Cytochrome P450

1. Cytochrome P450 Lecture 8 Modified from textbooks, journals and internet sources

2. Introduction • Cytochrome P450 (P450)  very large and diverse superfamily of hemoproteins • range of proteins • found in all domains of life • P450  use a plethora of both exogenous and endogenous compounds as substrates in enzymatic reactions • The most common reaction catalysed by cytochrome P450 = a monooxygenase reaction • insertion of one atom of oxygen into an organic substrate (RH) while the other oxygen atom is reduced to water

3. continued • RH + O2 + 2H+ + 2e– → ROH + H2O • CYP enzymes have been identified from all lineages of life (mammals, birds, fish, insects, worms, sea squirts, sea urchins, plants, fungi, slime molds, bacteria and archaea) • more than 7700 distinct CYP sequences are known

4. Definition of hemoprotein • Or heme protein = a metalloprotein containing a heme prosthetic group bound to the protein itself • the iron in the heme is capable of undergoing oxidation and reduction • A prosthetic group = a non-protein (non-amino acid) component of a conjugated protein that is important in the protein's biological activity • prosthetic group  be organic (such as a vitamin, sugar, or lipid) or inorganic (such as a metal ion)

5. continued • name cytochrome P450 derived from the fact that these are colored ('chrome') cellular ('cyto') proteins • a "pigment at 450 nm“  formed by absorbance of light at wavelengths near 450 nm when the heme iron is reduced and complexed to carbon monoxide

6. Names • Genes encoding CYP enzymes, and the enzymes themselves, are designated with the abbreviation "CYP“ (followed by an Arabic numeral indicating the gene family, a capital letter indicating the subfamily, and another numeral for the individual gene) • E.g. CYP2E1 is the gene that encodes the enzyme CYP2E1  one of the enzymes involved in paracetamol (acetaminophen) metabolism • current nomenclature guidelines suggest that members of new CYP families share >40% amino acid identity

7. CytP450Oxidase-1OG2

8. Mechanism of the P450 catalytic cycle • The active site of cytochrome P450 contains a heme iron center • The iron is bound to the P450 protein via a thiolate ligand derived from a cysteine residue • There is vast variety of reactions catalyzed by CYPs • In general, the P450 catalytic cycle proceeds as follows:

9. The P450 catalytic cycle • 1: The substrate binds to the active site of the enzyme (close to the heme group) • The bound substrate induces a change in the conformation of the active site, displacing a water molecule • This gives rise to a change in the spectral properties of the enzyme (increase in absorbance at 390~nm and a decrease at 420~nm)

10. continued • 2: The change in the electronic state of the active site favors the transfer of an electron from NAD(P)H • this takes place via the electron transfer chain • 3: Molecular oxygen binds to the heme iron • The "decoupling reaction", releases a reactive superoxide radical • 4: A second electron is transferred via the electron-transport system • reducing the dioxygen adduct to a negatively charged peroxo group

11. continued • 5: The peroxo group formed in step 4 is rapidly protonated twice by local transfer from surrounding amino-acid side chains, releasing one mole of water, and forming a highly reactive iron(V)-oxo species

12. continued • 6: Depending on the substrate and enzyme involved, P450 enzymes can catalyse any of a wide variety of reactions • hypothetical hydroxylation is shown in the following illustration • after the product has been released from the active site, the enzyme returns to its original state • water molecule returns to occupy the distal coordination position of the iron nucleus • C: If carbon monoxide (CO) binds to reduced P450, the catalytic cycle is interrupted • this reaction yields the classic CO difference spectrum

14. continued • most CYPs require a protein partner to deliver one or more electrons to reduce the iron (and eventually molecular oxygen) • CYPs are, properly speaking, part of P450-containing systems of proteins • Five general schemes are known:

15. continued • CPR/cyb5/P450 systems  employed by most eukaryotic microsomal CYPs involve the reduction of cytochrome P450 reductase by NADPH (Nicotinamide adenine dinucleotide, abbreviated NAD+, coenzyme found in all living cells) • FR/Fd/P450 systems which are employed by mitochondrial and some bacterial CYPs

16. continued • CYB5R/cyb5/P450 systems in which both electrons required by the CYP come from cytochrome b5 • FMN/Fd/P450 systems originally found in Rhodococcus sp. in which a FMN-domain-containing reductase is fused to the CYP • P450 only systems, which do not require external reducing power

17. P450s in humans • Human CYPs  primarily membrane-associated proteins • located either in the inner membrane of mitochondria or in the endoplasmic reticulum of cells • CYPs metabolize thousands of endogenous and exogenous compounds • Most CYPs can metabolize multiple substrates • central importance in metabolizing the extremely large number of endogenous and exogenous molecules

18. continued • In the liver  these substrates include drugs and toxic compounds as well as metabolic products such as bilirubin (a breakdown product of hemoglobin) • Cytochrome P450 enzymes  present in most other tissues of the body, and play important roles in hormone synthesis and breakdown (including estrogen and testosterone synthesis and metabolism), cholesterol synthesis, and vitamin D metabolism • The Human Genome Project  has identified 57 human genes coding for the various cytochrome P450 enzymes

19. Drug metabolism • CYPs  the major enzymes involved in drug metabolism (accounting for about 75% of the total metabolism) • P450  the most important element of oxidative metabolism (also known as phase I metabolism) • (Metabolism in this context is the chemical modification or degradation of drugs)

