Molecular Biochemistry II

Eicosanoid Synthesis

Copyright 1999-2008 by Joyce J. Diwan. All rights reserved.

Prostaglandins and related compounds are collectively known as eicosanoids.

Most are produced from arachidonic acid, a 20-carbon polyunsaturated fatty acid (5,8,11,14-eicosatetraenoic acid). The eicosanoids are considered "local hormones." They have specific effects on target cells close to their site of formation. They are rapidly degraded, so they are not transported to distal sites within the body. But in addition to participating in intercellular signaling, there is evidence for involvement of eicosanoids in intracellular signal cascades.

 Examples of eicosanoids: prostaglandins prostacyclins thromboxanes leukotrienes epoxyeicosatrienoic acids. They have roles in: inflammation fever regulation of blood pressure blood clotting immune system modulation control of reproductive processes & tissue growth regulation of sleep/wake cycle.

O COOH

PGE2 (prostaglandin E2) is an example of a prostaglandin, produced from arachidonic acid.

HO

OH

PGE2

COOH

Arachidonic acid

O COOH

PGE2 (prostaglandin E2).

HO

OH

PGE2

Prostaglandins all have a cyclopentane ring.

A letter code is based on ring modifications (e.g., hydroxyl or keto groups).

A subscript refers to the number of double bonds in the two side-chains.

Thromboxanes are similar but have instead a 6-member ring.

Prostaglandin receptors:

Prostaglandins & related compounds are transported out of the cells that synthesize them.
Most affect other cells by interacting with plasma membrane G-protein coupled receptors. Depending on the cell type, the activated G-protein may stimulate or inhibit formation of cAMP, or may activate a phosphatidylinositol signal pathway leading to intracellular Ca++ release. Another prostaglandin receptor, designated PPARg, is related to a family of nuclear receptors with transcription factor activity.

 Prostaglandin receptors are specified by the same letter code. E.g., receptors for E-class prostaglandins are EP. Thromboxane receptors are designated TP. Multiple receptors for a prostaglandin are specified by subscripts (E.g., EP1, EP2, EP3, etc.).

Different receptors for a particular prostaglandin may activate different signal cascades.
Effects of a particular prostaglandin may vary in different tissues, depending on which receptors are expressed. E.g., in different cells PGE2 may activate either stimulatory or inhibitory or G-proteins, leading to either increase or decrease in cAMP formation.

The fatty acid arachidonate is often esterified to OH on C2 of glycerophospholipids, especially phosphatidyl inositol.

Site of cleavage by Phospholipase A2

O R2 C O H2C C O H

O C O P O OH OH H OH OH H H R1 H OH H

CH2O

Site of cleavage by OPhospholipase C H

Phosphatidyl inositol

Arachidonate is released from phospholipids by hydrolysis catalyzed by Phospholipase A2. This enzyme hydrolyzes the ester linkage between a fatty acid and the OH at C2 of the glycerol backbone, releasing the fatty acid & a lysophospholipid as products.

Corticosteroids are anti-inflammatory because they prevent inducible Phospholipase A2 expression, reducing arachidonate release. There are multiple Phospholipase A2 enzymes, subject to activation via different signal cascades.

The inflammatory signal platelet activating factor is involved in activating some Phospholipase A2 variants.
Attempts have been made to develop drugs that inhibit particular isoforms of Phospholipase A2, for treating inflammatory diseases. Success has been limited by the diversity of Phospholipase A2 enzymes, and the fact that arachidonate may give rise to inflammatory or anti-inflammatory eicosanoids in different tissues.

O O R1 C O H2C CH H2C O O C O P O OH H OH OH H H H OPO32 O H OPO32 R2

Phosphatidyl inositol signal cascades may lead to release of arachidonate.

cleavage by Phospholipase C

PIP2

phosphatidylinositol4,5-bisphosphate

After PI is phosphorylated to PIP2, cleavage via Phospholipase C yields diacylglycerol (and IP3).

Arachidonate release from diacylglycerol is then catalyzed by Diacylglycerol Lipase.

leukotrienes

Linear pathway Lipoxyganase

phospholipids arachidonate diacylglycerol

Two major pathways of eicosanoid metabolism.

Cyclic pathway PGH2 Synthase Prostacyclin prostaglandin H2 Thromboxane Synthase Synthase

prostacyclins thromboxanes other prostaglandins

Cyclic pathway:

Prostaglandin H2 Synthase catalyzes the committed step in the cyclic pathway that leads to production of prostaglandins, prostacyclins, & thromboxanes. Different cell types convert PGH2 to different compounds.

COOH

PGH2 Synthase is a heme-containing dioxygenase, bound to ER membranes.

