Structure Of P450 Research Articles

Changes in secondary structure and salt links of cytochrome P-450cam induced by photoreduction: a Fourier transform infrared spectroscopic study.

Tris(2,2'-bipyridyl)ruthenium(II) was used as a light-induced artificial electron donor for the transfer of the first electron to cytochrome P-450(cam) bound with (1R)-camphor and camphane substrates, and in the substrate-free form. Fourier transform infrared spectroscopy was used to detect changes of the amide I' band and the CO ligand stretch vibration of heme-bound carbon monoxide associated with the heme redox transition. The reduced-minus-oxidized difference spectra show that not only the heme group but also the protein backbone and individual amino acid side chains were affected by the redox transition. Observed secondary structure changes were almost identical for (1R)-camphor-bound and camphane-bound cytochrome P-450(cam), with a remarkable negative signal at 1724.3 cm(-)(1) and a positive signal at 1716.0 cm(-)(1). These signals were not observed in substrate-free P-450(cam). On the basis of known crystallographic data, we assign these signals to a change of hydrogen bonds of a salt link between Arg112, His355, and the heme 6-propionic group.

How Similar Are P450s and What Can Their Differences Teach Us?

Cytochromes P450 form a very large superfamily of proteins which metabolize substrates from steroids to fatty acids to drugs and are found in organisms from protists to mammals. P450s all appear to take on a similar structural fold, yet frequently having less than 20% sequence identity and having vastly different substrates. Within the structural fold there appears to be a highly conserved core, as determined from the comparison of the structures of the six crystallized, soluble P450s. There are also variable regions which by and large appear to be associated with substrate recognition, substrate binding, and redox partner binding. Molecular dynamics simulations of motion in P450cam and P450BM-3 indicate that substrate binding and product release require substantial motion around the “substrate access channel.” Additionally, at the 11th International Conference on Cytochrome P450 Biochemistry, Biophysics, and Molecular Biology and briefly here, the first structure of a microsomal eukaryotic P450 will be presented and compared to the already determined structures by Drs. Johnson and McRee. Finally, with a better understanding of the structure/function relationship of P450s, one will be better able to modify P450s to metabolize the substrates of choice or produce needed valuable chemicals.

Structure, evolution, and liver-specific expression of sterol 12alpha-hydroxylase P450 (CYP8B).

The rat CYP8B cDNA encoding sterol 12alpha-hydroxylase was cloned and sequenced. The amino acid sequence of the heme-binding region of CYP8B was close to those of CYP7A (cholesterol 7alpha-hydroxylase) and CYP7B (oxysterol 7alpha-hydroxylase). Molecular phylogenetic analysis suggests that CYP8B and the CYP7 family derive from a common ancestor. The P450s of the CYP7 and CYP8 families, except for CYP8A (prostacyclin synthase), catalyze the oxygenation of sterols from an alpha surface in the middle of the steroid skeleton. These facts suggest that CYP8B is a P450 closely linked to those of the CYP7 family. CYP8B was expressed specifically in liver. Hepatic CYP8B mRNA level and the 12alpha-hydroxylase activity were altered by cholestyramine feeding, starvation, streptozotocin-induced diabetes mellitus, and administration of clofibrate, dexamethasone or thyroxin, indicating the pretranslational regulation of CYP8B expression. The enhanced CYP8B mRNA expression in streptozotocin-induced diabetic rats was significantly decreased by insulin within 3 h of its administration. These facts demonstrate a regulatory role of insulin in CYP8B expression as a suppressor.

