Cytochrome P450

Question 1

What are the major categories of P450 function?

Answer 1

1) synthesis and elimination of cholesterol,
2) regulation of blood hemostasis,
3) steroid and arachidonic acid metabolism,
4) drug metabolism

Question 2

Why is induction of some P450s (i.e. by cigarette smoking) is a risk factor in several cancers ?

Answer 2

because these enzymes can convert procarcinogens to carcinogens.

Question 3

Are Mammalian cytochrome P450s are membrane bound?

Answer 3

yes (and have a heme group)

Question 4

P450s catalyze many types of reactions, but the one that is most important for us is what?

Answer 4

hydroxylation. (adding an oxygen to a lipophilic substrate). Oxygen acts as a “handle” for attachment of other components that will make it more water soluble in Phase II metabolism

Question 5

P450s are called mixed function oxidases or monooxygenases. What does that mean?

Answer 5

they incorporate one atom of molecular oxygen into the substrate and one atom into water.

They differ from dioxygenases that incorporate both atoms of molecular oxygen into the substrate

Question 6

Foreign chemicals or drugs are also called what?

Answer 6

xenobiotics. Cytochrome P450s play an important role in xenobiotic metabolism, especially for lipophilic drugs

Question 7

The metabolism of these compounds takes place in two phases. What are these two phases?

Answer 7

Phase I is chemical modification to add a functional group that can be used to attach a conjugate. The conjugate makes the modified compound more water soluble so it can be excreted in the urine. Many P450s add a hydroxyl group in a Phase I step of drug metabolism. The hydroxyl then serves as the site for further modifications in Phase 2 drug metabolism.

Thus, P450s are primarily Phase I enzymes

Question 8

What do cytochrome P450s need to function?

Answer 8

a source of electrons

Question 9

What role do electrons play in P450?

Answer 9

The addition of two electrons (reduction) to the heme iron makes the difficult chemistry of breaking the oxygen-oxygen bond possible

Question 10

Where do the electrons for P450 come from?

Answer 10

The electrons are donated by another protein that binds briefly to the P450 and passes an electron from a prosthetic group.

Question 11

The transfer of electrons from one protein to the heme in P450 is called what?

Answer 11

This handoff of electrons between proteins is called an electron transfer chain, and it is similar to the electron transfers that go on in complexes I to IV of the electron transfer chain in mitochondria. (However, this is not the same electron transfer chain.)

Question 12

There are two different kinds of electron transfer chains for cytochrome P450s. What do these types depend on?

Answer 12

These depend on the location of the enzyme in the cell. Some P450s are found in the mitochondrial inner membrane and some are found in the endoplasmic reticulum (ER).

Question 13

Are both the inner mitochondrial membrane and ER P450s membrane bound?

Answer 13

Yes

Question 14

The protein that donates electrons to P450s in the ER is called

Answer 14

NADPH cytochrome P450 reductase

Question 15

Describe the positioning of NADPH cytochrome P450 reductase

Answer 15

It is also membrane bound (like ER P450) by an N-terminal tail that crosses the ER membrane once. The bulk of this protein is on the cytosolic side of the ER membrane

Question 16

How are electrons passes from NADPH cytochrome P450 reductase to ER P450?

Answer 16

This protein has two domains that each contain one flavin. Two electrons are acquired from NADPH and migrate from FAD to FMN, then to the P450 heme iron.

Question 17

What is the immediate donor of electrons to the P450s in mitochondria?

Answer 17

Ferredoxin (called adrenodoxin in the adrenals)

Question 18

What is the source of electrons that flow from ferrodoxin to mitochondrial P450?

Answer 18

NADPH

Question 19

How do electrons flow from ferrodoxin to mitochondrial P450?

Answer 19

NADPH electrons flow from ferredoxin reductase to ferredoxin and then to P450.

Ferredoxin has an iron sulfur cluster instead of a flavin, however, ferredoxin is reduced by ferredoxin reductase (or adrenodoxin reductase in the adrenals) that does contain a flavin.

Question 20

What is cytochrome b5? Where does it get electrons from?

Answer 20

A few P450s also can accept electrons from cytochrome b5. This is a small membrane bound heme containing protein that gets its reducing equivalents (electrons) from NADH.

