Anabolism - Biosynthesis of Fatty Acids and Lipids

ANABOLISM OUTLINE: Photosynthesis Carbon Assimilation – Calvin Cycle Carbohydrate Biosynthesis in Animals Gluconeogenesis Glycogen Synthesis Pentose-Phosphate Pathway Regulation of Carbohydrate Metabolism Anaplerotic reactions Biosynthesis of Fatty Acids and Lipids Fatty Acids contrasts location & transport Synthesis acetyl-CoA carboxylase fatty acid synthase ACP priming 4 steps Diversification of fatty acids Eicosanoids Prostaglandins and Thromboxane Triacylglycerides Membrane lipids Control of fatty acid metabolism Glycerophospholipids Sphingolipids Isoprene lipids: Cholesterol ANABOLISM II: Biosynthesis of Fatty Acids & Lipids 1 ANABOLISM II: Biosynthesis of Fatty Acids & Lipids 1. Biosynthesis of fatty acids 2. Regulation of fatty acid degradation and synthesis 3. Assembly of fatty acids into triacylglycerol and phospholipids 4. Metabolism of isoprenes a. Ketone bodies and Isoprene biosynthesis b. Isoprene polymerization i. Cholesterol ii. iii. Steroids & other molecules Regulation iv. Role of cholesterol in human disease ANABOLISM II: Biosynthesis of Fatty Acids & Lipids Lipid Biosynthesis Fatty Acid Synthesis Isoprene Biosynthesis Fat Catabolism Fatty Acid Degradation Ketone body Utilization 2 Fatty Acid Biosynthesis Catabolism Anabolism • Contrast with Sugars – Lipids have have hydro-carbons not carbo-hydrates – more reduced=more energy – Long-term storage vs short-term storage – Lipids are essential for structure in ALL organisms: membrane phospholipids • Catabolism of fatty acids –produces acetyl-CoA –produces reducing power (NADH, FADH2) –takes place in the mitochondria • Anabolism of fatty acids –requires acetyl-CoA and sufficient carbohydrates –requires reducing power from NADPH –takes place in cytosol in animals, chloroplast in plants Anabolism of fat will occur only when there is excess, carbon, electrons, and DG Acetyl-CoA & carbohydrates How is this monitored in the cell? NADPH ATP Fatty Acid Biosynthesis Cytosol for animals Fatty Acid Synthesis Chloroplast for plants Both of these compartments are where there are ample Sources of NADPH: in adipocytes: pentose phosphate pathway and malic enzyme NADPH is made as malate converts to pyruvate + CO2. in hepatocytes and mammary gland: pentose phosphate pathway NADPH is made as glucose-6-phosphate converts to ribulose 6-phosphate. in plants: photosynthesis 3 Fatty Acid Biosynthesis EXAMPLE: Synthesis of Palmitate (16:0) Where do the carbons come from? Acetyl-CoA 1. 8 acetyl-CoA x 2 carbons = 16 carbons (palmityl-CoA) 2. Longer fatty acids and desaturases use palmityl-CoA 3. 1 acetyl-CoA ”primes” the enzyme a. b. other “acetyl-CoA-derived units” are ACTIVATED by carboxylation (recall gluconeogenesis) Used to make 7 malonyl-CoAs… using? ATP & CO2 7 acetyl-CoA + 7 CO2 + 7 ATP à 7 malonyl-CoA + 7 ADP + 7 Pi ….more later 2. Seven 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+ à palmitate (16:0) + 7 CO2 + 8 CoA + 14 NADP+ + 6 H2O Fatty Acid Biosynthesis •In non-photosynthetic eukaryotes…Acetyl-CoA is made in the mitochondria •But fatty acids are made in the cytosol •So acetyl-CoA is smuggled (transported) into the cytosol (This antiporter not used as much because it moves NADH into mitochondria) – cost of 2 ATPs –Converted back to acetylCoA by citrate lyase –Therefore, cost of FA synthesis is 3 ATPs per 2-C And where does the NADPH and need for carbohydrates come from? unit….. Where is third? 