Lipid Synthesis - Control of Fatty-Acid Synthesis

Fatty Acid Biosynthesis Control of Fatty-Acid Synthesis • Acetyl CoA carboxylase (ACC) catalyzes the committal step. • Allosteric Control – Inhibited when energy is needed, fatty acids are acylated for degradation, and inhibit ACC. • ACC is feedback-inhibited by palmitoyl-CoA. – ACC is activated by citrate. • Citrate is made from acetyl-CoA in mitochondria (acetyl-CoAmt). • Citrate signals excess energy to be converted to fat. – When [acetyl-CoA]mt ­ it is converted to citrate… citrate is exported to cytosol. • Hormonal Control – Glucagon and epinephrine: leads to activation of AMP-dependent Protein Kinase (AMPK) • Phosphorylation inactivates ACC • Phosphorylation reverses the polymerization à monomers (inactive) –Insulin: leads to activation of Protein Phosphatase 2A • Dephosphorylation activates ACC • When dephosphorylated, ACC polymerizes into long filaments (active) • Changes in gene expression – example: excess of certain polyunsaturated fatty acids (eicosanoids) bind to transcription factors called peroxisome proliferator-activated receptors (PPARS). 1 ANABOLISM II: Biosynthesis of Fatty Acids & Lipids Fatty Acid Biosynthesis Recall how b-oxidation starts: Acyl-Carnitine/Carnitine Transport: • b oxidation of fatty acids occurs in mitochondria. • If fatty acyl-CoAs are not transported in, they cannot be degraded • Transport is via carnitine transporter. • Blocking b oxidation with initial committed product of fatty-acid synthesis: malonyl-CoA -S Co A Malonyl-CoA b-oxidation Allosteric Reciprocal Control of FattyAcid Degradation versus Fatty-Acid Synthesis ensures that fat synthesis and oxidation don’t occur simultaneously 2 Fatty Acid Biosynthesis Diversification of Palmitate: Elongation Fatty Acid Elongation Systems • Elongation systems in the endoplasmic reticulum and mitochondria create longer fatty acids. • Use CoA derivatives • As in palmitate synthesis, each step adds units of 2 C. Use malonyl-CoA. • Stearate (18:0) is the most common product. Plants can produce: –linoleate 18:2(D9,12 ) –a-linolenate 18:3 (D9,12,15 ) • Plants make linoleate (Ω6) and α-linolenate (Ω3), animals can’t; But they can elongate these ingested essential FA • These fatty acids are “essential” to humans. –Polyunsaturated fatty acids (PUFAs) help control membrane fluidity. –PUFAs are precursors to eicosanoids elongation • The ratio of omega-6 to omega-3 fatty acids in the diet, if too high, can lead to cardiovascular disease Eicosanoids elongation PG = Prostaglandins TX = Thromboxanes LT = Leukotrienes Fatty Acid Biosynthesis Diversification of Palmitate: Fatty Acyl-CoA Desaturase Desaturation • Looks like a mixed-function oxidase: OXIDASE • Humans have D4, D5, D6, and D9 desaturases but cannot desaturate beyond D9. FOR EXAMPLE: for a D9-desaturation Palmitate(16:0) à palmitoleate(16:1, D9) Stearate (18:0) à oleate (18:1, D9) catalyzed by fatty acyl-CoA desaturase in animals red ox ox ox ox red (Fe3+ ) m = 7, 4, 3, 2 (D9 , D6 , D5 , D4 ) red ox In plants & bacteria the desaturases work on PL, not fatty-acyl CoA; needed for rapid changes in fluidity (Fe2+ ) red red O2⇀ 2H2O red ox requires NADPH; enzyme uses cytochrome b5 and cytochrome b5 reductase Both donate electrons to O2 (total of 4) 3 Fatty Acid Biosynthesis Animals can do, plants can do Omega 3 D15 Eicosanoids: Synthesis and Classes D6 desaturation elongation desaturation • Linoleate (18:2(Δ9,12) gets desaturated on both sides = γ-linolenate (18:3(Δ6,9,12) & α-linolenate (18:3(Δ9,12,15). • Elongation to C20, and further desaturation of γ-linolenate (18:3(Δ6,9,12) gives Arachidonate, 20:4(Δ5,8,11,14) hydroxylation elongation desaturation hydroxylation hydroxylation • Elongation to C20, and TWO further desaturation of α-linolenate (18:3(Δ9,12,15) gives EPA, 20:5(Δ5,8,11,14,17). More of the same gets DHA, 22:6(Δ4,7,10,13,16,19) • These desaturations are all going closer to the carboxylate and can be accomplished by animals. Eicosanoids (20C Polyunsaturated fatty acids (PUFAs)) bioactive paracrine From these signaling molecules are made: PG = Prostaglandins TX = Thromboxanes LT = Leukotrienes elongation • Created from the arachidonate or EPA, which are incorporated into membranes phospholipids. • In response to stimuli (hormone, etc.), phospholipase A2 is activated • The unsaturated C-2 fatty acid is released elongation Fatty Acid Biosynthesis Animals can do, plants can do Omega 3 D15 Eicosanoids: Synthesis and Classes D6 desaturation elongation desaturation (EET) • Elongation to C20, and further desaturation of γ-linolenate (18:3(Δ6,9,12) gives Arachidonate, 20:4(Δ5,8,11,14) hydroxylation (ARA) elongation desaturation hydroxylation • Linoleate (18:2(Δ9,12) gets desaturated on both sides = γ-linolenate (18:3(Δ6,9,12) & α-linolenate (18:3(Δ9,12,15). hydroxylation • Elongation to C20, and TWO further desaturation of α-linolenate (18:3(Δ9,12,15) gives EPA, 20:5(Δ5,8,11,14,17). More of the same gets DHA, 22:6(Δ4,7,10,13,16,19) • These desaturations are all going closer to the carboxylate and can be accomplished by animals. Eicosanoids (20C Polyunsaturated fatty acids (PUFAs)) bioactive paracrine From these signaling molecules are made: PG = Prostaglandins TX = Thromboxanes LT = Leukotrienes • Created from the arachidonate or EPA, which are incorporated into membranes phospholipids. • In response to stimuli (hormone, etc.), phospholipase A2 is activated • The unsaturated C-2 fatty acid is released 4 Fatty Acid Biosynthesis Eicosanoids: Cyclic Pathway • PGH2 synthase (cyclooxygenase, or COX) has isozymes • COX-1 is ubiquitous and catalyzes synthesis of prostaglandins that regulate gastric mucin secretion and other smooth muscle functions • COX-2 is inducible (except in brain) by immune response and acts at the sites of inflammation. It catalyzes synthesis of prostaglandins that mediate pain, inflammation, and fever. Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit COX isozymes • Aspirin (acetylsalicylate) is an irreversible inhibitor. – acetylates a Ser in active site – blocks active site in both COX isozymes PGH2 synthase is a cyclooxygenase/peroxidase enzyme that functions in the smooth ER. • Ibuprofen, naproxen, and indomethacin are competitive inhibitors. •Step 1: PGH2’s cyclooxygenase activity adds 2 O2 to form PGG2. – resemble substrate; also block active site in both isozymes •Step 2: PGH2’s peroxidase activity converts peroxide to alcohol, creates PGH2. Bengt Samuelsson Nobel Prize 1982 Celebrex Indomethacin • Celebrex and Vioxin are specific for COX-2 Fatty Acid Biosynthesis Eicosanoids: Cyclic Pathway • PGH2 synthase (cyclooxygenase, or COX) has isozymes • COX-1 is ubiquitous and catalyzes synthesis of prostaglandins that regulate gastric mucin secretion and other smooth muscle functions • COX-2 is inducible (except in brain) by immune response and acts at the sites of inflammation. It catalyzes synthesis of prostaglandins that mediate pain, inflammation, and fever. Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit COX isozymes • Aspirin (acetylsalicylate) is an irreversible inhibitor. – acetylates a Ser in active site – blocks active site in both COX isozymes PGH2 synthase is a cyclooxygenase/peroxidase enzyme that functions in the smooth ER. • Ibuprofen, naproxen, and indomethacin are competitive inhibitors. •Step 1: PGH2’s cyclooxygenase activity adds 2 O2 to form PGG2. – resemble substrate; also block active site in both isozymes •Step 2: PGH2’s peroxidase activity converts peroxide to alcohol, creates PGH2. Bengt Samuelsson Nobel Prize 1982 Celebrex Indomethacin • Celebrex and Vioxin are specific for COX-2 5 Fatty Acid Biosynthesis Different Series EET (3) ARA(4) PGE1 Prostaglandin & Thromboxane: Synthesis and Classes PGF1 a Different Classes PGE2 PGF2 a EPA (5) Series 1 Series 2 PGF3 a PGE3 PG = Prostaglandins/TB=Thromboxane –letters-after are for number and oxidation state of oxygens and ring –subscript is the number of double bonds Series 2 prostaglandins have important roles in the immediate response to stress or injury, including inflammation, Fatty Acid Biosynthesis Thromboxane: Synthesis and Classes TB=Thromboxane –letters after are for number and oxidation state of oxygens and ring –subscript is the number of double bonds Platelet aggregation and wound healing 6 Fatty Acid Biosynthesis Eicosanoids: Linear Pathway Synthesis of Leukotrienes /peroxidase (LOX) 3-lipoxygenase (3-LOX) LOX LTA4 Hepoxilin A3 GSH glutathione-Stransferase Psoriasis •O2 added to arachidonate via lipoxygenases (mixedLTB function oxidases) (LOX) •Creates species that differ in the position of the OOH group •LOX enzymes are found in leukocytes and in heart, brain, lung, and spleen, are mixed-function oxidases of the cytochrome P-450 family. •Inflammatory chemicals released after contact with an allergen; airways constrict, excess mucus and fluid produced (e.g., asthma; difficult to breathe). 4 LCC4 g-glutamyl transpeptidase Glu LCD4 Gly cysteinyl-glycine dipeptidase LTE4 ANABOLISM II: Biosynthesis of Fatty Acids & Lipids Lipid Biosynthesis Fatty Acid Synthesis Fat Catabolism Fatty Acid Degradation Ketone body Utilization 7 Lipid Biosynthesis Biosynthesis of Triacylglycerol Fat (Triacylglycerol) and Phospholipids in Animals, Plants, and Bacteria have a common intermediate: Phosphophatidic Acid Usually saturated FA Lysophosphatidate Usually unsaturated FA • Animals and plants store fat for fuel. – plants: in seeds, nuts – typical 70-kg human has ~15 kg fat • enough to last 12 weeks • compare with 12 hours worth of glycogen in liver and muscle • All organisms make glycerophospholipids for membranes. • Both molecules contain glycerol from Glycerol 3-P • Most Glycerol 3-P comes from dihydroxyacetone phosphate (DHAP) in glycolysis. Phosphatidate – via glycerol 3-phosphate dehydrogenase 1,2 Diacylglycerol • Minor amount from glycerol (liver and kidney). – via glycerol kinase • Phosphatidic acid is the precursor to fat and glycerophospholipids. – fatty acids attached by acyl transferases – releases CoA Triacylglycerol Lipid Biosynthesis Biosynthesis of Triacylglycerol Fat (Triacylglycerol) and Phospholipids in Animals, Plants, and Bacteria have a common intermediate: Phosphophatidic Acid Usually saturated FA Lysophosphatidate Usually unsaturated FA • Animals and plants store fat for fuel. – plants: in seeds, nuts – typical 70-kg human has ~15 kg fat • enough to last 12 weeks • compare with 12 hours worth of glycogen in liver and muscle • All organisms make glycerophospholipids for membranes. • Both molecules contain glycerol from Glycerol 3-P • Most Glycerol 3-P comes from dihydroxyacetone phosphate (DHAP) in glycolysis. – via glycerol 3-phosphate dehydrogenase • Minor amount from glycerol (liver and kidney). Phosphatidate 1,2 Diacylglycerol – via glycerol kinase • Phosphatidic acid is the precursor to fat and glycerophospholipids. – fatty acids attached by acyl transferases – releases CoA Triacylglycerol 8 Lipid Biosynthesis Regulation of Triacylglycerol Synthesis by Insulin • Secretion of Insulin results in stimulation of triacylglycerol synthesis. • Lack of insulin results in: Fat ▲ –increased lipolysis –increased fatty acid oxidation Fatty Acids • And when citric acid cycle intermediates (oxaloacetate) are depleted (as in diabetes when glucose cannot get in), acetylCoA builds up and ketones bodies are made –Repression of fatty acid synthase (PP2A) –ACC is not active –No malonyl-CoA means boxidation is active b-oxidation Fatty Acid Synthase Lipid Biosynthesis Regulation of Triacylglycerol Synthesis by Insulin • Secretion of Insulin results in stimulation of triacylglycerol synthesis. • Lack of insulin results in: Fat ▲ –increased lipolysis –increased fatty acid oxidation Fatty Acids • And when citric acid cycle intermediates (oxaloacetate) are depleted (as in diabetes when glucose cannot get in), acetylCoA builds up and ketones bodies are made –Repression of fatty acid synthase (PP2A) –ACC is not active –No malonyl-CoA means boxidation is active b-oxidation Fatty Acid Synthase Diabetics prone to metabolic ketosis and acidosis 9 Lipid Biosynthesis Biosynthesis of Membrane Phospholipds Glycerophospholipid Biosynthesis requires Attach head group by condensation here: Activation by CTP • Begin with phosphatidic acid (microorganisms) or 1,2 Diacylglycerol (mammals) • Both activate precursors using CTP • Bacteria use phosphatidate and attach head group to C-3 phosphate group mammals bacteria – Make CDP-diacylglycerol from CTP and phosphatidic acid • Mammals use CDP-alcohol and attach Example: head group to diacylglycerol – Make CDP-alcohol from CTP and choline or ethanolamine Lipid Biosynthesis Biosynthesis of Membrane Phospholipds Glycerophospholipid Biosynthesis requires Attach head group by condensation here: Activation by CTP • Begin with phosphatidic acid (microorganisms) or 1,2 Diacylglycerol (mammals) • Both activate precursors using CTP • Bacteria use phosphatidate and attach head group to C-3 phosphate group mammals bacteria – Make CDP-diacylglycerol from CTP and phosphatidic acid • Mammals use CDP-alcohol and attach Example: head group to diacylglycerol – Make CDP-alcohol from CTP and choline or ethanolamine 10 Lipid Biosynthesis Glycerophospholipid Synthesis in E. coli • For Phosphatidylserine and phosphatidylinositol, the free alcohol does a nucleophilic attack on the CDP-activated phosphatidate. • Phosphatidylserine is decarboxylated to phosphatidylethanolamine. • enzyme is phosphatidylserine decarboxylase • Phosphatidylethanolamine acted on by S-adenosylmethionine (SAM) adds three methyl groups to amino group à phopshatidylcholine (lecithin). • catalyzed by methyltransferase • Cardilipin is from the condensation of two phosphatidyl glycerols, which are formed from glycerol 3-phosphate. Lipid Biosynthesis Glycerophospholipid Synthesis in E. coli • For Phosphatidylserine and phosphatidylinositol, the free alcohol does a nucleophilic attack on the CDP-activated phosphatidate. • Phosphatidylserine is decarboxylated to phosphatidylethanolamine. • enzyme is phosphatidylserine decarboxylase • Phosphatidylethanolamine acted on by S-adenosylmethionine (SAM) adds three methyl groups to amino group à phopshatidylcholine (lecithin). • catalyzed by methyltransferase • Cardilipin is from the condensation of two phosphatidyl glycerols, which are formed from glycerol 3-phosphate. 11