lecture20_11_13_08 - Lecture 20 Overview •...

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Unformatted text preview: Lecture 20 Overview: • triacylglycerol - energy storage • fatty acids as fuel - three stages • unsaturated and odd-chain fatty acids • synthesis of fatty acids • acetyl CoA carboxylase • elongation and unsaturated of fatty acids - accessory enzymes HW: 22.1, 22.3, 22.6, 22.8, 22.16, 22.17 Chapter 22: Fatty Acid Metabolism Fatty acids C12-C18 CH3(CH2)nCOOH from Ch. 12 CH3(CH2)16COOH C18 - stearic acid saturated CH3(CH2)7CH=CH(CH2)7COOH C18 - oleic acid (mono)unsaturated - cis configuration Biological roles of lipids: energy storage & membranes Lipid metabolism from Ch. 12 Triacylglycerol adipose tissue (adipocytes/fat cells) storage sites of triacylglycerols in animals low water content function is to store fatty acids in the form of triacylglycerols provide energy and insulation Entry of glycerol into glycolytic pathway glycerol can be catabolized, but only represents about 5% of the energy from the triacylglycerols complete oxidation of fatty acid yields 38 kJ/g of energy compared to 17 kJ/g for carbohydrates and proteins see step 6 of glycolysis Steps of fatty acid degradation and synthesis Lipid processing in vertebrates Structure of chylomicron (B-48 predominant) chylomicron: lipoprotein aggregrate ~200 nm in diameter Hormones trigger mobilization of stored triacylglycerols epinephrine or glucagon binding to their receptors activate adenylyl cyclase, increase [cAMP], activate PKA (kinase), phosphorylate and activate triacylglycerol lipase, hydrolyze triacylglycerols, transport fatty acids Lipases: hydrolysis of ester bonds of triacylglycerols triacylglycerol lipase diacylglycerol + 1 fatty acid lipase glycerol + 2 fatty acids Digestion of dietary lipids by pancreatic lipases Chylomicron formation packaging triacylglycerols into lipotransport systems: chylomicrons Degradation of triacylglycerols to fatty acids and glycerol bile salt facilitates digestion Glycerol formed by lipolysis is absorbed by the liver and phosphorylated Catabolic fate of fatty acids after triacylglycerol hydrolysis • fatty acids are oxidized in the mitochondrial matrix glycolysis; gluconeogenesis • entry of fatty acids into the matrix requires a special translocase protein • fatty acid is activated as fatty acyl-CoA • fatty acids undergo !-oxidation in 4 steps (repeated) • fatty acids with unsaturation or odd-number of C’s require additional enzymes for complete oxidation Fatty acid activation different isozymes for fatty acids with short, intermediate, and long alkyl chain Fatty acid activation: another view This reaction takes place outside of the mitochondrial matrix. What drives the reaction? RCOO– + CoA + ATP + H2O -> RCO–CoA + AMP + 2Pi + 2H+ Shuttling fatty acids into the mitochondrial matrix requires carnitine Role of carnitine carnit ine + CH3 -N-CH2-CH-CH2 -COOCH3 OH CH3 -carnitine acyltransferase I exchanges carnitine for CoA to form fatty acid (oxygen) ester (transesterification reaction) -fatty acid-carnitine conjugate passes through transporter -carnitine acyltransferase II exchanges CoA for carnitine to generate 'activated fatty acid' (thioester) 3 remaining steps for complete oxidation of a fatty acid 1) "-oxidation 2) CAC 3) oxidative phosphorylation (respiratory chain) (compare to glycolysis) Common mechanisms in metabolic pathways Recall: oxidation-reduction FAD FADH2 -CH2-CH2OH NAD+ NADH -CH=CHO H2O -CH-CH2-C-CH2seen in citric acid cycle, fatty acid oxidation, amino acid catabolism alkane -> carbonyl Step 1 " ! C16; palmitoyl-CoA Overview: 4 steps in "-oxidation pathway acyl-CoA dehydrogenase **shortened acyl-CoA reenters path, repeats; each cycle yields one NADH and one FAHD2 alkene Step 2 " alkene Step 3 " ! alcohol enoyl-CoA hydratase !