Notes
Cytochrome P450 Catalytic Cycle
Sections
General Information
Overview
- Here, we'll learn about the fundamental biochemistry of the cytochrome P450 catalytic cycle.
- To begin, start a table.
- Cytochrome P450 (aka CYP, CYP450, or P450) (hereafter we'll use: P450) plays an important role in both the metabolism of exogenous and also endogenous substances.
- In regards to exogenous P450 metabolism, P450 is fundamental for the conversion of foreign substances (xenobiotics) into polar entities that can be readily excreted, rather than build-up to toxic levels within the tissues.
- Denote that P450 intermediary metabolites can be beneficial; the P450 system can be exploited for the conversion of inert prodrugs to their active metabolites; eg, the metabolism of codeine to morphine (which has 200 times the analgesic effect as codeine (it exhibits 200 times the affinity to mu opioid receptors)).
- Unfortunately, however, P450 metabolites can be toxic, such as strengthening the carcinogenic effect of foreign compounds (eg, cigarette smoke): benzo[a]pyrene.
- Benzo[a]pyrene by itself is a weak carcinogen but via P450 metabolism, it is transformed over a series of steps to benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide, which is a much stronger carcinogen.
- In regards to endogenous P450 metabolism, P450 metabolism includes the biosynthesis of key endogenous compounds, such as the metabolism of steroid hormones (eg, testosterone and progesterone), cholesterols, bile acids, amines (eg, tyramine), and lipid-soluble vitamins (eg, the liver metabolism of vitamin A and vitamin D3).
- And the generation of potentially toxic mediators from endogenous precursors. For instance, P450 is involved in the oxidation of arachidonic acids (a long chain fatty acid) to eicosanoids.
- Eicosanoids play a mix injurious/protective role in stroke. It is believed that 20-hydroxyeicosatetraenoic acid (20-HETE) is destructive whereas epoxyeicosatrienoic acids (EETs) may be protective role.
Hydroxylation Reaction Stoichiometry
Overview
- To begin, let's address the basic stoichiometry of P450-mediated hydroxylation, the most common form of drug biotransformation; we address its biochemistry in detail elsewhere.
- P450 is a mixed function oxidase (aka monooxygenase), which means that it transfers one ("mono") oxygen to a substrate; they add a single oxygen into a bond. We highlight this definition because it is the over-arching mission of the catalytic cycle.
Reactants
Indicate that the reactants are:
- NADPH, which serves as a reducing agent; it is involved in electron transfer via P450 reductase.
- Hydrogen, which serves a key role in oxygen activation.
- Molecular oxygen; as mentioned, the reaction involves the insertion of an oxygen atom into a bond.
- And the substrate with a hydrogen bond (R-H).
- Consider that NAPDH is an important co-factor, so it is present in the basic stoichiometry; whereas the enzymes and prosthetic groups are not.
Summary
- Reactants: NADPH + H+ + O2 + R-H
- Consider that NAPDH is an important co-factor, so it is present in the basic stoichiometry; whereas the enzymes and prosthetic groups are not.
Products
And show that the products are:
- NADP+, which is the result of NADPH oxidation
- H2O, which results from the introduction of protons.
- And R-OH, which is the result of hydroxylation of the substrate.
Summary
- Products: NADP+ + H2O + R-OH
- We see that an oxygen is inserted between the carbon-hydrogen bond to make this an hydroxylation and two hydrogens bind with the other oxygen to form water.
CATALYTIC CYCLE Overview
- Let's also generate a short-hand version of the major steps of the catalytic cycle, which we learn in detail elsewhere.
Reactants
First, the key reactants:
- Drug (which we draw as a pill) with hydrogen bond
- 2 protons
- Molecular oxygen
Summary
- Substrate-H + 2H+ + O2
Products
Next, the products:
- We show the drug with an hydroxyl.
- And water.
Summary
- Substrate-OH + H2O
Co-factors/Enzymes
- Show that NADPH is oxidized to NADP+ via P450 reductase which transfers the electron from this oxidation to the P450 enzyme.
- We show that electron transfer occurs two times during the catalytic cycle.
- Note that one of these electron transfers can occur via cytochrome b5 (rather than NADPH reductase).
- Finally, show that the major catalytic enzyme is P450, which contains a central ferric iron (Fe3+) atom.
HISTOLOGY
So, now, let's take a look at where the reaction actually occurs in the cell.
Cell
- First, draw a portion of a cell: the nucleus (with a nucleolus and chromatin) and include endoplasmic reticulum (ER). * Show that rough ER is lined with ribosomes (rough ER is essential for protein synthesis). Then, show smooth ER is void of ribosomes (it has a smooth surface) and is the location of P450; it's anchored to the ER membrane.
Smooth ER
- Show a magnified view of the smooth ER membrane.
- Indicate the ER lumen and the cell cytoplasm.
- Then, show that P450 is anchored to the smooth ER membrane, facing into the cell cytoplasm.
- Also show its neighboring P450 reductase, which provides electrons to P450.
- And cytochrome b5 (CYB5), which can provide the second electron in the catalytic cycle (instead of P450 reductase).
