LipMetab_I_palmitate_synthesis_NOTES_2022 PDF

S.H. Ackerman, PhD Synthesis of Palmitate & Related Molecules 1 [email protected] 4213 Scott Hall LECTURE TITLE: SYNTHESIS OF PALMITATE & RELATED MOLECULES LEARNING OBJECTIVES 1. Describe the mechanism used to bring acetyl-CoA from the mitochondria...

S.H. Ackerman, PhD Synthesis of Palmitate & Related Molecules 1 [email protected] 4213 Scott Hall LECTURE TITLE: SYNTHESIS OF PALMITATE & RELATED MOLECULES LEARNING OBJECTIVES 1. Describe the mechanism used to bring acetyl-CoA from the mitochondrial matrix to the cytoplasm. 2. Explain the relationship between the acetyl-CoA carboxylase reaction and condensation catalyzed by the ketoacyl synthase activity of the fatty acid synthase. 3. Recognize the names of enzymes and cofactors used to put double bonds in the FA hydrocarbon chain. 4. Explain the significance of pyrophosphatases with respect to group transfer reactions. 5. Explain the significance of the omega-3 and omega-6 fatty acids 6. Compare/contrast the properties of cyclooxygenases 1 & 2 OUTLINE I. Dogma: all lipids are derived from acetyl-CoA II. Review of fatty acid structure/nomenclature III. Key point about fatty acid synthesis IV. Transfer of acetyl-CoA from mitochondrial matrix to cytosol. V. Acetyl-CoA carboxylase: activation of the acetyl methyl carbon. VI. Palmitate (C16:0 FA) synthesis by the multifunctional fatty acid synthase (FAS) A. Phosphopantetheine as an acyl group carrier B. FAS domain structure C. FAS reaction cycle 1. Priming with acetyl-CoA 2. Recursive rounds of elongation and reduction 3. Hydrolytic release D. Alternate FA products of the FAS. VII. Fatty acid neutralization & activation for group transfer reactions. VIII. Fatty acid elongation and desaturation IX. Requirement for the essential fatty acids, Linoleate and a-Linolenate X. Fatty acid-derived signaling molecules; eicosinoids A. Prostaglandin H2 synthases (COX-1 and COX-2) acids B. COX inhibitors  Synthesis of Palmitate & Related Molecules 2 All lipids are derived from Acetyl-CoA Acetyl-CoA is an activated form of acetic acid; high energy intermediate owing to thioester bond. Acetyl-CoA is to lipid metabolism what G6P is to carbohydrate metabolism, in terms of commonality as a pathway intermediate. §¨©ª§¨©ª§¨©ª§¨©ª§¨©ª§¨©ª§¨©ª§¨©ª§¨©ª§¨©ª§¨©ª§¨©ª§¨©ª§¨©ª§¨©ª§¨©ª Review of Fatty Acid (FA) structure and nomenclature Fatty acids are monocarboxylic hydrocarbons of variable carbon chain lengths (from 5 to 26); physiologically-significant FA’s typically range between 16-18C. Fatty acids are numbered in the direction going from the carboxyl end to the methyl end. Greek symbol designations; C2 is a, C3 is b, and the terminal carbon is w. The hydrocarbon chain of a fatty acid can be saturated with hydrogen (no double bonds) or partially oxidized with one or more double bonds; defined as either a monounsaturated fatty acid, MUFA or a polyunsaturated fatty acid, PUFA. Short-hand nomenclature indicates the # of carbons * Linoleic acid, LA (left of colon), the # of double bonds (right of colon), and, 18:2(D 9,12) when present, the position of the double bond(s) Alternate name is w-6, “omega-6” FA (number(s) following the “delta” symbol shown in parentheses). * Linolenic acid, ALA (a -linolenic acid) 18:3(D 9,12,15) Alternate name is w-3 “omega-3” FA Asterisks denote two essential FAs that must be supplied by diet. Linoleic acid (LA) is an “omega-6” FA; precursor to arachidonic acid, which is the precursor to eicosinoids. Eicosinoids activate platelets (clotting) and leukocytes (inflammation). (a)Linolenic acid (ALA) is an “omega-3” FA; precursor to bioactive lipids, (EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid)), that exert anti-inflammatory effects. w-6 FA’s are pro-inflammatory w-3 FA’s are anti-inflammatory §¨©ª§¨©ª§¨©ª§¨©ª§¨©ª§¨©ª§¨©ª§¨©ª§¨©ª§¨©ª§¨©ª§¨©ª§¨©ª§¨©ª§¨©ª§¨©ª  Synthesis of Palmitate & Related Molecules 3 Overview of fatty acid synthesis When nutrients are ingested in excess to what can be catabolized to ATP, stored in glycogen, or used to meet requirements for new macromolecules in the cell, the material is converted to triacylglycerol (TAG) and stored. The de novo pathway of fatty acid synthesis is the principal destination for excess hydrocarbon material (fatty acids make up the bulk of TAG). The two main sites of fatty acid synthesis in the human body are the liver and adipose tissue. Lactating mammals synthesize fatty acids in mammary glands. Palmitate (16:0) is the principal end-product of de novo FA synthesis Palmitate synthesis requires: § Acetyl-CoA (all palmitate carbons originate from acetyl-CoA, added on 2 carbons at a time). § Energy ATP (required for creating new carbon-carbon bonds). NADPH (required to saturate the hydrocarbon chain). § Two enzymes Acetyl-CoA Carboxylase (increases the chemical reactivity of acetyl-CoA). Fatty Acid Synthase (builds & reduces the hydrocarbon chain). Fatty acid synthesis is cytoplasmic Acetyl-CoA is produced inside mitochondrial matrix, and while there is no membrane carrier for acetyl-CoA, there is a carrier for citrate. (recall that citrate is built from acetyl-CoA and OAA) The Citrate Shuttle provides the means for the continuous production of citrate inside mitochondria coupled to its cleavage in the cytoplasm. Fatty acid elongation is recursive (repeating process whose output at each stage is applied as input in the succeeding stage). For palmitate synthesis, four enzyme-catalyzed steps are repeated 7 times. § Condensation of the growing chain with an activated acetyl unit. § Reduction of a ketone to an alcohol. § Dehydration of the alcohol to a trans-alkene. § Reduction of the trans-alkene to an alkane.  Synthesis of Palmitate & Related Molecules 4 The citrate shuttle, acetyl-CoA carboxylase reaction, and fatty acid synthase reactions encompass the 3 major steps of de novo fatty acid synthesis. Citrate shuttle ACC reaction FAS reactions Step 1: Citrate shuttle transfers acetyl units out of mitochondria, refurbishes the TCA cycle with carbon (to make more citrate), and produces 8 of the 14 NADPH that are required to make palmitate. 1. Citrate synthase condenses Acetyl-CoAmito with OAAmito to make citratemito. 2. Citrate transporter brings citrate out of mitochondria. 3. ATP citrate lyase cleaves citrate yielding acetyl-CoAcyt & OAAcyt. 4. Cytosolic MDH isozyme reduces OAAcyt to malatecyt. Malate takes 1 of 2 paths 5. Malate/aKG exchange carrier brings malate into mitochondria. 6. Mitochondrial MDH isozyme oxidizes malatemito to OAAmito. or 7. Cytosolic malic enzyme oxidizes and decarboxylates malate yielding pyruvate & NADPH. 8. Pyruvate enters mitochondria via its transporter. 9. Pyruvate carboxylase converts Pyrmito to OAAmito.  Synthesis of Palmitate & Related Molecules 5 Step 2: Acetyl-CoA activation (Acetyl-CoA carboxylase reaction). Palmitate is built 2 carbons at a time in a condensation reaction that joins, what was formerly, the methyl carbon of an acetyl unit with the carbonyl carbon at the acid terminus of the nascent fatty acyl chain. Acetyl-CoA is not used as the substrate for the condensation reactions because the methyl carbon is chemically inert. The actual substrate for the condensation reactions is a carboxylated, high-energy derivative of acetyl-CoA called malonyl-CoA. The methyl C of acetyl-CoA is the methylene C of malonyl-CoA Acetyl-CoA carboxylase (ACC) synthesizes malonyl-CoA from acetyl-CoA and CO2 in the rate- limiting step of FA synthesis. The reaction is analogous to oxaloacetate synthesis from pyruvate and CO2, catalyzed by pyruvate carboxylase, and to