-hydroxyacyl-CoA dehydrogenase " ! alcohol " ! ketone (L-"-hydroxy-acyl-CoA) Step 4 " ! repeat steps 1–4 acyl-CoA acetyltransferase (thiolase) + 1 NADH + 1 FADH2 " C14 ! C2 C2 remember from last lecture: NADH + H+ + 1/2O2 -> NAD+ + H2O ~1.5 ATP per e- pair from FADH2; ~2.5 ATP per e- pair from NADH ex.: palmitic acid (C16) (7 cycles) ATP yields from complete oxidation of one molecule of palmitoyl-CoA C16 -> C14-> C12 -> C10 -> C8 -> C6 -> C4 -> C2 (acetyl-CoA) acetyl-CoA -> CAC palmitoyl-CoA + 7 CoA + 7 FAD + 7 NAD+ + 7 H2O -> 8 acetyl-CoA + 7 FADH2 + 7 NADH + 7 H+ palmitoyl-CoA + 7 CoA + 7 O2 + 28 Pi + 28 ADP -> 8 acetyl-CoA + 28 ATP + 7 H2O CAC 4 ATP/cycle compare to # ATPs derived from one glucose under aerobic or anaerobic glycolysis palmitoyl-CoA + 23 O2 + 108 Pi + 108 ADP -> CoA + 108 ATP + 23 H2O + 16 CO2 (after CAC) Oxidation of monounsaturated fatty acids Oxidation of poly-unsaturated fatty acids 2nd enzyme needed only need one extra enzyme an isomerase converts cis to trans alkene (an intermediate in the "-oxidation pathway) Oxidation of odd-number fatty acids requires three extra reactions biotin Coenzyme B12 i i. propionyl-CoA carboxylase ii. methylmalonyl-CoA epimerase iii. methylmalonyl-CoA mutase ii iii coenzyme B12 CAC Rearrangement of atoms The methylmalonyl CoA mutase reaction begins with the homolytic cleavage of the bond joining Co3+ to a carbon atom of the ribose of the adenosine moiety. The cleavage generates a 5'deoxyadenosyl radical and leads to the reduction of Co3+ to Co2+. Formation of succinyl CoA by a rearrangement reaction A free radical abstracts a hydrogen atom in the rearrangement of methylmalonyl CoA to succinyl CoA. Active site of methylmalonyl CoA mutase Note: important role of the histidine (his) residue peroxisome: membrane-enclosed organelle of animal and plant cells; fatty acid oxidation here halts at octanoyl CoA - may serve to shorten long chains to make them better substrates of ! oxidation in mitochondria. The reaction involves a flavoprotein dehydrogenase and transfer of electrons from O2 to yield H2O2. H2O2 is subsequently degraded by the catalase enzyme. Initiation of peroxisomal fatty acid degradation "Ketone bodies" The acetyl CoA from fatty acid oxidation enters CAC only if fat and carbohydrate degradation are appropriately balanced. Acetyl CoA must combine with oxaloacetate to gain entry to CAC (see p. 482). The availability of oxaloacetate depends on the level of carbohydrate (see p. 459; formed from pyruvate). In fasting, oxaloacetate is consumed to form glucose by gluconeogenesis and is unavailable for CAC. Under these conditions, acetyl CoA is diverted to form acetoacetate and !-hydroxybutyrate, or "ketone bodies" (including acetone). H3 C C O CH3 acet one O H3 C C O CH2 C O- acet oaceta te H C OH D-!