Weill Cornell Medicine-Qatar Principles of Biochemistry Lecture 23b PDF

Principles of Biochemistry SPRING 2024 Professor: Moncef LADJIMI [email protected] Office: C-169 As faculty of Weill Cornell Medical College in Qatar we are committed to providing transparency for any and all external relationships prior to giving an academic presentation. I, Moncef LADJ...

Principles of Biochemistry SPRING 2024 Professor: Moncef LADJIMI [email protected] Office: C-169 As faculty of Weill Cornell Medical College in Qatar we are committed to providing transparency for any and all external relationships prior to giving an academic presentation. I, Moncef LADJIMI DO NOT have a financial interest in commercial products or services. Lecture 23b Lipid Metabolism: Fatty Acid Biosynthesis Additional material for this lecture may be found in: § Lehninger’s Biochemistry (8th ed), chapter 21:p. 744-771 OULINE OF FATTY ACIDS BIOSYNTHESIS § Origin of cytoplasmic acetyl-CoA § The acetyl-CoA carboxylase (ACC) reaction § Fatty acid synthesis § The mammalian fatty acid synthase § Regulation of fatty acids biosynthesis § Synthesis of long-chain fatty acids § Desaturation of fatty acids CATABOLISM AND ANABOLISM OF FATTY ACIDS PROCEED VIA DIFFERENT PATHWAYS Catabolism (oxidation) of fatty acids – produces acetyl-CoA – produces reducing power (NADH) – takes place in the mitochondria Anabolism (synthesis) of fatty acids – requires acetyl-CoA and malonyl-CoA – requires reducing power from NADPH – takes place in cytosol in animals, chloroplast in plants OVERVIEW OF FATTY ACID SYNTHESIS Fatty acids are built in several passes, processing one acetate unit at a time (by the fatty acid synthase complex in the cytoplasm where NADPH/NADP is high). The acetate is coming from activated malonate in the form of malonyl-CoA (a 3-Carbons intermediate) Formation of malonate is catalyzed by Acyl CoA Carboxylase (ACC) Each pass involves reduction of a carbonyl carbon to a methylene carbon. Synthesis may be viewed as b-oxidation in reverse, but – uses different enzymes and cofactors – Takes place in a different subcellular location The end-product is palmitoyl-CoA which inhibits Acyl CoA Carboxylase COMPARISON OF STEPS IN FATTY ACID SYNTHESIS & β-OXIDATION β-oxidation SPLIT synthesis CONDENSATION OXIDATION REDUCTION HYDRATION DEHYDRATION OXIDATION REDUCTION ACETYL-COA IS TRANSPORTED FROM MITOCHONDRIA INTO THE CYTOSOL AS CITRATE Acetyl-CoA is made in the mitochondria But lipid synthesis occurs in the cytosol, and there is no way for acetyl-CoA to cross mitochondrial inner membrane to the cytosol So, acetyl-CoA is converted to citrate. – Acetyl-CoA + oxaloacetate à citrate (in mitochondria) Same reaction as that of the CAC Catalyzed by citrate synthase Citrate is then shuttled to the cytosol: Citrate passes through the citrate transporter CITRATE IN THE CYTOSOL IS CLEAVED TO REGENERATE ACETYL-COA Citrate (now in cytosol) is cleaved by citrate lyase – Regenerates acetyl-CoA and oxaloacetate (in cytosol) – The reaction requires 1 ATP – Acetyl-CoA can now be used to make malonyl-CoA for lipid synthesis – What happens to the cytoplasmic oxaloacetate? (there is no oxaloacetate transporter in mitochondria) OXALOACETATECYT IS CONVERTED TO MALATE, WHICH HAS TWO FATES Malate dehydrogenase in cytosol reduces oxaloacetate to malate Two potential fates for malate: – Can be converted to pyruvatecyt and NADPHcyt via the malic enzyme (major route) NADPH used for lipid synthesis Pyruvatecyt sent back to mitochondria via pyruvate transporter Pyruvate is converted back in mitochondria to oxaloacetatemt by pyruvate