(A dioxygenase incorporates O2 into a substrate). PGH2 Synthase exhibits 2 activities: cyclooxygenase & peroxidase.

arachidonic acid
2 O2 O

Cyclooxygenase
COOH

PGG2
2 e O

OOH

Peroxidase
COOH

PGH2

OH

COOH

PGH2 Synthase (expressing both cyclooxygenase & peroxidase activities) is sometimes referred to as Cyclooxygenase, abbreviated COX.

arachidonic acid
2 O2 O

Cyclooxygenase
COOH

PGG2
2 e O

The interacting cyclooxygenase and peroxidase reaction pathways are complex.

OOH

Peroxidase
COOH

PGH2

OH

A peroxide (such as that generated later in the reaction sequence) oxidizes the heme iron. The oxidized heme accepts an electron from a nearby tyrosine residue (Tyr385). The resulting tyrosine radical is proposed to extract a H atom from arachidonate, generating a radical species that reacts with O2.

COOH

arachidonic acid
2 O2 O

Cyclooxygenase
COOH

PGG2
2 e O

OOH

Peroxidase
COOH

PGH2

OH

The signal molecule NO (nitric oxide) may initiate prostaglandin synthesis by reacting with superoxide anion (O2) to produce peroxynitrite, which oxidizes the heme iron enabling electron transfer from the active site tyrosine. Prostaglandin synthesis in response to some inflammatory stimuli is diminished by inhibitors of Nitric Oxide Synthase.

PDB 1PGE Membranebinding domain: heme 4 short amphipathic ibuprofen a-helices that analog insert into one membrane leaflet of the bilayer, facing binding domain Prostaglandin H2 Synthase the ER lumen. Arachidonate, derived from membrane lipids, approaches the heme via a hydrophobic channel extending from the membrane-binding domain. In the image above, the channel is occupied by an inhibitor, an ibuprofen analog.

Non-steroidal anti-inflammatory drugs (NSAIDs), such as aspirin and derivatives of ibuprofen, inhibit cyclooxygenase activity of PGH2 Synthase. They inhibit formation of prostaglandins involved in fever, pain, & inflammation. They inhibit blood clotting by blocking thromboxane formation in blood platelets. Ibuprofen and related compounds block the hydrophobic channel by which arachidonate enters the cyclooxygenase active site.

H3C CH

CH3

CH2

CH H3C COOH

Ibuprofen

COOH O

O C CH3

Enz-Ser

CH2

OH

Aspirin

PGH2 Synthase (active)

COOH OH O

+
Salicylic acid

Enz-Ser

CH2 O

CH3

Acetylated PGH2 Synthase (inactive)

Aspirin acetylates a serine hydroxyl group near the active site, preventing arachidonate binding. The inhibition by aspirin is irreversible. However, in most body cells re-synthesis of PGH2 Synthase would restore cyclooxygenase activity.

Thromboxane A2 stimulates blood platelet aggregation, essential to the role of platelets in blood clotting. Many people take a daily aspirin for its anti-clotting effect, attributed to inhibition of thromboxane formation in blood platelets.

This effect of aspirin is long-lived because platelets lack a nucleus and do not make new enzyme.

Two isoforms of PGH2 Synthase: COX-1 & COX-2 (Cyclooxygenase 1 & 2): COX-1 is constitutively expressed at low levels in many cell types. COX-2 expression is highly regulated. Transcription of the gene for COX-2 is stimulated by growth factors, cytokines, and endotoxins. COX-2 expression may be enhanced by cAMP, and in many cells PGE2 produced as a result of COX-2 activity itself leads to changes in cAMP levels.

Both catalyze PGH2 formation, but differing localization within a cell, & localization of enzymes that convert PGH2 into particular prostaglandins/ thromboxanes, may result in COX-1 & COX-2 yielding different ultimate products.

 COX-1 is essential for thromboxane formation in blood platelets, and for maintaining integrity of the gastrointestinal epithelium. COX-2 levels increase in inflammatory diseases such as arthritis. Inflammation is associated with up-regulation of COX-2 & increased amounts of particular prostaglandins. COX-2 expression is increased in some cancer cells. Angiogenesis (blood vessel development), which is essential to tumor growth, requires COX-2. Overexpression of COX-2 leads to increased expression of VEGF (vascular endothelial growth factor). Regular use of NSAIDs has been shown to decrease the risk of developing colorectal cancer.

Most NSAIDs inhibit both COX I & COX II. Selective COX-2 inhibitors have been developed, e.g., Celebrex and Vioxx. COX-2 inhibitors are anti-inflammatory & block pain, but are less likely to cause gastric toxicity associated with chronic use of NSAIDs that block COX-1. A tendency to develop blood clots when taking some of these drugs has been attributed to:

decreased production of an anti-thrombotic (clot blocking) prostaglandin (PGI2) by endothelial cells lining small blood vessels lack of inhibition of COX-1-mediated formation of pro-thrombotic thromboxanes in platelets.

