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Chapter 19 Lipid Metabolism II: Membrane Lipids, Steroids, Isoprenoids, and Eicosanoids 19 - 1 Copyright © 2013 Pearson Canada Inc. Biochemistry, 4th Edition Chapter 19 Outline: • • • • • Metabolism of Glycerophospholipids Metabolism of Sphingolipids Steroid Metabolism Other Isoprenoid Compounds Eicosanoids: Prostaglandins, Thromboxanes, and Leukotrienes Copyright © 2013 Pearson Canada Inc. 19 - 2 Biochemistry, 4th Edition Metabolism of Glycerophospholipids Intracellular synthesis and transport of membrane phospholipids: • The most abundant phospholipids are those derived from glycerol. • These glycerophospholipids are found primarily as components of membranes. Copyright © 2013 Pearson Canada Inc. 19 - 3 Biochemistry, 4th Edition Metabolism of Glycerophospholipids • Phosphatidic acid is a branch metabolite between triacylglycerol biosynthesis and phospholipid biosynthesis. Copyright © 2013 Pearson Canada Inc. 19 - 4 Biochemistry, 4th Edition Metabolism of Glycerophospholipids Copyright © 2013 Pearson Canada Inc. 19 - 5 Biochemistry, 4th Edition Metabolism of Glycerophospholipids Copyright © 2013 Pearson Canada Inc. 19 - 6 Biochemistry, 4th Edition Metabolism of Glycerophospholipids Regulation of membrane phospholipid composition in bacteria: Copyright © 2013 Pearson Canada Inc. 19 - 7 Biochemistry, 4th Edition Metabolism of Glycerophospholipids Copyright © 2013 Pearson Canada Inc. 19 - 8 Biochemistry, 4th Edition Metabolism of Glycerophospholipids • In eukaryotes, phosphatidic acid has three different origins: o Glycerol-3-phosphate o Dihydroxyacetone phosphate o Diacylglycerol Copyright © 2013 Pearson Canada Inc. 19 - 9 Biochemistry, 4th Edition Metabolism of Glycerophospholipids Synthesis of phosphatidylcholine and phosphatidylethanolamine in mammals: Copyright © 2013 Pearson Canada Inc. 19 - 10 Biochemistry, 4th Edition Metabolism of Glycerophospholipids • Copyright © 2013 Pearson Canada Inc. Salvage pathways to phospholipids, starting with choline or ethanolamine, are quantitatively important in eukaryotic cells. 19 - 11 Biochemistry, 4th Edition Metabolism of Glycerophospholipids Metabolic activation of phosphocholine: • Copyright © 2013 Pearson Canada Inc. CTP:phosphocholine cytidylyltransferase catalyzes nucleophilic attack at the a-phosphate of CTP by a phosphoryl oxygen of phosphocholine, displacing the b,g-phosphates (pyrophosphate) of CTP, forming CDP-choline. 19 - 12 Biochemistry, 4th Edition Metabolism of Glycerophospholipids • The second pathway to PE and PC begins with the conversion of phosphatidylserine (PS) to PE, which is catalyzed by either of two different enzymes: • Phosphatidylserine decarboxylase is a mitochondrial enzyme that, like the corresponding bacterial enzyme, decarboxylates PS to PE. • The second enzyme is a calcium-activated transferase, phosphatidylethanolamine serinetransferase, which exchanges free ethanolamine Copyright © 2013 Pearson Canada Inc. 19 - 13 Biochemistry, 4th Edition Metabolism of Glycerophospholipids • S-Adenosyl-L-methionine (AdoMet) is the methyl group donor in synthesis of phosphatidylcholine and numerous other methylated metabolites. Copyright © 2013 Pearson Canada Inc. 19 - 14 Biochemistry, 4th Edition Metabolism of Glycerophospholipids • AdoMet is formed from methionine and ATP in an unusual reaction catalyzed by methionine adenosyltransferase, in which ATP is cleaved to yield inorganic triphosphate (PPPi) plus an adenosyl moiety linked directly via the ribose C5 to the methionine sulfur. • The triphosphate is hydrolyzed by the enzyme to pyrophosphate(PPi) and orthophosphate (Pi), drawing the reaction to completion. Copyright © 2013 Pearson Canada Inc. 19 - 15 Biochemistry, 4th Edition Metabolism of Glycerophospholipids • Fatty acid chains in phospholipids are remodeled through the concerted actions of phospholipases and specific lysophospholipid acyltransferases to meet the needs of the organism. • Specificities