Cytochromes P450: Overview Mary V. Relling 26 Jan 05 Objectives
Know the subcellular localization and a basic schema for recovery of microsomes from liver. Write the overall stochiometry of the mixed function oxidase function of P450. List at least two types of reactions catalyzed by P450 other than mixed function oxidation. Describe why plants might have more P450s than humans List three methods for measuring P450 and understand the basic distinction among the three methods.
Basic Biochemistry * highest concentration in liver * located in ER (microsomes); about 2.5% of total hepatic microsomal protein
*Cytochrome: electron-transporting protein that contains a heme-prosthetic group. Iron alternates between ferrous and oxidized (ferric, Fe3+) state during electron transport. The reduced (ferrous, Fe2+) form binds with CO that gives abs at 450 nm.
Dual spectrophotometer to detect the difference spectrum between 2 cuvettes (with and without CO). Mixed function oxidase (oxidative) function of P450 requires 3 components: P450, reductase, and lipid. The reaction: NADPH + H+ + O2 + RH NADP+ + H2O + ROH Note: NADPH + H+ is a 2 x 1 electron donor; P450 accepts 2 electrons (1 at a time); NADPHcytochrome 450 reductase is the "transducer" of those electrons by transferring them sequentially (1 at a time) to P450. Catalytic cycle of oxidation:
Figure 3-1. Proposed catalytic reaction cycle involving cytochrome P450 in the oxidation of xenobiotics.
Uncoupling reactions: formation of reactive oxidative species; worse for P450s with promiscuous substrate specificity Reductive reactions with P450 e.g., during anaerobic reduction of substrate, reducing equivalents are used to form radical species of drug, rather than to "activate" O2. Because of high affinity of P450 for O2, it's hypothesized that oxygen tension of particular tissue may affect "balance" between oxidative and reductive reactions for P450.
Figure 2.17. Reductive dehalogenation of halothane. (a) Halothane metabolism forming 2-chloro1,1,1-trifluoroethane and 2-chloro-1,1-difluoroethylene. (b) Proposed reaction scheme of cytochrome P450 in the dehalogenation of halothane. Abbreviations used: Fe3+ and Fe2+, the oxidized and reduced haem of cytochrome P450;FPT, NADPH-cytochrome P450 reductase; cyt.b5, cytochrome b5; FPD, NADH-cytochrome b5 reductase. Derived from Ahr et al., (1982) Biochemical Pharmacology 31:383-390.
(Note: NADPH-cytochrome P450 reductase can donate its electrons to quinones and nitrosoureas, instead of to P450, and thereby directly reduce drugs.)
Peroxidative Function of P450 RH + R'OOH ROH + R'OH where RH and ROH represent substrate and product R'OOH is the peroxy compound that serves as the oxygen donor (e.g., cumene hydroperoxide) and R'OH is the resulting decomposition product (e.g. cumenol) (Blake & Coon, JBC, 255:4100, 1980; Estabrook et al, Xenobiotica 14:87,1984; Marnett et al, Chapter 2, Ortiz de Montellano book)
Figure 6. Proposed mechanism for catalytic action of liver microsomal cytochrome P450.
P4508A and P4505A: both use peroxide as O donor: with peroxide, no reductase is required.
P450 proteins and genes: * heme-protein: heme = iron-containing porphyrin (protoporphyrin IX) * protein is apoprotein; MW 45,000 - 55,000 Da
Figure 3-2. A simplified depiction of the proposed "activated oxygen" cytochrome P450 substrate complex. Note the simplified apoprotein portion and the heme (protoporphyrin IX) portion of cytochrome P450 and the close proximity of the substrate RH undergoing oxidation.
Figure 2.2. Putative functional domains of cytochrome P450. Many aspects of these functional domains remain to be clarified and this model is based on the CYP2B1 isoenzyme. Numbers represent the amino acid residues. Derived, in part, from Waxman DJ and Azaroff L (1992) Biochemical Journal 281:577-592.
Figure 2.3. The conserved haem-binding domain in cytochrome P450s. The cysteine residue that binds the haem prosthetic group as the 5 th ligand is shown towards the carboxyl terminus of the polypeptide chain. The important cysteine residue is shown (C) and other amino acids are abbreviated by their one letter codes. The amino acid residues that are common between the various isoforms are boxed in.
Genomics of P450 ~ 2400 P450s - 450 genes in rice - 272 genes in arabidopsis 57 human P450s ~ 85 genes in mice Why do plants have more P450s than animals? Eukaryotes: all have sterols in membranes One of the most conserved P450s: CYP51 (makes cholesterol)
Figure 1. A P450 protein phylogenetic tree. The divergence times were calculated as described by Nelson and Strobel (312). A total of 69 P450 sequences were compared.
Figure 5. Scheme depicting evolution of a P450 gene subfamily in two species. Gene conversion and gene loss are illustrated for species 1 and species 2, respectively.