Aldehydic products of lipid peroxidation inactivate cytochrome P-450

The effects of aldehydic products of lipid peroxidation, malondialdehyde (MDA) and 4-hydroxynonenal (HNE), on the structure of rat liver microsomal membrane and cytochrome P-450 was studied. MDA (15-30 microM) similarly to p-chlormercuribenzoate decreased the cytochrome P-450 content by 50 % and lowered microviscosity of lipid surrounding of the spin label OTMB bound to SH-groups of membrane proteins. OTMB was effectively reduced by K3Fe(CN)6 in microsomes preincubated with MDA (20 (M), but not in native microsomes. HNE (10 microM) decreased the cytochrome P-450 content by 90 %. Reduced glutathione and cysteine (5 mM) prevented the decrease of cytochrome P-450 under influence of both MDA or HNE, whereas cytochrome P-420 formation remains unchanged. MDA and HNE decreased activities of NADPH oxidase and NADPH cytochrome c reductase. HNE increased microviscosity of the OTMB lipid environment. The further increase of HNE concentration did not affect this parameter. Both MDA and HNE increased the absorbance at 420 nm, which indicated inactivation of cytochrome P-450 by changes in hydrophobicity of lipid surrounding. We suggest that HNE and aliphatic aldehydes at low concentrations can enter into hydrophobic environment of cytochrome P-450 binding to its SH-groups, which led to inactivation of cytochrome P-450. At the same time, the modification of membrane surface layer and subsequent decrease of hydrophobicity of cytochrome P-450 environment preceded the binding of MDA to SH-groups of cytochrome P-450 to develop its inactivating effect.

Comparison of Two Types of Cytochrome P-450 Models. Effect of Steric Hindrance near Active Site

Two types of iron porphyrin complexes were synthesized as models to elucidate the relation of the function and structure of cytochrome P-450. One was a “picket fence-type” complex having amino acid residues in specified conformation as a chiral field. The other was an “etioporphyrin-type” complex with peptide chains, on meso position, resembling closely proteins of cytochrome P-450. When the former complex, αβαβ or ααββ-atropisomeric complex, was used in the epoxidation of styrene as a model reaction of cytochrome P-450, the increase in enantiomeric excess of the product, styrene oxide, depended on the bulkiness of amino acid residues, and a high enantiomeric excess of 54% resulted using αβαβ-type complex modified with L-Phe-OMe. The turnover, however, was considerably low. The latter model, on the other hand, enhanced remarkably the reaction efficiency along with high enantiomeric excess, when the reaction was conducted in similar conditions.

Inhibition of cytochrome P450 by nitric oxide

Nitric oxide (NO) reacts with iron, superoxide, thiols and oxygen. Although NO reversibly interacts with the heme-iron of P450, the pathophysiological role of this interaction remains to be elucidated. We found that hepatic levels of P450 markedly decreased in endotoxemic rats, particularly when the rate of NO generation was increased. To determine the possible role of NO in the modulation of the structure and function of P450, changes in the levels and activities of P450 isozymes were determined in liver microsomes from normal and endotoxemic rats. Electron spin resonance analysis revealed that incubation of microsomes with the NO donor NOC-7 rapidly generated NO-P450 adducts. Microsomal levels of NO-P450 adducts increased and peaked at 10 min after incubation and decreased thereafter; it disappeared completely within 60 min. In contrast, microsomal levels of the low-spin ferric form and CO-differential spectrally detectable P450 rapidly decreased during the initial 10 min; the signal intensity for P450 recovered thereafter. Western blot analysis using specific antibodies against CYP3A2 and CYP2C11 isozymes revealed no detectable degradation of these isoforms. Effect of NO on the catalytic activity of the enzymes was also determined by using testosterone as the substrate. The hydroxylation activity in microsomes rapidly decreased during the initial 10 min and disappeared slowly thereafter. These results suggested that NO might form dissociable complexes with the heme moiety of P450 and irreversibly inactivate them. The mechanism for P450 inactivation by NO and the role of NO-P450 interaction in the pathogenesis of liver injury in endotoxemia are discussed.

Determination of genotoxicity using a chloroperoxidase-mediated model of PAH-DNA adduct formation.

