Cytochrome P450 Enzymes

4.04 Cytochrome P450 Enzymes F P Guengerich, Vanderbilt University School of Medicine, Nashville, TN, USA ª 2010 Elsevier Ltd. All rights reserved.

4.04.1 4.04.2 4.04.3 4.04.4 4.04.5 4.04.5.1 4.04.5.2 4.04.5.3 4.04.6 4.04.7 4.04.7.1 4.04.7.2 4.04.7.3 4.04.8 4.04.8.1 4.04.8.2 4.04.8.3 4.04.9 4.04.10 4.04.11 References

Historical Perspective Nomenclature Gene Organization and Relationships Polymorphisms Regulation of Gene Expression Transcriptional Regulation Post-Transcriptional Regulation Post-Translational Modification Protein Structure Catalytic Mechanisms Generalized Mechanisms Rates of Individual Catalytic Steps ‘Alternate’ Reaction Mechanisms Catalytic Selectivity of P450s General Features Discrimination of Catalytic Specificities of Individual P450s Drugs and Non-Invasive Assays Roles of P450S in Biotransformation of Toxic Chemicals Clinical Significance Future Directions

43 50 51 52 53 53 54 55 55 58 58 62 63 64 64 65 67 68 70 72 72

Glossary (17) ethynylestradiol The estrogenic component of most oral contraceptives. acetaminophen (paracetamol, sold as Tylenol) A common analgesic drug. active oxygen species The form of oxygen involved in an oxygenation reaction. aflatoxin B1 A potent mycotoxin produced by various Aspergillus species. allele Variant forms of a gene. antipyrine An analgesic and antipyretic drug. benzo[a]pyrene A prototypical polycyclic aromatic hydrocarbon (carcinogen). bioactivation The biological conversion of a compound to a more toxic chemical. biomimetic (models) Chemical systems that mimic a biological mechanism. cation radical A species with a positive charge and an unpaired electron.

chlorzoxazone Muscle relaxant drug used in cytochrome P450 phenotyping. compound I A form of a peroxidase (or cytochrome P450) with the formal electronic structure (FeO)3þ. coumarin Natural product used as a cytochrome P450 substrate. desaturation The process of oxidizing an alkane by removal of two electrons and two protons. FAD Flavin adenine dinucleotide (prosthetic group). FMN Flavin mononucleotide (prosthetic group). group migration The transfer of a chemical group from one atom to the adjacent one in the course of a reaction. haloalkanes Alkyl compound containing halogens.

41

42 Cytochrome P450 Enzymes

heteroatom dealkylation Enzymatic cleavage of a molecule (by oxidation) between a carbon and a heteroatom (atom other than carbon). heterocyclic amines Compounds (produced by combustion) that contain primary amine and heteroatomic rings. hydrazines Molecules with the grouping –NHNH–. hydroperoxides Molecules containing –OOH. hydroxylation The addition of a single oxygen atom to form an alcohol product. immunoinhibition Inhibition (of an enzyme) by an antibody. iodosylbenzene A compound with the structure Ph-I¼O (where Ph is phenyl). kinetic isotope effects The effect (inhibitory) of substituting an atom in a molecule with a heavier isotope. nifedipine A common 1,4-dihydropyridine hypotensive drug, used in the early characterization of cytochrome P450 3A4. nitrosamines Chemicals containing –N¼O (N-nitrosamines are generally carcinogenic). non-invasive assays Assays using body fluids (usually blood, urine, saliva, etc.) to gain inference about enzymatic processes in the body (as opposed to requiring surgery or biopsy). oxidation–reduction potential The electrical valve (in volts, relative to the H2  2Hþ reaction) at which the oxidized and reduced forms of a compound are at the same concentration. oxygen rebound The second part of a cytochrome P450 oxygenation, considered to be FeOH2þ reacting with an incipient radical to produce an alcohol as a product. oxygen surrogate In cytochrome P450 reaction, compounds such as hydroperoxides or iodosylbenzene that can be added directly to the enzyme–substrate complex to effect an oxygenation. peripheral blood cells Blood cells other than erythrocytes, for example, lymphocytes and leucocytes. phenacetin An analgesic drug. phospholipid Glycerol lipids containing phosphate, including phosphotidylcholine, phosphotidylserine, phosphotidylethanolamine. polycyclic aromatic hydrocarbons Chemicals having three or more fused aromatic rings (many are carcinogens).

polyhalogenated biphenyls Biphenyl compounds (two attached benzene rings) substituted with halogens, usually chlorine or bromine. Used industrially as flame retardants, of concern as toxicants. post-translational regulation Regulation of protein concentrations (in a cell) by processes that occur after protein synthesis. rate-limiting step The slowest step in a series, for example, with the catalytic cycle of an enzyme. rifampicin An antibiotic drug, of interest as an enzyme inducer. single nucleotide polymorphisms Differences in individual bases in a gene, among individuals. site-directed mutagenesis The process of changing individual amino acids in proteins to explore the effects on biological activity. St. John’s wort An herbal medicine used for anti-depressant effects, of interest as an enzyme inducer and cause of changes in drug metabolism. substrate recognition sequence Site regions in cytochrome P450 proteins considered to be involved in substrate binding. taxol A drug that prevents tubulin polymerization; used to treat cancer. terfenadine The first non-sedating antihistamine drug marketed, later withdrawn. testosterone A major androgenic steroid. thiophene Five-membered ring aromatic compounds with a single sulfur (furan with sulfur replacing oxygen). transcription The copying of a gene (DNA) to yield RNA. transcriptional regulation Regulation of protein concentrations in a cell by effects on rates of transcription. vinyl halides Alkanes substituted with halogen atoms. vitamins Compounds important to health but not produced in the body, must be procured from food. warfarin A synthetic coumarin used as a drug (anticoagulant) and rodenticide. X-ray crystallography A process of obtaining 3-dimensional structures of proteins (crystals) by measuring scattering when placed in a source of X-rays.

Cytochrome P450 Enzymes

Abbreviations AF Ah ARNT B[a]P b5 CAR CYP DEN DMN E1/2 Em,7 FDA FMO FXR Glu P-1 HNF IND IQ kcat Kd

aflatoxin aryl hydrocarbon aryl hydrocarbon receptor nuclear transferase benzo[a]pyrene cytochrome b5 constitutive androgen receptor cytochrome P450 gene N,N-diethylnitrosamine N,N-dimethylnitrosamine oxidation potential oxidation–reduction potential Food and Drug Administration (US) flavin-containing monooxygenase farnesoid X receptor 2-amino-6-methyldipyrido[1,2-a:3,29-d]imidazole hepatocyte nuclear receptor Investigational New Drug (application) 2-amino-3-methylimidazo-[4,5-f]quinoline maximum catalytic rate dissociation constant

4.04.1 Historical Perspective Reactions catalyzed by cytochrome P450 (P450) enzymes were already known in the 1940s (Mueller and Miller 1948), even when the enzyme was not defined as such. These mixed-function oxidations (and also some reductions) involved drugs and chemical carcinogens. The stoichiometry of mixed-function oxidases was developed in the 1950s largely through work by Mason and Hayaishi with other enzymes (Hayaishi 1974; Mason 1957). The significance of this general group of enzymes increased when their role in the oxidation of steroids was noted. However, the nature of the system remained largely unknown. In 1956, Williams observed an unusual pigment in liver microsomes that bound CO and formed an unusual spectrum with a peak near 450 nm, an observation first reported by Klingenberg and Garfinkel (Garfinkel 1958; Klingenberg 1958). In 1962 Omura and Sato further characterized this entity and termed it ‘P-450,’ simply indicating a

Km LXR MAO MOCA -NF NNAL NNK NNN P450 PCR PhIP PPAR PXR RXR SNP SRS UGT VDR XRE

43

Michaelis constant lithocholic acid X receptor monoamine oxidase 4,49-methylene-bis(2-chloroaniline) -naphthoflavone 4-(methylnitrosamino)-1-(3-pyridyl)-1butanol 4-(methylnitrosamino)-1-(3-pyridyl)-1butanone nornitrosonicotine cytochrome P450 polymerase chain reaction 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine peroxisome proliferator activator receptor pregnane X receptor retinoid X receptor single nucleotide polymorphism substrate recognition sequence uridine diphosphoglucuronic acid glucuronosyl transferase vitamin D receptor xenobiotic response element

‘pigment’ (‘P’) with an absorbance maximum at 450 nm (Omura and Sato 1962). The association between this protein and the hydroxylation reaction was first established in studies by Cooper et al. (Cooper et al. 1965) in which they showed that the CO inhibition of steroid 21-hydroxylation in adrenal cortex microsomes could be reversed most efficiently by illumination with light at 450 nm, that is, the photochemical action spectrum (for light reversal of CO inhibition) matched the absorbance spectrum. In the 1950s, evidence for the inducibility of the system had been demonstrated in terms of both drug and carcinogen metabolism. In the 1960s, several lines of investigation provided evidence that multiple forms of P450s could be present in a single animal (Conney 1967). Despite these important studies, knowledge about the P450 systems was still very meager at this time, because the enzymes had not been isolated for more detailed studies. Gunsalus and his associates studied a bacterial model, in which a pseudomonad was isolated that could use camphor as a sole carbon source. The

44 Cytochrome P450 Enzymes

purified components of the system that catalyzed the first step in camphor degradation (5-exo hydroxylation) included a flavoprotein, a ferredoxin (putidaredoxin), and a P450 (commonly termed P450cam or, now more systematically, P450 101A1) (Tyson et al. 1972). This system has long served as a reasonable model for work on other P450s, although it seems more related to the mitochondrial than the microsomal P450s. The bacterial P450 101A1 is a soluble protein but the eukaryotic P450s are almost exclusively bound in membranes and proved difficult to isolate. A seminal contribution was the solubilization of rabbit liver microsomes with detergents, separation of the lipid, flavoprotein, and P450 components, and reconstitution of lauric acid !-hydroxylation activity by Lu and Coon (Lu and Coon 1968). In the mid-1970s, several P450 enzymes had been purified from livers of experimental animals, and the number increased in the 1980s (Guengerich 1987). Other major advances in the early 1980s included the elucidation of complete P450 primary sequences by amino acid and nucleotide analysis and the determination of the 3-dimensional structure of P450 101A1 (Ortiz de Montellano 1986). Much of the current understanding of the general catalytic mechanism of P450 catalysis was developed in this period. Although the clinical significance of P450s had been appreciated in the field of pharmacology, it was in the 1980s that the major human P450 enzymes were characterized by purification and cDNA cloning (Distlerath and Guengerich 1987; Gonzalez 1989; Nebert and Gonzalez 1987).

Today the total number of known P450 sequences is already >8500. This number is somewhat misleading, since it includes all species, and will continue to climb with the interest in plants and insects and the ease of obtaining new sequences through polymerase chain reaction (PCR), whole genome sequencing, and other technologies. Within each mammalian organism, the number of known P450 proteins is generally in the range of 50–100 (Nelson et al. 1993). The mammalian P450s can be considered to have two general functions. Some of the enzymes have restricted functions and are often quite specific. These include the P450s involved in the metabolism of steroids, eicosanoids, and fat-soluble vitamins (Table 1). The regulation of this subset of the enzymes seems to be very tight and, in general, there is not much variation in their levels. Indeed, genetic deficiencies of these enzymatic activities can be severely debilitating and even fatal (Nebert and Russell 2002). The main group of P450s of relevance in toxicology is the group in Table 1 that utilizes xenobiotic substrates (see also Table 2 for characteristics of human P450s). These are less specific in terms of catalytic specificity, often inducible, and show considerable variation in levels among individuals. These P450s may be considered to be present for the purpose of removing unwanted natural products that are consumed in the diet (Jakoby 1980). This group of P450s is also involved in the metabolism of drugs, carcinogens, pesticides, and diverse pollutants (Tables 3 and 4). Individuals seem to be

Table 1 Classification of human P450s based on major substrate class Sterols

Xenobiotics

Fatty acids

Eicosanoids

Vitamins

Unknown

1B1 7A1 7B1 8B1 11A1 11B1 11B2 17A1 19A1 21A2 27A1 39A1 46A1 51A1

1A1 1A2 2A6 2A13 2B6 2C8 2C9 2C18 2C19 2D6 2E1 2F1 3A4 3A5 3A7

2J2 4A11 4B1 4F12

4F2 4F3 4F8 5A1 8A1

2R1 24 26A1 26B1 26C1 27B1

2A7 2S1 2U1 2W1 3A43 4A22 4F11 4F22 4V2 4X1 4Z1 20A1 27C1

This classification is somewhat arbitrary in some cases, for example, P450s 1B1 and 27A1 could be grouped in either of two different categories.