4 Fatty Acid Biosynthesis •In non-photosynthetic eukaryotes…Acetyl-CoA is made in the mitochondria •But fatty acids are made in the cytosol •So acetyl-CoA is smuggled (transported) into the cytosol (This antiporter not used as much because it moves NADH into mitochondria) – cost of 2 ATPs –Converted back to acetylCoA by citrate lyase –Therefore, cost of FA synthesis is 3 ATPs per 2-C And where does the NADPH and need for carbohydrates come from? unit ….. Where is third? Fatty Acid Biosynthesis • Fatty acids are built in several passes, processing one acetate unit at a time. • The acetate is coming from activated malonate in the form of malonyl-CoA. Each pass involves reduction of a carbonyl carbon to a methylene carbon. • • Making the Malonyl-CoA: – Reaction carboxylates acetyl-CoA – Catalyzed by acetyl-CoA carboxylase 5 Fatty Acid Biosynthesis Acetyl-CoA Carboxylase Reaction: –The enzyme has three subunits: • One unit has biotin covalently linked to Lys. • Another subunit is biotin carboxylase • The third subunit is a transcarboxylase • Biotin carries CO2. • In animals, all three subunits are on one polypeptide chain. –HCO3− (bicarbonate) is the soluble source of CO2. – Two-step Rx similar to carboxylations catalyzed by pyruvate carboxylase and propionyl-CoA carboxylase • CO2 binds to biotin. - Reaction with ATP produces carboxy-phosphate. - Activated CO2 is attached to N in ring of biotin. • CO2 attaches to acetyl-CoA and leaves active site • Enzyme undergoes conformational change to carry carbamoyl to transcarboxylase site Fatty Acid Biosynthesis Fatty Acid Synthase (FAS) • Catalyzes a repeating four-step sequence that elongates the fatty acyl chain by two carbons at each step • NADPH as as the electron donor • Condensation with acetate – b-ketoacyl-ACP synthase (KS) • Reduction of carbonyl to hydroxyl – b-ketoacyl-ACP reductase (KR) • Overall goal: attach acetate unit (2carbon) from malonyl-CoA to a growing chain and then reduce it. • Reaction involves cycles of four enzymecatalyzed steps: • Dehydration of alcohol to alkene – b-hydroxyacyl-ACP dehydratase (DH) • Reduction of alkene to alkane –enoyl-ACP reductase (ER) – 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. • Chain transfer/charging –malonyl/acetyl-CoA ACP transferase (M/AT) 6 Fatty Acid Biosynthesis FAS I vs. FAS II FAS I • Single polypeptide chain in vertebrates • Leads to single product: palmitate 16:0 • C-15 and C-16 are from the acetyl-CoA used to prime the Rx • FAS I in vertebrates and fungi FAS II • Made of separate, diffusible enzymes • Makes many products (saturated, unsaturated, branched, many lengths, etc.) • Mostly in plants and bacteria FAS I What you can’t see is the ACP…... Fatty Acid Biosynthesis FAS I vs. FAS II FAS I • Single polypeptide chain in vertebrates • Leads to single product: palmitate 16:0 • C-15 and C-16 are from the acetyl-CoA used to prime the Rx • FAS I in vertebrates and fungi FAS II • Made of separate, diffusible enzymes • Makes many products (saturated, unsaturated, branched, many lengths, etc.) • Mostly in plants and bacteria FAS I What you can’t see is the ACP…... 7 Fatty Acid Biosynthesis Acyl Carrier Protein (ACP) •The E. coli ACP is a small 77-residue protein with a a covalently attached prosthetic group, 4’phosphopantetheine, at a Ser residue. – In vertebrate FAS, it’s a domain with a flexible arm to tether the growing acyl chain •Delivers acetate (in the first step) or malonate (in all the next steps) to the fatty acid synthase enzymes •Shuttles the growing chain from one active site to another during the four-step reaction Priming FAS • Two thiols must be charged with the correct acyl groups before the condensation reaction can begin. H– H- – thiol from 4-phosphopantethine in ACP – thiol from Cys b-ketoacyl-ACP synthase (KS) M A/T • The