Enzymes from cytochrome P450 superfamily, which are components of monooxygenase systems, are thought to play a major role in carcinogenic activation. The physicochemical properties of PAH’s and the catalytic properties of cytochrome P450 suggest that PAH’s are generally activated by two major mechanisms: One-electron oxidation with formation of radical cations and monooxygenation (two-electron oxidation) to yield bay-region diol epoxides (Devanesan et al. 1992; Wellemans et al 1994). Hemoproteins such as peroxidases, cytochromes, and hemoglobin are able to catalyze PAH’s oxidation in vitro (Vazquez-Duhalt et al. 1994; Torres et al. 1995; Ortiz-Leon et al. 1995). The reaction products are mainly quinones and the mechanism involves free radical formation. Chloroperoxidase (CPO) from Caldaryomyces fumago, an enzyme with three different catalytic activities (halogenase, peroxidase and catalase), catalyzes phenol oxidation (Carmichael et al. 1985). On the other hand, chloroperoxidase shows close structural (Dawson 1988) and catalytic similarities with the cytochrome P450 (Sundaramoorthy et al. 1995). The similarities of both proteins could help to provide a better understanding of the biological activities and structure of cytochrome P450 in vivo. The primary activation of PAH’s via one-electron by cytochrome P450 could be similar to the PAH-DNA binding mediated by chloroperoxidase.

Construction of a 3D model of cytochrome P450 2B4.

A three-dimensional structural model of rabbit phenobarbital-inducible cytochrome P450 2B4 (LM2) was constructed by homology modeling techniques previously developed for building and evaluating a 3D model of the cytochrome P450choP isozyme. Four templates with known crystal structures including cytochrome P450cam, terp, BM-3 and eryF were used in multiple sequence alignments and construction of the cytochrome P450 2B4 coordinates. The model was evaluated for its overall quality using available protein analysis programs and found to be satisfactory. The model structure was stable at room temperature during a 140 ps unconstrained full protein molecular dynamics simulation. A putative substrate access channel and binding site were identified. Two different substrates, benzphetamine and androstenedione, that are metabolized by cytochrome P450 2B4 with pronounced product specificity were docked into the putative binding site. Two orientations were found for each substrate that could lead to the observed preferred products. Using a geometric fit method three regions on the surface of the model cytochrome P450 structure were identified as possible sites for interaction with cytochrome b5, a redox partner of P450 2B4. Residues that may interact with the substrates and with cytochrome b5 have been identified and mutagenesis studies are currently in progress.

Immunochemical relationships of cytochrome P4503A-like proteins in teleost fish

Multiple P450 proteins have been purified from several teleost species, including rainbow trout (Oncorhynchus mykiss), scup (Stenotomus chrysops) and Atlantic cod (Gadus morhua). Identity, relationships and/or functions have been established in these fish species for the cytochrome P4501 As. Information about the structure, function, regulation and relationships of other piscine cytochrome P450 (CYP) proteins is sparse. In the present study we have focused on constitutively expressed CYP forms, P450con and LMC5 isolated from rainbow trout, P450A from scup, and P450b from Atlantic cod, and we consider evidence for the relationship of these proteins to mammalian members of the CYP3A subfamily. Reciprocal western blot analysis shows that P450con and LMC5, isolated from rainbow trout in two different laboratories, are closely related and ostensibly identical proteins. These trout proteins show specific reciprocal cross-reactivity with scup P450A, and polyclonal antibodies (PAb) to the trout and scup proteins both recognize cod P450b, indicating that rainbow trout P450con/LMC5, scup P450A and cod P450b are immunochemically-related proteins. In analyses of liver microsomes of trout, scup and cod, PAb to trout P450con/LMC5 and scup P450A recognize only bands that are identical in migration to the CYP proteins purified from these species, and which were used as immunogens. These CYP proteins purified from fish are each immunochemically-related to mammalian CYP3A proteins, showing recognition by PAb to human CYP3A4 and to rat CYP3A1. PAb to the mammalian CYP3As also recognize the same bands in liver microsomes from these fish species as seen by PAb to the fish proteins. These results strongly suggest that these fish proteins are members of theCYP3 gene family and probably theCYP3A subfamily. Although sequence analysis is required before their designation in the CYP3A subfamily can be confirmed and specified, we refer to these as CYP3A-like. Immunoblot analyses of hepatic microsomes from other fish species with PAb to scup P450A and trout P450con show that multiple CYP3A-like proteins are expressed in liver of several species, including killifish (Fundulus heteroclitus) and winter flounder (Pleuronectes americanus). Important questions still remain to be addressed concerning CYP3A structure, multiplicity, physiological function, regulation and metabolism of endogenous as well as exogenous substrates in fish.