Table 2 Characteristics of human P450 enzymes

P450

Chromosome location

1A1

15q24.1

1A2

15q24.1

1B1

2p22.2

2A6 2A7 2A13 2B6 2C8 2C9

19q13.2 19q13.2 19q13.2 19q13.2 10q23.33 10q23.33

2C18 2C19 2D6 2E1 2F1 2J2 2R1 2S1 2U1

10q23.33 10q23.33 22q13.1 10q26.3 19 1p32.1 11p15.2 19q13.2 4q25

2W1 3A4

7q22.3 7q22.1

3A5 3A7 3A43 4A11 4A22 4B1 4F2 4F3 4F8

7q22.1 7q22.1 7q22.1 1p33 1p33 1p33 19p13.2 19p13.2 19q13.12

Tissue

Known inducers

% total of hepatic P450

Extent of variability in level

Location

Many extrahepatic Liver

Polycyclic hydrocarbons

<1

100

ER

Smoking, polycyclic hydrocarbons, charred food Polycyclic hydrocarbons

12

40

ER

<1

Barbiturates, rifampicin

4 ? ? 1 1 20 (total 2C)

30 ? ? 50 ? 25 (total 2C)

(ER) ER

<1 3? 4 6 ?

? 100 >1000 20 ? ? ? ? ?

(ER) ER ER ER ER ER ER ER ER

? 20

ER ER

>100 ? ? ? ? ? ? ? ?

ER ER ER ER ER ER ER ER ER

Many extrahepatic Liver, others Liver (?) Liver, others Liver Liver Liver Kidney (Liver?) Liver Liver Liver, others Lung Heart, others Liver Skin, lung Thymus, heart, brain Tumors Liver, small intestine, others Liver, placenta Fetal liver Fetal liver ? Liver Lung, liver Liver, others Liver, others Seminal vesicles

Barbiturates, rifampicin (none) Ethanol, isoniazid

?

Barbiturates, rifampicin, dexamethasone

Clofibrate

28

0-8 <1 <1 ? ? ? ? ?

Validated non-invasive clinical markers

Caffeine, theophylline

ER ER (ER) (ER)

Coumarin

Diclofeanc, tolbutamide

(S)-Mephenytoin Debrisoquine, dextromethorphan Chlorzoxazone

Erythromycin, midazolam, nifedipine, (lidocaine, 6-hydroxycortisol)

(Continued )

Table 2

(Continued)

P450

Chromosome location

4F11 4F12 4F22 4V2 4X1 4Z1 5A1 7A1 7B1 8A1 8B1 11A1

19q13.12 19q13.12 19q13.12 4q35.2 1p33 1p33 7q34 8q12.1 8q12.3 20q13.13 3p22.1 15q24.1

11B1

8q24.3

11B2

8q24.3

17A1

10q24.32

19A1

15q21.2

20A1 21A2

2q33.2 6p21.33

24A1 26A1 26B1 26C1 27A1 27B1 27C1 39A1 46A1 51A1

20q13.2 10q23.33 12q14.1 10q23.33 2q35 12q14.1 2q14.3 6p13.3 14q32.2 7q21.2



Tissue

Liver, others Liver, others Eye Brain Breast cancer Platelets Liver Brain? Aorta Liver Steroidogenic tissues Steroidogenic tissues Steroidogenic tissues Steroidogenic tissues Steroidogenic tissues Liver, brain Steroidogenic tissues Kidney (Several) Brain liver Kidney Liver, kidney Liver Brain Liver, testes

Known inducers

% total of hepatic P450

Extent of variability in level

? ? ?

? ? ? ? ? ? ? ? ? ? ?

? ?

? –



Location ER ER ER ER ER ER ER ER ER ER ER Mito

–



Mito

–



Mito

–



ER

–



ER

?

ER ER

–



? ? ? ? ?



?

? ? ? ?



?

Validated non-invasive clinical markers

Mito ER ER ERE Mito Mito Mito ER ER ER

The levels of these P450s are relatively constant; when the activity is deficient (due to inactive enzyme or enzyme deficiency) the result is usually a disease state. ER, endoplasmic reticulum; Mito, mitochondria. Source: Guengerich, F. P. In Cytochrome P450: Structure, Mechanism, and Biochemistry, 3rd ed.; Ortiz de Montellano, P. R., Ed.; Kluwer Academic Press: New York, NY, 2005; pp 377–530.

Table 3 Chemicals activated to toxic forms by P450s Chemical class

Examples

Type of transformation

Toxic products

Biological effects

Hexane Ethyl carbamate, aflatoxin B2 CHCl3, CCl4, halothane 1,2-Dibromoethane 1,2-dichloroethane, 1,2-dibromo-3- chloropropane Benzylcyclo-propylamine

C-Hydroxylation Desaturation to olefins (vide infra) Heteroatom release, reduction Heteroatom release, conjugation

Diketones Epoxides Acyl halides, radicals -Halocarbonyls, half mustards/ episulfonium ions Methylene radicals

Neurotoxicity Carcinogenicity Toxicity Toxicity, carcinogenicity ?

Olefinic carbon systems Olefins

Styrene, acrylonitrile

Epoxidation, oxidative group transfer, suicide inactivation

Epoxides

Haloalkenes

Vinylidene chloride, trichloroethylene

Epoxides?

Dihydrofurans

Aflatoxins

Epoxidation, oxidative group transfer, suicide inactivation Epoxidation

Toxicity, carcinogenicity, porphyria Toxicity

Epoxides

Toxicity, carcinogenicity

Benzene

Epoxidation

Epoxides, phenols, quinones?

Benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, naphthalene Bromobenzene, pentachlorophenol Polychlorinated biphenyls, polychlorinated biphenyls Acetaminophen (paracetamol, Tylenol)

Epoxidation, others

Epoxides, diol epoxides

Toxicity, carcinogenicity Carcinogenicity

Epoxidation, oxidative group transfer Epoxidation, others?

Epoxides, quinones, others? Epoxides (?)

Toxicity Toxicity

Quinoneimines, semiquinone radicals (?) Michael acceptors

Toxicity Toxicity

Pyrroles Thiophenes

4-Ipomeanol, perillaketone, 3-methylfuran Pyrrole, 3,4-dimethylpyrrole Thiophene

Formal dehydrogenation, 1-electron oxidation Heteroatom oxidation?, epoxidation? ? S-Oxides

Maleimides? ?

Toxicity Toxicity

Nitrogen-based systems N-Nitrosamines

Dimethylnitrosamine

C-hydroxylation, heteroatomrelease

Pyrrolizidine alkaloids (lasiocarpaine, monocrotaline) 2-Aminofluorene

Heteroatom release

-Hydroxy N-nitrosamines,alkyldiazohydroxides Pyrroles

Toxicity, carcinogenicity Toxicity, carcinogencity Carcinogenicity

Alkyl carbon systems Alkanes Substituted alkanes Haloalkanes Vicinal dihaloalkanes Heteroatom-substituted cyclopropyls

Aromatic carbon systems Monocyclic aromatic hydrocarbons Polycyclic aromatic hydrocarbons Halobenzenes Poly-halogenated biphenyls Acetanilides Furans

Pyrrolines Amino-fluorenes

1-Electron oxidation, inactivation

N-Oxidation, one electron oxidation

Hydroxylamines and esters, radicals, nitroso compounds

(Continued )

Table 3

(Continued)

Chemical class

Examples

Type of transformation

Toxic products

Biological effects

Aromatic amines Azo dyes Hydrazines

2-Naphthylamine, benzidines, Trp-P-2 N,N-Dimethyl-4-aminoazo-benzene Procarbazine, dimethylhydrazine

Hydroxylamines and esters Hydroxylamines and esters CH3, CH4þ, diazomethane?

Nitro compounds

Ronidazole

N-Oxidation N-Oxidation Dehydrogenation, N-oxidation, C-oxidation, carbene formation Reduction

Carcinogenicity Carcinogenicity Toxicity, carcinogenicity Toxicity

Sulfur-based systems Thiocarbonyls Thiophosphone compounds

Thioacetamide Parathion, other insecticides

S-Oxygenation S-Oxygenation

Sulfenes, sulfines P¼O compounds (paraoxon)

Toxicity Toxicity

Tetramethyltin

Dealkylation

Triethyltin?

Toxicity

Metal-based systems Alkyl tins

Nitro anion radicals, hydroxylamines, oxygen radicals

Table 4 Human P450 enzymes involved in the activation of carcinogens P450 1A1

P450 1A2

P450 1B1

P450 2A6

P450 2A13

P450 2E1

P450 3A4

Benzo[a]pyrene (B[a]P) and other polycyclic hydrocarbons, including diols 2-Amino-1-methyl6-phenylimidazo[4,5-b]pyridine (PhIP) and some other heterocyclic amines

PhIP, 2-Amino-3methylimidazo-[4,5f]quinoline (IQ), and some other heterocyclic amines 2-Amino-6-methyldipyrido[1,2-a:3,29d]-imidazole(Glu P-1) 2-Aminodipyrido[1,2-a:3,29d]imidazole(Glu P-2) 4-Aminobiphenyl 2-Naphthylamine, and other arylamines 4-(Methylnitrosamino)1-(3-pyridyl)-1butanone (NNK)

B[a]P and other polycyclic hydrocarbons and diols IQ and other heterocyclic amines 2-Aminoanthracene and other arylamines 1-Nitropyrene and other nitropolycyclic hydrocarbons 17-Estradiol and other estrogens

N,N-Diethylnitrosamine (DEN) NNK 4-(Methylnitrosamino)1-(3-pyridyl)-1butanol (NNAL) Nornitrosonicotine (NNN)

NNK

N,N-Dimethylnitrosamine (DMN) Benzene Styrene Acrylonitrile Butadiene Vinyl carbamate

Aflatoxin B1 and other aflatoxins 7,8-Dihydroxy-7,8-B[a]P and some other polycyclic hydrocarbon diols Senecionine 6-Aminochrysene 4,49-Methylene-bis (2chloroaniline)(MOCA) tris(2,3-Dibromopropyl) phosphate

Vinyl chloride and other vinyl halides Ethyl carbamate Carbon tetrachloride Chloroform NNK NNAL NNN

50 Cytochrome P450 Enzymes

able to exist without some of these P450s in the absence of a particular stress. For instance, humans lacking P450 2D6 appear to be normal unless confronted with a drug that has serious side effects (Gonzalez and Nebert 1990). However, the line of demarcation between the ‘endogenous-substrate’ and the ‘xenobiotic-substrate’ groups of P450s may not be so sharp as sometimes thought. Some of the P450s in the latter group are also involved in the oxidation of endogenous compounds to entities with marked physiological activities (e.g., eicosanoid epoxides (Capdevila et al. 2005)), and the steroid-hydroxylating P450 1B1 has many xenobiotic substrates (Shimada et al. 1996). In plants and insects, certain P450s have critical roles in defense systems and provide them with the ability to withstand pests, digest certain foodstuffs, etc. P450s are also involved in processes such as color development in flowers (Holton et al. 1993). Finally, it should be pointed out that the functions of many P450s are unknown, with regard to both endogenous and xenobiotic chemicals (Table 1).

4.04.2 Nomenclature ‘Cytochrome P450’ is an operational definition. The term ‘heme thiolate protein’ has been recommended by The Enzyme Commission (Palmer and Reedijk 1992) (E.C. 1.14.14.1, ‘non-specific oxygenase,’ is the assigned number, but E.C. numbers 1.14.13.11, 1.14.13.12, 1.14.13.17, 1.14.13.30, 1.14.13.39, 1.14.15.1, 1.14.15.3, 1.14.15.4, 1.14.15.6, 1.14.99.9, 1.14.99.10, 1.14.99.22, 1.14.99.28, 4.2.1.92, 5.3.99.4, and 5.3.99.5 designate certain individual P450s, primarily for various historic reasons). In a strict sense, P450 is not a cytochrome in that it does not transfer electrons from one protein to another. The term applies to all heme proteins having heme cysteine thiolate axial ligation. However, some heme proteins that meet this definition are not included in the listings (Nelson et al. 1993) because of low sequence similarity (e.g., fungal chloroperoxidase (Blanke and Hager 1988) and nitric oxide synthases (White and Marletta 1992)). This cysteinal thiolate ligand is the basis of the Fe2þ–CO spectral absorption maximum near 450 nm. Thus, P450s are defined either by these spectral properties or, if spectra are not available, by a high degree of sequence similarity in the cysteine region known to bind the heme. The generally accepted designation is ‘P450,’ without a hyphen. The prefix ‘CYP’ is usually used to designate the genes (‘cyp’ for the mouse

genes), although sometimes also applied to proteins (however, cyclophilins also use this same acronym). In the course of discovery and characterization of the P450 enzymes, many were given different designations by individual investigators and, with the growing number of P450s, comparisons became more difficult. A systematic nomenclature was developed, based upon primary sequence identity. The overall similarity of the different P450s constituting the superfamily (all P450s) is rather low (20%). P450s with >40% identity are grouped in the same family, indicated by the first numeral in the designation (i.e., 1, 2. . .). Two sequences with >59% identity are grouped in the same subfamily, designated by a letter (i.e., 1A, 2A. . .) (a small letter is used for mouse genes and proteins). The last number in the designation refers to the individual gene or protein. In this regard the system is not totally consistent. In the case of P450s where function is generally considered to be relatively constant, a single designation is used for all species (e.g., 1A1, 1A2, 2E1). If a single gene is in a family, it is designated ‘A1.’ However, in complex gene families there is often considerable difference in functional properties in a subfamily, both within a species and across species. Thus, individual numbers are assigned to the P450s in different species. These are often not continuous and eventually the system may have to be revised to accommodate all the P450s in different species. For example, in the P450 3A family, 3A1 and 3A2 are rat genes, 3A4, 3A5, 3A7, and 3A43 are human genes, and 3A6 is a rabbit gene (Nelson et al. 1993). Alleles are genetic variants (usually only small base changes) that occur within populations and may or may not affect their function. These are not given separate designations within the system but authors are simply asked to define the differences in their publications. Lists the accepted polymorphisms and their nomenclature. Also, there is no provision for introducing changes made by site-directed mutagenesis into the system. Some of the family numbers were designated on the basis of functions (e.g., P450 17A1 catalyzes steroid 17-hydroxylation). In general, the ‘early’ family numbers 1–50 are for mammals and insects, 50–100 are for yeast and plants, and those above 100 are for bacteria (Nelson et al. 1993). At this point it is of interest to consider the functions of P450s prior to further discussion. Although the classification of P450s by sequence alignment is highly useful in the nomenclature, conclusions about function cannot be drawn from this. Another

