Sec_5 - 5-1 Lipid Synthesis Fatty acids are a more...

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Unformatted text preview: 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 -oxidation). However, as work progressed a number of significant differences between synthesis and degradation were noted including: 1. -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 C SCoA O Biotin: common component cofactors with c arboxylases H2 C -OOC C SCoA ATP biotin ADP + Pi O CO2 AcetylCoA carboxylase acetyl CoA 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 Acyl carrier protein multimer - citrate (active) s trong analogy 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 O H2 C CH3 C CH3 OH C H O C H N H2 C H2 C O C H N H2 C H2 C SH 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. Syn is the synthase it self we're responsible for only SH, the active part 3 H 3C C SCoA O 4 CoA-SH H 3C C O ACP-SH -Ketoacyl-ACP synthase Syn-SH O C H 3C C O H+ acetyl-CoA COOH 2C C Acetyl-CoA-ACP Acetyl-CoA-ACP transacetylase (once per fatty acid) SACP ACP-SH 6 -Ketoacyl-ACP synthase O S-Syn acetyl-ACP -O acetyl-Synthase O ACP-SH 5 CoA-SH H 2C C SCoA Malonyl-CoA-ACP transacetylase CO2 + Syn-SH H 3C C H 2C C SACP O O SACP malonyl-CoA was formed in the first step * then, the CoA is replaced with ACP, the s ame one from acetyl-ACP, to form malonyl ACP malonyl-ACP combined acetoacetyl-ACP ( -ketoacyl-ACP) 5-3 H 3C C H 2C C SACP O 7 NADPH + H O + NADP + H 3C CH H 2C OH 8 H2O O H 3C CH HC O C SACP -Ketoacyl-ACP reductase C SACP -Hydroxyacyl-ACP dehydratase acetoacetyl-ACP ( -ketoacyl-ACP) -hydroxybutyryl-ACP ( -hydroxyacyl-ACP) butenoyl-ACP (trans- 2-enoyl-ACP) NADPH + H+ Enoyl-ACP reductase NADP+ CH3 CH2 H 2C C SACP O The butyryl-ACP is now "equivalent" to acetyl-CoA and returns to step #4 where it is transfered onto the -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 C O 2NADP+ S-ACP 2NADPH + 2H+ MalonylACP CO2 (CH2)2CH3 9 Steps 5 to 9 C S-Syn O Step 4 -Ketoacyl-ACP synthase hexanoyl-ACP (C6) C8 C10 C12 C14 C16 S-ACP (CH2)14CH3 C O butyryl-ACP (acyl-ACP) 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. H2O ACP-SH (CH2)14CH3 C O Palmitoyl-ACP thioesterase O palmitoyl-ACP Overall 7ATP 7 acetyl-CoA 7 ADP + 7Pi palmitate (palmitic acid if protonated) 14 NADPH + 14 H+ 14 NADP+ palmitate acetyl-CoA 8 CoASH 7 malonyl-CoA 5-4 or 8 AcCoA + 7 ATP + 14 NADPH large amnt of energy required to make this palmitate CH3-CH2-CH2-CH2- - - - - - - - - - - - - - - - - - - - CH2-COOacetate malonate malonate synthesis degradation 2. Sources of NADPH is the major route for generatiting NADPH liver ofr fat. 1. from the Pentose Phosphate Pathway in liver cells 2. from malic enzyme in fat cells malate NADP+ pyruvate + CO2 NADPH + H+ 3. Sources of AcCoA ethanol pyruvate amino acids acetate citrate lipids AcCoA fatty acids 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. lyse: cuttss ATP-citrate lyase citrate + CoASH Acetyl-CoA + OAA Citrate required: (so it activates ACCoA Acetyl CoA carboxylase has a monomer, a single subunit is inactive, but if you add citrate, it promotes multimerization to become active. *Where malonyl CoA is produced, it is the rate determining step. ATP ADP + Pi 5-5 4. Unsaturated fatty acid synthesis most fatty acids are unsaturated 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 NADH + H+ acting as e- donor one step will convert a saturated fattya, into an unsatureated fatty acid ( with cis, double bond) palmitoleyl-CoA (cis 9-) 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: in bacteria, one additional enzyme is required Essentially, the unsaturation is introduced in a configuration that cannot be reduced during subsequent steps (bacteria). OH H3C(H2C)5 C H2 CH C H2 O C SACP H2O H C HC C H2 3 O C SACP -hydroxy decanoylACP dehydratase Not a substrate for the enoyl-ACP reductase and is taken directly to step 4 (transferred to the synthase. H3C(H2C)5 -Hydroxyacyl-ACP dehydratase H C C H2 C H cis -decenoyl-ACP Syn-SH H2O O H3C(H2C)5 C SACP treateed as a fully s aturated FA -Ketoacyl-ACP synthase ACP-SH O normal pathway O H3C(H2C)11 C H2 H2 C C H2 C SACP H C HC H3C(H2C)5 C H2 C S-Syn palmitoyl-ACP three rounds of synthesis adding 6 carbons O H C HC H3C(H2C)5 C (CH2)7 S-ACP 5-6 palmitoleyl-ACP 5. Control