of phospholipases A1, A2, C, and D: Copyright © 2013 Pearson Canada Inc. 19 - 16 Biochemistry, 4th Edition Metabolism of Glycerophospholipids Structure of phospholipase A2: •An enzyme that metabolizes membrane phospholipids. •Catabolism of phospholipids at membrane–water interfaces is important both in modification of membrane structure and as a source of second messengers and other regulators. •The crystal structure of the porcine pancreas phospholipase A2 reveals the catalytic triad (D99-H48-water) and the active site Ca2+ ion, liganded to a second water molecule (W2). Copyright © 2013 Pearson Canada Inc. 19 - 17 Biochemistry, 4th Edition Metabolism of Glycerophospholipids Copyright © 2013 Pearson Canada Inc. 19 - 18 Biochemistry, 4th Edition Metabolism of Glycerophospholipids • Phosphatidylinositol and its phosphorylated derivatives, collectively termed phosphoinositides, play important roles as precursors of second messengers. • PIP3 plays an important role as a second messenger in transmembrane signaling, the transmission of an extracellular signal to some element of the intracellular metabolic apparatus. Copyright © 2013 Pearson Canada Inc. 19 - 19 Biochemistry, 4th Edition Metabolism of Glycerophospholipids Biosynthetic route to alkyl ether phospholipids: Copyright © 2013 Pearson Canada Inc. 19 - 20 Biochemistry, 4th Edition Metabolism of Glycerophospholipids Copyright © 2013 Pearson Canada Inc. 19 - 21 Biochemistry, 4th Edition Metabolism of Glycerophospholipids • An unusual ether lipid, called platelet-activating factor, has the structure 1-alkyl-2-acetylglycerophosphocholine. • Physiologically, this compound is perhaps the most potent compound known. Copyright © 2013 Pearson Canada Inc. 19 - 22 Biochemistry, 4th Edition Metabolism of Glycerophospholipids Synthesis of a plasmalogen from a glyceryl ether: • Desaturation of 1-alkylacylglycerophosphoethanolamine (the alkyl analog of phosphatidylethanolamine) yields the corresponding vinyl ether, or plasmalogen. Copyright © 2013 Pearson Canada Inc. 19 - 23 Biochemistry, 4th Edition Metabolism of Sphingolipids • Membranes are assembled by membrane vesicles moving from synthesis sites in the endoplasmic reticulum and Golgi complex to existing membranes and fusing with them. Copyright © 2013 Pearson Canada Inc. 19 - 24 Biochemistry, 4th Edition Metabolism of Sphingolipids Copyright © 2013 Pearson Canada Inc. 19 - 25 Biochemistry, 4th Edition Metabolism of Sphingolipids Copyright © 2013 Pearson Canada Inc. 19 - 26 Biochemistry, 4th Edition Metabolism of Sphingolipids A myelinated axon from the spinal cord: • Copyright © 2013 Pearson Canada Inc. Myelin, an insulating layer wrapping about the axon, is rich in sphingomyelin. 19 - 27 Biochemistry, 4th Edition Metabolism of Sphingolipids Copyright © 2013 Pearson Canada Inc. 19 - 28 Biochemistry, 4th Edition Metabolism of Sphingolipids Copyright © 2013 Pearson Canada Inc. 19 - 29 Biochemistry, 4th Edition Steroid Metabolism • We turn now to an extraordinarily large and diverse group of lipids, the isoprenoids, or terpenes. • These compounds are built up from one or more fivecarbon activated derivatives of isoprene. • The family includes: o o o o o o o o o o Copyright © 2013 Pearson Canada Inc. Steroids Bile acids Lipid-soluble vitamins Dolichol and undecaprenol phosphates Phytol Gibberellins Insect juvenile hormones Components of rubber Coenzyme Q And many more compounds 19 - 30 Biochemistry, 4th Edition Steroid Metabolism Copyright © 2013 Pearson Canada Inc. • Ring identification system (a) • Carbon numbering system (b) used for steroids. • Structural conventions (c), with cholestanol as the example. • a-Substituents project below the plane of the • The hydrogens at positions 5, 9, and 14 have the a-configuration, whereas the hydroxyl, the two methyl groups, the hydrogen at C-8, and the aliphatic side chain at C-17 are all b-substituents. steroid ring system (blue dashed wedge), and bsubstituents project above that plane (red solid wedge). 