Cytochrome P450 Enzymes

approach is to group these by the nature of the substrates (Table 1). Thus the 57 human P450s are grouped in terms of function. This classification has some caveats, for example, P450 1B1 has a role in estrogen metabolism (Hayes et al. 1996) and appears to be important in the eye (Stoilov et al. 1997, 1998) but is also involved in the metabolism of many carcinogens (Shimada et al. 1996), and P450 24A1 is involved in both steroid and vitamin metabolism (Guengerich 2005). A number of P450s have little, if any, information available about them and are termed ‘orphans’ at the present time (Guengerich et al. 2005). Similar classifications could be made with rodent P450s or those from other experimental animals. The available information from studies with transgenic mice (vide infra) exemplifies that the P450s involved in the metabolism of steroids, vitamins, and at least some of the eicosanoids are critical to normal physiology but the P450s involved in xenobiotic metabolism are not. This conclusion is consistent with the fact that many humans are missing certain P450s due to polymorphisms (vide infra) but function normally unless exposed to the wrong drugs or other chemicals. In considering the metabolism of marketed drugs, P450s account for 75% of the metabolism (Figure 1a), and five of the P450s are responsible for 95% of the drug oxidations (Figure 1b) (Wienkers and Heath 2005; Williams et al. 2004a). A similar analysis has not been done with carcinogens with regard to percentages, but P450s 1A1, 1A2, 1B1, 2A6, 2A13, 2E1, and 3A4 appear to be most important in carcinogen metabolism (Table 4). In considering (a)

NAT FMO MAO

51

toxicity as a whole, drugs and carcinogens together constitute a major fractionof the potentially toxic chemicals. One question that arises frequently is how useful animal models are for humans, in terms of similarity of their P450s. The appropriateness varies with the case, but a few general comments can be made. The similarities of P450s have been considered elsewhere, although less information was available at the time (Guengerich 1997a). With regard to similarity of a P450 across species, P450 2E1 seems to have the most conserved properties. More caution is advised in extrapolating across species in the 1A1, 1A2, 1B1, 4A, and 17A1 subfamilies. Even more caution is needed in the 2D and 3A subfamilies. Major problems occur in extrapolations with the 2A, 2B, and 2C subfamilies. As an example of a change in the P450s involved in a reaction, there is the case of mephenytoin (Shimada and Guengerich 1985): in rats a 3A P450 is involved but in humans the polymorphic 2C19 is the main catalyst (de Morais et al. 1994b). Another example is the toxic natural product 4-ipomeanol: in rabbits the lung-specific P450 4B1 is involved in activation but in humans the liver-selective P450 3A4 is, thus causing some clinical toxicity problems (Czerwinski et al. 1991).

4.04.3 Gene Organization and Relationships Chromosomal assignments (Table 2) can be made readily with various hybrids or through genomic sequence analysis. The individual genes within a (b) 1A1 1A2

2B6

Esterases

UGT 2C9

3A4 P450

2C19 2D6

2E1 Figure 1 a, Fraction of drugs that undergo biotransformation by individual enzyme systems (UGT, UDP glucuronosyl transferase (Chapter 4.20); FMO, flavin-containing monooxygenase (Chapter 4.05); NAT, N-acetyl transferase (Chapter 4.19); MAO, monoamine oxidase (Chapter 4.05)). b, Contribution of individual P450s to biotransformation of drugs. The five major P450s are underlined (Wienkers and Heath 2005; Williams et al. 2004a).

52 Cytochrome P450 Enzymes

subfamily are clustered together in a particular region of a chromosome (Nelson et al. 1993). In a very few cases, different P450 subfamily genes are clustered together in a certain chromosomal region, for example, human P450s 2A, 2F, and 2G (Nelson et al. 1993). In general the P450s within a given subfamily have similar patterns of intron/exon organization (Gonzalez 1989; Nelson et al. 1993). This similarity has given rise to the concept that many of the P450 genes are the result of gene duplication, defined as events involving non-reciprocal exchange of nucleotide sequences between similar genes. In some instances there may be advantages associated with such events, giving rise to P450 proteins with new functions to provide selective advantages. Some genes may be considered somewhat residual and there is probably not much selective pressure to either maintain or eliminate them at this point in history. However, it is possible that these P450s might have played important roles in the processing of particular natural products earlier, at least in some populations. Another point of interest is that the conserved exons seem to correspond, in general, to important structural domains within the P450s. This is a relatively short treatise of genomic organization and postulation of evolutionary relationships; for further discussion see (Nelson et al. 2004) and references therein.

4.04.4 Polymorphisms Polymorphisms exert dramatic effects on activities of P450s, and many have now been well characterized in humans and experimental animals. As discussed in Chapter 4.02, these are generally defined as occurring at an incidence of >1% in the population. The molecular bases and some of the implications are also discussed in that chapter. Several P450 polymorphisms have been defined and studied in experimental animal models, for example, rat P450 2B1 and rabbit P450 2C3. Strain differences are seen in rat P450 2B1, with residue 478 being variant (Gly or Ala) (Kedzie et al. 1991). The change makes no difference with regard to some catalytic activities but has an effect on others (e.g., androstenedione 16-hydroxylation). With rabbit P450 2C3, Johnson and his associates defined two populations of animals that differ in their abilities to catalyze progesterone 6-hydroxylation. Studies with chimeras in which regions of the proteins were switched led to the conclusion that the Ser/Thr polymorphism at residue 364 underlies the difference

in catalytic activity, as well as inhibition by 16methylprogesterone (Tsujita and Ichikawa 1993). An Ile/Met difference at position 178 has a dramatic effect on the Michaelis constant, Km. In humans a number of important polymorphisms have been recognized, usually after observations made regarding in vivo drug metabolism. Some polymorphisms occur with all of the P450 genes, although in many cases non-functional ones may not have been identified. P450 2D6 was the first P450 enzyme demonstrated to be under monogenic control (Smith et al. 1978; Tucker et al. 1977). Studies were prompted by the enhanced sensitivity to the side effects of debrisoquine in some individuals due to slow 4hydroxylation. The polymorphism is racially linked: in Caucasians 5–10% of the population is considered to be ‘poor metabolizers’ while in Asians only 1% is deficient, although the mean level of activity is somewhat less in the total population of the latter group. A number of single nucleotide polymorphisms (SNPs) and other genotypic variations that give rise to the poor metabolizer phenotype have been identified through techniques such as restriction fragment length polymorphism, PCR, and total sequencing. The most common defect is a G-to-A transition mutation at an intron/exon boundary that does not allow proper processing of the initial RNA transcript to mRNA. Thus no mRNA or protein is produced in these individuals. Other defects include a gene deletion and some mutations within the protein coding sequence that generate a protein with less than normal activity. It is also of interest to note that the total population varies considerably in the level of P450 2D6 and its catalytic activities, even in the ‘extensive metabolizer’ group, due to all of the various polymorphisms (>110 alleles). With this P450, there is no evidence for induction by xenobiotic chemicals or endogenous compounds, although some cell-specific regulatory elements must be involved. Finally, some individuals have very high catalytic activity and this can be accounted for, at least in some cases, by gene duplication. Thirteen copies of the P450 2D6 gene were found in some members of a Swedish family, as the result of a base change that promotes and/or stabilizes the duplication (Johansson et al. 1993). Thus, there are a number of mechanisms accounting for the polymorphism, which now seems to be the general case with P450s and other enzymes discussed in this volume. Of the >110 allelic variants of P450 2D6 now recognized, a few are most predominant and give rise to major phenotypic differences. A rough estimate with any gene is that 10% of the

Cytochrome P450 Enzymes

SNPs yield significant changes in functional activity (Shen et al. 1998). Another now classic human P450 polymorphism involved the 49-hydroxylation of the (S)-enantiomer of the drug mephenytoin (Goldstein and Demorais 1994). Although the involvement of P450 2C enzymes in the polymorphism had been known for some time the specific assignment within the subfamily had been unclear until the characterization of P450 2C19 (Goldstein et al. 1994). The most common change responsible for the poor metabolizer phenotype is a G-to-A transition in exon 5, which creates an aberrant splice site (de Morais et al. 1994b). In Caucasians the incidence of the defective metabolizer phenotype is considerably less (2%) than in Asian populations (15–20%). The polymorphism cited above is seen in both Caucasians and Asians but another base pair mutation creating a premature stop site in exon 4 is seen almost exclusively only in Asians (de Morais et al. 1994a). Although at least 27 different alleles have been identified, these two variants can explain 90% of the poor metabolizer phenotypes in all races examined to date. Another means by which a polymorphism might alter enzyme activity is a mutation in a 59 upstream or other regulatory region. For instance, it is known that single mutations can often abolish binding of transcription factors and proteins that bind to enhancers (Nguyen et al. 1994). Polymorphisms of this type tend to be less common.

The other major means of altering enzyme activity through polymorphism is through mutations in the coding sequences of regulatory proteins. Such changes may affect the affinities of these proteins for either their ligands or their DNA. As an example, it has been shown that only five amino acid differences in the aryl hydrocarbron (Ah) receptor protein (vide infra) increase its dissociation constant, Kd, for polycyclic hydrocarbons dramatically, and strains of mice with the low affinity receptor are relatively insensitive to induction of P450s 1A1 and 1A2 (Chang et al. 1993).

4.04.5 Regulation of Gene Expression There are numerous points at which activities (Kedzie et al. 1991) of individual P450 enzymes may be regulated. We will focus on regulation of expression of (active) protein here and not consider aspects of cofactor supply, dietary inhibitors, etc. 4.04.5.1

Transcriptional Regulation

This type of regulation, alluded to above, would appear to be the most common type of regulation of the P450s, at least to date. Transcriptional regulation generally involves receptors that bind ligands and then interact with DNA. The basic mechanism is outlined in Figure 2 and follows the general model of several of the classic steroid hormone receptors.

R′ L +

L R

R

L R

R′

(Movement to nucleus?) Coactiv L

R

R′

L R

R′

P450 gene

P450 gene DNA

53

DNA RNA pol (Increased access to promoter, start site)

Increased transcription Figure 2 Generalized mechanism of P450 induction by enhanced transcription. L, ligand; R, receptor; R9, heterodimer component for binding the receptor R; Coactiv, co-activator protein; pol, polymerase.

