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Fat from diet (chylomicrons) is immediately stored (adipocytes) PowerPoint Presentation
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Fat from diet (chylomicrons) is immediately stored (adipocytes)

Fat from diet (chylomicrons) is immediately stored (adipocytes)

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Fat from diet (chylomicrons) is immediately stored (adipocytes)

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  1. Fat from diet (chylomicrons) is immediately stored (adipocytes) • excess of replenishing glycogen are made into FA (liver, adypocytes) and sent to tissues for use and to adipocytes for storage. • Most of FA synthesis in animals occurs in adipocytes and liver.

  2. The products of FA biosynthesis (stage I anabolic processes). • can be exported into blood (where they travel bound to albumin) to serve as fuel for other tissues (through beta-oxidation: stage II catabolic) or • they are esterified • with glycerol to result in TAG for storage; or • with phosphatidic acid to make phospholipids (membrane constituents).

  3. When circulating glucose levels are elevated, as after a meal rich in carbs, insulin secretion from the pancreas is stimulated. • Insulin activates hormone sensitive glucose transporter (GLUT4) by stimulating its translocation from cytosol to plasma membrane. • Glucose enters the cells (liver and adipocytes) and following glycolysis plenty AcetylCoA is made which will be STORED as fatty acids through the FA biosynthetic pathway. • Note that some tissues (heart, resting muscle, liver) prefer fats as a source of energy. Recall that fats generate more energy than carbs of same carbon content.

  4. Ac-CoA is a common metabolite in CARB and FA pathways. • Ac-CoA is an intermediate in FA biosynthesis and degradation BUT it can only be a product of carbohydrate metabolism. • Ac-CoA cannot be used for de novo glucose synthesis in animals because the reaction catalyzed by Pyr-DH complex is irreversible and catabolism through TCA cycle fully oxidizes both acetyl carbons to CO2.

  5. Fatty acid biosynthesis is not a simple reversal of fatty acid degradation • Degradation of fatty acids takes place in mitochondria • Synthesis of fatty acids occurs in the cytosol • Intermediates in fatty acid breakdown are bound to the -SH group of coenzyme A • Intermediates in fatty acid synthesis are linked covalently to the sulfhydryl groups of acyl carrier proteins • 3. Degradative enzymes are not associated as a complex • Enzymes of fatty acid synthesis (in animals) are components of one long polypeptide chain, the fatty acid synthase (plants and bacteria have separate enzymes that form the fatty acid synthase) • 4. In fatty acid degradation the coenzyme for the oxidation-reduction reactions involves NAD+/NADH • In fatty acid synthesis the coenzyme for the oxidation-reduction reactions is NADP+/NADPH

  6. Transport of 2C units (acetyl CoA) out of the mitochondria Production of e- (NADPH) for FA synthesis

  7. NADPH Sources for FA Synthesis

  8. Transport of 2C units and Reducing Equivalents • OAA cannot pass inner mitochondrial membrane either malate or pyruvate are exchanged 8

  9. same process different diagram… MRC MITOCHONDRION CYTOSOL AcetylCoA is made in the mitochondrion; FA biosynthesis occurs in cytosol; and inner mitochondrial membrane is impermeable to AcetylCoA or OAA. Net transport of acetyl-units (C2) can result by transporting citrate (C6) into the cytosol and returning C4 units as malate (C4) or as pyruvate (C3) and CO2. In the process reducing equivalents are also transported.

  10. Transport of Acetyl units Enzymes (locations) and reactions involved: Citrate synthase( mit) Acetyl CoA +OAA citrate + CoASH Citrate lyase (cyt): citrate + ATP + CoASH  acetylCoA +ADP +Pi + OAA Malate DH (cyt) OAA + NADH + H+ malate + NAD+ Malic enzyme (cyt) malate + NADP+  pyruvate + CO2 + NADPH + H+ Pyr carboxylase (mit) pyruvate + CO2 + ATP + H2O  OAA + ADP + H+ -------------------------------------------------------------------- ATP is expended. In addition to transporting one acetyl unit from mit to cyt, the process results in: NAD+  NADH and NADH NAD+ (malate DH) (malate DH) NADP+  NADPH (malic E) (mitochondria) (cytosol) • Inner mitochondrial membrane transporters used: • Citrate: tricarboxylic acid transporter (citrate out) • Malate: dicarboxylic acid transporter (malate in) • Pyruvate: pyruvate transporter (pyruvate in) GP_Fall2007 10