methylmalonyl-CoA synthesis from propionyl- CoA and CO2, catalyzed by propionyl-CoA carboxylase. + ADP As shown in the adjacent figure for ACC, all of these carboxylases catalyze a 2-step reaction in which CO2 (from bicarbonate in solution) is transferred first, at the expense of ACC ACC ATP hydrolysis, to a covalently bound biotin cofactor (panel A), followed by transfer of the “activated” carboxylate group (circled)to the substrate (panel B). *In a subsequent step catalyzed by the fatty acid synthase, ACC ACC cleavage of the carboxylate bond in malonyl-CoA (high-energy metabolite) releases the energy originating from the ATP that was hydrolyzed by ACC in order to create the carboxylate bond. Step 3: Synthesis of palmitate by FAS The multifunctional fatty acid synthase (FAS) uses a phosphopantetheine prosthetic group to move fatty acyl intermediates between multiple different catalytic sites to make the end-product, palmitate (C16:0) The fatty acid synthase (FAS) synthesizes palmitate by catalyzing rounds of reactions in which a 2-C acetyl unit from malonyl-CoA is attached to a growing acyl chain and reduced. Phosphopantetheine is a universal carrier for acyl groups In general, acetate (2-C acid), propionate (3-C acid), acetoacetate (4-C acid), and fatty acids (≥ 5-C’s) never exist as free molecules inside cells; they are, instead, found as covalent adducts to the thiol group of a carrier molecule. The most common small molecule thiols used to carry acyl groups in the cells are phosphopantetheine (vit. B5 derivative) and lipoamide, with phosphopantetheine (Pan-SH) used most frequently.  Synthesis of Palmitate & Related Molecules 6 Unliganded carrier abbreviated Pan-SH (free thiol) Acylated carrier abbreviated Pan-S Pan-SH is covalently bonded to one of two anchors in nature: § 3’-phospho-adenosine, to create the soluble carrier, Coenzyme A Coenzyme A § the serine hydroxyl in a protein, giving rise to a covalently attached cofactor (prosthetic group). In bacteria, Pan-S is attached to a serine of a unique gene product called “acyl Ser carrier protein (ACP)”. In mammals, Pan-S is attached to a serine in the “ACP domain” of a multifunctional protein (see ACP below). Mammalian Fatty Acid Synthase (FAS) FAS is a dimer of identical polypeptides. Each polypeptide contains 6 different active sites (abbreviated MAT, KS, KR, DH, ER, TE), a Pan-S attachment site (ACP domain), and catalyzes 7 different reactions Substrate entry (acetyl-CoA to prime the pathway, malonyl-CoA for elongation) occurs in the MAT site and product (palmitate) is released from the TE site. The other 4 active sites participate in elongating and reducing the fatty acyl chain. During these reactions, acyl intermediates are transferred back and forth between two critical thiol groups (circled in above figure): a cysteine in the KS active site and the Pan-SH component of ACP. The latter are referred to as ACP derivatives. Fatty acids synthesis includes 4 chemical steps: condensation, reduction, dehydration, reduction For comparison, the opposing reactions (oxidation, hydration, oxidation, cleavage) are used in the b-oxidation pathway to degrade fatty acids. Decarboxylation of the malonyl unit in drives the condensation of an acetyl unit. Decarboxylation of malonyl unit leaves behind ONLY the acetyl carbons originating from acetyl-CoA, and these are the only carbons that get incorporated in the palmitate product. The energy for condensation originates from the ATP that was hydrolyzed in order make malonyl-CoA from acetyl-CoA and CO2.  