54 Cytochrome P450 Enzymes

The P450 1A1 system is probably the best understood among models of P450 regulation (Chapter 4.02) (Williams et al. 2005). The Ah receptor binds polycyclic hydrocarbons or even indolo[3,2-b]carbazole, a product of dietary indole-3-carbinol formed in the acidic environment of the stomach (Bjeldanes et al. 1991). In the process of ligand binding, two heat-shock proteins (hsp90) are removed from the Ah receptor and the receptor then binds to the aryl hydrocarbon receptor nuclear transferase (ARNT) protein; it is now ‘active’ and moves into the nucleus. The heterodimeric Ah receptor–ARNT complex bonds to xenobiotic regulatory elements (XREs) on the DNA; there are several 59 XREs upstream of the TATA box at which transcription begins. Such binding somehow facilitates transcription, apparently by altering gene structure in chromatin through ‘longrange’ effects. These basic tenets of the system seem to be well accepted but there are still more things to learn about the system. For instance, it has been shown that high concentrations of the Ah receptor and ARNT proteins (together) can activate in the absence of ligand (Matsushita et al. 1993). Also, interleukins downregulate P450 1A1 transcription (Barker et al. 1992) and a possible explanation is that a binding site for an interleukin receptor is ‘close’ to an XRE on the DNA and causes interference (Robertson et al. 1994). Several groups have reported evidence for the existence of at least one negative regulatory element (Jones et al. 1985). Since the first edition of this series, considerable information has become available about the roles of some of the proteins of the so-called steroid receptor superfamily in the regulation of P450s. In the early 1990s, the role of the peroxisome proliferator activator receptor  (PPAR) in P450 4A subfamily regulation was demonstrated (Issemann and Green 1990; Muerhoff et al. 1992). Mining of the ‘orphan’ receptors in the steroid receptor superfamily led to the discovery of two more important receptors, the pregnane X receptor (PXR) and constitutive androgen receptor (CAR) (Honkakosski et al. 1998; Kliewer et al. 1998). These two receptors, like PPAR, form heterodimers with the retinoid X receptor (RXR). PPAR and PXR require ligand binding for activation; the identity of the ligand (retinoid) bound to one of the RXR isoforms also regulates transcriptional activity of the heterodimer. Ligands for PXR and PPAR vary among animal species. PXR ligands include steroids and a variety of drugs and other xenobiotics (Watkins

et al. 2001). PPAR ligands include fatty acids and some derivatives, including some eicosanoids, and a variety of xenobiotic chemicals, including drugs (e.g., fibrates), plasticizers, and various carboxylic acids (Rao and Reddy 1991). CAR is an unusual regulatory protein. While there is evidence that some strong inducers are ligands for CAR (e.g., TCPOBOPOP (Swales et al. 2005)), the major mode of action involves the constitutively active mode of CAR regulation. Barbiturates, for example, the classic being phenobarbital, somehow cause phosphorylation of CAR and enhanced nuclear transport. PXR and CAR show some ‘cross-talk’ in their action with consensus sites (Willson and Kliewer 2002). In general, PXR appears to be the major regulator of P450 3A enzymes and CAR of several P450 subfamily 2B and 2C enzymes. However, the cross-talk can explain the inducibility of rat P450 3A enzymes by barbiturates. PXR also shows some cross-talk with several other members of the steroid nuclear receptor superfamily, including the vitamin D receptor (VDR), farnesoid X receptor (FXR), and lithocholic acid X receptor (LXR) (Jung et al. 2006; Pascussi et al. 2003). The complexity of the systems is considerable in that recent studies have shown that Ah receptor activation can regulate CAR levels in mouse and human liver (Patel et al. 2007) and PPAR induces nuclear translocation of CAR (Guo et al. 2007). With each of the receptor systems, binding of the liganded heterodimer to its cognate recognition sequence leads to the recruitment of co-activator proteins that bind to the DNA–heterodimer complex to alter the chromatin structure and open the promoter region for RNA polymerase binding and enhanced rates of transcription. Both the receptors and the co-activators show cell-and tissue-specific expression and therefore regulate the tissue-specific expression of individual P450s. For instance, hepatocyte nuclear factor 4 (HNF-4) is a co-activator of P450 3A4 (Tirona et al. 2003) and the human P450 1A2 gene contains a (half) HNF-1 consensus site presumably contributing to the liver-selective expression of these P450s. Accordingly, only in vitro cell models expressing the appropriate co-activator proteins (Tirona et al. 2003) show P450 3A4 induction. 4.04.5.2

Post-Transcriptional Regulation

As pointed out in Chapter 4.02, these mechanisms are generally harder to characterize than those involved in transcriptional regulation. In many cases

Cytochrome P450 Enzymes

researchers use mRNA levels as surrogates for protein, which is not always reliable. One proposed mechanism is enhancement of mRNA stability, which has been suggested for P450 2E1 (Eliasson et al. 1990). Another possibility is selective stimulation of translation of individual mRNA transcripts (Kim et al. 1990). Further evidence for such mechanisms of P450 regulation is not yet available. 4.04.5.3

Post-Translational Modification

Some attention has been given to this possibility of modulating P450 enzyme activity and also enzyme stability to degradation. Obviously, the insertion of heme is an obligatory post-translational modification of all P450s. When certain bile pigments accumulate in abnormal conditions or follow mechanism-based inactivation of P450 heme, these can interfere with -aminolevulinic acid synthase, a key step in heme biosynthesis. The degradation of heme involves the enzyme heme oxygenase(s), which forms biliverdin IX and CO, except in certain circumstances. The heme in P450s is generally thought to be well sequestered within the protein structure (at least as judged by the structures of the bacterial P450s) and only available for degradation after release, despite the view sometimes seen in the literature that heme oxygenase induction leads to P450 loss. Phosphorylation is a current theme for regulation of many proteins and has also been considered to modify P450 stability and activity. Most evidence has come with in vitro studies involving either kinases or hepatocytes in which radiolabeling/inhibitor studies are done. It seems clear that it is possible to phosphorylate certain P450s and that such modification may affect catalytic activity at least somewhat. A correlation has been made with the tendency of the different P450s to interact with cytochrome b5 (b5). What is still not clear is the extent to which this is a significant mechanism in the normal in vivo state. Some of the strongest evidence for the role of phosphorylation had been obtained with P450 2E1, a protein known to show some types of post-transcriptional regulation. The presence of substrates or inducers (which are very overlapped for this enzyme) has been thought to block phosphorylation of Ser 129, thus leading to protein stabilization (Eliasson et al. 1990). However, site-directed mutagenesis of Ser 129 to Ala (which cannot be phosphorylated at this site) did not affect the half-life of P450 2E1 in a cell culture system (Freeman and Wolf 1994).

55

Phosphorylation of P450 2E1 also targets the protein to mitochondria (Robin et al. 2002). Little evidence has been obtained for any significance of P450 glycosylation. In early studies with some rat and rabbit P450s, no evidence for glycosylation was obtained (Armstrong et al. 1983; Guengerich et al. 1982). Many functionally active mammalian P450s have now been expressed in bacteria, presumably without modification. P450 19A1 (aromatase) appears to be glycosylated near the N-terminus and this may be related to its membrane localization (Shimozawa et al. 1993). However, a functional enzyme can be expressed in Escherichia coli (Kagawa et al. 2003) and apparently this is not glycosylated. Few, if any, other post-translational modifications of P450s have been identified. However, searches have not been systematic since more than 100 different types of modifications are now possible. Fortunately, the newer methods of mass spectrometry offer the possibility of general searches for structural modifications with small amounts of material.

4.04.6 Protein Structure Among the characterized bacterial P450s are 15 common helices – A, B, B9, and C through L – plus five -sheet regions (Figure 3). Some of these P450s contain additional helical regions. The most conserved region is near the conserved Cys that serves as the axial ligand to the heme iron. The ‘I’ and ‘L’ helices make contact with the heme, and these two moieties are thought to be general. General agreement exists that the N-terminal hydrophobic tail present in most of the eukaryotic P450s is involved in helping to anchor these in the membrane, and there is evidence for the roles of signal recognition particle and ‘halt-transfer’ elements in the primary sequence to aid in proper membrane insertion. However, the removal of the N-terminal hydrophobic segment from some P450s has not led to a loss of binding to bacterial membranes. Although one must exercise caution in comparing bacterial and mammalian membranes, these results would suggest that there may be other previously unrecognized elements within at least some of the P450 sequences that aid in membrane binding (Hasemann et al. 1995). Along the I helix there is usually (but not always) a Thr corresponding to Thr 252 in P450 101. This residue has been postulated to have various

56 Cytochrome P450 Enzymes

NH2

β2

C

β1 A

I

B′

B

G

G′

G′

H

B′ F′

E

β1 G

F

β4

F

NH2

C

E

D

I

β3

K

β2

L D

COOH A B

J

K J

β3 COOH

Figure 3 Crystal structure of human P450 1A2 (-NF complex) showing two views (Sansen, S.; Yano, J. K.; Reynald, R. L.; Schoch, G.; Griffin, K.; Stout, C. D.; Johnson, E. F. J. Biol. Chem. 2007, 282, 14348–14355).

functions. Initially, site-directed mutagenesis studies with P450 101A1 led to the view that this Thr donates a proton to the Fe2þ–O2 complex and is important in proper coupling of oxygen with electron flow (Imai et al. 1989; Martinis et al. 1989). However, the high pKa of Thr is a problem for this explanation and others have suggested that it serves as part of a triad system for protonation. It has also been postulated to play a more structural role, perhaps by forming a ‘bubble’ for O2 binding in the I helix. At least three P450s appear not to contain this Thr in all reasonable alignments used, so this amino acid cannot be considered essential (Hasemann et al. 1995). It has been postulated that this Thr is one of several ways that P450s use to protonate molecular oxygen and reduce the abortive use of oxygen (which admittedly is not very efficient with many of the eukaryotic P450s) (Hasemann et al. 1995). Studies using site-directed mutagenesis with unnatural amino acids also indicate that the Thr of P450 101A1 can be replaced by a Thr-methyl ether without a dramatic effect (Kimata et al. 1995). Since the publication of the previous edition of this series (Guengerich 1997b), our information about P450 has advanced considerably with the acquisition of many bacterial P450 structures, several structures of two rabbit P450 family 2 structures, and structures of human P450s 1A2 (Sansen et al. 2007), 2A6 (Yano et al. 2005), 2C8 (Schoch et al. 2004), 2C9

(Wester et al. 2004; Williams et al. 2003), 2C19 (Johnson et al. 2007), 2D6 (Rowland et al. 2006), 3A4 (Ekroos and Sjo¨gren 2006; Williams et al. 2004b; Yano et al. 2004), and 2R1 (Strushkevich et al. 2008). Thus, the structures of most of the major drug metabolizing P450s (Table 1, Figure 1b) are available (Figure 4). The reader is referred to original articles and some reviews (Johnson et al. 2007; Poulos and Johnson 2005; Rowland et al. 2006; Sansen et al. 2007; Schoch et al. 2004; Wester et al. 2004; Williams et al. 2003; Yano et al. 2004, 2005) (Figures 3 and 4). Several general conclusions can be made from the available knowledge. P450s generally exist in an open configuration in the absence of ligand and then change to a closed form when the ligand (substrate) is bound. Therefore, ligand-free structures are not particularly informative. Twelve helices (A–L) and four -sheets (1–4) are generally conserved in both prokaryotic and eukaryotic P450s. The polypeptide chains forming the catalytic domains of eukaryotic P450s are generally longer than those of the prokaryotic P450s. Although the overall structural organization of the P450s is similar, there are major differences in the lengths of helices and loops, as well as their placement. Thus the shapes and sizes of the active sites differ considerably (Figure 4), even within families and subfamilies. Substrates bind in a cavity above the heme surface (Figures 3 and 4). The active site volume varies at

Cytochrome P450 Enzymes

(a)

(b)

(c)

1A2 G′

2A6 G′

G

3A4 G′

G

G

F′ B′

B′

F

B–C loop

F

F′ F′

F

I

heme

(d)

D293 F114

SRS5

heme

(e) 2D6

Gln244

2C9

Glu216

V292

R108

heme

SRS5

M240 V237

F

I

I

SRS5

57

N204 L208 G296

I205

A297 V113

Glycerol Asp301 Phe120

L366

T301

Figure 4 Some crystal structures of human P450s showing the binding sites (Poulos and Johnson 2005). The calculated volumes of the active sites are listed for each. a, P450 1A2 (375 A˚3) (Sansen et al. 2007); b, P450 2A6 (260 A˚3) (Sansen et al. 2007; Yano et al. 2005); c, P450 3A4 (1385 A˚3) (Sansen et al. 2007; Yano et al. 2004); d, P450 2D6 (540 A˚3) (Rowland et al. 2006); e, P450 2C9 (with flurbiprofen, 470 A˚3) (Wester et al. 2004).

least 5-fold, as seen in the comparisons of P450 2A6 (smallest) and P450 3A4 (largest) (Figure 4). The shapes of the active sites also vary, so size is not the only factor to consider in docking. Although the six ‘substrate recognition sites’ (SRSs) developed by Gotoh (1992) are useful in comparing P450s, the size of the sequence regions varies, and in the P450s with the larger active sites (e.g., 3A4) the boundaries of the SRSs are distributed beyond those of the six regions defined originally on the basis of the small active site structure of bacterial P450 1A1 to include regions between helices F and G, the first turn in -sheet 1, and the region surrounding the N-terminal end of helix A (Poulos and Johnson 2005). Some insight is possible in docking substrates into known structures of P450-ligand complexes, at least in terms of permissible size and what

amino acids might form ionic or hydrogen bonds with substrates. Prediction can be made with some accuracy, but one issue is the flexibility of the active site (Ekroos and Sjo¨gren 2006; Guengerich 2006; Sansen et al. 2007). Another issue with several of the crystal structures is that in some cases the substrate is bound at a site too far away to be catalytically competent (He et al. 2006; Williams et al. 2003, 2004b), and it is not clear if the structure is an artifact of crystallization or represents a viable intermediate in the entry of the substrate in moving toward the active site (Isin and Guengerich 2006). One structure of P450 3A4 (Ekroos and Sjo¨gren 2006) has two molecules of substrate bound and may be relevant to issues of catalytic cooperativity, although this point is still not exactly clear (Isin and Guengerich 2007). Multiple substrate/effector

58 Cytochrome P450 Enzymes

4.04.7 Catalytic Mechanisms

occupancy is thought to play a role in both the homo- and the heterotropic cooperativity of P450s. The problem of predicting catalysis from structures is exemplified by the P450 1A2?naphthoflavone (-NF) complex (Figure 3). -NF is slowly oxidized, forming only the 5,6-epoxide (Sohl et al. 2008). However, this part of the molecule is positioned furthest from the iron in the crystal structure of the complex (Sansen et al. 2007). Thus, a repositioning of the ligand must be necessary for catalysis to occur. Another point to be made is that catalytic function of a P450 is determined by amino acid changes outside of the active site, in that these residues act as hinges to modulate protein motion (Kim et al. 2005). Even though most P450s have a rather buried active site, the rates of substrate binding and release are much more rapid than overall rates of catalysis, so the entire protein must open and close rapidly. Thus conclusions about the roles of particular amino acids in catalysis are often based only on analysis of reactions. In the past, the reasoning has been somewhat circuitous in that often homology models were proposed, site-directed mutagenesis was done, an attenuation (often modest) was observed, and the results were used to validate the original model. Clearly the crystal structures available today have not only provided more direct insight but have also raised the standard for interpretation.