-hydr oxybutyra te O CH2 C O- H3 C Formation of ketone bodies Pathway integration: delivery of ketone bodies Acetoacetate is formed from acetyl CoA in three steps: 1) condensation of 2 acetyl CoA to from acetoacetylCoA (catalyzed by 3-ketothiolase; reverse of thiolysis step in oxidation of fatty acids), 2) reaction with water to give HMG-CoA and CoA (hydroxymethylglutaryl CoA synthase) 3) hydroxymethylglutaryl CoA cleavage enzyme. Acetoacetate is then reduced by D-3hydroxybutyrate dehydrogenase to form D-3-hydroxybutyrate or it spontaneously decarboxylates to form acetone. Utilization of acetoacetate as a fuel Diabetic ketosis when insulin is absent CAC Fatty acids are synthesized and degraded by different pathways • synthesis takes place in the cytoplasm; degradation takes place primarily in the mitochondrial matrix • intermediates in fatty acid synthesis are covalently linked to the acyl carrier protein (ACP), whereas intermediates in fatty acid breakdown are linked to coenzyme A (both though sulfhydryl groups) • the enzymes of fatty acid synthesis are joined by a single polypeptide chain called fatty acid synthase. The degradation enzymes do not seem to be associated. • the growing fatty acid chain is elongated by sequential addition of 2-carbon units derived from acetyl CoA. The activated donor of 2carbon units is malonyl ACP. The elongation reaction is driven by the release of CO2. • The reductant in fatty acid synthesis is NADPH. The oxidants in fatty acid degradation are NAD+ and FAD. • Elongation by fatty acid synthase stops at C16, palmitate; further elongation and formation of double bonds requires other enzymes. The formation of malonyl CoA is the committed step in fatty acid synthesis Step 1 Carboxylation of acetyl CoA to malonyl CoA by acetyl CoA carboxylase. Can you guess which coenzyme is involved? biotin-enzyme + ATP + HCO3– CO2-biotin-enzyme + acetyl CoA CO2-biotin-enzyme + ADP + Pi malonyl CoA + biotin-enzyme Intermediates in fatty acid synthesis are attached to ACP 77 amino acids Reactions: condensation, reduction, dehydration, and reduction acetyl CoA + ACP malonyl CoA + ACP acetyl ACP + CoA malonyl ACP + CoA acetoacetyl ACP + ACP + CO2 acetyl ACP + malonyl ACP 'macro CoA' Step 6 Step 4 Step 5 Step 7 Single chain of animal fatty acid synthase domain 3 TE = thioesterase ex.: palmitic acid synthesis (C16) (7 cycles) C2 -> C4 -> C6 -> C8-> C10 -> C12 -> C14 -> C16 acetyl CoA + 7 malonyl CoA + 14 NADPH + 20 H+ -> palmitate + 7 CO2 + 14 NADP+ + 8 CoA + 6 H2O 7 acetyl CoA + 7 CO2 + 7 ATP -> 7 malonyl CoA + 7 ADP + 7 Pi + 14 H+ Overall: domain 1 domain 2 AT = acetyltransferase MT = malonyltransferase CE = condensing enzyme ACP = acyl carrier protein KR = !-ketoacylreductase DH = dehydratase ER = enoyl reductase 8 acetyl CoA + 7 ATP + 14 NADPH + 6 H+ -> palmitate + 14 NADP+ + 8 CoA + 6 H2O + 7 ADP + 7 Pi Citrate carries acetyl groups from mitochondria to the cytoplasm Fatty acids are synthesized in the cytoplasm, whereas acetyl CoA is formed from pyruvate in mitochondria. Mitochondria are not permeable to acetyl CoA. Citrate carries acetyl groups. oxaloacetate + NADH + H+ malate + NAD+ malate + NADP+ pyruvate + CO2 + NADPH pyruvate + CO2 + ATP + H2O oxaloacetate + ADP + Pi + H+ Sum: NADP+ + NADH + ATP + H2O NADPH + NAD+ + ADP + Pi + H+ Pathway integration citrate + ATP + CoA + H2O -> acetyl CoA + ADP + Pi + oxaloacetate Control of acetyl CoA carboxylase another example of regulation through phosphorylation acetyl CoA carboxylase: catalyzes committed step in fatty acid synthesis – production of malonyl CoA; AMPK activation by AMP and inhibition by ATP Elongation and unsaturation of fatty acids by accessory enzymes stearoyl CoA + NADH + H+ + O2 -> oleoyl CoA + NAD+ + 2 H2O insertion of a cis !9 double bond by oxidase, a three-enzyme complex NADH-cytochrome b5 reductase, cytochrome b5 and desaturase citrate can partly activate the phosphorylated carboxylase by allosteric regulation ...
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