Some evidence suggests the existence of a third isoform of PGH2 Synthase, designated COX-3, with roles in mediating pain and fever, and subject to inhibition by acetaminophen (Tylenol). Acetaminophen has little effect on COX-1 or COX-2, and thus lacks anti-inflammatory activity. Explore the structure of PGH2 Synthase-1 (COX-1) crystallized with bound iodosuprofen, a derivative of ibuprofen.

leukotrienes

Linear pathway Lipoxyganase

phospholipids arachidonate diacylglycerol

Cyclic pathway PGH2 Synthase Prostacyclin prostaglandin H2 Thromboxane Synthase Synthase

prostacyclins thromboxanes other prostaglandins

The 1st step of the Linear Pathway for synthesis of leukotrienes is catalyzed by Lipoxygenase. Mammals have a family of Lipoxygenase enzymes that catalyze oxygenation of various polyunsaturated fatty acid at different sites. Many of the products have signal roles.

Catalyzed by 5-Lipoxygenase:
COOH

E.g., 5-Lipoxygenase, found in leukocytes, catalyzes conversion of arachidonate to 5-HPETE (5-hydroperoxyeicosatetraenoic acid). 5-HPETE is converted to leukotriene-A4, which in turn may be converted to various other leukotrienes.

O2 OOH

arachidonate

COOH

5-HPETE
H2O O COOH

leukotriene-A4

A non-heme iron is the prosthetic group of Lipoxygenase enzymes. Ligands to the Fe include 3 His N atoms & the C-terminal carboxylate O. The arachidonate substrate binds in a hydrophobic pocket, adjacent to the catalytic iron atom. O2 is thought to approach from the opposite side of the substrate than the iron, for a stereospecific reaction.
15-Lipoxygenase

membranebinding domain

substrate analog adjacent to Fe (green)

PDB 1LOX

COOH

Lipoxygenase reaction starts with extraction of H from arachidonate, with transfer of the e to the iron, reducing it from Fe3+ Fe2+. The resulting fatty acid radical reacts with O2 to form a hydroperoxy group.

O2 OOH

arachidonate

COOH

5-HPETE

Which H is extracted, & the position of the hydroperoxy group, varies with different lipoxygenases (e.g., 5-Lipoxgenase shown here, 15-Lipoxygenase, etc.) Additional reactions then yield the various leukotrienes.

Leukotrienes have roles in inflammation. They are produced in areas of inflammation in blood vessel walls as part of the pathology of atherosclerosis. Leukotrienes are also implicated in asthmatic constriction of the bronchioles. Some leukotrienes act via specific G-protein coupled receptors (GPCRs) in the plasma membrane. Anti-asthma medications include: inhibitors of 5-Lipoxygenase, e.g., Zyflo (zileuton) drugs that block leukotriene-receptor interactions. E.g., Singulair (montelukast) & Accolate (zafirlukast) block binding of leukotrienes to their receptors on the plasma membranes of airway smooth muscle cells.

5-Lipoxygenase requires the membrane protein FLAP (5-lipoxygenase-activating protein). FLAP binds arachidonate, facilitating its interaction with the enzyme.

Translocation of 5-Lipoxygenase from the cytoplasm to the nucleus, and formation of a complex including 5-Lipoxygenase, FLAP, & Phospholipase A2 in association with the nuclear envelope has been observed during activation of leukotriene synthesis in leukocytes.

A b-barrel domain at the N-terminus of Lipoxygenase enzymes may have a role in membrane binding. Explore the structure of Lipoxygenase, with a substrate analog present at the active site.
15-Lipoxygenase

membranebinding domain

substrate analog adjacent to Fe (green)

PDB 1LOX

COOH

COOH

Arachidonic acid

14,15-EET

Cytochrome P450 epoxygenase pathways: Epoxyeicosatrienoic acids (EETs) and hydroxyeicosatrienoic acids are formed from arachidonate by enzymes of the cytochrome P450 family.

Other members of the cytochrome P450 family participate in a variety of oxygenation reactions, including hydroxylation of sterols.

Shown is an EET produced from arachidonate by activity of a cytochrome P450 epoxygenase.

COOH

14,15-EET

(14,15-epoxyeicosatrienoic acid)

EETs are modified by additional enzyme-catalyzed reactions to produce many distinct compounds. They may be incorporated into phospholipids, and released by action of phospholipases.

EETs have roles in regulating cellular proliferation, inflammation, peptide hormone secretion, & various signal pathways relevant to cardiovascular and renal functions. E.g., EETs inhibit apoptosis in endothelial cells.