Synthesis of Palmitate & Related Molecules 7 For your reference, the FAS reaction scheme is summarized here. FAS Overview *The intermediates that are shown attached to phosphopantetheine (Pan-S) in the figure are referred to as ACP derivatives in the reactions listed below. Condensation reaction ENERGY See inset for expanded view 1. Acetyl Transferase: Acetyl group transferred from CoA to KS site cysteine in 2 sub-steps Acetyl-CoA + ACP ⇆ Acetyl-ACP + CoA (occurs in MAT active site) Acetyl-ACP + KS-Cys ⇆ Acetyl-KS + ACP (occurs in KS active site) 2. Malonyl Transferase: Malonyl group transferred from CoA to ACP Malonyl-CoA + ACP ⇆ Malonyl-ACP + CoA (occurs in MAT active site) 3. (b)-Ketoacyl Synthase (aka condensing enzyme): The methylene carbon of malonyl-CoA stages nucleophilic attack on carbonyl carbon of Acetyl-KS. The carboxylate bond is broken, releasing CO2. The thioester bond to the KS site cysteine breaks, leaving a b-ketoacyl intermediate attached to ACP that has been elongated 2 carbons. Acetyl-KS + Malonyl-ACP ⇆ b-ketoacylC4-ACP + CO2 + KS-Cys (occurs in KS site) 4. (b)-Ketoacyl Reductase transfers reducing equivalents from NADPH to the fatty acyl intermediate, which reduces the b- ketone to a b-hydroxyl group. b-ketoacylC4-ACP + NADPH + H+ ⇆ b-hydroxyacylC4-ACP + NADP+ (occurs in KR site) 5. (b)-Hydroxyacyl Dehydratase removes water from the fatty acyl intermediate, which creates an enoyl derivative. b-hydroxyacylC4-ACP ⇆ b-enoylC4-ACP + H2O (occurs in DH site) 6. (b)-enoyl reductase transfers reducing equivalents from NADPH to the fatty acyl intermediate, which reduces the C=C double bond leaving a fully saturated fatty acyl intermediate. b-enoylC4-ACP + NADPH + H+ ⇆ AcylC4-ACP + NADP+ (occurs in ER site) Six more rounds are necessary to extend the hydrocarbon chain to C16. 7. Thioesterase hydrolyzes the thioester bond between ACP and palmitate (AcylC16) AcylC16-ACP + H2O ⇆ Palmitate + ACP  Synthesis of Palmitate & Related Molecules 8 The FAS builds fatty acids in the direction from the methyl end to the acyl end of the molecule. The methyl end is established in the priming step, which is the only time carbons are brought in to the FAS as the substrate “acetyl-CoA”. Malonyl-ACP, a 3-carbon metabolite, is used as the substrate for all of the condensation steps. However, it is still true that ALL of the carbons in palmitate originate from cytoplasmic acetyl-CoA. The malonyl unit is decarboxylated during the condensation reaction leaving behind the two carbons of the original acetyl-CoA molecule, which are incorporated into the growing acyl chain. Alternate fatty acids produced by the mammalian FAS Medium-chain fatty acid synthesis in lactating mammary gland Approximately 20% of the fatty acids in milk fat are derived by endogenous synthesis in the epithelial cells of the lactating mammary gland. Fatty acid synthase isozyme in this tissue has an atypical thioesterase domain that favors the early termination of the synthetic cycle, releasing medium-chain FA (8-14 carbons long) for incorporation into milk fat. Medium-chain FA are easier on the digestive systems of infants. Odd-chain fatty acid synthesis FAS produces odd-chain fatty acids (15C maximum) by priming the pathway with the three-carbon acyl unit provided by propionyl-CoA.  Synthesis of Palmitate & Related Molecules 9 Fatty acid neutralization & activation (attachment to Coenzyme A) In addition to palmitate and shorter chain or odd-chain FA’s released from the FAS, other sources of free fatty acids, include FA’s delivered from blood and FA’s released during the remodeling of membrane lipids or cleavage of lipidated proteins. None of these remain free for very long. Under normal conditions, the level of free fatty acids is quite low in cells b/c fatty acyl-CoA synthetases catalyze the thioester linkage of free fatty acids to Coenzyme A as soon as they appear. The action of fatty acyl-CoA synthetases is significant for two reasons: 1. Eliminates the negative charge on the fatty acid. 2. Creates a high-energy metabolite for use as the