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5-1 Lipid Synthesis Fatty acids are a more efficient form of energy storage than carbohydrates because they are less hydrated, as result of fewer hydroxyl groups being available for hydrogen bonding. The energy content of fat tissue is 38 kJ/gm compared to 17 kJ/gm for carbohydrates. The processes of fatty acid degradation had been worked out prior to fatty acid synthesis, and there was some conjecture that perhaps synthesis might simply be the reverse of degradation. Indeed, it was realized early on that [14C] acetate was a direct precursor for fatty acids providing some substance to the conjecture (since acetate was the product of β-oxidation). However, as work progressed a number of significant differences between synthesis and degradation were noted including: 1. β-oxidation occurs in the mitochondria and synthesis occurs in the cytoplasm; 2. citrate is required for synthesis as an activator; and 3. CO2 is required for synthesis but not incorporated. Ultimately, the principal enzyme fatty acid synthase was isolated and characterized. From eucaryotes it was found to be a single large protein with several activites whereas in bacteria, it was a complex of several proteins each with a different activity. Ultimately, the individual activities in the larger single protein were correlated with those of the separate enzymes, and it was realized that the overall processes were the same. The simpler bacterial system allowed for a dissection of the system. 1. Fatty acid synthesis As with all processes there is a "preparatory" phase in which substrates are prepared to enter the synthase complex. For lipids, that step involves the carboxylation of acetate in a process that explains the roles of both citrate and CO2. 1 H 3C O ATP C SCoA acetyl CoA biotin ADP + Pi H2 C -OOC CO2 AcetylCoA carboxylase O C SCoA malonyl CoA 1. CO2 is incorporated explaining where it is involved in the process (we will see its release shortly). 2. The carboxylase step is the slowest or rate determining reaction of the synthesis pathway and the enzyme requires citrate for activity through its involvement in stabilizing the multimeric form of the enzyme. By controling the slowest reaction, citrate controls fatty acid synthesis. 5-2 monomer (inactive) 2 multimer - citrate (active) Acyl carrier protein While not specifically part of a preparatory phase, it is necessary to introduce an unusual protein, acyl carrier protein. In bacteria, it is a small ~10 kDa protein with a 4-phosphopantetheine chain attached to a specific serine. The 4-phosphopantetheine chain is also a component of CoASH and its active portion is the -SH. In eucaryotic enzymes, the 4-phosphopantetheine chain is attached to the larger protein. It is best viewed as a flexible arm that "carries" the growing fatty acid chain from enzyme to enzyme (bacteria) or active site to active site (eucaryotes). O Ser O P O H2 C O CH3 OH O C C H C O H2 C H N H2 C C H2 C H N H2 C SH CH3 As with the SH of CoASH, its main role is to form thiol esters with growing fatty acid chains. This gives rise to the nomenclature, ACP-SH and fatty acyl-ACP. At this point we can get into the actual series of reactions involved in the synthesis. 3 H 3C O ACP-SH 4 CoA-SH H 3C C β-Ketoacyl-ACP synthase O C Acetyl-CoA-ACP transacetylase (once per fatty acid) SCoA acetyl-CoA SACP Syn-SH H 2C O malonyl-CoA S-Syn 6 O β-Ketoacyl-ACP synthase C CoA-SH H 2C C SCoA ACP-SH O CO2 + Syn-SH C Malonyl-CoA-ACP transacetylase H+ acetyl-Synthase -O ACP-SH O C acetyl-ACP COO- 5 H 3C SACP malonyl-ACP H 3C O C H 2C O C SACP acetoacetyl-ACP (β-ketoacyl-ACP) 5-3 H3C 7 O NADP+ NADPH + H+ C H2C H3C O H C 8 OH H2C C β-Ketoacyl-ACP reductase HC butenoyl-ACP (trans-Δ2-enoyl-ACP) β-hydroxybutyryl-ACP (β-hydroxyacyl-ACP) C CO2 NADPH + H+ Enoyl-ACP reductase Steps 6 to 9 2NADP+ S-ACP 2NADPH + 2H+ MalonylACP hexanoyl-ACP (C6) β-Ketoacyl-ACP synthase S-Syn CoASH C O SACP Step 5 butyryl-ACP (acyl-ACP) MalonylCoA Once palmityl-ACP is formed the last step in this series of reactions involves the cleavage of palmitic acid. Longer chains are produced usually by a different series of reactions. (CH2)14CH3 