9. –ROH

Fe3+

Fe3+ ROH

4.04.7.1

Generalized Mechanisms

Inference about the range of reactions P450s can be involved in has its basis in understanding the chemistry of catalysis. The overall catalytic cycle is generally accepted to be that depicted in Figure 5 for most P450 oxidation reactions. The substrate is bound near (but not to) the iron atom of the heme (Figures 3 and 4). The first step is generally thought to be addition of substrate to the enzyme (Figure 5, step 1). In some cases this binding changes the configuration of the iron d5 orbitals from low spin to high spin (Fisher and Sligar 1985) but this is not universally the case (Guengerich 1983; Huang et al. 1986); indeed, some P450s are normally isolated in the high-spin state in the absence of any ligands (Guo et al. 1994; Sandhu et al. 1994). In step 2, one electron is transferred from the flavoprotein NADPH-P450 reductase to the substrate-bound P450. Electrons from NADPH enter the FAD flavin of the reductase, in the form of hydride in equivalents (a formal 2e process). Electrons then flow to the FMN component and subsequently, one at a time, to the P450. The reductase has a number of possible oxidation–reduction states and it is difficult to evaluate which predominates during turnover with P450. However, there is evidence to indicate that the reductase cycles primarily between FAD?/FMNH2 and FAD?/FMNH.

1. RH Fe3+ RH

NADPH-P450 reductasered

1e–

8.

2. NADPH-P450 reductaseox

FeOH3+ R• 7.

Fe2+ RH

FeO3+ RH 6.

O2 3.

–H2O FeII–OOH RH

H+ 5.

Fe2+–O2 RH Fe2+–O2– RH

1e–

NADPH-P450 reductasered

4.

NADPH-P450 reductaseox Figure 5 General mechanism of P450 catalysis.

Cytochrome P450 Enzymes

forms in reducing ferric P450, and probably between FADH?/FMNH2 and FADH?/FAMNH? in reducing the Fe2þ–O2 complex (Guengerich 1983; Iyanagi et al. 1981; Oprian and Coon 1982; Vermilion et al. 1981). In the case of P450 101A1 there is considerable evidence that the binding of the substrate camphor changes the iron spin state and also raises the Em,7 (oxidation–reduction potential) of the P450 from 340 to 170 mV (Sligar and Gunsalus 1976). However, such events do not seem to occur to this extent or be coupled in many of the eukaryotic P450s (Guengerich 1983), although this has now been demonstrated with one substrate with (human) P450 3A4 (Denisov et al. 2007). As in the case of other hemoproteins (e.g., hemoglobin), O2 binds only to the ferrous enzyme (step 3). This ferrous–O2 complex has been observed in bacterial and mammalian P450s (Bonfils et al. 1979; Guengerich et al. 1976; Ishimura et al. 1971) but is unstable and can decompose to generate superoxide anion (O2 ?) or, if protonated, H2O2 (Oprian et al. 1983). A second electron enters the system in step 4. The Em,7 for this step has been estimated to be 0 mV, considerably higher than in step 2 (Guengerich 1983). NADPH-P450 reductase can contribute the electron, although in some cases this second electron comes from b5. This aspect is discussed below. After this point, all intermediates are unstable and only limited evidence for defined intermediates is available. Much of our knowledge concerning steps 5–8 in Figure 5 comes by inference from biomimetic models. The complex formed following step 4 must be protonated for function. It is possible for the complex to decompose at this point to generate H2O2, as an alternative to H2O2 generation from O2 ? derived from the Fe2þ–O2 complex (Ortiz de Montellano 1986). The next step (6) involves the heterolytic scission of the O–O bond. This step is critical for most P450-catalyzed oxidations (Hasemann et al. 1995; Vaz et al. 1991). Such scission generates a formal (FeO)3þ entity, which is generally accepted to be the actual oxygenating species and will be considered below in terms of mechanism (i.e., steps 7 and 8). In the early days of P450 research, considerable attention was given to the nature of the ‘active oxygen species’ involved in P450 reactions, and possibilities were superoxide anion, singlet oxygen, etc. (Strobel and Coon 1971). However, the concept has developed that an iron oxygen complex, analogous to peroxidase Compound I, is the oxygenating entity. Step 9 is the release of product to complete the reaction cycle.

59

Part of the cycle may be bypassed experimentally with the use of ‘oxygen surrogates.’ The most developed of these are cumene hydroperoxide and iodosylbenzene (Kadlubar et al. 1973; Lichtenberger et al. 1976), which have helped provide evidence for the catalytic mechanisms discussed here. However, in most cases these compounds rapidly destroy the P450 heme (Lichtenberger et al. 1976; Ortiz De Montellano 1986). Iodosylbenzene is particularly notorious in this respect, so reaction time must usually be limited to seconds. Some P450s are resistant to the detrimental action of cumene hydroperoxide and other alkyl hydroperoxides (Brian et al. 1990; Shimizu et al. 1994; Zanger et al. 1988). A drawback of the use of alkyl hydroperoxides (ROOH) is that homolytic scission can occur in some instances to give alkoxide radicals (RO), which can dominate the oxidation chemistry (Mansuy et al. 1982). Although hydroperoxides are found in cells, there is no evidence that the mechanism shown here contributes substantially to the oxidation of other P450 substrates. The actual oxygenation steps, 8 and 9, are considered to be common to the normal cycle of Figure 5 and the ‘shunt’ involving oxygen surrogates. Although P450 enzymes seem to catalyze a diverse group of reactions, most of the chemistry can be rationalized in a common mechanism (Figure 6). The initial step is the formal abstraction of a hydrogen atom or a non-bonded or  electron from the substrate. The second reaction is ‘oxygen rebound,’ or radical recombination (Figure 6) (Guengerich and Macdonald 1984, 1993). Thus it is possible to explain reactions such as C-hydroxylation, heteroatom oxygenation and dealkylation, and the epoxidation, group migration, and mechanism-based inactivation reactions seen with olefins, acetylenes, and aromatic molecules in such a context. Evidence for the mechanism of C-hydroxylation comes from the very high kinetic hydrogen isotope effects (often 6–10 for intramolecular effects) and the scrambling of stereochemistry in the products (Groves et al. 1978; Ortiz de Montellano 1986). However, results on the estimated rates of rearrangement of strained cyclopropyl substrates led to questions as to whether the process is really stepwise or concerted (Newcomb et al. 1995a, 2003). One issue is consideration of the effects of the protein in retarding rates in such systems (Frey 1997). A further complication is the possibility that an Fe–O2 or FeOOH complex (Figure 5) might be acting as the oxidizing species

60 Cytochrome P450 Enzymes

Carbon hydroxylation FeO3+

[FeOH3+] •C

HC

Fe3+

HOC

Heteroatom release FeO3+

•

FeO2+ +NHCH2R

:N–CH2R

FeOH3+

•

•

N– – CH2R

:N–CH2R

OH Fe3+

:N–CHR

:NH

O

+

CHR

Heteroatom release FeO3+

–O–X +

•

Fe3+

R

R

FeO2+ +X

:X

Epoxidation and group migration R FeO3+

•

O N N Fe4+ N N

O

+

Fe3+

R +• FeO2+

R

O

O

R

HO

R

R

N N

HO

N N

Figure 6 Rationalization of various P450 oxidation reactions with a unified chemical mechanism (Guengerich and Macdonald 1984, 1993).

(Chandrasena et al. 2004). The conclusions about peroxy intermediates and stepwise reaction also have evidence against them (Auclair et al. 2002; Austin et al. 2006). However, with nitrogen and sulfur compounds, where the oxidation potentials of the heteroatoms are low, there is evidence to support the view that the initial event may be the abstraction of an electron from the heteroatom. This mechanism (abstraction) could be applied to the dealkylation reactions as well, because the

products are unstable and would degrade to the observed carbonyls. Such reactions have considerable precedent in the case of peroxidases (Chapter 4.09), and it can be argued that the Em,7 for the (FeO)3þ/(FeO)2þ pair is at least as high in P450 as in horseradish peroxidase (Hayashi and Yamazaki 1979; Lee et al. 1985; Macdonald et al. 1989). Evidence for 1-electron oxidation includes the rearrangements of certain substrates (Bondon et al. 1989), the trapping of alkyl radicals from

Cytochrome P450 Enzymes

1,4-dihydropyridines (Augusto et al. 1982) and other studies with these substrates (Guengerich and Bo¨cker 1988), the mechanism-based inactivation of P450s by cycloalkylamines (Bondon et al. 1989; Hanzlik and Tullman 1982; Macdonald et al. 1982), and linear free energy relationships with substrates of differing potential (Burka et al. 1985; Macdonald et al. 1989). A stable cation radical accumulates during the oxidation of 1,2,4,5-tetramethoxybenzene, with an E1/2 of 1 V (Sato and Guengerich 2000). A central role of a cation radical also provides a satisfying explanation for the association of dealkylation and oxygenation reactions often seen with nitrogen and sulfur compounds. With -bonds, a number of reactions are observed. Studies with biomimetic models have not provided a single mechanism that can explain all reactions of olefins. One possibility, which has a basis in some of the biomimetic model chemistry (Ostovic and Bruice 1989), is that a common initial event is the formation of a -complex which can rearrange to any of a series of -complexes to give the various reaction products, depending upon the particular substrate and its orientation in the individual P450 active site (Guengerich and Macdonald 1990). This generalized scheme may also find application in oxidation reactions of aromatic molecules (Guengerich and Macdonald 1993).

61

The above framework (Figure 6) can explain many of the basic P450 reactions. However, within each there are also some further options available. For instance, desaturation reactions are seen and have been shown not to involve dehydration of an alcohol. They are always accompanied by some Chydroxylation and can be explained by partitioning (Figure 7) (Guengerich and Kim 1991; Guengerich and Macdonald 1993; Rettie et al. 1987). Ether Odealkylation (Harada et al. 1984) and the oxidative cleavage of carboxylic acid esters (Guengerich et al. 1982, 1988) can also be understood in terms of the basic C-hydroxylation mechanisms (Figure 7). Oxides of nitrogen and sulfur compounds are usually stable (Guengerich 1984) except in certain cases (e.g., Cope elimination) (Cashman 1989). Their mechanism of formation may not be so simple as direct oxygenation of a radical cation (Bondon et al. 1989; Guengerich and Macdonald 1984; Hammons et al. 1985) and further electron transfer events may proceed the rebound step (Seto and Guengerich 1993). Evidence for oxygenation of organic iodine (Guengerich 1989) and even chlorine (He et al. 2005) has been presented. The basic proposal for amine dealkylation is considered to be applicable to the dehydrogenation of 1,4dihydropyridines, a vinylogous system (Guengerich

(a)

OH Fe3+

CH CH2 •

CH

CH2 CH2

CH2

FeO3+

FeOH3+

–H2O

H C C H

(b)

(FeO)3+ ROCH2R′ Fe3+ ROCH(OH)R′

(c)

•

(FeOH)3+ ROCHR′ Fe3+ ROH OHCR′

O (FeO)3+

Fe3+

O

RC–OCH2R′ O

Fe3+

(FeOH)3+

OH

RC– OCHR′

Fe3+

•

RC–OCHR′ O

O

RCO–

HCR′

Figure 7 Variations on the general theme of carbon hydroxylation. a, Desaturation; b, ether cleavage; c, ester cleavage (Guengerich, F. P. Chem. Res. Toxicol. 2001, 14, 611–650).