19 - 31 Biochemistry, 4th Edition Steroid Metabolism • Copyright © 2013 Pearson Canada Inc. Cholesterol, the precursor to all steroids, derives all of its carbon atoms from acetate. 19 - 32 Biochemistry, 4th Edition Steroid Metabolism • The five carbons of isoprene could be derived metabolically from three molecules of acetate, and the prediction that cholesterol was a product of the cyclization of the linear hydrocarbon squalene. • Squalene contains six isoprene units (delineated by red marks on the structures below), and its configuration makes it a plausible steroid precursor. Copyright © 2013 Pearson Canada Inc. 19 - 33 Biochemistry, 4th Edition Steroid Metabolism Cholesterol biosynthesis can be considered as three distinct processes: 1.Conversion of C2 fragments (acetate) to a C6 isoprenoid precursor (mevalonate). 2.Conversion of six C6 mevalonates, via activated intermediates, to the C30 squalene. 3.Cyclization of squalene and its transformation to the C27 cholesterol. Copyright © 2013 Pearson Canada Inc. 19 - 34 Biochemistry, 4th Edition Biosynthesis of mevalonate and conversion to isopentenyl pyrophosphate and dimethylallyl pyrophosphate: • The two carbons of the third acetyl group are shown in red. Copyright © 2013 Pearson Canada Inc. 19 - 35 Biochemistry, 4th Edition Steroid Metabolism Stage 2: Synthesis of Squalene from Mevalonate • The next several reactions occur in the cytosol. • First, mevalonate is activated by three successive phosphorylations. • The first two are simple nucleophilic substitutions on the g-phosphorous of ATP. • The third phosphorylation, at position 3, sets the stage for a decarboxylation to give the five-carbon isopentenyl pyrophosphate Copyright © 2013 Pearson Canada Inc. 19 - 36 Biochemistry, 4th Edition Steroid Metabolism Conversion of isopentenyl pyrophosphate and dimethylallyl pyrophosphate to farnesyl pyrophosphate: • Both of these head-to-tail condensations are catalyzed by the same prenyltransferase (farnesyl pyrophosphate synthase). Copyright © 2013 Pearson Canada Inc. 19 - 37 Biochemistry, 4th Edition Steroid Metabolism Conversion of farnesyl pyrophosphate to squalene: •Catalyzed by squalene synthase. •Dissociation of the PPi group on one of the molecules of farnesyl-PPi yields an allylic carbocation. •The double bond nucleophilically attacks the carbocation, forming a tertiary cation at C3 of the first farnesyl-PPi. •Loss of a proton from C1 gives the activated cyclopropane intermediate, presqualene-PPi. •Dissociation of the second pyrophosphate leads to rearrangement and formation of another tertiary carbocation intermediate. •Hydride transfer from NADPH completes the rearrangement, giving squalene. Copyright © 2013 Pearson Canada Inc. 19 - 38 Biochemistry, 4th Edition Steroid Metabolism Copyright © 2013 Pearson Canada Inc. 19 - 39 Biochemistry, 4th Edition Steroid Metabolism Regulation of HMG-CoA reductase by ubiquitinmediated proteolysis: • Sterol binding leads to rapid degradation of the enzyme. Copyright © 2013 Pearson Canada Inc. 19 - 40 Biochemistry, 4th Edition Steroid Metabolism Insig-mediated regulation of SREBP activation: •When cholesterol levels in the cell are low, Scap transports SREBP to the Golgi. •Proteolytic processing by membranebound proteases S1P and S2P releases SREBP’s transcription factor domain (bHLH), which enters the nucleus and binds to sterol regulatory elements (SRE) in the promoters of target genes, stimulating their transcription. •High levels of cholesterol block this process by Insig-mediated retention of Scap–SREBP in the ER. Copyright © 2013 Pearson Canada Inc. 19 - 41 Biochemistry, 4th Edition Steroid Metabolism Copyright © 2013 Pearson Canada Inc. • Once the rate-limiting role of HMG-CoA reductase in cholesterol biosynthesis was understood, specific inhibitors were sought to lower blood cholesterol levels. • The compounds discovered are collectively called statins. • They act by competitively inhibiting HMG-CoA reductase. • Lovastatin and simvastatin are fungal polyketides and atorvastatin (Lipitor®) is synthetic. • Each statin carries a mevalonate-like moiety (blue), explaining the competitive nature of their activity. • Inhibition of HMG-CoA reductase depresses de novo cholesterol biosynthesis and, hence, intracellular cholesterol levels. • This in turn leads to increased production of LDL receptors, allowing more rapid clearance of extracellular cholesterol from the blood, thus lowering blood cholesterol levels. 