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Molecular Biochemistry II

Synthesis of Eicosanoids

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Prostaglandins and related compounds are "local hormones" that are synthesized from the polyunsaturated fatty acid arachidonate. 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.

Examples include prostaglandins, prostacyclins, thromboxanes, leukotrienes, and epoxyeicosatrienoic acids. They have 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 and related compounds are collectively known as eicosanoids. They are produced from arachidonic acid, a 20-carbon polyunsaturated fatty acid (5,8,11,14- eicosatetraenoic acid).

Molecular Biochemistry II Synthesis of Eicosanoids Contents of this page: <a href=Prostaglandins and related compounds Cyclic pathway: Prostaglandin-H Synthase (cyclooxygenases) Linear pathway (leukotriene synthesis): Lipoxygenase EET synthesis: Cytochrome P epoxygenase Prostaglandins and related compounds are " local hormones " that are synthesized from the polyunsaturated fatty acid arachidonate . 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. Examples include prostaglandins, prostacyclins, thromboxanes, leukotrienes, and epoxyeicosatrienoic acids . They have 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 and related compounds are collectively known as eicosanoids . They are produced from arachidonic acid , a 20-carbon polyunsaturated fatty acid (5,8,11,14- eicosatetraenoic acid). ∑ 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 E (PGE ) 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 PPAR , is related to a family of nuclear receptors with transcription factor activity. " id="pdf-obj-0-36" src="pdf-obj-0-36.jpg">

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

Molecular Biochemistry II Synthesis of Eicosanoids Contents of this page: <a href=Prostaglandins and related compounds Cyclic pathway: Prostaglandin-H Synthase (cyclooxygenases) Linear pathway (leukotriene synthesis): Lipoxygenase EET synthesis: Cytochrome P epoxygenase Prostaglandins and related compounds are " local hormones " that are synthesized from the polyunsaturated fatty acid arachidonate . 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. Examples include prostaglandins, prostacyclins, thromboxanes, leukotrienes, and epoxyeicosatrienoic acids . They have 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 and related compounds are collectively known as eicosanoids . They are produced from arachidonic acid , a 20-carbon polyunsaturated fatty acid (5,8,11,14- eicosatetraenoic acid). ∑ 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 E (PGE ) 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 PPAR , is related to a family of nuclear receptors with transcription factor activity. " id="pdf-obj-0-53" src="pdf-obj-0-53.jpg">

Prostaglandin E 2 (PGE 2 ) 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 PPAR , is related to a family of nuclear receptors with transcription factor activity.

Prostaglandin receptors are specified by the same letter code. For example:

Receptors for E-class prostaglandins are designated EP. Thromboxane receptors are designated TP. Multiple receptors for a prostaglandin are specified by subscripts (e.g., EP 1 , EP 2 , EP 3 , etc.). Different receptors for a particular prostaglandin may activate different signal cascades. Effects may vary in different tissues, depending on which receptors are expressed.

The fatty acid arachidonate is frequently esterified at the hydroxyl on C-2 of glycerophospholipids, especially phosphatidyl inositol, shown at right with arachidonate in blue.

Arachidonate is released from phospholipids by hydrolysis. Sites of hydrolytic cleavage by Phospholipases A 2 and C are shown at right.

Phospholipase A 2 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.

∑ Prostaglandin receptors are specified by the same letter code. For example: ∑ Receptors for E-classlysophospholipid as products. There are multiple Phospholipase A enzymes, subject to activation via different signal cascades. ∑ The inflammatory signal molecule platelet activating factor is involved in ∑ activating some variants of Phospholipase A . Corticosteroids are anti-inflammatory because they inhibit Phospholipase A , reducing arachidonate release. " id="pdf-obj-1-50" src="pdf-obj-1-50.jpg">

There are multiple Phospholipase A 2 enzymes, subject to activation via different signal cascades.

The inflammatory signal molecule platelet activating factor is involved in

activating some variants of Phospholipase A 2 . Corticosteroids are anti-inflammatory because they inhibit Phospholipase A 2 , reducing arachidonate release.

Phosphatidyl inositol signal cascades may lead to release of arachidonate. After phosphatidyl inositol is phosphorylated to PIP 2 , cleavage of PIP 2 via Phospholipase C yields diacylglycerol (and IP 3 ). Arachidonate release from diacylglycerol is then catalyzed by Diacylglycerol Lipase.

Phosphatidyl inositol signal cascades may lead to release of arachidonate. After phosphatidyl inositol is phosphorylated to

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 H 2 Synthase (PGH 2 Synthase) catalyzes the committed step in the "cyclic pathway" that leads to production of prostaglandins, prostacyclins, and thromboxanes. Different cell types convert PGH 2 to different compounds.

Phosphatidyl inositol signal cascades may lead to release of arachidonate. After phosphatidyl inositol is phosphorylated to

Prostaglandin H 2 Synthase is a heme- containing dioxygenase, bound to endoplasmic reticulum membranes. (A dioxygenase incorporates O 2 into a substrate.) PGH 2 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 O 2 .

The signal molecule ·NO (nitric oxide) may initiate prostaglandin synthesis by reacting with superoxide anion (O 2 · - ) 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 PGH 2 Synthase consists of 4 short amphipathic -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.