62 Cytochrome P450 Enzymes

1990; Guengerich and Bo¨cker 1988). A hydroxylamine can also be oxidized to a nitrone (Bondon et al. 1989). 4.04.7.2

Rates of Individual Catalytic Steps

In the early research, there was also considerable discussion about what the limiting step in P450 reactions is. With the growing number of P450s under study and more mechanistic information, there is evidence that a single kinetic scheme will not be applicable to all P450s. At this point it is useful to consider some more salient kinetic aspects of P450 reactions. Substrate binding (step 1 in Figure 5) is generally considered to be fast. Experimental evidence comes from measurements of rates of changes in spin state associated with substrate binding to (bacterial) P450 101A1 (Tyson et al. 1972) and (rabbit) P450 2B1 (Ristau et al. 1978). Since substrate binding often enhances rates of P450 reduction (vide infra), it is assumed to generally be the initial reaction. However, this is not necessarily always the case and the binding and dissociation of the substrate can be observed with ferrous P450 (Yun et al. 2005). Another point to be made is that with some of the P450s known to have large active sites, multi-step kinetics can be observed in the binding and progression of substrates and other ligands toward the heme (Isin and Guengerich 2006, 2007; Sohl et al. 2008) P450 reduction (step 2) is probably rate-limiting in some cases (Peterson and Prough 1986). With purified P450s, rates of reduction increase with the addition of more NADPH-P450 reductase (Oprian et al. 1979). In liver microsomes the ratio of total P450 to NADPH-P450 reductase is about 20:1 (Peterson and Prough 1986), and rates of reduction sometimes approximate the faster rates of oxidation of substrates (Peterson and Prough 1986). Step 3 (O2 binding) is probably very fast, as judged by some direct measurements (Ortiz de Montellano 1986) and the lack of a lag in the reoxidation kinetics (Guengerich et al. 1976, 2004). The addition of the second electron to the system (step 4) may be rate-limiting. Cytochrome b5 (b5) can contribute this electron in some instances, and the stimulation of catalytic activity by b5 can be quite dramatic (Imaoka et al. 1992; Miki et al. 1980). The b5 effect is often rather dependent upon the nature of the lipid/detergent environment and can also be stimulated by high ionic strength or the presence of divalent metal cations (Gillam et al. 1995; Imaoka et al. 1992; Yamazaki et al. 1995). The b5 effect seems to be

more common with P450s in the 2C, 2E, and 3A subfamilies (Guengerich et al. 1986; Levin et al. 1986; Yamazaki et al. 1995). Another line of evidence for the involvement of b5 in microsomes (as opposed to the artificial reconstituted enzyme systems) is the immunoinhibition of certain catalytic activities (Noshiro et al. 1980). The role of b5 is rather unwieldy, however, in that even with a single P450 differing reactions may show differential effects of b5. A kinetic explanation has been proposed by Pompon (1987). If step 4 is too slow then superoxide anion appears to dissociate from the Fe2þ?O2 complex. However, many P450 reactions can be stimulated by apo-b5 (devoid of heme) instead of b5, indicating that electron transfer is not obligatory (Yamazaki et al. 2001). This, however, is not the case with P450 2E1 (Yamazaki et al. 2002). Rates of protonation of the formal Fe–O22þ complex and heterolytic scission are unknown but assumed to be relatively rapid. It should be pointed out that in many cases the rates of oxidation measured in reactions supported by oxygen surrogates (e.g., cumene hydroperoxide and iodosylbenzene) are considerably higher than seen in the reactions supported by NADPH, NADPH-P450 reductase, and O2, even in the presence of b5 (Macdonald et al. 1989; Yamazaki et al. 1995). Iodosylbenzene tends to give very high rates when caution is taken to be sure that initial rates are being measured. The specific reasons for the differences are not known, but usually the higher activity is attributed to the rate-limiting nature of reduction steps. However, it is conceivable that events in oxygen activation following the reduction steps are limiting reaction rates; these would be circumvented by mono-oxygen donors such as iodosylbenzene. Step 7 involves the breaking of a C–H bond in the case of some substrates and is amenable to experiments involving kinetic hydrogen isotope effects. These are not usually seen in C-hydroxylation reactions when intermolecular isotope effects are measured and intramolecular experimental designs are usually necessary to see the high isotope effects used as evidence for radicaloid pathways (Groves et al. 1978; Ortiz de Montellano 1986; Ullrich 1969). Thus, the C–H bond breaking step is usually not limiting in the overall reaction cycle. There are some notable exceptions, however. The oxidative cleavage of esters shows a dramatic intermolecular isotope effect on maximum catalytic rate (kcat  8), clearly indicating that C–H bond breakage is ratelimiting (Guengerich et al. 1982, 1988), as do some

Cytochrome P450 Enzymes

reactions catalyzed by P450 1A2 (Guengerich et al. 2004) and 2A6 (Yun et al. 2005). Several substrates of P450 2E1 enzymes show substantial (intermolecular) isotope effects on (kcat/Km) (5) but not on kcat (Bell and Guengerich 1997; Bell-Parikh and Guengerich 1999). Thus, the isotope effect is on Km. This phenomenon should not be interpreted in terms of altered substrate binding but is the reflection of a contribution of the rate of step 7 to the denominator of the Km expression. Although the rate of C–H bond breaking would not affect kcat, the ratio kcat/Km is influenced by the rate of step 7 and such an effect has been manifested in in vivo experiments (Keefer et al. 1973; Swann et al. 1983). A variation of step 7 is the abstraction of an electron from a nitrogen or sulfur compound (vide supra). The ease of withdrawal of the electron is a function of the E1/2 of the amine or sulfur. paraSubstitution of N,N-dimethylanilines has been used to alter E1/2 within the series. Increasing the electronwithdrawal tendency has the effect of raising E1/2 and also decreasing rates of N-demethylation (Burka et al. 1985). The results may be fitted to a Marcus plot and used to estimate an E1/2 of 1.8 V for the (FeO)3þ/(FeO)2þ couple (Macdonald et al. 1989). If a P450 abstracts an electron from an amine, an aminium radical results. The -protons of aminium radicals are not very acidic; for example, the pKa of the N,N-dimethylaniline cation radical has been estimated to be 9 (Dinnocenzo and Banach 1989). Evidence has been presented that P450s are able to catalyze the removal of the -protons from aminium radicals and generate carbon radicals for incipient oxygen rebound (Okazaki and Guengerich 1993). P450s and even biomimetic models appear to be able to do this because of the inherent basicity of the (FeO)2þ complex (Okazaki and Guengerich 1993). In classical peroxidases (Chapter 4.09) the distal ligand position of the heme is shielded (by the apoprotein) and this mode of catalysis is excluded; the aminium radicals yield not only N-dealkylation products but also coupled products (Marnett et al. 1986). Step 8 (oxygen rebound) appears to be very fast. With C-hydroxylation reactions, it is possible to see rearrangements of the radical intermediates prior to rebound, in cases where ring strain facilitates rapid rearrangement. Such substrates have been used as ‘clocks’ to time this event and an estimated rate constant for rebound is nearly 1012 s1 in some cases (Atkinson et al. 1994; Ortiz de Montellano and Stearns 1987). Obviously this is not a rate-

63

limiting step in P450 reactions, and it follows that neither carbon radicals (nor N- or S-radicals) have been detected in P450 reactions. As mentioned above, one interpretation is that the process is concerted; that is, steps 7 and 8 are combined (Newcomb et al. 1995b). The oxidation of polycyclic hydrocarbons has been reported to yield radicals that react with DNA bases (Devanesan et al. 1992). Direct evidence for (quasi-stable) radical products has been obtained with 1,2,4,5-tetramethoxybenzene, a low potential model with an E1/2 value near those of polycyclic aromatic hydrocarbons (Sato and Guengerich 2000). In some instances product release (step 9) may be rate-limiting. With some substrates in which multiple oxidations normally occur, the rates of the individual reactions increase and the available evidence is probably consistent with the view that the product does not dissociate (e.g., aromatase (P450 19A1), side chain cleavage (P450 11A1)). In the case of toluene, evidence involving kinetic isotope effects has been used to argue that product formation is rate-limiting (Ling and Hanzlik 1989). With P450 2E1, one explanation for the effect of deuterium on Km but not kcat is that a step following product formation is rate-limiting (Guengerich et al. 1995). However, direct measurements of the rate of product dissociation have been made and this appears not to be the case, so presumably the slow step is a protein conformational change (Bell-Parikh and Guengerich 1999). In summary, the ratios of the various generalized steps can vary among the different P450s and, even with a given P450, among its different reactions. One point that should be emphasized is that P450s should be expected to have complex expressions for kcat and Km. Depending on the ratios of the various rate constants, these will simplify to different expressions in different situations. In particular, Km should not be simply regarded as a substrate affinity constant. 4.04.7.3

‘Alternate’ Reaction Mechanisms

Although the general steps cited above can explain many of the P450 reactions, there are also some alternate modes (Guengerich 2001). The first is reduction (Wislocki et al. 1980). This involves the simple transfer of one electron from ferrous P450 to a substrate. The substrates must compete with oxygen and electron transfer must occur in sequential fashion in those cases where multiple electron reduction products are found. Reductions are often seen with halogenated

64 Cytochrome P450 Enzymes

hydrocarbons (e.g., CCl4, halothane). They also occur with metals (e.g., Cr) (Mikalsen et al. 1991) and have been reported for N-oxides (Seto and Guengerich 1993), nitro compounds (Wislocki et al. 1980), and epoxides (Sugiura et al. 1980). P450s can react with N-oxides, although these reactions seem to be slow. Although mechanisms involving oxygen transfer to metalloporphyrins are known (Nee and Bruice 1982), with P450s there is really no evidence for this. With ferric P450, the reaction is considered to involve binding of the oxygen to the iron, homolytic N–O bond scission, and rearrangement of the aminium ion in the same manner proposed for N-dealkylation reactions (Sugiura et al. 1980). The product is a dealkylated amine. When the reaction is catalyzed by reduced iron, the reaction is thought to resemble Polonowski chemistry, and the net result is reduction of the N-oxide to the amine (Seto and Guengerich 1993). In general the P450s generate a reactive intermediate bearing a single oxygen atom, according to the current prevailing thought. However, in some cases the Fe-OOH entity is now believed to react directly with the substrate. The best example seems to be the third step in the aromatization of androgenic steroids to estrogens (Cole and Robinson 1991). This reaction is selectively catalyzed by P450 19A1 and termed ‘aromatase’; it follows two preceding oxidations in the process that are thought to proceed by the more general catalytic mechanisms depicted in Figure 7. Evidence for this type of chemistry is also seen in a model C-demethylation reaction catalyzed by rabbit P450 2B4 (Vaz et al. 1991). However, even with this reaction an alternate C-1 H-atom abstraction mechanism has been proposed to be preferred on the basis of density functional theory calculations (Hackett et al. 2005). Oxygen surrogates (vide supra) are hypervalent (highly oxidized) molecules that can transfer an oxygen directly to P450 (Lichtenberger et al. 1976). In general, these are considered artificial and do not contribute to the bulk of P450 oxidations, even though H2O2 and alkyl hydroperoxides are found in cells and could conceivably be utilized. However, some rather specialized P450s simply rearrange specific hypervalent substrates to products. Among these are the (plant) allene oxide synthase and the (mammalian) prostacyclin and thromboxane synthases (Hecker and Ullrich 1989; Song and Brash 1991). P450s can carry out other reactions with alkyl hydroperoxides, including lipid hydroperoxides (Chang et al. 1996; Mansuy et al. 1982; Song et al. 1993; Vaz

et al. 1990). For instance, the reduction of an alkyl hydroperoxide by (ferrous) P450 yields an alkane and a carbonyl. Homolytic scission yields a formal (FeO)2þ complex and an alkoxy radical (RO), which may dominate the observed chemistry (Mansuy et al. 1982). Cleavage of hydroperoxides of unsaturated fatty acids can generate allene oxides or epoxy alcohols (Chang et al. 1996; Song et al. 1993; Weiss et al. 1987).

4.04.8 Catalytic Selectivity of P450s 4.04.8.1

General Features

In the early years of P450 research, the general concept held was that the mammalian enzyme(s) had very broad catalytic specificity. This view was predicated on the belief that only one or two P450s existed and there was a need to explain catalytic reactions involving substrates of very great diversity. As more P450s were characterized, this view changed. Indeed, the use of the term ‘isozymes’ (to describe the P450s) has generally been dropped, in that a better term is enzymes (in that isozymes are defined as catalyzing the same reaction). Today we recognize that some of the P450s have rather limited catalytic specificity, such as those catalyzing specific aspects of steroid anabolism (e.g., P450 17A1, 19A1, 21A1). Even among these there is often the capability for more than one substrate and reaction, though. For instance, P450 17A1 enzymes in some animal species catalyze both 17-hydroxylation and a lyase reaction. Other P450s tend to have more diversity in the substrates they will use and the reactions they catalyze. These enzymes, which are usually found in the 1, 2, and 3 families, are often associated with xenobiotic metabolism and are most prominent in the degradation of drugs, carcinogens, pollutants, pesticides, etc. One view is that these enzymes are present for the express purpose of degrading ingested dietary and other materials. This is not an unreasonable view of function, although it is certainly not possible to state that some of these P450s do not have important roles in normal homeostasis. Demonstration of catalytic activity toward a compound normally found in the body may or may not be of use in ascertaining function, especially if a clear case for appropriate tissue localization cannot be made. Individual humans seem to vary quite considerably in their levels of some of the hepatic and extrahepatic P450s, apparently without dramatic consequences save for possible drug interactions.

