Cytochrome P450 Flashcards
What oxidise Xenobiotics?
What cofactor do these proteins contain?
Cytochrome P450s oxidise xenobiotics. They directly incorporate molecular oxygen to the carbon chain of a substrate. They contain haem.
What are the roles of cytochrome P450s?
Drug and xenobiotic metabolism (activation, degradation, solubilisation, detoxification).
Steroid hormone synthesis (fatty acid metabolism).
Fat soluble vitamin metabolism.
Carcinogen activation.
How many different genes encode cytochrome P450s?
How many cytochrome P450 genes do humans have?
What sequence homology must be shared for P450s to be in the same family or subfamily?
How many families and subfamilies of cytochrome P450s do humans have?
Over 4000 genes of cytochrome P450s have been identified.
Humans have 57 sequenced cytochrome P450 genes.
40% sequence homology to be in the same family, 55% to be in the same subfamily.
Humans have 18 families and 43 subfamilies.
Can cytochrome P450s be inhibited? If so what can they be inhibited by?
Multiple drugs can cause inhibition. Other xenobiotics such as St Johns Wort and Grapefruit juice can inhibit CYP450 3A4 causing increased drug levels.
The enzyme group of cytochrome P450s was initially identified as what in what year?
Where was this enzyme extracted from?
Pyrocatecholase in 1958.
It was extracted from homogenised liver microsomes.
What are the types of reactions catalysed by cytochromes P450 for the drugs Pentobarbital, Phenobarbital and Aldrin?
Pentobarbital: aliphatic hydroxylation: cyclohexane -> cyclohexanol,
Phenobarbital: aromatic hydroxylation: benzene -> phenol,
Aldrin: alkene epoxidation: cyclohexene -> cyclohexene oxide.
The active site of cytochrome P450s contains what?
Compare the active sites if CYP450 3A4 and CYP450 2C19.
A hydrophobic cavity with a haem and a cysteine ligand.
3A4 is phenylalanine rich and is also more flexible than 2C19 as due to a shorted F helix. The 3A4 site is also capable of binding more than one substrate.
What model of substrate/enzyme interaction do cytochrome P450s obey?
Induced fit
Describe the initial binding of Camphor to cytochrome P450s.
In the resting state P450s have water as the haem ligand. The resting state becomes structure 2 with the addition of crystal soaked in Camphor. The Camphor binds to the ferric P450 active site, displacing water. The haem iron becomes a 5-coordinate and thus easier to reduce.
Describe the initial reduction of P450 structure 2.
P450 is reduced with the addition of e- making the Fe^III have a transition state of II instead, this state has a higher affinity for iron and is now structure 3.
The reduced crystals are now saturated with oxygen. The oxygen binds to give a oxyferrous intermediate. An electron is transferred to O2, reverting Fe^II to Fe^III. This is now structure 4.
Describe the second electron reduction of P450s and subsequent Protonation.
Structure 4 is reduced again with the addition of another electron (e-) which makes the ferrous ion Fe^II again and an overall charge of 2-. A proton is added to this intermediate, hydroxylation the O2 ligand and reverting the ferrous ion back to Fe^III.
Describe how structure 5 forms in P450s.
After the second reduction and Protonation, the complex has another proton added and water dissociates leaving the ferrous ion at a transition state of IV and double bonded to one oxygen atom. This is structure 5 and it finally anneals to the crystal structure to form 5-hydroxycamphor (structure 6).
How does P450 avoid oxidising itself?
The thiolate ligand makes the haem environment very basic therefore favouring oxidation rather than auto-oxidation.
What are xenobiotics?
These are chemicals not normally produced by the organism or expected to be within the organism. These include drugs, antibiotics, ingested toxins and pollutants. They are often harmful. Because they are hydrophobic and accumulate at the cell membrane.
