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

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

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

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)

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

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

CytP450Oxidase-1OG2

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:

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)

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

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

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

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:

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

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

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

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

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)

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

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

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

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

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

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)

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

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

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

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

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

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

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

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

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)