Cytochrome P450 Enzymes

4.04.8.2 Discrimination of Catalytic Specificities of Individual P450s There are several ways to discern which P450s are most prominent in a particular catalytic activity (Table 2). In this regard we need to discern between (1) intrinsic catalytic activity and (2) contribution to the total activity in a tissue. The distinction between the two is important but not always distinguished. Two basic ways to compare the intrinsic catalytic activities are to (1) purify the P450 from a tissue source and measure its catalytic activity in the presence of NADPH-P450 reductase and phospholipid and (2) express a recombinant P450 in a heterologous vector system and measuring the catalytic activity of the recombinant P450. An inherent problem with both of these approaches is that the conditions in the system may not be optimal for catalytic activity. For instance, the nature of the membrane/phospholipid milieu and the concentrations of NADPH-P450 reductase and b5 may not be ideal for optimal activity. This can be considered a deficiency of all enzymology, but the problem is more serious in a system where accessory proteins and elements of membranes are required. Some P450s are notorious for difficulties in reconstitution. For instance, many P450s in the 3A family show needs for b5 and mixtures of phospholipids (Imaoka et al. 1992). With many of the P450s optimal activity can be obtained in the presence of only added L--dilauroyl-sn-glycero-3phosphocholine, a simple lecithin with short saturated acyl chains, and NADPH-P450 reductase. Some other P450s routinely require b5 for all of their catalytic activities (e.g., P450 2E1) (Levin et al. 1986). What is most problematic is that a single P450 may sometimes show different requirements for different reactions. For instance, some of the reactions catalyzed by P450 3A4 do not require b5 or phospholipid (Gillam et al. 1995; Shet et al. 1993). Also, human P450 17A1 shows steroid 17-hydroxylation in the absence of b5 but additionally has 17,20-lyase activity when b5 is present (Katagiri et al. 1995). Another problem is that different P450s may compete for the limited amount of NADPH-P450 reductase in the endoplasmic reticulum (Kaminsky and Guengerich 1985) and this competition is neglected when only a single enzyme is considered. One means of estimating the effectiveness of the reconstitution system is to compare the rate with that measured in microsomes, particularly after consideration of the concentration of the enzyme in the microsomes.

65

The other type of information is what extent of a particular reaction is catalyzed by a particular form of P450. Qualitative estimates may be made by comparing rates measured with different purified or recombinant enzymes (vide infra). One major approach is to use a crude tissue preparation, usually microsomes, and add inhibitors. These can be either chemicals or antibodies and are usually used in varying levels to ‘titrate’ the activity. If antibodies are used they are usually selective for subfamilies (1A, 2B, 2D, etc.). That is, the individual enzymes within a subfamily are all usually inhibited by an antibody. Sometimes monoclonal antibodies can distinguish between individual subfamily P450s, or the same end may be achieved by cross-adsorption of polyclonal sera (Thomas et al. 1979). It should be emphasized that the same assay as that used in a test must be used as criterion for P450 antibody specificity,that is, immunoinhibition of accepted marker reactions as a marker for immunoinhibition of a new reaction of interest. Chemical inhibitors are also useful (Table 5). Many are quite selective and have been reviewed elsewhere (Halpert et al. 1994; Newton et al. 1994). Chemical inhibitors have the advantage of being generally easier to obtain (and renewable). They also have a distinct advantage in that they can be used with whole cell preparations, tissue slices, and even in vivo. Some can even be administered to humans. Another means of assessing the contributions of different P450s is to do correlation experiments (Beaune et al. 1986; Guengerich and Shimada 1991). If the levels of a particular P450 vary considerably among a set of microsomal preparations, then the catalytic activity may be measured and compared to markers of individual P450s (either a catalytic activity (Table 6) or immunochemically determined P450 levels). If a certain P450 is catalyzing the test reaction, then there should be a high correlation with a particular marker. In principle, the correlation coefficient r2 is an estimate of the fraction of the variance accounted for (in the entire set) by the particular relationship. Evidence about the involvement of particular P450s in experimental animals can be obtained using induction data. For instance, several rat enzymes are known to be induced by certain chemicals (e.g., P450s 1A1, 1A2, and some others by certain polycyclic hydrocarbons; P450 2E1 by ethanol and isoniazid, etc.). Advantage can also be made of known gender differences in rodents (e.g., P450s 2C11 and 3A2 are

66 Cytochrome P450 Enzymes Table 5 Useful selective inhibitors of human P450 enzymes P450 1A1

P450 1A2

P450 1B1 P450 2A6 P450 2C9 P450 2D6 P450 2E1

P450 3A4

7,8-Benzoflavone (but see (Shimada et al. 1998) regarding P450 1A2) Ellipticine 1-(1-Propynyl)pyrene 2-(1-Propynyl)phenanthrene 7,8-Benzoflavone Furafylline Fluvoxamine 7,8-Benzoflavone 2-Ethynylpyrene Diethyldithiocarbamate (see Yamazaki et al. 1992) Sulfaphenazole Tienilic acid Quinidine Aminoacetonitrile 4-Methylpyrazole Diethyldithiocarbamate (see Yamazaki et al. 1992) Troleandomycin Ketoconazole Gestodene

Source: Correia, M. A.; Ortiz de Montellano, P. R. In Cytochrome P450: Structure, Mechanism, and Biochemistry, 3rd ed.; Ortiz de Montellano, P. R., Ed.; Kluwer Academic/Plenum Press: New York, NY, 2005; pp 247–322.

Table 6 In vitro marker activities for some human P450s involved in toxicology P450

Tissue sites

Typical reaction

1A1

Benzo[a]pyrene 3-hydroxylation

2A6 2A13

Lung, several extrahepatic sites, peripheral blood cells Liver Many extrahepatic sites, including lung and kidney Liver, lung, and several extrahepatic sites Nasal tissue

2B6 2C8 2C9 2C19 2D6 2E1 3A4 3A5 3A7 4A22

Liver, lung Liver Liver Liver Liver Liver, lung, other tissues Liver, small intestine Liver, lung Fetal liver Liver

1A2 1B1



Caffeine N3-demethylation, phenacetin O-deethylation 17-Estradiol 4-hydroxylation Coumarin 7-hydroxylation Activation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) (S)-Mephenytoin N-demethylation Taxol 6-hydroxylation Tobutamide methyl hydroxylation (S)-Mephenytoin 49-hydroxylation Debrisoquine 4-hydroxylation Chlorzoxazone 6-hydroxylation Testosterone 6-hydroxylation Testosterone 6-hydroxylation Testosterone 6-hydroxylation Fatty acid !-hydroxylation

For a comparison with rats see Chovan et al. (2007).

male-specific while P450 2C12 is female-specific). In humans there seems to be little gender specificity, if any, but induction experiments can be done with barbiturates (2C9, 3A4), rifampicin (2C9, 3A4), isoniazid (2E1), and even charbroiled meat (1A1, 1A2). Obviously the in vivo situation is more complex in that some pharmacokinetic parameter must be measured and that a specific reaction may be obscured by further metabolism, blood flow, protein binding, etc.

Finally, it should be emphasized that all of these procedures can be applied to other enzyme families, with some modification. However, the total battery of approaches has been applied more extensively in the case of the P450s than any of the other enzymes treated in this volume. As a final note, the point should be made that the most reliable conclusions are reached when several lines of evidence support the same finding.

Cytochrome P450 Enzymes

4.04.8.3

Drugs and Non-Invasive Assays

Historically a number of in vivo assays have been used as measures of human P450 function. These have included antipyrine clearance, hexobarbital metabolism, and phenacetin O-deethylation (Distlerath and Guengerich 1987). With the development of knowledge about which human P450 enzymes are involved in the oxidation of particular drugs, it has become possible to develop non-invasive assays that predict levels of individual P450 enzymes (Table 2), particularly the major ones involved in the metabolism of drugs and protoxicants/procarcinogens. This information is usually derived from in vitro studies on particular P450s coupled with pharmacokinetic work in humans. General conclusions are presented in Table 6. Several comments are in order. First, extrapolation from in vitro assays to the in vivo situation is not trivial and validation is important (Emoto et al. 2006; Proctor et al. 2004; Shiran et al. 2006; Wienkers and Heath 2005). Moreover, the type of in vivo studies that can be done with humans is limited for ethical reasons. While evidence has been obtained for roles of individual human P450 enzymes in certain oxidations of drugs and other compounds (vide supra), in vivo evidence may be lacking. Sometimes the expected inter-individual variation in an activity is not seen in vivo. There are several possible reasons, all somehow related to pharmacokinetics. If metabolism is limited by blood flow or protein binding rather than enzymatic transformation, then these factors will alternate any differences in P450 levels. Another possibility involves reactions in which more than one P450 can contribute to the reaction. Individuals who lack the major P450 involved in the reaction may compensate with other P450s that play minor roles. Most drugs are (ideally) administered orally, and in recent years there has been a developing appreciation of the role of intestinal metabolism that occurs before drugs reach the liver (Kolars et al. 1991). P450 3A4 is very abundant in the small intestine and can make a major contribution to altering bioavailability in some cases (Kaminsky and Fasco 1992). This enzyme is inducible in the small intestine, but the comparative regulation of human P450s in different tissues still requires considerably more investigation. Another problem is that in vivo studies with young healthy volunteers may not reflect the total variation in the general population. A few comments are in order about some of the non-invasive assays most widely used today (Table 6).

67

P450 1A1 is not generally considered a hepatic enzyme (Table 2); levels of extrahepatic protein may be estimated by analysis of activity in peripheral blood cells. However, direct correlation of the regulation of the enzyme in different tissues has not been established. P450 1A2 appears to be essentially only a hepatic enzyme (Table 2). Levels of the enzyme may be estimated measuring the metabolism of caffeine or theophylline, although there is some disagreement over which parameters are most valid (Butler et al. 1992; Kalow and Tang 1991). Antipyrine clearance had been used as an in vivo measurement of P450 activity in the past and it now appears that P450 1A2 contributes to the formation of some of the products (Engel et al. 1996). Phenacetin O-deethylation had also been used (in vivo) in the past (Conney et al. 1976) and it now appears to be a marker of P450 1A2 (Butler et al. 1989); however, phenacetin is no longer cleared for use in the United States because it is a cancer suspect. Tolbutamide (methyl) hydroxylation appears to be a reasonable predictor of P450 2C9 levels (Brian et al. 1989; Knodell et al. 1987), and the 7-hydroxylation of (S)-warfarin also seems to be a useful assay in this regard (Rettie et al. 1992). Probably the most popular assay today is diclofenac 4-hydroxylation (Yasar et al. 2001). No non-invasive assays for P450 2C8 have been developed yet. (S)-Mephenytoin 49hydroxylation was used as an in vivo assay before 2C19 was identified as the enzyme responsible for the polymorphism and is the only assay used (Goldstein et al. 1994). P450 2D6 is another polymorphic enzyme for which assays had been developed prior to characterization of the enzyme. Among the more widely used in vivo assays are debrisoquine 4-hydroxylation, and particularly dextromethorphan O-demethylation (Evans et al. 1983, 1989), with the latter being the most widely used. Chlorzoxazone 6-hydroxylation has been developed as a useful assay for P450 2E1 (Kim et al. 1994). P450 3A4 is, on the average, the most abundant P450 in human liver and small intestine and is of interest because of its prominence in the oxidation of a great variety of drugs, procarcinogens, and steroids (Guengerich et al. 1994). Many years went into the development of assays, and some proved better than others. The most widespread assays in use today are (i. v.) erythromycin N-demethylation for hepatic P450 3A4 (Paine et al. 2002; Watkins et al. 1992) and oral midazolam 4-hydroxylation for P450 3A4 in the small intestine (Lin et al. 2001). No selective

68 Cytochrome P450 Enzymes

non-invasive assays have been developed to completely distinguish the activities of P450 3A5 and P450 3A4, although there is considerable evidence that the latter generally has a more dominant role. With the other P450s non-invasive measurements have not yet been developed. In some cases alterations of steroidogenic P450s can be inferred by analysis of levels of the relevant steroids.

