Download Cytochrome P450

Survey
yes no Was this document useful for you?
   Thank you for your participation!

* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project

Document related concepts

Cold-Food Powder wikipedia , lookup

Pesticide degradation wikipedia , lookup

Triclocarban wikipedia , lookup

Transcript
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-domaincontaining 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 22hydroxylase, 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 18methyloxidase 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 21hydroxylase 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 nonhuman 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 drugmetabolizing 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 (Rwarfarin 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)