Microsomal Enzymes
- Note that P450s are commonly referred to as microsomal enzymes; this is because, in general, scientists do not study these enzymes in their in vivo environments but rather in broken-down in vitro in samples.
- During centrifugation, tissue samples are fractionated and then allowed to reform into microsomes (vesicles).
- The microsomes retain certain morphological and functional properties of their un-fractionated in vivo origins, however – smooth microsomes are derived from smooth ER and rough microsomes are derived from rough ER. And it is the smooth microsomes that contain the P450s whereas the rough microsomes contain the materials for protein synthesis.
P450 STRUCTURE
- Now, finally, before we address the catalytic cycle, let's learn the three-dimensional structure of the P450 heme protein, which is a porphyrin structure.
Porphyrin
- Write that porphyrin comprises four pyrrole rings bridged by methine groups to form a planar structure.
Heme protein
- Show that the heme protein active site of cytochrome P450 (aka CYP, CYP450, or P450) comprises:
- A central iron atom with six binding sites (we show the iron in its ferric state).
- Four of these go to the nitrogen (N) of porphyrin (protoporphyrin (protoporphyrin IX)).
- Pyrrole is a 5-membered ring that consists of 1 nitrogen, 4 carbon atoms, and 5 hydrogens (C4H5N).
- Show that the 5th bond goes to cysteine.
- And then show that the final bond is available to bind up oxygen. As discussed, oxygen plays a key role in P450 oxidation reactions.
- Note that we learn about heme proteins (aka hemoproteins) in detail in our hemoglobin tutorials, as well.
*P450 CATALYTIC CYCLE
Overview
- With all of that as a background, we are ready to tackle the fundamentals of the cytochrome P450 catalytic cycle; note that it is richly afforded with numerous organic chemistry reactions that are far beyond our scope.
General Features of the Hydroxylation Reaction
- To begin, draw our drug (the substrate) and show a carbon-hydrogen bond (C-H).
- Indicate that the drug is lipophilic in its present state, which allows it to pass freely across cell membranes.
- Then, show that, ultimately, this cycle will catalyze an hydroxylation reaction (most commonly in the liver) wherein the carbon-hydrogen bond on the drug will be oxidized to a carbon-hydroxyl bond (C-OH), which is a polar moiety that can be renally excreted (we show a kidney).
Key P450 Reactions
- Although we focus on hydroxylation, here, P450 catalyzes numerous oxidation reactions and also various reduction reactions; three reactions predominate, let's list them now:
- Hydroxylation (our focus, here) – the insertion of oxygen into a carbon (or non-carbon) hydrogen bond.
- Heteroatom oxidation – oxygen addition to a heteroatom (a non-carbon or hydrogen atom, such as nitrogen or sulfur).
- Epoxidation – the addition of an oxygen across a carbon-carbon double bond (C=C) to form an epoxide (a cyclic ether).
Two Key Parts of the Cycle
Show that the catalytic cycle will involve two key parts:
- Part 1 is the activation of molecular oxygen; this part will take up the bulk of the steps in the cycle; it is basically just preparing oxygen for part 2.
- Part 2 is the oxidation of the substrate (the drug); this is the key portion of the reaction and it's the simpler of the two parts.
The P450 Catalytic Cycle
- To begin, represent the cytrochrome P450 enzyme as P450 bound to ferric iron (its 3+ oxidation state).
Drug-P450 Binding
- Show that the drug binds to the active site of P450.
1st Electron Transfer
- Next, show via a P450 reductase catalyzed reaction, NADPH is oxidized to NADP+ and an electron is transferred to the P450 iron, which reduces the iron to its ferrous state (2+ oxidation).
- As a reminder: "ox"idation is the "loss" of electrons and reduction is a gain of electrons.
O2 Binding
- Now, show that molecular oxygen binds to the iron.
- Indicate that in the process, an electron is transferred from the iron to the oxygen.
- Thus, iron is oxidized back to its 3+ state and oxygen becomes negatively charged.
2nd Electron Transfer
- Next, show that another electron is transferred to the enzyme.
- This can occur either via another P450 reductase reaction (involving the conversion of another NADPH to NDAP+) or via a lesser discussed enzyme: cytochrome b5; either way, an electron is transferred to P450.
Proton Binding/H2O Release
- Indicate that subsequently, two proton ions bind to the enzyme complex and perform a heterolytic dioxygen bond cleavage, which allows the protons to bind with one of the oxygen atoms, and in the process water is formed and released.
Ferryl Intermediate
- This step forms the most reactive intermediate in the cycle, a ferryl intermediate, which has a double bond to oxygen.
- We indicate that it is in a 5+ oxidation state (2 steps above the ferric iron state).
Hydroxylation
- From here, many different reactions can occur but the most common reaction (and the key one for our purposes) is that through two separate steps, the oxygen is transferred to the carbon-hydrogen bond on the substrate (the drug) (it is hydroxylated) and two electrons return to the ferryl iron, so that it returns to its original ferric iron oxidation state (3+).
Conclusion
- Show that the drug, then, leaves the enzyme active site.
- It is now in its hydroxylated, polar state, ready to be renally excreted.
- And the P450 enzyme is ready for the next reaction.