The Cytochrome P450 Enzymes of Hepatic Drug Metabolism: How are their Activities Assessed In Vivo, and what is their Clinical Relevance?

The cytochromes P450 comprise a superfamily of mixed function oxidases responsible for the oxidation of numerous endobiotics and thousands of xenobiotics. Though they are distributed in many organ systems, the hepatic cytochromes P450 are most prominent in both the detoxification and bioactivation of xenobiotics. They have been investigated intensively for over 30 years; however a systematic nomenclature did not evolve until it became clear that numerous isoforms existed. Based on similarities of amino acid sequences, the cytochrome P450 (CYP) enzymes have now been classified by family, subfamily, and isoform. As expression systems have been developed to assist in the determination of which isoforms mediate the oxidations of which substrates, it has become possible to model binding sites for some of the isoforms. This enterprise has been assisted by the determination of the amino acide sequence and the X-ray crystal structure of the bacterial cytochrome P450, P450(CAM). As the CYP enzymes play such a vital role in drug metabolism, and as the activities of various CYP isoforms have been implicated in cancer risk, there is a compelling need to develop methods for genotyping or phenotyping subpopulations with regard to CYP activity. It should be especially important to phenotype CYP activities in subpopulations anytime CYP enzyme activity can be regulated by environmental factors. A priori knowledge about CYP activity in various subpopulations (e.g., the aged, those with hepatic dysfunction, women, those occupationally exposed to certain environmental chemicals) will be helpful in predicting therapeutic outcomes for drugs whose elimination is CYP dependent. Additionally, cancer risk has been shown to correlate with various CYP activities, suggesting that a priori knowledge about select CYP activities may be useful in cancer risk assessment. Several noninvasive and minimally invasive strategies for phenotyping CYP activities have emerged in recent years. These procedures hold the promise of permitting investigators to phenotype select CYP isoform or subfamily activities within defined subpopulations through the use of endobiotic probes of CYP activity, xenobiotic probes of CYP activity, and noninvasive and minimally invasive measurements of those probes. This article reviews the CYP enzymes, the minimally invasive and noninvasive methods for characterizing their hepatic activities in humans in vivo, and the significance of being able to phenotype their activities in vivo.

Changes in chromatin structure and nuclear matrix association of the rat cytochrome P450 2B1/2 (CYP2B1/2) gene following induction with phenobarbital

Treatment of rats with phenobarbital has been shown to result in an enormous increase in CYP2B1/2 mRNA levels in the liver. This study was performed in order to relate this effect of phenobarbital treatment to changes in the chromatin structure of the CYP2B1/2 gene. Phenobarbital treatment was shown to be associated with a twofold increase in DNase I sensitivity of the CYP2B1/2 gene in the liver. In addition, induction of the CYP2B1/2 gene transcription by phenobarbital resulted in a marked enrichment in the liver of CYP2B1/2 sequences in the nuclear matrix as compared to the matrix-free fraction. Control studies with the CYP1A2 gene, of which the transcriptional activity is not influenced by phenobarbital, did not show any changes in both DNase I sensitivity and matrix association upon phenobarbital treatment. Furthermore, after phenobarbital treatment, no changes in DNasel sensitivity or nuclear matrix association of the CYP2B1/2 gene were observed in the kidney. These data indicate a direct, positive correlation between the structure of the rat CYP2B1/2 gene and its activity. © 1996 John Wiley & Sons, Inc.

Cytochrome P450 Structure, Mechanism, and Biochemistry, 2nd ed Edited by Paul R. Oritz de Montellano (University of California, San Francisco). Plenum: New York. 1995. xi + 631 pp. $125.00. ISBN 0-306-45141-7.