acetyl group of acetyl-CoA is transferred to ACP. 2 CoASH – catalyzed by malonyl/acetyl-CoA transferase (MAT) – ACP passes this acetate to the Cys of the KS domain of FAS 1. – ACP –SH group is recharged with malonate from malonyl-CoA again catalyzed by MAT ACP KS Synthase Fatty Acid Biosynthesis Acyl Carrier Protein (ACP) •The E. coli ACP is a small 77-residue protein with a a covalently attached prosthetic group, 4’phosphopantetheine, at a Ser residue. – In vertebrate FAS, it’s a domain with a flexible arm to tether the growing acyl chain •Delivers acetate (in the first step) or malonate (in all the next steps) to the fatty acid synthase enzymes •Shuttles the growing chain from one active site to another during the four-step reaction Priming FAS • Two thiols must be charged with the correct acyl groups before the condensation reaction can begin. H– H- – thiol from 4-phosphopantethine in ACP – thiol from Cys b-ketoacyl-ACP synthase (KS) M A/T • The acetyl group of acetyl-CoA is transferred to ACP. – catalyzed by malonyl/acetyl-CoA transferase (MAT) – ACP passes this acetate to the Cys of the KS domain of FAS 1. – ACP –SH group is recharged with malonate from malonyl-CoA again catalyzed by MAT 2 CoASH ACP KS Synthase 8 Fatty Acid Biosynthesis Note that malonyl-CoA and acetyl-CoA have already been attached to complex via thioester linkages to enzyme and have shed their CoA attachments. Step 1: Condensation reaction attaches two C from acetyl group (or longer fatty acyl chain) to two C from malonyl group. - Acetoacetyl-ACP release of CO2 activates malonyl group for attachment creates b-keto intermediate (acetoacetyl-ACP) Catalyzed by b-ketoacyl-ACP synthase (KS) Step 2: First reduction: NADPH reduces the b-keto intermediate to an alcohol. – – – carbonyl at C-3 reduced to form D-b-hydroxybutyryl-ACP NADPH is 2e− donor catalyzed by b-ketoacyl-ACP reductase (KR) D-b-hydroxybutyryl-ACP Step 3: Dehydration: OH group from C-2 and H from neighboring CH2 are eliminated, creating double bond (transalkene). – – 2 OH and H removed from C-2 and C-3 of b-hydroxybutyryl-ACP trans-D -Butenoyl-ACP (Crotonyl-ACP) to form trans-D2-butenoyl-ACP catalyzed by b-hydroxyacyl-ACP dehydratase (DH) Step 4: Second reduction: NADPH reduces double bond to yield saturated alkane. – – Butyryl-ACP NADPH is the electron donor to reduce double bond of transD2-butenoyl-ACP to form butyryl-ACP. catalyzed by enoyl-ACP reductase (ER) Fatty Acid Biosynthesis Palmitate (16:0) Thioesterase H2 O H2O 9 Fatty Acid Biosynthesis Condensation, reduction, dehydration, reduction, translocation, ACP-loading ① ② H2O H2O Palmitate (16C) Synthesis H2O What is this like? 16H+ + 16e– Protein synthesis; where the peptidyl-tRNA puts the growing peptide onto the incoming AA-tRNA 4CO2 7H2O 7 acetyl-CoA + 7 CO2 + 7 ATP à 7 malonyl-CoA + 7 ADP + 7 Pi acetyl-CoA + 7 malonyl-CoA + 14 NADPH + 14 H+ à palmitate (16:0) + 7 CO2 + 8 CoA + 14 NADP+ + 6 H2O 8 acetyl-CoA + 14 NADPH + 14 H+ + 7 ATP à palmitate (16:0) + 8 CoA + 14 NADP+ + 7 ADP + Pi + 6 H2O If you add in the transport (8x2=16): 23 ATP Fatty Acid Biosynthesis 1 2 3 4 5 6 Comparison of Fatty-Acid Degradation versus Fatty-Acid Synthesis 10 Fatty Acid Biosynthesis 1 2 3 4 5 6 Comparison of Fatty-Acid Degradation versus 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). 11 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. -S Co A Malonyl-CoA b-oxidation • Blocking b oxidation with initial committed product of fatty-acid synthesis: malonyl-CoA Reciprocal Control of FattyAcid Degradation versus Fatty-Acid Synthesis ensures that fat synthesis and oxidation don’t occur simultaneously 12