19 - 42 Biochemistry, 4th Edition Steroid Metabolism The protein prenylation pathway: • Copyright © 2013 Pearson Canada Inc. aa = amino acid residue 19 - 43 Biochemistry, 4th Edition Steroid Metabolism Copyright © 2013 Pearson Canada Inc. 19 - 44 Biochemistry, 4th Edition Steroid Metabolism • Copyright © 2013 Pearson Canada Inc. Pregnenolone is an intermediate en route from cholesterol to all other known steroid compounds. 19 - 45 Biochemistry, 4th Edition Biosynthetic routes from pregnenolone to other steroid hormones: Copyright © 2013 Pearson Canada Inc. 19 - 46 Biochemistry, 4th Edition Steroid Metabolism • Diethylstilbestrol, a synthetic estrogen, was widely used to promote growth of beef cattle, until it was found to be potentially carcinogenic at the levels found in meat from treated cattle. Copyright © 2013 Pearson Canada Inc. 19 - 47 Biochemistry, 4th Edition Steroid Metabolism • Oral contraceptives are formulated with compounds containing progesterone and estrogen activities. • Two widely used synthetic estrogens are norethynodrel and mestranol. Copyright © 2013 Pearson Canada Inc. 19 - 48 Biochemistry, 4th Edition Other Isoprenoid Compounds • There are three active forms of vitamin A: all-transretinol, -retinal, and –retinoic acid. Collectively, these are referred to as retinoids. • The vitamin can be either consumed in the diet as esterified retinol, or biosynthesized from b-carotene, a plant isoprenoid especially abundant in carrots. • b-carotene is cleaved in the intestine by a monooxygenase to form two molecules of all-transretinal (retinaldehyde), which are then reduced to retinol. • Copyright © 2013 Pearson Canada Inc. All-trans-retinol is the form which circulates in the blood and which has the highest biological activity. 19 - 49 Biochemistry, 4th Edition Other Isoprenoid Compounds Schematic drawing of a rod cell: 1. The outer segment is a stack of membranous disks, which contain the photoreceptive pigments. 2. This segment is connected by a thin cilium to the inner segment, which contains the cell nucleus, cytosol, and synaptic body. 3. The potential change produced in the outer segment travels to the synaptic body and is transmitted to one or more of the neurons of the retina. Copyright © 2013 Pearson Canada Inc. 19 - 50 Biochemistry, 4th Edition Other Isoprenoid Compounds The chemical changes in photoreception: Copyright © 2013 Pearson Canada Inc. • 11-cis-retinal and opsin in a rod cell combine to form rhodopsin. • Absorption of a photon of light leads to the chemical changes shown in steps 2 and 3. • Step 3 involves at least 3 distinct conformational changes that occur in ~1 ms. • Metarhodopsin II is the species that activates transducin (not shown) to initiate the visual cascade. • After about 1 second, metarhodopsin II dissociates into all-trans-retinal, which isomerizes as the cycle begins again. 