L8 Cytochromes P450 1

where are CYP450’s found

endoplasmic reticulum and mitochondria (eukaryotes)

what is the prosthetic group of CYP450s

single haem molecule

what do CYP450’s form complexes that show maximum absorbance at?

450nm when reduced and CO added

how do we name CYP450s

use root CYP followed by family, subfamily and form number e.g. CYP2D6

how many different mammalian CYP450 families are there

10

what is the sequence similarity between different families

less than 40%

describe subfamilies of CYP450

certain families have large numbers of subfamilies

• all members show between 40 and 70% sequence similarity

describe number

the individual sequence coding for an isoform

what are the allelic variants of CYP450s

95% sequence similarity and products of the same gene

CYP1A1 has a primordial substrate which is a…

UV degradation product of tryptophan (FICZ)

what is FICZ

• perfect ligand of the irlhydrocarbon setting which controls expression of CYP1A1

• good substrate for CYP1A1

what are the functions of CYP450

• Xenobiotic metabolism

• steroid, fatty acid and vitamin oxidation

• steroid biosynthesis

what do CYP450s do in xenobiotic metabolism

• particularly families 1,2,3

• mainly occurs in endoplasmic reticulum

what occurs in steroid, fatty acid and vitamin oxidation

• variety of enzyme isoforms both xenobiotic metabolising and more specialised (e.g. CYP4 fatty acid oxidation, CYP26 retinoic acid oxidation

• mainly occurs in the ER

what occurs in steroid biosynthesis

• CYP450 found in mitochondria

• concerned with biosynthesis of steroids from cholesterol

• e.g. aromatase (synthesis of oestrogen)

CYP4 role

endogenous, fatty acid oxidation

CYPs in shampoo

kill yeast and prevent dandruff

CYP1 number of subfamilies

2

CYP2 number of subfamilies

>10

CYP3 number of subfamilies

1

CYP1 reactions

xenobiotic metabolism

CYP2 reactions

xenobiotic and steroid metabolism

CYP3 reactions

Xenobiotic and steroid metabolism

CYP450 protein

monomeric

• MW 40,000 to 50,000

CYP450 haem group

• iron atom coordinated to the 4 nitrogens of the haem tetrapyrrole ring and to a cysteine residue at the C-terminal end of the protein

• Iron is in the Fe(III) state

the 6th coordination position of the haem prosthetic group is generally…

water in the absence of substrate binding

regions of the protein concerned with haem and oxygen binding tend to be…

conserved

areas of protein conserved with haem and oxygen binding important for substrate binding…

vary more in sequence

crystal structures of most human P450s are relevant to… are now available

xenobiotic metabolism

Iron 3 needs to be reduced to bind…

carbon dioxide

Iron coordinates to 4 nitrogen’s and has a link to…

Cys on P450 protein and normally water

Highly conserved aa in the…

cys binding domain

oxygen binding site

CYPB5

what is the CYP450 reaction

DH + NADPH + O2 DOH + NADP + H2O

what are the 3 components needed for the CYP450 reaction

• CYP450 enzyme

• NADPH-cytochrome P450 reductase and phosphatidylcholine

NADPH supplies 2 protons and 2 electrons in an…

electron transfer process

CYP450 surrounds the …in the endoplasmic reticulum

reductase

CYP3A4 is good at what reactions

alkylation

what is NADPH-cytochrome P450 reductase

essential enzyme in the electron transfer process of the CYP450 reaction in the ER

• important in recycling intermediates

NADPH-CYP450 reductase contains flavoprotein as…

prosthetic groups

• flavin adenine dinucleotide (FAD)

• flavin mononucleotide (FMN)

NADPH-CYP450 reductase interacts with the CYP450 enzyme by electrostatic interaction involving…

• carboxyl groups on the reductase

• amino groups (probably lysine residues) on the CYP450

mitochondrial P450s use a different protein called… for electron transfer

adrenodoxin

describe the reaction mechanism of CYP450

• an unsuspecting drug molecule and CYP450 binds the drug

• there’s a single electron reduction passed from the intermediates flavins (FMN/FAD)

• oxidation state of Fe changes to Fe²⁺

• molecular oxygen binds which forms a peroxide

• second electron is passed via intermediate flavins (FMN/FAD)

• complex changes to an O_2-

• two protons are added which releases H20

• this causes a change in oxidation state back to Fe3+

• the oxygen passes onto the drug

• the hydroxylated version of the drug is released

what is the origin of the electrons from the CYP450 mechanism

NADPH

electrons move between which carriers in P450 complex

• FADH2

• FMN

• Change in iron reduction oxidation status

some P450 isoforms can be induced by exposure to certain…

xenobiotics

where does induction usually take place

transcription

• increase in mRNA and protein present

give examples of CYP450 inducers

polycyclic aromatic hydrocarbons (PAH) and barbituates

debrisoquine is an anti-hypotensive agent

metabolised and the hydroxylated product is no longer able to exert its function

give an example of how CYP450s can activate carcinogens

benzo[a]pyrene is a component of tobacco smoke that becomes carcinogenic after several P450-mediated reactions

give an example of how CYP450s can produce toxic molecules from certain harmless drugs

paracetamol (NAPQI-reactive electrophilic molecule causing liver damage)

give an example of how CYP450s can activate prodrugs

cyclophosphamide

describe tissue distribution of CYP450s

• found at highest levels in liver

• detectable but generally at lower levels in kidney, lung, intestine, adrenals and brain

• some forms detected in extrahepatic tissue e.g. CYP1A1, some steroid biosynthetic P450s

what are the 4 most important reactions of CYP450s

• N-dealkylation (diazepam)

• O-dealkylation (codeine)

• Aliphatic hydroxylation (ibuprofen)

• aromatic hydroxylation (phenytoin)