carboxylase, requiring 1 ATP – Can be transported back to mitochondria via Malate-a-ketoglutarate transporter Malatemt is then reoxidized to oxaloacetatemt SHUTTLE FOR TRANSFER OF ACETYL GROUPS FROM MITOCHONDRIA TO THE CYTOSOL The mitochondrial outer membrane is freely permeable to all these compounds. Pyruvate derived from amino acid catabolism in the mitochondrial matrix, or from glucose by glycolysis in the cytosol, is converted to acetyl-CoA in the matrix. Acetyl groups pass out of the mitochondrion as citrate; in the cytosol they are delivered as acetylCoA for fatty acid synthesis. Oxaloacetate is reduced to malate, which can return to the mitochondrial matrix and is converted to oxaloacetate. The major fate for cytosolic malate is oxidation by malic enzyme to generate cytosolic NADPH; the pyruvate produced returns to the mitochondrial matrix. ACETYL-CoACYT AND BICARBONATE FORM MALONYL-CoA Reaction carboxylates acetyl CoA Catalyzed by Acetyl-CoA Carboxylase (ACC) (rate limiting step of fatty acid synthesis) – Enzyme has three subunits: One unit has Biotin to carry CO2 HCO3− (bicarbonate) is the source of CO2 Uses one ATP molecule In animals, all three subunits are on one polypeptide chain Therefore, the cost of malonyl CoA formation (precursor for fatty Acid synthesis) is 3 ATPs per 2-carbons unit (2 for shuttling acetyl-CoA to the cytosol and 1 for making malonyl-CoA) A REMINDER: BIOTIN CARRIES CO2 Two-step reaction similar to carboxylations catalyzed by pyruvate carboxylase and propionyl-CoA carboxylase CO2 binds to biotin - Bicarbonate react with ATP to produce carboxy phosphate as an intermediate - Carboxyphosphate breaks down to CO2 - CO2 is attached to N in ring of biotin - Biotin transports CO2 to acetyl CoA on another active site THE ACETYL-COA CARBOXYLASE (ACC) REACTION Malonyl-CoA is formed from Acetyl-CoA and Bicarbonate ACA has 3 functional regions: § Biotin carrier protein (gray) § Biotin carboxylase, which activates CO2 by attaching it to a nitrogen in the biotin ring in an ATP-dependent reaction § Transcarboxylase, which transfers activated CO2 (shaded green) from biotin to acetyl-CoA, producing malonyl-CoA The long, flexible biotin arm carries the activated CO2 from the biotin carboxylase region to the transcarboxylase active site. The active enzyme in each step is shaded blue FATTY ACID SYNTHESIS OCCURS IN CELL COMPARTMENTS WHERE NADPH LEVELS ARE HIGH Cytosol for animals, yeast Chloroplast for plants Major sources of NADPH*: – In adipocytes: pentose phosphate pathway and malic enzyme NADPH made as malate converts to pyruvate + CO2 – In hepatocytes and mammary gland: pentose phosphate pathway NADPH made as glucose-6phosphate converts to ribulose 6phosphate – In plants: photosynthesis Two routes to NADPH, catalyzed by (a) malic enzyme (b) the pentose phosphate pathway * NADP dependent Isocitrate dehydrogenase to a lesser extent SYNTHESIS OF FATTY ACIDS IS CATALYZED BY FATTY ACID SYNTHASE (FAS) Catalyzes a repeating four-step sequence that elongates the fatty acyl chain by two carbons at each step – Uses NADPH as as the electron donor – Uses two enzyme-bound -SH groups as activating groups FAS I in vertebrates Single polypeptide chain Leads to single product: palmitate 16:0 C-15 and C-16 are from the acetyl CoA used to prime the reaction FATTY ACID SYNTHESIS Overall goal: attach two-Carbon acetate unit from malonyl-CoA to a growing chain and then reduce it Reaction involves cycles of four enzyme-catalyzed steps – Condensation of the growing chain with activated acetate – Reduction of carbonyl to hydroxyl – Dehydration