Versatility due to overlapping substrate specificity and multiplicity of P450s. 1. Nomenclature system developed in a consensus paper by Nelson et al. (Pharmacogenetics 1996;6:1-42). www.imm.ki.se/CYPalleles/criteria.htm Families (Arabic numeral CYP1) share >40% protein sequence homology with each other and thus contain members from several species. Within subfamilies (indicated by a capital letter CYP1A), members share >55% homology with each other and also contain members from several species. Some antibodies have cross-reacted with different members of the same subfamilies. Molecular techniques have allowed cloning, expression and better characterization (generation of better antibodies, development of molecular probes) of individual members of CYP subfamilies. Each is indicated with an Arabic numeral (e.g., CYP1A2) and is numbered sequentially as it is characterized. Each member represents a single gene and a single protein.
Gene = italicized Final P = pseudogene Final x = discontinued cDNA, mRNA and ezs: all caps – no italics or hyphens CYP = human or non-mouse, non-Drosophila Cyp = Drosophila or mouse
Inclusion Criteria The main function of this Web page is to encourage scientists worldwide to speak the same language and to avoid "home-made" allelic designations that can confuse the nomenclature system and the scientific literature, and it contains the recommended nomenclature for the human polymorphic CYP genes. It adheres to the guidelines provided in Shows et al. (1987), Daly et al. (1996) and Antonarakis and the Nomenclature Working Group (1998) as detailed below: 1. 2. On this Web page only human CYP-alleles are considered. The gene and allele is separated by an asterisk followed by Arabic numerals and upper-case Roman letters with less than four characters to name the allele (e.g., CYP1A1*3, CYP1B1*22, CYP2D6*10B). A gene is considered as the sequence from 5 kb upstream from the transcription start site to 500 bp downstream of the last exon. However, if a regulatory element has been characterized at a more distant part of the gene, also this area belongs to the gene. To be assigned as a unique allele it should contain nucleotide changes that have been shown to affect transcription, splicing, translation, posttranscriptional or posttranslational modifications or result in at least one amino acid change. Additional nucleotide changes and combinations of nucleotide changes in the gene will be given letters (e.g., *21A, *21B). Thus, in cases where silent mutations occur or mutations are present in regulatory parts or introns with unclear function, the allelic name should adhere to the closest functionally characterized allele by subgroup assignments such as CYP2D6*4A. Allelic variants can be defined as combinations of up to three letters (e.g., CYP2D6*2ABC), thereby allowing room for 22 x 22 x 22 = 10,648 different variants for each allelic number. The letters I, O, X and Y are excluded because of indexing problems. For extra gene copies (n) placed in tandem the entire allelic arrangement should be referred to as e.g., CYP2D6*2Xn. Numbering of nucleotides in the allele should be as described in Antonarakis and the Nomenclature Working Group (1998). The base A in the initiation codon ATG is denoted +1 and the base before A is numbered -1. For reasons of indexing, the names for proteins should have a period between the name of the gene product and number (e.g., CYP2D6.2A). The wild type allele is defined as the sequence of the first alleles sequenced and should be designated as *1 (or *1A and *1B in case of slightly variant sequences). SNPs that are not easily assigned to a specific allele, will be listed at the bottom of the corresponding nomenclature page with relevant literature references. Submission of new alleles should be done with information sufficient to fulfill the criteria to be assigned a unique allele as under # 4 above or a letter as described under # 5 above. For incorporation into the Web page as a unique allele, all exons and exon-intron borders should have been sequenced. If a new allele has been detected on the cDNA level, verification of the mutation(s) on the genomic level is required. For acceptance of a new SNP given a separate letter, evidence for its presence on the genomic level is required. No temporary allelic numbers or letters are provided, and information about any new allele submitted, will continuously be published on the Web Page. In case an author does not want to release the information on the Web Page before publication, a provisional assignment NOT based on the asterisk system has to be given by the author in the first publication.
8. 9. 10. 11.