19 - 51 Biochemistry, 4th Edition Other Isoprenoid Compounds Structures of other important isoprenoids: Copyright © 2013 Pearson Canada Inc. 19 - 52 Biochemistry, 4th Edition Other Isoprenoid Compounds • Terpene is a generic term for all compounds that are biosynthesized from isoprene precursors. • Terpenes are biosynthesized ultimately from isopentenyl pyrophosphate (C5) and dimethylallyl pyrophosphate (C5). • When these combine to yield geranyl pyrophosphate (C10), any terpene formed thereby is called a monoterpene. • When a compound is formed from 1 mole of farnesyl pyrophosphate (C15), the product is called a sesquiterpene. • Triterpenes (C30) are formed from 2 moles of farnesyl pyrophosphate. • Geranylgeranyl pyrophosphate yields either diterpenes (C20) or tetraterpenes (C40). • The dolichols and undecaprenol, are examples of polyprenols (polyisoprenoid alcohols), which have more than 50 carbons Copyright © 2013 Pearson Canada Inc. 19 - 53 Biochemistry, 4th Edition Other Isoprenoid Compounds Some terpene compounds: • These examples are representative of an enormous class of natural products. Copyright © 2013 Pearson Canada Inc. 19 - 54 Biochemistry, 4th Edition Eicosanoids: Prostaglandins, Thromboxanes, and Leukotrienes Structures of the major prostaglandins and thromboxane A2: •The most abundant prostaglandins are those of the 2-series are shown. •They are derived from arachidonic acid, as is thromboxane A2. •Numbering of carbons begins with the carboxyl group as shown for the structure of PGG2. Copyright © 2013 Pearson Canada Inc. 19 - 55 Biochemistry, 4th Edition Eicosanoids: Prostaglandins, Thromboxanes, and Leukotrienes Summary of biosynthetic routes to the major prostaglandins and thromboxane A2: PLA2, phospholipase A2 PLC, phospholipase C DGL, diacylglycerol lipase MGL, monoacylglycerol lipase Copyright © 2013 Pearson Canada Inc. 19 - 56 Biochemistry, 4th Edition Eicosanoids: Prostaglandins, Thromboxanes, and Leukotrienes • COX-1 is constitutively expressed in most tissues, and is responsible for the physiological production of prostaglandins. • COX-2 is induced by cytokines, mitogens, and endotoxins in inflammatory cells and is responsible for the elevated production of prostaglandins during inflammation. • Both isoforms are covalently modified, and hence inactivated, by reaction with aspirin (acetylsalicylic acid). • As shown, aspirin acetylates a specific serine residue, which in turn blocks access of the fatty acid substrate to the cyclooxygenase active site. Copyright © 2013 Pearson Canada Inc. 19 - 57 Biochemistry, 4th Edition Eicosanoids: Prostaglandins, Thromboxanes, and Leukotrienes Nonsteroidal anti-inflammatory drugs (NSAIDs): •Ibuprofen and naproxen are examples of nonselective COX Inhibitors. •Rofecoxib (Vioxx®) and celecoxib (Celebrex®) are selective COX-2 inhibitors. o The phenylsulphonamide moieties, which contribute to their selectivity, are highlighted in red. Copyright © 2013 Pearson Canada Inc. 19 - 58 Biochemistry, 4th Edition Eicosanoids: Prostaglandins, Thromboxanes, and Leukotrienes Structural basis for selective inhibition of COX-2: Copyright © 2013 Pearson Canada Inc. • Ovine COX-1 with the nonselective NSAID flurbiprofen bound in the COX active site. • Arg120, which is important in binding the normal substrate arachidonic acid, extends behind flurbiprofen to interact with its carboxylate. • Mouse COX-2 with the selective inhibitor SC588 bound in the COX active site. • The phenylsulfonamide group of SC-588 extends into the side pocket made accessible by Val523. • The bulkier isoleucine at this position would prevent binding of SC-588 to COX-1. 19 - 59 Biochemistry, 4th Edition Eicosanoids: Prostaglandins, Thromboxanes, and Leukotrienes Biosynthesis of leukotrienes: •Leukotriene C was originally discovered in the class of white blood cells called polymorphonuclear leukocytes (PMNs) and was named after the source (leukocytes) and the triene structure. •It is a potent muscle contractant that is involved in the pathogenesis of asthma, through constriction of the small airways in the lung. •LTs are formed from the initial attack on arachidonate of a lipoxygenase, which adds O2 to C-5, giving 5-HPETE. •A dehydration to give the epoxide coupled with isomerization of double bonds gives leukotriene A4. •Hydrolysis of the epoxide ring yields leukotriene B4. •Transfer of the thiol group of glutathione yields leukotriene C4. •Subsequent modifications of the peptide chain yield related compounds, leukotrienes D and E. Copyright © 2013 Pearson Canada Inc. 19 - 60