ADVERTISEMENT RETURN TO ISSUEPREVBook ReviewNEXTCytochrome P450 Structure, Mechanism, and Biochemistry, 2nd ed Edited by Paul R. Oritz de Montellano (University of California, San Francisco). Plenum: New York. 1995. xi + 631 pp. $125.00. ISBN 0-306-45141-7.Helen L. HenryView Author Information University of California, RiversideCite this: J. Am. Chem. Soc. 1996, 118, 44, 10945Publication Date (Web):November 6, 1996Publication History Published online6 November 1996Published inissue 1 January 1996https://doi.org/10.1021/ja965506pCopyright © 1996 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views267Altmetric-Citations2LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit Read OnlinePDF (71 KB) Get e-AlertsSUBJECTS:Peptides and proteins,Protein structure Get e-Alerts

Molecular modelling of members of the P4502A subfamily: application to studies of enzyme specificity.

1. Using the recently published crystal structure of a bacterial P450, namely 102 (also termed P450bm3), as a template molecular models of mammalian 2A1, 2A4, 2A5 and 2A6 were constructed. 2. Substrate interaction studies demonstrated that in keeping with known catalytic activities the putative binding sites of mouse hepatic P4502A4 and 2A5 oriented testosterone for 15 alpha-hydroxylation and coumarin for 7-hydroxylation respectively. 3. Substrate interaction studies with the putative binding site of human liver P4502A6 demonstrated that coumarin was oriented for 7-hydroxylation. However, in keeping with previous site-directed mutagenesis studies with P4502A4 and 2A5, changing a single phenylalanine residue to leucine in 2A6 gave rise to a mutant enzyme, which could bind testosterone as a substrate for 15 alpha-hydroxylation rather than coumarin. 4. Substrate interaction studies with the putative binding site of rat hepatic P4502A1 suggested that this isoenzyme would hydroxylate coumarin at the 3- rather than at the 7-position. 5. The results of these molecular modelling studies demonstrate that apparently minor modifications to P4502A subfamily amino acid sequences can result in major alterations in enzyme specificity. 6. Molecular modelling is thus a useful technique that can aid in elucidating substrate specificities of P450 isoenzymes and species differences in xenobiotic metabolism. The technique can also be utilized to complement site-directed mutagenesis studies in order to identify critical structural features of P450s and other enzymes.

Relationship between hydrocarbon structure and induction of P450: effect on RNA levels.

1. Exposure to simple aromatic hydrocarbons has been shown to induce P450-dependent activities and the expression of particular P450 isozymes in a manner related to the molecular structure of the inducing hydrocarbon. In an attempt to identify the structural relationship controlling P450 induction, the effect of hydrocarbon treatment on the RNA levels for specific P450 isozymes was examined. 2. Rats were treated with daily injections of hydrocarbons (benzene, toluene, ethylbenzene, n-propylbenzene, m- and p-xylene) for 3 days, and the effects on specific RNA levels were examined by Northern blot hybridization. 3. Although P4502B1 mRNA was not elevated after hydrocarbon treatment, a significant elevation in 2B2 mRNA was observed after exposure to the larger aromatic hydrocarbons, ethylbenzene and m-xylene. It is interesting to note that despite the substantial elevation of P4502B protein levels, only a small elevation of P4502B1 and 2B2 RNA was observed. 4. P4502C11 mRNA was only suppressed by ethylbenzene administration, despite the depression of 2C11 protein levels by several hydrocarbons. 5. P4501A1 mRNA was not detectable and 2E1 mRNA was not changed by any aromatic hydrocarbon treatment investigated in this study. 6. The data indicate that the levels of mRNA species for a number of P450 isozymes are differentially regulated by exposure to hydrocarbons, and that small changes in hydrocarbon size or isomeric structure can influence the levels of these mRNA species.

Alterations of the regiospecificity of progesterone metabolism by the mutagenesis of two key amino acid residues in rabbit cytochrome P450 2C3v.