of alcohol to trans-alkene – Reduction of alkene to alkane The growing chain is initially attached to the enzyme via a thioester linkage During condensation, the growing chain is transferred to the acyl carrier protein (ACP) After the second reduction step, the elongated chain is transferred back to fatty acid synthase ACYL CARRIER PROTEIN (ACP) SERVES AS A SHUTTLE IN FATTY ACID SYNTHESIS Contains a covalently attached prosthetic group 4’-phosphopantetheine – Flexible arm to tether acyl chain while carrying intermediates from one enzyme subunit to the next Delivers acetate (in the first step) or malonate (in all the next steps) to the fatty acid synthase Shuttles the growing chain from one active site to another during the fourstep reaction The prosthetic group is 4-phosphopantetheine, which is covalently attached to the hydroxyl group of a Ser residue in ACP. Phosphopantetheine contains the B vitamin pantothenic acid, also found in the coenzyme A molecule. Its —SH group is the site of entry of malonyl groups during fatty acid synthesis. FATTY ACID SYNTHASE TYPE I (FAS I) Mammalian FATTY ACID SYNTHASE (FAS I): a mega synthase with multiple active sites Condensation with acetate – b-ketoacyl-ACP synthase (KS) Reduction of carbonyl to hydroxyl – b-ketoacyl-ACP reductase (KR) Dehydration of alcohol to alkene – b-hydroxyacyl-ACP dehydratase (DH) Reduction of alkene to alkane – enoyl-ACP reductase (ER) Chain transfer/charging – Malonyl/acetyl-CoA ACP transferase (MAT) Thioesterase – TE: releases the palmitate product from ACP ACP – Acyl Carrier Protein A homodimer of a single 240 kD polypeptide with all catalytic activities for the synthetic cycle FATTY ACID SYNTHESIS PROCEEDS IN A REPEATING REACTION SEQUENCE (1) Addition of two carbons to a growing fatty acyl chain: a four-step sequence Acyl Carrier Protein KS KS § Each malonyl group (on Acyl Carrier Protein; ACP) and acetyl (or longer acyl) group is activated by a thioester that links it to fatty acid synthase, then: 1. Condensation of an activated acyl group (an acetyl group from acetyl-CoA is the first acyl group) and two carbons derived from malonylCoA, with elimination of CO2 from the malonyl group, extends the acyl chain by two carbon 2. Reduction of the b-keto group to an alcohol KR KR FATTY ACID SYNTHESIS PROCEEDS IN A REPEATING REACTION SEQUENCE (2) 3. Dehydration (elimination of H2O) to create a double bond DH 4. Reduction of the double bond to create saturated fatty acyl group 5. Translocation of elongated product to SH on FAS and recharging of now free SH of ACP with another malonyl group (not shown here) ER THE OVERALL PROCESS OF PALMITATE SYNTHESIS: SEVEN CYCLES OF CONDENSATION AND REDUCTION The fatty acyl chain grows by two-carbon units donated by activated malonate, with loss of CO2 at each step. The initial acetyl group is shaded yellow, C-1 and C-2 of malonate are shaded pink, and the carbon released as CO2 is shaded green. After each two-carbon addition, reductions convert the growing chain to a saturated fatty acid of four, then six, then eight carbons, and so on. The final product is palmitate (16:0). STOICHIOMETRY OF SYNTHESIS OF PALMITATE (16:0) 1. Formation of 7 malonyl CoA : 7 acetyl-CoAs are carboxylated to make 7 malonyl-CoAs… using ATP: 7 Acetyl-CoA + 7 CO2 + 7 ATP -> 7 Malonyl-CoA + 7 ADP + 7 Pi 2. 