of fatty acid synthesis Lec #16 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 kinase and cAMP, the protein kinase, in turn, activates pancreatic lipases and -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 + AcCoA OAA + malonylCoA fatty acids (energy storage) citrate Citrate is important as its an (energy release) intermediate in TCA cycle, also r equred to activate malonylCoA, - under high energy, the buildup of c itrate will OAA isocitrate 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 oxidation occurs in the mitochondria and synthesis occurs in the cytoplasm Mitochondria TCA Cycle citrate AcCoA OAA malate fatty acyl CoA pyruvate Cytoplasm energy rich conditions citrate AcCoA OAA malate pyruvate fatty acids fatty acyl CoA 5-7 fatty acyl carnitine energy poor conditions fatty acyl carnitine complex lipids 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 CHOH ATP ADP CH2OH CHOH NAD+ NADH + H+ CH2OH C O base c ompoundCH2OH for lipids Glycerol kinase (liver) Glycerol phosphate acyl transferases glycerol O R Glycerol-3-phosphate 2 CH2OPO3 dehydrogenase CH2OPO3 (intestines) dihydroxyacetone glycerol phosphate phosphate 2 2C SCoA O O R O SCoA CoASH CH2O CHO CR1 C R2 C R3 O O C O CH2O CHO 2 2 CoASH O CR1 C R2 H2O Pi CH2O CHO CR1 C R2 O CH2OPO3 Phosphatidate phosphatase CH2OH Diacylglyceride acyl transferase CH2O diacyl phosphatidate diacyl glyceride triacyl glyceride Route A to phospholipids Route B to phospholipids (a) Route A to phospholipids O CH2O CHO 2 5-8 IPPase H2O 2 Pi O serine c uts her O CR1 C R2 NH2 N N O O CTP PPi CH2O CHO CR1 CR2 2 CH2OPO3 diacyl phosphatidate Phosphatidate cytidylyl transferase CH2OPO3PO2 O O H H OH H H OH CDP diacyl glycerol CDP diacyl glycerol serine-O-phosphatidyl transferase O CH2O CHO CR1 C R2 OH2C O serine CO2 CH2O NH3 CHO CH2 O O CMP NH3 CR1 C R2 CH2OPO2 Phosphatidyl serine decarboxylase CH2OPO2 OH2C COO phosphatidyl ethanolamine (b) Route B to phospholipids H2 C (H3C)3N C H2 phosphatidyl serine ATP OH ADP (H3C)3N H2 C C H2 2 OPO3 Choline kinase O choline start with alcohol (choline) O CH2O CHO CR1 C R2 OH2C O CH2O CHO N(CH3)3 CH2 phosphocholine energy source CTP Phosphocholine CR1 cytidylyl O transferase IPPase PPi 2 Pi C R2 H2O H2 C (H3C)3N C H2 2 CMP CH2OH OCDP CH2OPO2 phosphatidyl choline Phosphocholine transferase CDP-choline This route can also generate phosphatidyl ethanolamine. 7. Synthesis of terpenes and steroids. O C H 3C SCoA 5-9 acetyl-CoA CoASH O C O C C H2 SCoA acetyl-CoA -Ketoacyl-CoA thiolase H 3C acetoacetyl-CoA acetyl-CoA HMG-CoA synthase 2 NADP+ OH H 3C C H 2C COOH2 C H2 C OH 2 NADPH + 2 H+ OH H 3C C H2 C CoASH O C SCoA CoASH mevalonate 3 ATP 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) feed back "inhibition" by cholestrol 3 enzymes 3ADP +Pi + CO2 H 3C C H 2C 3 3 H2 C H2 C H 3C 3 H 2C C CH OPO3PO3 OPO3PO3 -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 2 Pi OPP 5-10 monoterpenes (C10) PPi IPPase OPP PPO OPP Prenyl transferase geranyl pyrophosphate sesquiterpenes (C15) carotenoids (C40) dimerize ubiquinones Prenyl transferase OPP diterpenes (C20) Squalene synthase (dimerize) farnesyl pyrophosphate hormones bile acids 23 steps squalene cholesterol HO triterpenes (C30) 5-11 Acetyl-CoA HMG-CoA reductase Cholesterol acylCoA-cholesterol acyl transferase Fatty acylcholesterol HDL (high density lipoprotein) excretion as bile acids Regulation LDL synthesis is inhibited by c holestrol genetic low production, causes low lelvels of LDL r eceptors (body cannt synthesize properly). - to work around the genetic production: drugs inhibit HMG CoA for lower cholestrol, often combined with ion exchange resins, which bind with bile acids and removes them. LDL receptors (uptake into tissue) LDL (low density lipoprotein) 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. body wants to cut down on c holestrol 8. Summary 1. Fatty acid synthesis 2. Unstaturated fatty acid synthesis 3. Regulation by and importance of citrate 4. Triglyceride and phospholipid synthesis 5. Terpene and steroid synthesis. Lec #17 ...
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This note was uploaded on 02/27/2011 for the course MBIO 2370 taught by Professor Spearman during the Winter '11 term at Manitoba.

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