Prostaglandin H Synthase is a <a href=heme - containing dioxygenase , bound to endoplasmic reticulum membranes. (A dioxygenase incorporates O into a substrate.) PGH 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 O . The signal molecule ·NO (nitric oxide) may initiate prostaglandin synthesis by reacting with superoxide anion (O · ) 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 PGH Synthase consists of 4 short amphipathic -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. " id="pdf-obj-3-45" src="pdf-obj-3-45.jpg">
Prostaglandin H Synthase is a <a href=heme - containing dioxygenase , bound to endoplasmic reticulum membranes. (A dioxygenase incorporates O into a substrate.) PGH 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 O . The signal molecule ·NO (nitric oxide) may initiate prostaglandin synthesis by reacting with superoxide anion (O · ) 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 PGH Synthase consists of 4 short amphipathic -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. " id="pdf-obj-3-47" src="pdf-obj-3-47.jpg">

Non-steroidal anti-inflammatory drugs (NSAIDs), such as aspirin and derivatives of ibuprofen, inhibit Cyclooxygenase activity of PGH 2 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.

Non-steroidal anti-inflammatory drugs ( NSAID s), such as aspirin and derivatives of ibuprofen, inhibit Cyclooxygenase activity

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 PGH 2 Synthase would restore cyclooxygenase activity.

Thromboxane A 2 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.

Non-steroidal anti-inflammatory drugs ( NSAID s), such as aspirin and derivatives of ibuprofen, inhibit Cyclooxygenase activity

Two isoforms of PGH 2 Synthase are designated COX-1 and COX-2 (Cyclooxygenase 1 & 2).

COX-1 is constitutively expressed at low levels in many cell types. COX-1 is essential for thromboxane formation in blood platelets, and for maintaining the integrity of the gastrointestinal epithelium.

COX-2 expression is stimulated by growth factors, cytokines, and endotoxins.

Inflammation is associated with up-regulation of COX-2 and increased

formation of prostaglandins. COX-2 levels increase in inflammatory disease states such as arthritis. Increased expression of COX-2 is seen in some cancer cells. Angiogenesis (blood vessel development) 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-1 and COX-2. More selective COX-2 inhibitors have been developed, e.g., Celebrex and Vioxx. COX-2 inhibitors are anti-inflammatory and block pain, but are less likely to cause the gastric toxicity often associated with chronic use of less specific NSAIDs. However, cardiovascular side effects have curtailed use of some of these drugs.

Some evidence suggests the existence of a third isoform of PGH 2 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 PGH 2 Synthase-1 (COX-1) crystallized with bound iodosuprofen, a derivative of ibuprofen.

Linear Pathway:

endothelial growth factor). Regular use of NSAIDs has been shown to decrease the risk of developing

PGH 2 Synthase

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.

5-Lipoxygenase, found in leukocytes, catalyzes conversion of arachidonate to 5-HPETE (5- hydroperoxyeicosatetraenoic acid). 5-HPETE is then converted to various leukotrienes that induce inflammation and asthmatic constriction of the bronchioles.

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 receptors on the plasma membranes of airway smooth muscle. A non-heme iron is the prosthetic group of Lipoxygenase enzymes. Ligands to the iron include 4 histidine nitrogen

endothelial growth factor). Regular use of NSAIDs has been shown to decrease the risk of developing

atoms and the C-terminal carboxylate oxygens. The structure has been described as resembling an octahedron with two unoccupied vertices. Presumably O 2 binds at one of these open ligand positions.

5-Lipoxygenase is overexpressed in cancer cells. As in the case of COX-2, 5-Lipoxygenase has a role in promoting angiogenesis essential to tumor growth.

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

atoms and the C-terminal carboxylate oxygens. The structure has been described as resembling an octahedron withcholesterol synthesis and metabolism). An example of an EET (14,15- epoxyeicosatrienoic acid), produced from arachidonate by activity of a cytochrome P 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 . Copyright © 1998-2005 by Joyce J. Diwan. All rights reserved. " id="pdf-obj-6-18" src="pdf-obj-6-18.jpg">

Lipoxygenase

5-Lipoxygenase requires the presence of the membrane protein FLAP (5-Lipoxygenase- activating protein). FLAP binds arachidonate, facilitating its interaction with the enzyme. 5-Lipoxygenase, FLAP, and Phospholipase A 2 (which catalyzes release of arachidonate from phospholipids) form a complex in association with the nuclear envelope during leukotriene synthesis in leukocytes. This is in contrast to other types of signal protein complexes that associate with the plasma membrane.

Cytochrome P 450 epoxygenase pathways:

Epoxyeicosatrienoic acids (EETs) and hydroxyeicosatrienoic acids are formed from arachidonate by enzymes of the cytochrome P 450 family. Other members of the cytochrome P 450 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 P 450 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.

atoms and the C-terminal carboxylate oxygens. The structure has been described as resembling an octahedron withcholesterol synthesis and metabolism). An example of an EET (14,15- epoxyeicosatrienoic acid), produced from arachidonate by activity of a cytochrome P 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 . Copyright © 1998-2005 by Joyce J. Diwan. All rights reserved. " id="pdf-obj-6-63" src="pdf-obj-6-63.jpg">

Copyright © 1998-2005 by Joyce J. Diwan. All rights reserved.

Additional material on Eicosanoids : Readings, Potential Test Questions & Tutorial
Additional material on Eicosanoids : Readings, Potential Test Questions & Tutorial
Additional
material on
Eicosanoids
:
Readings,
Potential
Test
Questions &
Tutorial
Additional material on Eicosanoids : Readings, Potential Test Questions & Tutorial