  11. Fatty Acid Metabolism

  12. Strategy Activation: specific carrier To store energy from carbs as FAT, the common intermediate, AcetylCoA, is directed into the Fatty Acid Synthesis pathway by exchanging the acyl-carrier CoASH with the acyl-carrier ACPat the expense of one ATP.This represents the ‘activation’ step in Fatty Acid Synthesis pathway Fatty Acid Synthesis FAS pathway is a spiral stage II anabolic pathway where 2C units are added sequentially to a growing chain of acyl-ACP to form C16 chains; same chemical strategy as beta-oxidation (in reverse): Condensation (SYNTHASE) Reduction (REDUCTASE) with NADPH Dehydration (DEHYDRATASE) Reduction (REDUCTASE) with NADPH Specific structural elements are introduced later: C16 Fatty Acids are elongated as necessary (to C18; C20; etc) Double bonds are introduced into saturated Fatty Acids as necessary.

  13. 1. Condensation:CO2 is eliminated from malonyl group. Net effect is extension of the acyl chain by two carbons. The beta group is then reduced in three more steps nearly identical to the reactions of beta oxidation, but in the reverse sequence. • 2. The beta-keto group is reduced to an alcohol. • 3. The elimination of H2O creates a double bond, and • 4. The double bond is reduced to form the corresponding saturated f.acyl group. • The f.a chain grows by two-carbon units donated by activated malonate, with loss of CO2. • After each two-carbon addition, reductions convert the growing chain to a saturated fatty acid of four, then 6,8, and so on. The final product is palmitate (16:0)

  14. FA Biosynthesis: First Committed Step • Acetyl-CoA + HCO3— + ATP  Malonyl-CoA + ADP + Pi • Principal point of regulation for FA synthesis. • Catalyzed by acetyl CoA carboxylase.

  15. Carboxylation of acetyl CoA to form malonyl CoA by Acetyl-CoA carboxylase • Acetyl-CoA carboxylase has 3 functional regions: • 1. Biotin carrier protein • 2. Biotin carboxylase, which activates CO2 by attaching it to a nitrogen in the biotin ring in an ATP-dependent reaction. • 3. Transcarboxylase, which transfers activated CO2 from biotin to acetyl-CoA producing malonyl CoA. The long-flexible biotin arm makes this.

  16. Reaction #1: synthesis of malonyl-CoA by acetyl CoA carboxylase Acetyl CoA carboxylase has three functional regions 1. Biotin carrier protein 2. Biotin carboxylase which activates CO2 by attaching it to a nitrogen in the biotin ring in an ATP dependent reaction 3. Transcarboxylase which transfers activated CO2 from biotin to acetyl-CoA producing malonyl CoA 21:15

  17. Fatty Acid Synthesis Overview • The two carbon atoms at the methyl terminus in each FA molecule derive from acetyl-CoA. • Malonyl-CoA supplies the remaining carbons. • No carbon atoms in FA derive from bicarbonate. • FA are assembled two carbon atoms at a time by the fatty acid synthase complex.

  18. The stage is set for lipogenesis by two reactions: • (1) transfer of an acetyl group from acetyl-SCoA to an acyl carrier protein (ACP) • (2) conversion of acetyl- SCoA to malonyl-SCoA in a reaction that requires investment of energy from ATP. The malonyl-SCoA is then transferred to the acyl carrier protein (ACP).

  19. Fatty acid synthesis requires ACP • acetyl carrier protein (ACP) • Small protein 77 kda • Contain the prosthetic group (4’-phosphopantetheine)

  20. ACP is the Carrier of Acyl Groups for FA Biosynthesis • The use of separate carriers: • prevents futile cycles; • prevents recognition of intermediates by enzymes used for the opposite pathway; • allows for independent flux regulation. will be swinging arm in FA Synthase ACYL-CARRIER PROTEIN: business endof carriers 21

  21. Entry into FA Synthesis: Acetyl-CoA CarboxylaseActivation of C2 units for FA biosynthesis Acetyl-CoA Carboxylase Both transacylases (2, 3) are activities on the FA Synthase complex • activation step (ATP) : metabolic principle • Commits acetyl-CoA to FA synthesis; • Bicarbonate requirement • Major control point:metabolic principle • Hormonal control: insulin activation by de-phosphorylation 22

  22. acetyl transacylase malonyl transacylase Formation of acetyl ACP and malonyl ACP • acetyl CoA + ACP acetyl ACP + CoA 2. malonyl CoA + ACP malonyl ACP + CoA

  23. Multifunctional enzyme complex • A dimer of 260 kd subunits • 3 domains • Domain 1 : the substrate entry and condensation unit • Acetyl transferase, malonyl transferase, b-ketoacyl synthase • Domain 2: the reduction unit • ACP, b-ketoacyl reductase, 3-Hydroxylacyl-ACP dehydratase, enoyl reductase • Domain 3: the palmitate release unit • Thioesterase • These enzymes are covalently linked..