7 Cycles of condensation, reduction dehydration and reduction… using NADPH to reduce the b-keto group and trans-double bond: Acetyl-CoA + 7 Malonyl-CoA + 14 NADPH + 14 H+ -> Palmitate + 7 CO2 + 8 CoA + 14 NADP+ + 6 H2O 3. Sum: 8 Acetyl-CoA + 7 ATP + 14 NADPH + 14 H+ -> Palmitate + 8 CoA + 6 H2O + 7 ADP + 7 Pi + 14 NADP+ Note: Eukaryotes have one additional energy cost (the two ATP needed to shuttle acetyl CoA to the cytosol). Therefore, the cost of Fatty Acid synthesis is 3 ATPs per 2-Carbons unit (2 for shuttling acetyl-CoA to the cytosol and 1 for making malonyl-CoA) minus 1 ATP for the first acetyl CoA (which is not made from malonyl CoA). FATTY ACID SYNTHESIS IS TIGHTLY CONTROLLED VIA ALLOSTERIC REGULATION OF ACC Acetyl CoA carboxylase (ACC) catalyzes the rate-limiting step – ACC is feedback-inhibited by palmitoyl-CoA – ACC is activated by citrate Remember citrate is made from acetyl-CoAmt When [acetyl-CoA]mt increases (with concomitant increase in ATP), signaling energy excess, acetylCoA is converted to citrate…citrate is exported to cytosol, and ACC is activated. Thus, citrate signals excess energy to be converted to fat. Note: Citrate inhibits also glycolysis by inhibiting PFK-1 ACC IS ALSO HORMONALLY REGULATED BY COVALENT MODIFICATION ACC is inhibited when energy is needed (low blood glucose) through: – Glucagon and epinephrine: reduce sensitivity of citrate activation lead to phosphorylation and inactivation of ACC (phosphorylated monomers of ACC are inactive) ACC is activated when energy is plenty (high blood glucose) through: – Insulin: leads to dephosphorylation of ACC monomers which polymerize into long active ACC filaments REGULATION OF FATTY ACID SYNTHESIS IN VERTEBRATES Both allosteric regulation and hormone-dependent covalent modification influence the flow of precursors into malonyl-CoA Regulation of fatty acid synthesis: Allosteric Hormonal Insulin triggers dephosphorylation and activation (ACC polymerization) (ACC depolymerization) Active form of ACC (a) In the cells of vertebrates, both allosteric regulation and hormone-dependent covalent modification influence the flow of precursors into malonyl-CoA. (b) Filaments of acetyl-CoA carboxylase (the active, dephosphorylated form) as seen with the electron microscope. COORDINATED REGULATION OF FATTY ACIDS SYNTHESIS AND BREAKDOWN * *Glucagon also triggers mobilization of fatty acids from lipid droplets in adipose tissue (through phosphorylation of HSL) REGULATION TARGETS Synthesis: Acetyl-CoA Carboxylase (ACC) Oxidation: Carnitine Acyl Transferase I (CAT I) When the diet provides a ready source of carbohydrate as fuel, β oxidation of fatty acids is unnecessary and is therefore downregulated. Two enzymes are key to the coordination of fatty acid metabolism: acetyl-CoA carboxylase (ACC), the first enzyme in the synthesis of fatty acids), and carnitine acyltransferase I, which limits the transport of fatty acids into the mitochondrial matrix for β oxidation. Ingestion of a high-carbohydrate meal raises the blood glucose level and thus 1 triggers the release of insulin. 2 Insulin-dependent protein phosphatase dephosphorylates ACC, activating it. 3 ACC catalyzes the formation of malonyl-CoA (the first intermediate of fatty acid synthesis), and 4 malonyl-CoA inhibits carnitine acyltransferase I, thereby preventing fatty acid entry into the mitochondrial matrix. When blood glucose levels drop between meals, 5 glucagon release activates cAMP-dependent protein kinase (PKA), which 6 phosphorylates and inactivates ACC. The concentration of malonyl-CoA falls, the inhibition of fatty acid entry into mitochondria is relieved, and 7 fatty acids enter the mitochondrial matrix and 8 become the major fuel. Because glucagon also triggers the mobilization of fatty acids in adipose tissue, a supply of fatty acids begins arriving in the blood. Also, transcriptional