substrate for a group transfer reaction in which the target molecule is fatty acylated. Synthetase reaction + pyrophosphate cleavage Attachment of a fatty acid to Coenzyme A activation of fatty acid Fatty acyl-CoA synthetase: 2 stage reaction Stage 1: ATP is cleaved at the a phosphate. PPi is released as the product and the adenylate is attached to the fatty acid generating a reactive intermediate (Fatty acyl adenylate). Stage 2: CoA-SH binds and there is an isoenergetic exchange of a thioester bond for the mixed anhydride bond and the final products (fatty acyl-CoA and AMP) are released from the active site of the synthetase. Pyrophosphatase cleaves PPi to two Pi molecules. The fatty-acyl-CoA synthetase reaction is freely reversible. The formation of fatty acyl-CoA is made irreversible through the action of pyrophosphatase, which by eliminating PPi prevents the reversal of the synthetase reaction.  Synthesis of Palmitate & Related Molecules 10 Beyond the FAS: Fatty acid elongation & desaturation Newly synthesized palmitoyl-CoA and CoA derivatives of the essential FA’s provided by the diet (LA, linoleic acid; ALA, a-linolenic acid) are substrates for modifying enzymes of the smooth endoplasmic reticulum and mitochondrial membranes. Fatty acids may be lengthened by addition of 2C at a time. Humans have four different desaturase enzymes for the introduction of a double at either position C4, C5, C6 or C9 in the alkyl chain. Each desaturase constitutes a mini electron transfer chain with two additional proteins: cytochrome b5 and cytochrome b5 reductase. O2 is reduced with electrons originating from NADPH and the saturated FA substrate. 2 electrons, originating from NADPH, are transferred, first, to the FAD prosthetic group in Cytochrome b5 reductase, and then to the heme iron in Cytochrome b5. Upon binding the saturated FA substrate and molecular oxygen to the active site, fatty acyl-CoA desaturases oxidize the acyl chain at the relevant position and reduce O2, releasing an unsaturated FA and 2 waters as products. Reduction of molecular oxygen to 2 waters requires 4 electrons and 4 protons; 2 electrons are donated by 2 reduced Cyt b5’s, another 2 electrons plus 2 protons come from the fatty acid substrate, and the final 2 protons are picked up from the aqueous solvent. The essential fatty acids, Linoleic acid (LA) and a-Linolenic acid (ALA) Relevance to biological membranes The vast majority of activities occurring in biological membranes is favored by the lamellar crystalline (“fluid”) state of the lipid component. The temperature at which a lipid bilayer transitions from a lamellar gel to lamellar crystalline state (melting temperature) decreases with an increase in the number of double bonds and their distribution along the length of the fatty acid chains. The majority of fatty acids contain 16 or 18 carbons, and can even be as long as 26 carbons, yet the desaturase pathway of humans cannot insert a double bond beyond carbon-9 in the acyl chain. Limiting the position of double bonds to only half the length of fatty acids would be expected to increase the lipid melting temperature, disfavoring bilayer fluidity.  Synthesis of Palmitate & Related Molecules 11 Linoleic acid (LA) (linoleate) and a-Llinolenic acid (ALA) (Linolenate) are plant fatty acids that w6 FA provide the solution to the problem b/c they contain double bonds distal to carbon-9. LA and ALA are substrates for the human enzymes w that catalyze elongation and desaturation proximal to C-9. Hence, they permit humans to synthesize w3 FA the full complement of PUFAs required by the body, w making them essential components of our diet. LA and ALA are commonly referred to using an alternative nomenclature for fatty acids that designates the position of the most distal double bond in the