Question 21

How are steroid P450s regulated by hormones?

Answer 21

One example is the induction of steroid biosynthetic P450s by ACTH (adrenocorticotropic hormone). ACTH stimulates production of cAMP that activates a cAMP dependent protein kinase that phosphorylates diaclyglycerol kinase theta (DGK), This enzyme increases the production of phosphatidic acid (PA) that competes for sphingosine on the transcription factor steroidogenic factor 1 (SF1) leading to an increase in gene transcription for steroid producing P450s like CYP17A1.

Question 22

P450 enzymes catalyze specific reactions that can be monitored by sampling the urine, blood or breath of patients given a noninvasive marker. What is a marker for CYP1A2?

Answer 22

Caffeine. It is demethylated, and the rate at which it is demethylated is related to the amount of CYP1A2 in a person’s liver.

Question 23

What is the function of the The CYP1 family of P450s?

Answer 23

They can hydroxylate estrogen (CYP1A2 and 1B1) and oxidize uroporphyrinogen to uroporphyrin (CYP1A2) in heme metabolism, but they may have additional undiscovered endogenous substrates

Question 24

What induces CYP1 family of P450s (aka CYP1A2 and 1B1)?

Answer 24

These enzymes are inducible by some polycyclic hydrocarbons, some of which are found in cigarette smoke and charred food.

Question 25

What is clinically significant about the CYP1 P450s?

Answer 25

These enzymes are of interest, because in assays, they can activate compounds to carcinogens. High levels of CYP1A2 have been linked to an increased risk of colon cancer. Since the 1A2 enzyme can be induced by cigarette smoking, this links smoking with colon cancer.

Question 26

What is the largest P450 family in humans?

Answer 26

2. 28% of human P450s are in this family.

Question 27

What do family 2 P450s do?

Answer 27

Many of these proteins can hydroxylate steroids, and some of them are expressed in a sex specific manner. This would be expected for enzymes that only act on sex specific steroids. Some of these may also be drug metabolism enzymes that are defensive, to protect us from toxins in our food. Plants especially make many toxic components that are probably defensive for the plants. Since we eat almost anything, it is necessary to have a detoxification system coded in our genes. This idea has been called plant animal warfare on the chemical level.

Question 28

What is the big picture of the fact that polymorphisms are common in metabolizers like P450?

Answer 28

the prevalence of polymorphisms that lead to poor metabolism is related to race (i.e. uncommon in whites, common in asians) so one must be careful of administering a drug in high dosage depending on the probability of a patient being a poor metabolizer

Question 29

How does up regulation of CYP4A1 work?

Answer 29

peroxisome proliferators like clofibrate stimulates it. These drugs act through a binding protein called the PPAR or peroxisome proliferator activated receptor. When drug is bound to this protein it migrates to the nucleus, heterodimerizes with retinoid X receptor (RXR) and binds to specific DNA sequences in the regulatory region of genes that are needed for peroxisome generation. The CYP4A1 gene is turned on by this mechanism. Peroxisomes oxidize fatty acids and the CYP4A1 is a known fatty acid hydroxylase.

Question 30

What are the members of the CYP1 family induced by? How?

Answer 30

aromatic hydrocarbons.

The activation involves a specialized receptor called the Ah receptor. Ah stands for aryl hydrocarbon. This receptor protein binds the aromatic hydrocarbon, but it cannot reach the nucleus to activate gene transcription without another protein called arnt for Ah receptor nuclear translocator. These two proteins bind and together, they enter the nucleus, then bind DNA and activate transcription.

Question 31

What induces the CYP2E enzymes?

Answer 31

ethanol

Question 32

What kind of fatty acid oxidation does P450 do?

Answer 32

omega

Question 33

What are CYP3A4 and CYP2D6 involved in?

Answer 33

drug metabolism

Question 34

What is CYP51 involved in?

Answer 34

lipid biosynthesis (it is a drug target for anti-fungal drugs)

Question 35

What causes the 450nm spectrum of P450?

Answer 35

negatively charged cystine

Question 36

What is congenital glaucoma caused by?

Answer 36

CYP1B

Question 37

What is the most abundant P450 in the liver?

Answer 37

CYP3A4