20. Phase I reactions • (also termed nonsynthetic reactions) may occur by oxidation, reduction, hydrolysis • Oxidation  involves the enzymatic addition of oxygen or removal of hydrogen, carried out by mixed function oxidases, often in the liver • These oxidative reactions  typically involve a cytochrome P450 haemoprotein, NADPH and oxygen

21. continued • If the metabolites of phase I reactions are sufficiently polar  they may be readily excreted at this point • many phase I products  not eliminated rapidly and undergo a subsequent reaction in which an endogenous substrate combines with the newly incorporated functional group to form a conjugate

22. Drug interaction • Many drugs may increase or decrease the activity of various CYP isozymes in a phenomenon known as enzyme induction and inhibition  • a major source of adverse drug interactions, since changes in CYP enzyme activity may affect the metabolism and clearance of various drugs • E.g. if one drug inhibits the CYP-mediated metabolism of another drug, the second drug may accumulate within the body to toxic levels, possibly causing an overdose

23. continued • these drug interactions may necessitate dosage adjustments or choosing drugs which do not interact with the CYP system • Such drug interactions  extra important to take into account when using drugs of vital importance to the patient, drugs with important side effects and drugs with small therapeutic windows • any drug may be subject to an altered plasma concentration due to altered drug metabolism

24. continued • A classical example: anti-epileptic drugs • Phenytoin  induces CYP1A2, CYP2C9, CYP2C19 and CYP3A4 • Substrates for the latter may be drugs with critical dosage  amiodarone or carbamazepine, whose blood plasma concentration may decrease because of enzyme induction

25. Interaction of other substances • naturally occurring compounds may cause a similar effect • E.g. bioactive compounds found in grapefruit juice and some other fruit juices, including bergamottin, dihydroxybergamottin, and paradisin-A  inhibit CYP3A4-mediated metabolism of certain medications  leading to increased bioavailability  strong possibility of overdosing • Saint-John's wort (common herbal remedy)  induces CYP3A4 • Tobacco smoking  induces CYP1A2 (example substrates are clozapine/olanzapine)

26. A subset of cytochrome P450 enzymes play important roles in the synthesis of steroid hormones • (steroidogenesis) by the adrenals, gonads, and peripheral tissue • CYP11A1 in adrenal mitochondria effects “the activity formerly known as 20,22-desmolase” (steroid 20α-hydroxylase, steroid 22-hydroxylase, cholesterol side chain scission) • CYP11B1 (encoding the protein P450c11β) found in the inner mitochondrial membrane of adrenal cortex has steroid 11β-hydroxylase, steroid 18-hydroxylase, and steroid 18-methyloxidase activities

27. continued • CYP11B2 (encoding the protein P450c11AS), found only in the mitochondria of the adrenal zona glomerulosa, has steroid 11β-hydroxylase, steroid 18-hydroxylase, and steroid 18-methyloxidase activities • CYP17A1 in endoplasmic reticulum of adrenal cortex has steroid 17α-hydroxylase and 17,20-lyase activities. • CYP21A1 (P450c21) in adrenal cortex conducts 21-hydroxylase activity. • CYP19A (P450arom, aromatase) in endoplasmic reticulum of gonads, brain, adipose tissue, and elsewhere catalyzes aromatization of androgens to estrogens

28. CYP Families in Humans CYP1: drug and steroid (especially estrogen) metabolism CYP2: drug and steroid metabolism CYP3: drug and steroid (including testosterone) metabolism CYP4: arachidonic acid or fatty acid metabolism CYP5: thromboxane A2 synthase

29. continued • CYP7: bile acid biosynthesis 7-alpha hydroxylase of steroid nucleus • CYP11: steroid biosynthesis • CYP24: vitamin D degradation • CYP51: cholesterol biosynthesis

30. P450s in animals • classes of CYPs most often investigated in non-human animals  those involved in either development (e.g. retinoic acid or hormone metabolism) or involved in the metabolism of toxic compounds (such as heterocyclic amines or polyaromatic hydrocarbons) • there are differences in gene regulation or enzyme function of CYPs in related animals that explain observed differences in susceptibility to toxic compounds

31. continued • CYPs have been extensively examined in mice, rats, and dogs, and less so in zebrafish, in order to facilitate use of these model organisms in drug discovery and toxicology • CYPs have also been heavily studied in insects, often to understand pesticide resistance

32. Clinical importance • Gene Information for CYP2C9 • Gene Common Name: CYP2C9 • CYP2C9  a major phase 1 drug-metabolizing CYP450 isoform and one of several CYP2C genes • CYP2C9  primarily expressed in the liver

33. continued • CYP2C9  the enzyme responsible for the metabolism of the S-isomer of warfarin (R-warfarin is mainly metabolized by other CYP450 enzymes) that is principally responsible for the anticoagulant effect of the drug • CYP2C9  also metabolizes most NSAIDs, COX-2 inhibitors, tolbutamide, phenytoin, glipizide, fluvastatin • It is induced by rifampin and inhibited by amiodarone

34. continued • Two variants within CYP2C9  produce a phenotype of poor metabolism • Persons with the genotype of poor metabolism require lower doses of warfarin to achieve an anticoagulant effect similar to that in patients with a *1 (wildtype) genotype • CYP2C9 genotype can account for only part of the variability in warfarin sensitivity (age, weight, etc)