A Ser/Thr difference at position 364 underlies a phenotypic difference between naturally occurring variants of microsomal cytochrome P450 2C3 in their capacity to catalyze the 6 beta-hydroxylation of progesterone, as well as in their sensitivity to the inhibitor 16 alpha-methyl-progesterone. Position 364 of P450 2C3 maps to a substrate contacting domain suggested by models for mammalian P450 enzymes based on the structure of P450,101. In this study, Thr-364 of P450 2C3v, a progesterone 6 beta- and 16 alpha-hydroxylase, was replaced by Gly, Asp, Asn, Val, Leu, or Ile. The latter three amino acids did not alter the regiospecificity of P450 2C3v, whereas the Gly, Asp, and Asn substitutions each produced enzymes with properties that correspond closely to the Ser mutant that catalyzes 16 alpha-hydroxylation but not 6 beta-hydroxylation. The former are distinguished from the latter amino acids by their greater hydrophobicity and size. In contrast, the 16 alpha-hydroxylase activity could be greatly diminished by the introduction of an alanine replacement for Val-113. This mutation conferred progesterone 21- and 17 alpha-hydroxylase activity to P450 2C3v at the expense of 16 alpha-hydroxylase activity, leaving the 6 beta-hydroxylase activity largely unaffected. Compound mutants displayed the additive effects of the two mutations. These results are consistent with two distinct orientations for the binding of progesterone to P450 2C3v, resulting in 6 beta- and 16 alpha-hydroxylation, respectively. The binding of progesterone in these two orientations can be modulated relatively independently by modifications of the two key amino acid residues at 113 and 364.

On the Domain Structure of Cytochrome P450 102 (BM-3): Isolation and Properties of a 45-kDa FAD/NADP Domain

Cytochrome P450 102 is a catalytically self-sufficient monooxygenase isolated from barbiturate-induced Bacillus megaterium. The enzyme contains FAD, FMN, and heme in a single polypeptide chain of 1048 residues, and each of the cofactors is believed to be located in a separate domain. In the present study we have used exhaustive endogenous proteolysis to produce a 45 kDa fragment of the cytochrome. This fragment bound the 2′,5′-adenosine diphosphate moiety of NADP(H) strongly, with approximately the same dissociation constant as in the native enzyme, and contained only FAD (0.93 equivalents per polypeptide, ϵ453nm = 11,200 M−1cm−1). Reduction of the flavin by sodium dithionite proceeded quite slowly to yield FADH2, but no stable semiquinone species was produced upon air re-oxidation. In contrast, NADPH rapidly reduced this FAD/NADP(H) domain aerobically to produce the FADH· semiquinone radical. At a 75:1 molar ratio of the FAD/NADP(H) domain to the P450 102 heme domain, no laurate hydroxylase activity was observed. Gas-phase sequence analysis showed the presence of two major sequences beginning at Phe646 (403 residues, MW 45,033) and Asp652 (397 residues). These data are in agreement with the crystal structures of related enzymes and closely define the boundary of the FAD/NADP+ domain in P450 102.

Application of 3-Dimensional Homology Modeling of Cytochrome P450 2B1 for Interpretation of Site-Directed Mutagenesis Results

Abstract Three-dimensional structures of cytochrome P450 2B1 were modeled based on the crystallographic structure of P450(cam). The effect of the alignment, loop choice, and minimization with or without water was assessed. Although final models were similar in overall structure, the identity of active site residues depended upon the alignment. An example is Phe-206, which may or may not form part of the active site. The choice of the loop conformation had a lesser effect, while including water in the final minimization step was essential for preserving the shape and size of the active site. The best model (model 2) was in good agreement with the data from site-directed mutagenesis studies, and correctly predicted the effect of substitutions at 9 out of 10 amino acid positions. Thus, residues important for P450 2B1 activity, such as Ile- 114, Phe-206, Ile-290, Thr-302, Val-363, and Gly-478, constitute part of the active site and are able to interact with the substrate androstenedione through hydrophobic interactions. On the other hand, Ser-303, Ser-360 and Lys-473 are far from the active site and/or cannot interact with the substrate, in agreement with experimental data. The model indicates other residues likely to be important for enzyme function, such as Tyr- 111, Leu-209, Ile-477, and Ile- 480, which can be tested experimentally. The substrate may assume numerous binding orientations consistent with observed patterns of hydroxylation at C(5) and C(6). The replacement in the model of certain amino acid residues to mimic residue substitutions from site-directed mutagenesis studies and docking of the substrate into the modified active site allowed a plausible explanation for alterations in regio- and stereospecificities of some mutants of P450 2B1, such as Gly-478 → Ala or Val-363 Ala.