  24. FA Synthase Complex cycle begins 1 a-transacylase 2 synthase 3 m-transacylase 4 k-reductase 5 dehydrase 6 e-reductase P-pantheteine swinging arm GP_Fall2007 25

  25. Fatty Acid Synthase Fatty acid synthase is the name of the complex of six enzymatic activities that performs biosynthesis of fatty acids in cytosol. The enzyme is composed of two multifunctional polypeptide chains, which contain the six enzymatic activities below: (2) Malonyl-CoA-ACP Transacylase: swaps ACP with CoA in malonyl-CoA (3) Acetyl-CoA-ACP Transacylase: swaps ACP with CoA in acetyl-CoA (4) b-Ketoacyl-ACP Synthase: C2 is added to malonyl-ACP  ketoacyl-ACP (5) b-Ketoacyl-ACP Reductase: reduces D-3-keto D-3-hydroxy, stereospecific (6) 3-Hydroxylacyl-ACP Dehydrase: dehydration  trans-2-enoyl (7) Enoyl-ACP Reductase: reduction with NADPH

  26. REACTIONSsynthase; reductase; dehydrase; reductase GP_Fall2007 28

  27. FA Synthase: Cycle of 4 rxns. The 2C from Malonyl-CoA are added to the carboxy terminus of the FA.

  28. Synthesis of 16:0 via 7 cycles by Fatty Acid Synthase.

  29. Comparison of fatty acid synthase systems 7 separate proteins a-subunit = 3 activities ß-subunit = 4 activities 1 polypeptide = 7 activities MW= 240,00, functions as a dimer arranged head-to-tail.

  30. -Carbon CH3C- ACP ACP ACP O O O O O H D isomer CH3C- HO H CH2C~S- = C- C~S- CH2C~S- CH3C- H CH3CH2CH2C~S- ACP Elongation Reduction NADPH -Ketoacyl-ACP reductase Dehydration -H2O  -Hydroxyacyl-ACP dehydrase NADPH Reduction Enoyl-ACP reductase

  31. Condensation and Reduction Inreactions 1 and 2 of fatty acid synthesis: • Condensation by a synthase combines acetyl-ACP with malonyl-ACP to form acetoacetyl-ACP (4C) and CO2 (reaction 1). • Reduction converts a ketone to an alcohol using NADPH (reaction 2).

  32. Dehydration and Reduction Inreactions 3 and 4 of fatty acid synthesis: • Dehydration forms a trans double bond (reaction 3). • Reduction converts the double bond to a single bond using NADPH (Reaction 4).

  33. Lipogenesis Cycle Repeats Fatty acid synthesis continues: • Malonyl-ACP combines with the four-carbon butyryl-ACP to form a six-carbon-ACP. • The carbon chain lengthens by two carbons each cycle.

  34. Lipogenesis Cycle Completed • Fatty acid synthesis is completed when palmitoyl ACP reacts with water to give palmitate (C16)and free ACP.

  35. Summary of Lipogenesis

  36. O O O CH3-CH -CH2-C-S CH3-CH2-CH2-C-S S-C-CH2-CH2-CH3 Acetyl-CoA -SH -SH S HS CoA-SH C=O KS KS KS KS KS CH2 NADP+ O C=O NADPH H+ CH3-CH=CH-C-S CH3 O O OH -C-CH3 SH Malonyl-CoA S-C-CH3 ACP O CoA-SH -C-CH2-COO- S NADP+ NADPH H+ S Fatty Acid Synthase -Ketoacyl -ACP synthase Acetyl-CoA- ACP transacylase Initiation or priming Enoyl-ACP reductase -Hydroxyacyl-ACP dehydrase H2O Malonyl-CoA- ACP transacylase -Keto-ACP synthase (condensing enzyme) CO2 -Ketoacyl -ACP reductase Elongation

  37. Step 4: Acetyl and Malonyl condense to form -ketoacyl-ACP bound to ACP reaction is catylzed by -ketoacyl-ACP synthase in this reaction the acetyl group is transferred to the malonyl group simultaneously a molecule of CO2 is produced The decarboxylation facilitates the nucleophilic attack of the methylene carbon on the thioester linking the acetyl group to -ketoacyl-ACP synthase Coupling the condensation to the decarboxylation makes the overall reaction highly exergonic 21:19

