Molecular Biochemistry II

Synthesis of Eicosanoids

Contents of this page:
Prostaglandins and related compounds

Cyclic pathway: Prostaglandin-H2 Synthase (cyclooxygenases)
Linear pathway (leukotriene synthesis): Lipoxygenase
EET synthesis: Cytochrome P450 epoxygenase

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 are prostaglandins, prostacyclins, thromboxanes, leukotrienes, and epoxyeicosatrienoic acids. They have various roles in inflammation, fever, regulation of blood pressure, blood clotting, immune system modulation, control of reproductive processes and tissue growth, and regulation of the sleep/wake cycle.

Prostaglandins

  • Prostaglandins all have a cyclopentane ring, and are designated by a letter code, 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 six-member ring
Prostaglandin E2 (PGE2) is shown at right. 

Prostaglandin receptors: Prostaglandins and 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. 

The fatty acid arachidonate is often esterified to the hydroxyl on C2 of glycerophospholipids, especially phosphatidyl inositol, shown at right with arachidonate in blue.

Arachidonate is released from phospholipids by hydrolysis catalyzed by Phospholipase A2. This enzyme hydrolyzes the ester linkage between a fatty acid and the hydroxyl at carbon 2 of the glycerol backbone, releasing the fatty acid (e.g., arachidonate) and 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.

Phosphatidyl inositol signal cascades may lead to release of arachidonate. After phosphatidyl inositol is phosphorylated to PIP2, cleavage of PIP2 via Phospholipase C yields diacylglycerol (and IP3). Arachidonate release from diacylglycerol is then catalyzed  by Diacylglycerol Lipase.
Two major pathways of eicosanoid metabolism are summarized at right. Structures of examples of the compounds listed are shown on p. 962 of Biochemistry, by Voet & Voet, 3rd Edition.

Cyclic pathway:

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

Prostaglandin H2 Synthase is a heme-containing dioxygenase, bound to endoplasmic reticulum membranes. (A dioxygenase incorporates O2 into a substrate.) PGH2 Synthase exhibits two catalytic activities, Cyclooxygenase and Peroxidase. The enzyme expressing both activities is sometimes referred to as Cyclooxygenase, abbreviated COX.

The interacting cyclooxygenase and peroxidase reaction pathways are complex. 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 hydrogen atom from arachidonate, generating a radical species that reacts with O2

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.

The membrane-binding domain of PGH2 Synthase consists of 4 short amphipathic a-helices that insert into one leaflet of the lipid bilayer, facing the lumen of the endoplasmic reticulum. 

Arachidonate, derived from membrane lipids, approaches the heme via a hydrophobic channel extending from the membrane-binding domain of the enzyme. In the image at right, 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 and inflammation. They inhibit blood clotting by blocking thromboxane formation in blood platelets. 

Ibuprofen and related compounds act by blocking the hydrophobic channel by which arachidonate enters the Cyclooxygenase active site. An iodinated analog of ibuprofen is seen in the structural diagram above, between the membrane-binding domain and the heme.

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 are designated COX-1 and COX-2 (Cyclooxygenase 1 & 2).

Though both cyclooxygenase isoforms catalyze PGH2 formation, differing localization within a cell, and localization of enzymes that convert PGH2 into particular prostaglandins or thromboxanes, may result in COX-1 and COX-2 yielding different ultimate products.

Most NSAIDs inhibit both COX-1 and COX-2.

Selective COX-2 inhibitors have been developed (e.g., Celebrex and Vioxx).

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 at right the structure of PGH2 Synthase-1 (COX-1) crystallized with bound iodosuprofen, a derivative of ibuprofen.


PGH2 Synthase

Linear Pathway:

The first step of the linear pathway  for synthesis of leukotrienes is catalyzed by Lipoxygenase. Mammalian organisms have a family of Lipoxygenase enzymes that catalyze oxygenation of various polyunsaturated fatty acids at different sites. Many of the products have signal roles. 

For example, 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 (diagrams p. 966, 968).

A non-heme iron is the prosthetic group of Lipoxygenase enzymes. Ligands to the iron include 4 histidine nitrogen atoms and the C-terminal carboxylate oxygen. 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 side facing the iron, for a stereospecific reaction.

The reaction starts with extraction of a hydrogen from arachidonate, with transfer of the electron to the iron, reducing it from Fe3+ to Fe2+. The fatty acid radical reacts with O2 to form a hydroperoxy fatty acid. Which hydrogen is extracted, & the position of the resulting hydroperoxy group, varies with different lipoxygenases (e.g., 5-Lipoxgenase shown at right, 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 in the plasma membrane.

Anti-asthma medications include inhibitors of 5-Lipoxygenase, such as Zyflo (zileuton), and drugs that interfere with leukotriene-receptor interactions. For example, Singulair (montelukast) and Accolate (zafirlukast) block binding of leukotrienes to their receptors on the plasma membranes of airway smooth muscle.

 5-Lipoxygenase requires the presence of 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, and Phospholipase A2 (which catalyzes release of arachidonate from phospholipids) 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 binding to membranes.

Explore at right the structure of Lipoxygenase, with a substrate analog present at the active site.


Lipoxygenase

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 (to be discussed in the section on cholesterol synthesis and metabolism).

An example of an EET (14,15-epoxyeicosatrienoic acid), produced from arachidonate by activity of a cytochrome P450 epoxygenase, is shown at right.

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, and various cellular signal pathways relevant to cardiovascular and renal functions. For example, EETs have been found to inhibit apoptosis (programmed cell death) in endothelial cells.

Copyright 1998-2008 by Joyce J. Diwan. All rights reserved.

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