Carbohydrate Biosynthesis in Animals

OUTLINE: ANABOLISM I: Carbohydrates Introduction and review Transport Glycogenolysis Glycolysis Other sugars Pasteur: Anaerobic vs Aerobic Exam-1 material Fermentations Pyruvate Krebs’ Cycle Oxidative Phosphorylation Exam-3 material Fatty acid Catabolism Stage One – Rubisco Carboxylase Know mechanism Oxygenase Glycolate cycle Stage Two – making sugar Stage Three - remaking Ru 1,5P2 Exam-2 material Electron transport Chemiosmotic theory: Phosphorylation Fat Catabolism Carbon Assimilation – Calvin Cycle Mobilization from tissues (mostly adipose) Activation of fatty acids Transport; carnitine Oxidation: b-oxidation, 4 steps: Protein Catabolism Amino-Acid Degradation Dealing with the nitrogen; Urea Cycle Dealing with the carbon; Seven Families Nucleic Acid & Nucleotide Degradation Overview and regulation Calvin cycle connections to biosynthesis C4 versus C3 plants Kornberg cycle - glyoxylate Carbohydrate Biosynthesis in Animals precursors Cori cycle Gluconeogenesis reversible steps irreversible steps – four energetics 2-steps to PEP in mitochondria: Pyr carboxylase-biotin & PEPCK FBPase G6Pase PHOTOSYNTHESIS: Overview of Photosynthesis Key experiments: Light Reactions energy in a photon pigments HOW Light absorbing complexes-”red-drop experiment” Reaction center Photosystems (PS) PSII – oxygen from water splitting PSI – NADPH Proton Motive Force – ATP Overview of light reactions Glycogen Synthesis UDP-Glc Glycogen synthase branching Pentose-Phosphate Pathway oxidative-NADPH non-oxidative-Ribose 5-P Regulation of Carbohydrate Metabolism Acetyl-CoA/Pyruvate Pyruvate/PEP F6P/FBP: Fru 2,6P2 Glc/Glc6P: sequestration Glycogen: PKA/PP1 Insulin signaling Anaplerotic reactions End of Exam-4 material ANABOLISM II OUTLINE: Biosynthesis of Fatty Acids and Lipids Fatty Acids contrasts location & transport Synthesis acetyl-CoA carboxylase fatty acid synthase ACP priming 4 steps Control of fatty acid metabolism Diversification of fatty acids elongation desaturation Eicosanoids Membrane lipids Prostaglandins and Thromboxane Triacylglycerides Glycerophospholipids Sphingolipids Isoprene lipids: Cholesterol Ketone body synthesis Mevalonate Cholesterol bile acids steroids metabolism control of cholesterol biosynthesis 1 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 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 How is the acetyl-CoA transported out CO2 + 8 CoA + 14 NADP+ + 6 H2O of mitochondria? 2 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. here is the third ATP! • OMSGAP • Making the Malonyl-CoA: – Reaction carboxylates acetyl-CoA – Catalyzed by acetyl-CoA carboxylase (ACC) 3 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 Rxn 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. • Enzyme undergoes conformational change to carry carbamoyl to transcarboxylase site • CO2 attaches to acetyl-CoA, which leaves active 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) • Chain transfer/charging –malonyl/acetyl-CoA ACP transferase (M/AT) – 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. What is this “ACP”? …let’s look at the structure. 4 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 Rxn • 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 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 5 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 6 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 (acyl-ACP) puts the growing peptide onto the incoming AA-tRNA (malonyl-ACP) 4CO2 4H2O 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 7 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). • High [Citrate] signals excess energy to be converted to fat. – When [acetyl-CoA]mt ­, [citrate] mt ­ ….. 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). 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). • High [Citrate] signals excess energy to be converted to fat. – When [acetyl-CoA]mt ­, [citrate] mt ­ ….. 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). 8 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 Allosteric • 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 Fatty Acid Biosynthesis Diversification of Palmitate: Elongation Fatty Acid Elongation Systems • Elongation systems in the endoplasmic reticulum and mitochondria create longer fatty acids. Plants can produce: –linoleate 18:2(D9,12 ) –a-linolenate 18:3 (D9,12,15 ) • 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 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. elongation –Polyunsaturated fatty acids (PUFAs) help control membrane fluidity. –PUFAs are precursors to eicosanoids • The ratio of omega-6 to omega-3 fatty acids in the diet, if too high, can lead to cardiovascular disease elongation Eicosanoids PG = Prostaglandins TX = Thromboxanes LT = Leukotrienes 9 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) 10