4.04.9 Roles of P450S in Biotransformation of Toxic Chemicals Some of the key studies leading to the discovery of the P450 enzymes were related to the enzymatic activation of toxic and carcinogenic chemicals (Mueller and Miller 1948). As the characterization of P450 enzymes has progressed, so has the information about their roles in the overall processes of bioactivation and detoxication. With a single chemical, P450s (or even a single P450) can often transform it into both activated and detoxicated products. Distinguishing between activation and detoxication is not always trivial. Formation of electrophilic products and covalent adducts with macromolecules is the usual criterion, in the absence of knowledge of which specific biological events are related to the transduction of alkylation damage into a toxic response. A representative (although not comprehensive) list of bioactivation reactions catalyzed by P450s is presented in Table 3. Only a few examples are presented in each category. More extensive reviews on the roles of P450s in toxicity have been presented elsewhere (Brodie et al. 1958; Guengerich and Liebler 1985; Nebert 1989; Nelson and Harvison 1987). Carcinogens known to be activated by particular human P450s are presented in Table 4. This section of the text will include only a few examples of situations where toxicity has been shown to be influenced by alteration of certain P450 activities, usually in studies with experimental animal models. The literature contains a number of examples of interactions of chemicals with ethanol. For instance, the toxicities of vinyl chloride, vinylidene chloride, N,N-dimethylnitrosamine (DMN), and a number of other low molecular weight compounds can be exacerbated by pretreatment of animals with ethanol. These changes are now attributed to induction of P450 2E1, the enzyme most prominently involved in their oxidation (Guengerich et al. 1991). However, if ethanol administration is continued the observed

effects may be altered because of the other role of ethanol as a competitive inhibitor. Disulfiram (Antabuse) and its reduced form diethyldithiocarbamate are selective inhibitors of this P450 (but also inhibit P450 2A6) (Yamazaki et al. 1992). Their actions had previously been interpreted in terms of effects on dehydrogenases but it is now clear that the explanation is usually in terms of P450 2E1 (Guengerich et al. 1991). These effects of ethanol and disulfiram are seen not only in alteration of gross toxicities but also carcinogenesis. For instance, the dramatic exacerbation of ethylene dibromideinduced liver tumors by disulfiram (Wong et al. 1982) can be explained by inhibition of oxidation by P450 2E1 and shunting of the compounds into the glutathione conjugation pathway for activation (Kim and Guengerich 1990). The importance of P450 2E1 in the bioactivation of acetaminophen was convincingly demonstrated by Gonzalez’s laboratory using transgenic (knockout) mice (Lee et al. 1996). The literature is abundant with examples of how toxicities of certain chemicals can be altered by changes in the expression of P450 1A1 (Nebert 1989). The conclusion that P450 1A1 is good or bad can depend upon which tissue expression occurs in and its relationship to the toxicity (Nebert et al. 2004). This system has been very amenable to manipulation because of the availability of strains of mice with a mutant Ah receptor that does not have high affinity ligand binding (Chang et al. 1993). Mice that do not express P450 1a1 are more likely not to develop polycyclic hydrocarbon-induced tumors at a number of sites (Nebert 1989). However, they are more prone to other problems such as aflatoxin (AF)-induced liver cancer because the induced enzymes are involved in detoxication reactions. These studies raise the question of the importance of P450 1A2, which seems to be an almost exclusively hepatic enzyme and which is expressed at significant levels in the absence of Ah/ARNT inducers. ‘Knock-out’ transgenic mice have been bred, in which the gene has been disrupted (Fernandez-Salguero et al. 1995; Pineau et al. 1995). This deletion is debilitating but not always lethal; immune function is severely compromised. Acetaminophen (Tylenol, paracetamol) has been studied considerably over the years. It is widely used as an analgesic and is generally quite safe unless a great overdose occurs, being extensively metabolized by sulfation and glucuronide formation. Activation involves oxidation to the iminoquinone,

Cytochrome P450 Enzymes

69

Figure 8 P450-catalyzed oxidation of acetaminophen and related reactions.

a Michael acceptor that can react with nucleophilic sulfhydryls (i.e., glutathione and proteins) (Figure 8) (Dahlin et al. 1984). This oxidation has been shown to be catalyzed by human P450 2E1, 1A2, and 3A4 enzymes (Patten et al. 1993). Mice devoid of P450 2e1 show considerably less acetaminophen toxicity (Lee et al. 1996), and deletion of both P450s 2e1 and 1a2 lowers the toxicity even further (Zaher et al. 1998). One mechanism for toxicity involves the reaction of the iminoquinone with critical protein sulfhydryl groups, although they have not been identified if they exist. Alternatively, the iminoquinone can be reduced (two electrons) back to acetaminophen, thus consuming glutathione or, indirectly, reduced pyridine nucleotides and creating an oxidized environment (Figure 8). Another case involves AFB1, a potent hepatotoxin and hepatocarcinogen produced by molds that grow on grains. AFB1 can be oxidized to a variety of products by P450s (Figure 9). The roles of the human P450 enzymes have been studied most extensively. P450 3A4 plays a prominent role in oxidation, forming the exo 8,9-epoxide and the 3-hydroxylation product AFQ1. AFQ1 does not readily undergo epoxidation at the 8,9-double bond and is considerably less genotoxic than AFB1. Even if the epoxide of AFQ1 is formed, it does not intercalate well into DNA and thus binds poorly (Raney et al. 1992).

Interestingly, addition of 7,8-benzoflavone (-naphthoflavone) directly to the enzyme shifts the pattern of oxidation from 3-hydroxylation to epoxidation (Raney et al. 1992; Ueng et al. 1995). The highly related P450 3A5 (85% sequence identity) shows a predominance of epoxidation over 3-hydroxylation (Gillam et al. 1995). P450 1A2 is another enzyme that has usually been assigned a protective role in AFB1 metabolism (Ueng et al. 1995). The human enzyme (recombinant) forms trace AFQ1, mostly AFM1 (also a detoxication product), and small amounts of both the exo and the endo 8,9-epoxide isomers. The distinction between the stereoisomers of the epoxide is critical, since the exo has 103 times the genotoxicity of the endo (Iyer et al. 1994). The difference is explained by the need for intercalation between the DNA bases and the necessity for SN2 attack of the guanyl N7 atom on the epoxide (Iyer et al. 1994). Exactly how changes in the levels of these enzymes relate to human liver cancer is unknown, since (1) the level of AFB1 intake is very low (less per day than amount of enzyme), (2) with oral ingestion the first enzymatic encounter should be with P450 3A4 in the small intestine, (3) activation in the small intestine could be considered a detoxication process since these cells are rapidly sloughed, (4) the balance between the activation (exo-epoxidation) and the detoxication

70 Cytochrome P450 Enzymes

Figure 9 Oxidation of aflatoxin B1 by human P450s.

(all others) reactions in vivo is unknown, and (5) numerous other factors such as hepatitis B viral status and DNA repair are also important.

4.04.10 Clinical Significance A considerable amount of literature on the clinical significance of P450s exists, with regard to both efficacy and toxicity. Much of this involves deficiencies in steroid hydroxylases and unexpected drug–drug interactions. In the United States, the Food and Drug Administration (FDA) expects information on the characterization of which human P450s are predominantly involved in the disposition of new drug candidates at an early stage for the approval of in vivo trials (Investigational New Drug (‘IND’) application). Deficiencies in steroid hydroxylases constitute some of the more well-studied inherited defects in metabolism. Among those known are partial or dramatic decreases in activities of P450s 11A1, 11B1, 17A1, 19A1, and 21A1 (Table 1) (Nebert and

Russell 2002). A defect in P450 21A1 occurs once in 104 children born. The molecular mechanisms are known in considerable detail and are dominated by cross-over events with a very similar pseudogene (Higashi et al. 1991). A number of problems with drug–drug interactions can now be understood in terms of the enzyme P450 3A4. For instance, the hypotensive agent nifedipine (Figure 10a) is rapidly oxidized by P450 3A4 (Guengerich et al. 1986) and subject to the influence of inducers and inhibitors. In the early 1970s, there were several reports of women in Germany who used oral contraceptives and experienced unexpected menstrual bleeding and pregnancies while using barbiturates or the antibiotic rifampicin. Bolt (Bolt et al. 1975) attributed the problem to the induction of the rates of 2-hydroxylation of 17-ethynylestradiol, the major estrogenic component of most oral contraceptives. Further studies showed that this reaction could be attributed to P450 3A4 (Guengerich 1988), which has been shown to be inducible by barbiturates and rifampicin (Morel et al. 1990; Watkins et al. 1985),

Cytochrome P450 Enzymes

(a)

(b) Barbiturates + Dexamethasone P450 3A4

Nifedipine (& other dihydropyridines)

–

P450 3A4 ≥3 oxidation producs (inactive)

Cyclosporin Erythromycin Ketoconazole

Pyridine product (inactive)

71

Immunosuppression

Renal toxicity

Hypotensive effect Erythromycin Ketoconazole

(c)

H+ N

X P450 3A4

CH3 CH3

CH3

H+ N

CH3 Terfenadine

+ H+ N

Dehydrogenases or P450 CH2OH

CH3 CH3

H+ N

CH3 CO2– CH3

CH3 CH3

Arrhythmia, QT

Antagonists of H1 receptor (antihistamines)

Zwitterionic; does not cross blood–brain barrier

Figure 10 Examples of the clinical significance of P450s: a, nifedipine; b, cyclosporine; c, terfenadine.

and, more recently, components of the herbal medicine St. John’s wort (Murphy et al. 2005). Another example of a situation in which the level of P450 is critical is with the use of cyclosporin during transplantation. P450 3A4 is the major enzyme involved in the oxidation of this drug (Figure 10b), and there is evidence that a considerable degree of inactivation occurs in the small intestine (Kolars et al. 1991). If P450 3A4 levels are low and the effective cyclosporin level is too high, renal toxicity is a problem. However, if P450 3A4 activity is too high and the effective cyclosporin level is too low, the immunosuppressive effect will not occur and graft rejection may be the result. There is little time to adjust cyclosporin dose levels, so the estimation of P450 3A4 levels in donors and recipients has been put into use (Turgeon et al. 1992). The antihistamine terfenadine (Seldane), which at one time had the 9th largest number of prescriptions in the world, was the first marketed one that was non-sedating. The parent drug is very rapidly oxidized, primarily by P450 3A4 (Yun et al. 1993), to an inactive, dealkylation product and to an alcohol

(formed on the tert-butyl group) (Figure 10c). The alcohol is rapidly oxidized to the carboxylic acid, apparently by dehydrogenases. The resulting zwitterionic product is charged and does not cross the blood–brain barrier to produce sedation, but it does bind the H1 receptor and has antihistaminic activity. The rapid oxidation is usually so extensive that no terfenadine itself is usually found in the plasma, and thus terfenadine is generally considered to be a ‘prodrug’ (Kivisto¨ et al. 1994). However, some individuals with low levels of P450 3A4 are sensitive to coadministration of inhibitors of the enzyme – for example, erythromycin and ketoconazole. If terfenadine itself accumulates in the plasma, it is much more likely to cause arrhythmia problems such as QT interval changes and torsades de pointes. Several deaths and other adverse reactions have been interpreted in terms of this paradigm, and the U. S. FDA withdrew approval in 1995. What is now known as the P450 2D6 polymorphism was originally discovered by Smith in the course of his personal participation in a drug study with the anti-hypotensive debrisoquine (Evans et al. 1983;

72 Cytochrome P450 Enzymes

Smith et al. 1978). He experienced unanticipated problems, and the 1975 episode led to his recognition that he was among a subset of the population deficient in the capability to clear the drug at the normal rate. This is one example of a P450 2D6 substrate that can cause side effects in poor metabolizers. Another example is perhexiline, an antihypertensive that can produce peripheral neuropathy in some individuals who do not have sufficient capability for metabolism (Shah et al. 1982). Most of the clinical attention has been focused on interactions involving P450s 2D6 and 3A4, but examples of the involvement of other P450s are known. For instance, induction of P450 1A2 can cause decreased effectiveness of theophylline as an antiasthmatic (Feldman et al. 1980) and deficiencies in P450 2C enzymes can prolong the sedative effects of barbiturates (Knodell et al. 1988) or affect the maintenance dose of the anticoagulant warfarin (Higashi et al. 2002), which has narrow therapeutic index.

4.04.11 Future Directions The 50-plus years of P450 research has revealed an extremely complex system having far more implications than ever originally imagined. The author takes this opportunity to speculate on what he considers to be those areas where considerable new knowledge will become available. Despite the emphasis that has been placed upon understanding the regulation of P450 genes, it is clear that much remains to be learned. Even with these, the identification of regulatory elements opens new questions about their interactions and their own regulation. Ultimately all of the information about various regulatory elements will need to be integrated. The general problem of roles of P450s in toxicity has many facets. Although it is often possible to identify individual reactions that generate reactive products, it is more problematic to relate these to the overall toxicity, particularly in the chronic situation. More information is needed at other levels to define the events most critical to permanent cell injury. Do P450s really make a difference? This question has certainly not been answered in many cases. Another open question is the role of partially reduced oxygen species generated by P450s. These are readily formed in in vitro settings, at least in microsomes and purified enzymes during NADPH oxidation, but the in vivo significance is not yet clear. With a rat model, only

induction of 2B subfamily enzymes produced oxidative stress, but induction of subfamily 1A, 2E, and 4A P450s did not (Dostalek et al. 2007). A number of opportunities also exist regarding the clinical applications. Better validation of many of the non-invasive probe systems is needed. Along with this there is a need for a better understanding of the significance of variations in P450 levels in the population and the significance for each drug. The whole use of relating differences in P450 levels to the etiology of diseases of unspecified origin (e.g., cancer, Parkinson’s disease) is still rather nebulous (d’Errico et al. 1996) and will require more study.

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Relevant Website http://www.cypalleles.ki.se – Human Cytochrome P450 (CYP) Allele Nomenclature Committee