C16 H2C ACP-SH C12 C14 CH2 O C8 C10 CH3 Step 4 C 9 NADP+ (CH2)2CH3 O C SACP The butyryl-ACP is now "equivalent" to acetyl-CoA and returns to step #4 where it is transfered onto the β-ketoacyl-ACP synthase. Then steps #5 to #9 are repeated to generate a C6 hexanoyl-ACP, and so on through five more rounds (7 in total) until C16 palmitoyl-ACP is formed. (CH2)4CH3 O β-Hydroxyacyl-ACP dehydratase SACP acetoacetyl-ACP (β-ketoacyl-ACP) CH O C SACP H3C H2O C H2O (CH2)14CH3 ACP-SH O C Palmitoyl-ACP thioesterase S-ACP O O palmitate (palmitic acid if protonated) palmitoyl-ACP Overall 7ATP 7 acetyl-CoA 7 ADP + 7Pi 14 NADPH + 14 H+ 14 NADP+ 7 malonyl-CoA acetyl-CoA palmitate 8 CoASH 5-4 or 8 AcCoA + 7 ATP + 14 NADPH palmitate CH3-CH2-CH2-CH2- - - - - - - - - - - - - - - - - - - - CH2-COOacetate malonate malonate synthesis degradation 2. Sources of NADPH 1. from the Pentose Phosphate Pathway in liver cells 2. from malic enzyme in fat cells malate pyruvate + CO2 NADP+ NADPH + H+ 3. Sources of AcCoA ethanol pyruvate amino acids acetate citrate lipids AcCoA CO2 + energy (TCA cycle) glyoxalate shunt (plants and bacteria) Citrate? Citrate synthase is considered irreversible ( a very large negative ∆G'o) in vivo and an alternate path is required to generate AcCoA from citrate. This involves ATP-citrate lyase and proceeds because of the involvement of ATP hydrolysis which provides the necessary energy. ATP-citrate lyase citrate + CoASH Acetyl-CoA + OAA ATP ADP + Pi 5-5 4. Unsaturated fatty acid synthesis Unsaturations can be introduced into fatty acids at two different stages: 1. after the saturated fatty acid is completed and 2. at a step during synthesis. 1. Into the completed fatty acid: Fattyacyl-CoA monoxygenase O2 2 H2O palmitoyl-CoA palmitoleyl-CoA (cis ∆9-) NADH + H+ NAD+ There are 4 such enzymes that introduce the unsaturations at C4, C5, C6 and C9. The enzymes are also sometimes called terminal desaturases and are part of an electron transport chain that includes cytochrome b5. 2. At an intermediate step of synthesis: Essentially, the unsaturation is introduced in a configuration that cannot be reduced during subsequent steps (bacteria). OH H3C(H2C)5 CH C H2 H2O O O H C C C H2 SACP β-hydroxy decanoylACP dehydratase HC C C H2 SACP H3C(H2C)5 β-Hydroxyacyl-ACP dehydratase Not a substrate for the enoyl-ACP reductase and is taken directly to step 4 (transferred to the synthase. H2O O H C H3C(H2C)5 C H2 C C H SACP cis ∆3-decenoyl-ACP Syn-SH β-Ketoacyl-ACP synthase ACP-SH O normal pathway H C O HC H2 C H3C(H2C)11 C H2 C C H2 palmitoyl-ACP SACP H3C(H2C)5 C C H2 S-Syn three rounds of synthesis adding 6 carbons 5-6 O H C HC C (CH2)7 S-ACP H3C(H2C)5 palmitoleyl-ACP Lec #16 5. Control of fatty acid synthesis Fatty acid synthesis is controlled at several levels including enzyme activity regulation, transcriptional control and hormonal control. In the case of hormonal control, adrenalin activates protein kinase via adenylate cyclase and cAMP, the protein kinase, in turn, activates pancreatic lipases and β-oxidation. Insulin formed under conditions of high glucose, activates synthesis as a means of storing energy in the form of fat. glycogen (energy storage) glc6P - + PEP pyr malonylCoA AcCoA + fatty acids (energy storage) + OAA citrate isocitrate (energy release) ATP This diagram illustrates the central role of citrate in the control of energy metabolism. Not only does it activate gluconeogenesis for energy storage in carbohydrates, but it activates energy storage in lipids and inhibits energy release in glycolysis. Under energy rich conditions it can also act as a source of AcCoA for additional lipid synthesis. Also in the diagram, the activation of pyruvate carboxylase by AcCoA is noted. In the following diagram, the complication of the mitochondrial barrier is imposed on the larger picture of lipid/carbohydrate synthesis and degradation. The main underlying point is that βoxidation occurs in the mitochondria and synthesis occurs in the cytoplasm Mitochondria 5-7 Cytoplasm energy rich conditions TCA Cycle citrate citrate AcCoA AcCoA OAA fatty acyl CoA OAA malate malate fatty acids pyruvate pyruvate fatty acyl carnitine fatty acyl carnitine fatty acyl CoA complex lipids energy poor conditions It is important to remember that free fatty acids do not exist in large amounts in free form but are rapidly assimilated into complex lipids, particularly triglycerides and phospholipids. 