I. II. III. IV. V.
PRINTS entries for structural domains of P450-containing systems OMIM entries related to P450-containing systems ( 9k ) MOLSCRIPT images of known 3D structures The table of age-dependent changes in liver microsomal P450 by Dr. A.L. Sukhodub ( 63k ) A tree of representative P450 sequences ( 1.6M PDF file) and legend provided by Osamu Gotoh. Here you can read Kirill's review Structural domains of P450-containing monooxygenase systems
The files mirrored from David Nelson's www site: An alignment of 192 P450 sequences ( 162k ) An alignment of 237 more P450 sequences ( 202k ) An alignment of 125 plant P450 sequences ( 106k ) An alignment of the C. elegans clan ( 36k ) A bibliography of new P450 sequence entries that have appeared since the 1993 nomenclature update: Part A: CYP1 to CYP2 ( 133k ) Part B: CYP3 to CYP9 ( 166k ) Part C: CYP10 to CYP69 and CYP501 to CYP507 ( 187k ) Part D: plant P450s ( 214k ) Part E: bacterial P450s ( 47k ) Part F: fish P450s (not up-do-date) ( 13k ) A table of P450 accession numbers ( 61k ) Familycount The number of genes in a given species A table of species distribution for the various CYP families Summary of P450 genes in C. elegans
Other related links The Arabidopsis P450 site at PlaCe The Drosophila P450 site at INRA Human Cytochrome P450 (CYP) Allele Nomenclature Committee Human P450 Metabolism Database by Prof. Slobodan Rendic A list of commercially available P450s and related products 14th International Symposium on Flavins and Flavoproteins. 14-18 July 2002, St John's College, Cambridge, England 6th European Conference on Biological Inorganic Chemistry (EUROBIC-6). 30 July - 3 August 2002, Lund, Sweden and Copenhagen, Denmark 6th International Symposium on P450 Biodiversity: Functional Biochemistry of P450 Cytochromes in Microorganisms, Plants and Insects. 20-25 August 2002, Los Angeles, USA. Contact: Dr. Armand J. Fulco 12th International Conference on Cytochrome P450: Biochemistry, Biophysics and Molecular Biology, 11-15 September 2001, La Grande Motte, France. Cytochrome P450 Family page from Gene Family Database Mirrored on our server ( 33k ) Cytochrome P450 Drug Interactions Table P450 Inhibiting Drugs Glaxo Wellcome Pharmacology Guide Individual Models of Cytochrome P450 The Nitric Oxide Home Page Pedro's collection of links to the Protein Families WWW Pages
P450 1A1 1A2 2A6 2B6 2C8 2C9/10
The major drug metabolizing human P450s are 1A, 2C, 2D, 2E, and 3A.
Substrates benzo(a)pyrene, phenacetin o-deethylation, PAH phenacetin o-deethylation, caffeine, ethoxyresorufin, APAP coumarin 7-hydroxylation, nicotine to cotinine Cyclophosphamide, ifosfamide TB (minor), retinoic acid, taxol TB (major), S-warfarin hydroxylation, phenytoin Inhibitors 7,8 benzoflavone, ellipticine, (-) maackiain acetate 7,8 benzoflavone, furafylline Inducers cig. smoke, benzo(A)pyrene omeprazole, 3MC barb barb relative of phenobarbinducible form in rat only 2 aa's different between 9/10; point mutations have huge effect catal. activity genetically regul. poly. genetically regul. polymorphism Comments prim. extra-hepatic
2C18 2C19 2D6
? S mephenytoin, omeprazole alprenolol, amiflamine, quinidine amitriptyline, bufuralol, clomipramine, codeine, debrisoquin, desipramine, dextromethorphan, encainide, flecainide, guanoxan, imipramine, indoramin, metiamide, metoprolol, methoxyamphetamine, methoxyphenamine, nortriptyline, N-propylajmaline, perhexiline, phenacetin, phenformin, propafenone, propranolol, sparteine, timolol N-nitrosodimethylamine disulfiram, 4-methylpyrazole N-demethylase, chlorzoxazone hydroxylation, APAP toxic met, dapsone emycin, cycloA, FK-506, nifed, midazolam, terfenadine, lidocaine N-deethylation, dapsone nhydroxylation, quinidine, triazolam, ethynylestradiol, TAO, etoposide, teniposide, lovastatin, alfentamil, tamoxifen; 6--hydroxylation of testosterone, cortisol, progesterone, taxol, androstenedione; benzo(a)pyrene 2, and 4 OH estradiol; progesterone, testosterone, androstenedione 6-hydroxylation; progesterone 16 hydrox, nifedipine, midaz, benzo(a)pyrene, cyA dehydroepiandrosterone, 3-sulfate 16-a-hydroxylase TAO, gestodene, quercetin, grapefruit juice (?naringenin), ketoconazole, sulfadimidine, erythromycin
?rif in Ems
TAO, rif, barbs, phenytoin, dex
3As not inducible by Pregnenolone carbonitrile (human); high content 3A4 in intestine; 3As const. up to 25% of liver P450 and > 75% intest. P450
prob. not inducible
Polymorphic, only 25% express, generally lower affinity than 3A3/4 (? midaz 1-OH), constit. in kidney ? predominantly fetal, extrahepatic (intestine)
Methods of Measuring P450(s) * immunodetection * spectrophotometry * catalytic activities Guengerich et al., JPET 256:1189,1991; Shimada et al., JPET 270:414, 1994; Beaune et al., DMD 14:437, 1986.
PHARMACOGENETICS ANTICANCER AGENTS RESEARCH GROUP
Relevant Links for Drug Metabolism and Pharmacogenomics Tables of Genetic Polymorphisms Affecting Human Drug Response Useful Links for CYP450s David Nelson's (the keeper of the CYP450 nomenclature) home page on CYP450 David Nelson's "What's New" Directory of P450 containing systems Bill Peterson's P450 Web site CYP450 allele nomenclature committee page (CYP450 polymorphisms) Human Gene Mutation Database CLASSIC DRUG TARGETS-Windows on the Human Genome Gene Trap Insertions – Has My Knockout Mouse Already Been Generated? Yeast Genome Deletion Project ABC Transporters – The Best Web Site The Environmental Genome Project National Center for Biotechnology Information CGAP Genetic Annotation Initiative (GAI) The National Human Genome Research Institute