hydrocarbon chain from the w-carbon at the methyl terminus. Relevance to the eicosanoid family of bioactive lipids Eicosinoids are a family of short-lived signaling molecules that are classified as “local hormones” b/c they exert effects on target cells in close proximity to their site of synthesis. There are 5 main sub-divisions of the eicosanoid super family: Prostaglandins Leukotrienes Thromboxanes Prostacyclins Epoxyeicosatrienoic acids. The eicosinoids are best known for their effects on inflammatory responses (predominantly those of the joints, skin and eyes), on the intensity and duration of pain and fever, and on reproductive function (including the induction of labor). Other important functions include inhibition of gastric acid secretion, regulation of blood pressure through vasodilation or constriction, and inhibition or activation of platelet aggregation and thrombosis. The starting point for eicosanoid synthesis is arachidonate, which is a 20-C, polyunsaturated w6 FA derived from linoleate.  Synthesis of Palmitate & Related Molecules 12 Eicosinoids biosynthesis 1. Mobilization of arachidonate Arachidonate is among the more common PUFA components of phosphoglycerol membrane lipids. Typically, arachidonate is esterified to the C2-OH of the backbone, in which cases it is released by Phospholipase A2. Corticosteroids (e.g. cortisone) are anti-inflammatory because they down- regulate gene expression for PLA2, which reduces the cell’s ability to release arachidonate. There are multiple PLA2 enzymes, which are subject to activation via different signal cascades. On-going work aims to develop drugs that inhibit particular isoforms of Phospholipase A2, for treating inflammatory disease. 2. Products of Linear and Cyclic pathways. Lipoxygenases catalyze the first step of the “linear pathway” from arachidonate for synthesis of leukotrienes. PGH2 synthase synthesizes prostaglandin H2 in the first step of the “cyclic pathway” from arachidonate. PGH2 is the starting material to make other prostaglandins, prostacyclins, and thromboxanes. PGH2 Synthase is a heme-containing dioxygenase, bound to ER membranes. (A dioxygenase incorporates O2 into a substrate). Even though PGH2 Synthase exhibits 2 activities: cyclooxygenase & peroxidase, it is typically referred to simply as Cyclooxygenase, abbreviated COX.  Synthesis of Palmitate & Related Molecules 13 There are 2 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. 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. Non-steroidal anti-inflammatory drugs (NSAIDs), such as aspirin and derivatives of ibuprofen, inhibit cyclooxygenase activity of PGH2 Synthases. Most NSAIDs inhibit both COX I & COX II. They inhibit formation of prostaglandins involved in fever, pain, & inflammation. They inhibit blood clotting by blocking thromboxane formation in blood platelets. The inhibition by aspirin is irreversible, though most cells in the body are capable of re-synthesizing new PGH2 Synthase and can overcome the effect of aspirin. An important exception is platelets, which lack a nucleus and cannot synthesize new COX enzymes. Hence, aspirin is highly effective for long-lived inhibition of thromboxane synthesis in platelets and is taken daily by many people for its anti-clotting action. 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 (bleeding stomach ulcers) 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: 1. Decreased production of an anti-thrombotic (clot blocking) prostaglandin (PGI2) by endothelial cells lining small blood vessels 2. Lack of inhibition of COX-1-mediated formation of pro-thrombotic thromboxanes in platelets.  Synthesis of Palmitate & Related Molecules 14 OPTIONAL READING Vioxx had a short (1999-2004) and scandalous history, which is summarized here for your reading enjoyment.