regulation of CAT, acyl CoA dehydrogenases... LONGER-CHAIN AND UNSATURATED FATTY ACIDS ARE SYNTHESIZED FROM PALMITATE Routes of synthesis of other fatty acids: Elongation of Palmitate (16:0) à stearate(18:0) – Catalyzed by elongase (works by adding two carbons at the COOH end, after activation, and using malonylCoA, just like FAS. (Elongases) (Desaturases) Desaturation of Palmitate (16:0) à palmitoleate (16:1; D9), Stearate (18:0) à oleate (18;1; D9): – Catalyzed by fatty acyl-CoA desaturase in animals – Requires NADPH; enzyme uses cytochrome b5 and cytochrome b5 reductase, see next slide. (Note that this is a D9-desaturase! It reduces the bond between C-9 and C-10). – Mammals cannot desaturate beyond, thus cannot convert oleate to linoleate or α-linolenate (shaded w3 precursor pink), which are therefore required in the diet as essential fatty acids. ALA – Conversion of a-linolenate (a-ALA, essential fatty acid) to other polyunsaturated fatty acids (EPA, 20:5(D5,8,11,14,17), DHA 22:6(D4,7,10,13,16,19) and eicosanoids by elongation/desaturation EPA, DHA is outlined. w6 precursor PLANTS, BUT NOT MAMMALS, CAN DESATURATE POSITIONS BEYOND C-9 Humans have D4, D5, D6, and D9 desaturases but cannot desaturate beyond D9 Plants can produce: – linoleate 18:2(D9,12) – a-linolenate 18:3 (D9,12,15) These fatty acids are “essential” to humans – Polyunsaturated fatty acids (PUFAs) help control membrane fluidity – PUFAs are precursors to eicosanoids VERTEBRATE FATTY ACYL DESATURASE IS A NON-HEME, IRON-CONTAINING, MIXED FUNCTION OXIDASE O2 accepts four electrons from two substrates – Two electrons come from saturated fatty acid – Two electrons come from ferrous state of cytochrome b5/NADPH Electron transfer in the desaturation of fatty acids in vertebrates: Blue arrows show the path of electrons as two substrates—a fatty acyl–CoA and NADPH—undergo oxidation by molecular oxygen. These reactions take place on the lumenal face of the smooth ER. A similar pathway, but with different electron carriers, occurs in plants. BIOSYNTHESIS OF TRIACYLGLYCEROLS 1/ Synthesis of Phosphatidic Acid 2/ Conversion of Phosphatidic from glycerol-P and acyl-CoAs Acid into Triacylglycerol Phosphatidic acid is the precursor of both TAGs and Glycerophospholipids Phosphatidic acid phosphatase (lipin) removes the 3phosphate from the phosphatidic acid – Yields 1,2diacylglycerol Third carbon is then acylated with a third fatty acid – Yields triacylglycerol BIOSYNTHESIS OF MEMBRANE LIPIDS PHOSPHODIESTER BOND OF PHOSPHOLIPIDS FROM CDP (See also next slide) 1/ DAG is activated with CDP 2/ the head group is transferred (Example: Inositol àphosphatidylinositol) 1/ Headgroup is activated with CDP 2/ DAG is transferred. (Example: CDPcholine à phosphatidylcholine) Two general strategies for forming the phosphodiester bond of phospholipids. In both cases, CDP supplies the phosphate group of the phosphodiester bond. SPHINGOLIPIDS ARE MADE IN FOUR STEPS 1) Synthesis of sphinganine from palmitoylCoA and serine 2) Attachment of fatty acid via amide linkage 3) Desaturation of sphinganine Yields N-acylsphingosine (ceramide) 4) Attachment of head group Can yield a cerebroside or ganglioside SUMMARY: BIOSYNTHESIS AND DEGRADATION OF FATTY ACIDS § Fatty acid synthesis and degradation: 4-step cycles to add/remove 2-carbon units; ~reverse of each other; in different compartments, using different enzymes. § Coordinately regulated; ACC and carnitine acyltransferase I are key enzymes that are the targets of regulation. Remember to prepare for next lecture: Lehninger’s Biochemistry (8th ed), §chapter 18: p. 625-644