6. Synthesis of triacylglycerides and phospholipids CH2OH ATP ADP CH2OH CHOH NAD+ NADH + H+ C CHOH 2 glycerol Glycerol phosphate acyl transferases O Glycerol-3-phosphate 2 CH2OPO3 dehydrogenase CH2OPO3 (intestines) dihydroxyacetone glycerol phosphate phosphate Glycerol kinase (liver) CH2OH CH2OH O 2 C R SCoA O O CH2O CR1 CHO C R2 O O 2 CoASH C R O 2 CH2OPO3 diacyl phosphatidate H2O Pi Phosphatidate phosphatase CH2O CR1 CHO C R2 CH2OH diacyl glyceride Route A to phospholipids SCoA CoASH CH2O CR1 CHO C R2 O Diacylglyceride acyl transferase CH2O C R3 O O triacyl glyceride Route B to phospholipids 5-8 (a) Route A to phospholipids 2 Pi IPPase O CR1 CH2O CHO H2O CTP O O NH2 CR1 CH2O PPi C R2 CHO 2 CH2OPO3 diacyl phosphatidate O N CR2 N 2 Phosphatidate cytidylyl transferase CH2OPO3PO2 O O CDP diacyl glycerol H H H OH H OH serine CDP diacyl glycerol serine-O-phosphatidyl transferase O CR1 CH2O CHO CH2O CR1 NH3 CHO C R2 CH2OPO2 O CO2 O Phosphatidyl serine decarboxylase CH2 OH2C O CMP O NH3 C R2 CH2OPO2 OH2C COO phosphatidyl serine phosphatidyl ethanolamine (b) Route B to phospholipids ATP H2 C (H3C)3N ADP C H2 (H3C)3N Choline kinase choline 2 H2 C OH OPO3 C H2 phosphocholine CTP Phosphocholine CR1 cytidylyl O transferase IPPase PPi 2 Pi C R2 H2O O CH2O O CH2O CR1 CHO C R2 CH2OPO2 CHO O N(CH3)3 CMP CH2OH CH2 OH2C phosphatidyl choline (H3C)3N Phosphocholine transferase 2 H2 C OCDP C H2 CDP-choline This route can also generate phosphatidyl ethanolamine. 5-9 7. Synthesis of terpenes and steroids. acetyl-CoA O CoASH C H 3C β-Ketoacyl-CoA thiolase SCoA acetyl-CoA O O C C H 3C C H2 SCoA acetoacetyl-CoA acetyl-CoA HMG-CoA synthase 2 NADP+ OH H 3C C CoASH H2 C OH H2 C H 3C OH H 2C COO- mevalonate 3 ATP CoASH 2 NADPH + 2 H+ C O H2 C C HMG-CoA H 2C reductase COOImportant site of regulation: (a) inhibited by a cholesterol metabolite hydroxymethyl (b) activated by insulin glutaryl-CoA (c) inhibited by glucagon (HMG-CoA) SCoA 3 enzymes 3ADP +Pi + CO2 3 H 3C C H2 C H2 C H 2C H 3C 3 C OPO3PO3 CH OPO3PO3 H 2C ∆3-isopentenyl pyrophosphate Isopentenyl pyrophosphate isomerase H 3C ∆2-isopentenyl pyrophosphate The isopentenyl pyrophosphates (isoprene units) are the building blocks for the larger terpenes and steroids. The steps on the next page outline generally how the larger molecules are generated. The overall process is as follows: AcCoA HMGCoA isopentenylPP terpenes steroids 5-10 monoterpenes (C10) 2 Pi PPi OPP IPPase OPP OPP PPO Prenyl transferase geranyl pyrophosphate sesquiterpenes (C15) ubiquinones Prenyl transferase carotenoids (C40) OPP dimerize diterpenes (C20) farnesyl pyrophosphate Squalene synthase (dimerize) hormones 23 steps squalene cholesterol HO triterpenes (C30) bile acids 5-11 Acetyl-CoA Cholesterol HMG-CoA reductase HDL (high density lipoprotein) Fatty acylcholesterol acylCoA-cholesterol acyl transferase LDL receptors (uptake into tissue) LDL (low density lipoprotein) excretion as bile acids Regulation 1. HMG-CoA reductase (a) insulin activates through dephosphorylation (b) glucagon inactivates through phosphorylation (c) cholesterol metabolites inhibit through stimulation of proteolysis (d) transcriptional regulation 2. AcylCoA cholesterol acyl transferase is activated by cholesterol 3. LDL receptor synthesis is repressed by intracellular cholesterol. A common cause of high blood cholesterol possibly leading to atherosclerosis is a genetic predisposition to low LDL receptor levels that result in diminished cholesterol uptake. Some control of this is possible through compactin or lovostatin, drugs which inhibit HMG-CoA reductase, or resins which bind to bile acids removing them from the metabolic system , thereby displacing the pathway to convert more cholesterol into bile acids. 8. Summary 1. Fatty acid synthesis 2. Unsaturated fatty acid synthesis 3. Regulation by and importance of citrate 4. Triglyceride and phospholipid synthesis 5. Terpene and steroid synthesis. Lec #17