Book Review: Relationships between Structure and Function of Cytochrome P‐450—Experiments, Calculations, Models. (Series: Frontiers in Biotransformation, Vol. 7.) Edited by K. Ruckpaul and H. Rein

Book Review: Relationships between Structure and Function of Cytochrome P‐450—Experiments, Calculations, Models. (Series: Frontiers in Biotransformation, Vol. 7.) Edited by K. Ruckpaul and H. Rein

Regulatory mechanisms in the activation of nitrogenous compounds by mammalian cytochrome P-450 isozymes.

Metabolic activation of nitrogenous compounds by the cytochrome P-450 system is a highly complex process. Inherent substrate factors, such as basicity, electronic state, lipophilicity, and conformation control binding of the diverse classes of amines to cytochrome P-450. Accommodation of these compounds in the enzyme cavity and proper orientation of the molecules are governed by intrinsic properties of the peptide structure of cytochrome P-450, which may be subject to modification by the action of effectors. On the membrane level, phospholipid might have some impact on substrate binding. On the other hand, bound amine substrate is beneficial to the productive interaction of the electron transport chains with the terminal acceptor, improving economy of the system. Certain amines appear to regulate O2 association with cytochrome P-450 and stabilize the various oxy species formed. Considering the selective prerequisites for oxidative attack by cytochrome P-450 at vulnerable nitrogen centers, many cytotoxic amines belonging to the category of relatively rigid, planar molecules undergo N-oxidative activation by the cytochrome P-450IA subfamily, while more bulky amines with flexible conformation are N-oxygenated preferentially by phenobarbital-inducible cytochromes P-450. Small differences in protein structure between the various cytochrome P-450 subforms might serve to stabilize aminium radicals to permit oxygen rebound. Collectively, the selective regulatory mechanisms operative in the bioactivation of nitrogen-containing compounds appear to be determined largely by the type of substrate used and the isozyme involved in catalysis. With respect to the latter, the interplay of the multiple cytochromes P-450 in the various organs of animal species thus serves to rationalize the differences in the particular selectivities for amine substrates. These are responsible for the mode and/or extent to which activation of nitrogenous compounds, including promutagens and procarcinogens, occurs, and this may explain the tissue-specific response to the tumorigenic action of these agents.

Functional domains of human aromatase cytochrome P450 characterized by linear alignment and site-directed mutagenesis.

The relationship of function to structure of aromatase cytochrome P450 (P450arom; the product of the CYP19 gene) has been examined by means of sequence alignment and site-directed mutagenesis. Comparison has been made between the sequence of P450arom and the two soluble bacterial cytochrome P450 isoforms, whose three-dimensional structure has been determined (P450BM3 and P450cam). From this comparison, it appears that although there is a similarity of overall structure in cytochromes P450, there is enough significant difference in the regions involved in substrate recognition and substrate binding that residues believed to be involved, even in the known structures, must be tested. With this in mind, we have generated a detailed alignment of P450arom, including the definition of putative alpha-helices and beta-sheets based on comparison of the alignments of P450BM3 and P450cam, generated from their three-dimensional structure, and have made mutations in regions we believe to be involved in substrate recognition at the solvent surface and orientation in the heme pocket. We have mutated F116 and F134 to determine if they are present in the heme pocket, and Q225 and L228 to determine if they are a part of the substrate recognition loop. Although F116E is essentially inactive and may be a folding mutant or may inhibit reductase binding, F134E is more active than the wild type and may be located in the heme pocket facilitating the hydrogen abstraction from C2 of androstenedione. Mutations at Q225 and L228 also result in the anticipated changes in the apparent Km and maximum velocity.(ABSTRACT TRUNCATED AT 250 WORDS)