  38. step #7: the synthesis of butyryl-ACP the double bond generated in the last reaction is reduced the enzyme is enoly-CoA reductase NADPH is the electron donor Step 6: dehydration of D-3-hyroxyacly-ACP to trans-∆2-enoyl-ACP water is removed from C-2 and C-3 to yield a double bond in the product the enzyme is 3-hydroxyacyl-ACP dehydrase (dehyratase) Step5: -ketoacyl-ACP is reduced (at the carbonyl group at C-3) to D-3-hydroxyacyl-ACP the enzyme is -keotacyl-ACP reductase the electron donor is NADPH note that in fatty acid oxidation, the 3-hydroxyacyl-CoAs produced have the L configuration 21:20

  39. One round of synthesis through the fatty acid synthase complex is complete A 4 carbon butyryl-ACP has been generated • The butyryl-ACP is now transferred from ACP to a Cys-SH of the • -ketoacyl-ACP synthase portion of fatty acid synthase complex (just like an acetyl group was transferred in the first round) To start a second cycle (of the last four reactions) in order to lengthen the chain by two more carbons, butyryl-ACP condenses with another molecule of malonyl-ACP butyryl acts just like acetyl did in the first cycle, and CO2 is lossed Now there would be six carbons total here, with the first two from malonyl CoA and the next four from butyryl the product of this second round of reactions is hexanoly-ACP; the pattern continues till seven cycles of condensation and reduction produce the 16 carbon saturated palmitoyl group (still bound to ACP) 21:21

  40. for reasons not well understood, chain elongation generally stops at this point and free palmitate is released from the ACP molecule by the action of a hydrolytic activity in the synthase complex small amounts of longer fatty acids such as stearate (18:0) are also formed in certain plants (cococut and palm) chain termination occurs earlier and up to 90% of the fatty acids in the oils of these plants are between 8 and 14 carbons long 21:22

  41. 21:23

  42. O Free to bind Malonyl-CoA -CH2CH2CH2C~S- ACP TERMINATION Ketoacyl ACP Synthase -KS Transfer to Malonyl-CoA Transfer to KS -S-ACP Split out CO2 CO2 When C16 stage is reached, instead of transferring to KS, the transfer is to H2O and the fatty acid is released

  43. After 7* Cycles

  44. FA Synthesis Stoichiometry • Review the requirements for NADPH and ATP. • Be able to identify where all carbon atoms in 16:0 come from.

  45. Synthesis of palmitate • Part 1) 7 acetyl CoA + 7 CO2 + 7 ATP 7 malonyl CO2 + 7 AMP + 7 Pi • Part 2) Acetyl CoA + 7 malonlyl CoA + 14 NADPH + 14 H+ palmitate + 7 CO2 + 8 CoA + 14 NADP + + 6 H2 0 • Overall process 8 Acetyl CoA + 14 NADPH + 14 H + palmitate + 8 CoA + 6 H2 0 + 7 ADP + 7 Pi +14 NADP +

  46. Regulation of fatty acid biosynthesis occurs at the ACCase rxn • ACCase (in animals) • rate limiting step for FA synthesis • dephosphorylated (polymerized, active form) • phosphorylated (depolymerized, inactive form) • ACCase (plants, bacteria) • Not regulated by citrate or by phosphorylation. • Activated in plants by elevated [Mg2+] and lower pH. • Reciprocal regulation of FA synthesis and -oxidation. • Elevated [Malonyl-CoA] inhibits carnitine acyltransferase I and thus blocks -oxidation of FA at the level of transport. Rxn1 Rxn2

  47. Long chain fatty acid biosynthesis • End product of FA biosynthesis in animals: • Palmitate (16:0) • Fatty acid elongation systems: • in smooth ER, mitochondria • ER mechanism similar to general FA synthesis • Condensing, reduction, dehydration, reduction • malonyl-CoA as 2 carbon donor • different enzymes and coenzyme A instead of ACP

  48. Elongationsynthase, reductase; dehydrase; reductase The fatty acid synthesis pathway involving cytosolic FA synthase leads to palmitate Synthesis of longer FA involves elongation; in EK this process occurs in mitochondria and in ER membranes (microsomes) Microsomal elongation is prevalent; it involves: acyl-CoA intermediates and separate enzymes similar enzymatic complex as cytosolic FA Synthase; several condensing enzymes and one set of the rest three activities; can act on unsaturated FA GP_Fall2007 50