel, and active site accessibility. These results indicate that binding affinity and catalytic turnover are fatty acid carbon-chain length dependent, and that the catalytic efficiency and the regioselectivity of fatty acid metabolism by BM-3 are determined by active site binding coordinates that control acceptor carbon orientation and proximity to the heme iron.     

Expression of a full-length cDNA encoding bovine adrenal cytochrome P450C21

Two full-length cDNA clones encoding bovine adrenocortical P450C21 have been constructed in a eukaryotic expression vector using partial-length cDNAs whose structures have been previously reported. Following expression of these cDNAs in COS 1 cells, the substrate specificity of P450C21 was determined. Of the 18 steroids tested, progesterone, 17 alpha-hydroxyprogesterone, and 11 beta,17 alpha-dihydroxyprogesterone were found to be the only steroids to serve as substrates for this adrenal enzyme, a much higher degree of substrate specificity than has been reported for a hepatic 21-hydroxylase. The Vmax for 17 alpha-hydroxyprogesterone was 2.5 times greater than that for progesterone, whereas delta 5-steroids were unable to serve as substrate for this enzyme. A difference between the two cDNAs is located at amino acid 401 where one resultant enzyme contains tyrosine while the other contains histidine. This amino acid difference appears to have no effect on the kinetic properties of adrenal P450C21.