Understanding Fatty Acid Synthesis Mechanisms

1. Fatty Acid Synthesis

2. Introduction • There are three systems for the synthesis of fatty acids • De novo synthesis of FAs in cytoplasm • Chain elongation in mitochondria • Chain elongation in microsomes

3. De novo synthesis of FAs • In mammals fatty acid synthesis occurs primarily in the cytosol of the liver and adipose tissues .It also occurs in mammary glands during lactation. • Acetyl-CoA is the starting material for FA synthesis. However, most acetyl-CoA in mitochondria(from the breakdown of sugars, some amino acids and other fatty acids). • So, acetyl-CoA must be transferred from the mitochondria to the cytosol • BUT Mitochondria not permeable to acetyl CoA

4. Continue.. • Citrate-malate-pyruvate shuttle provides cytosolic acetyl CoA and reducing equivalents NADPH for fatty acid synthesis. • Acetyl–CoA units are shuttled out of the mitochondrial matrix as citrate.

5. Continue.. RULE: Fatty acid synthesis is a stepwise assembly of acetyl CoA unit (mostly as malonyl CoA) ending with palmitate (16 C saturated) • 4 Steps repeating cycle, extension 2C: • Condensation • Reduction • Dehydration • Additional reduction

6. Formation of Malonyl-coenzyme A (Activation of acetate) • Is the committed step in fatty acid synthesis (Rate Limiting Reaction) • It takes place in two steps: 1. Carboxylation of biotin (involving ATP) 2. Transfer of the carboxyl to acetyl-CoA to form malonyl-CoA • Reactions are catalyzed by acetyl-CoA carboxylase (multienzyme)

7. Fatty acid synthase • It is a multi-enzyme complex consist of 7 enzymes linked covalently in a single polypeptide chain. • It is a dimer, and each monomer is identical, consisting of one chain (250 kD) containing all seven enzyme activities of fatty acid synthase and an acyl carrier protein (ACP) • ACP contains the vitamin pantothenic acid in the form of 4'-phosphopantetheine (Pant). ACP is the part that carry the acyl groups during fatty acid synthesis

8. Fatty acid synthase • It is a multi-enzyme complex consist of 7 enzymes linked covalently in a single polypeptide chain. • It is a dimer, and each monomer is identical, consisting of one chain (250 kD) containing all seven enzyme activities of fatty acid synthase and an acyl carrier protein (ACP) • ACP contains the vitamin pantothenic acid in the form of 4'-phosphopantetheine (Pant). ACP is the part that carry the acyl groups during fatty acid synthesis

9. Continue.. 1- A molecule of acetate is transferred from Acetyl CoA to the –SH group of ACP by acetyl CoA-ACP transacylase (initiation or priming). 2- Next, this 2C fragment is transferred to a cysteine residue in the active site of the condensing enzyme. 3-The now-empty ACP accepts a 3C malonate unit from malonyl CoA, malonyl CoA-ACP transacylase catalyzes this reaction

10. Continue.. 4- Acetyl unit (on the condensing enzyme) condenses with 2 carbon portion of malonyl unit on ACP forming acetoacetyl-S- ACP with release of CO2. This reaction is catalyzed by β-ketoacyl –ACP synthase  Active site on the condensing enzyme is free.

11. Continue.. 5-The β-ketone is reduced to an alcohol by e- transfer from NADPH. 6- Dehydration yields a trans double bond.7- Reduction by NADPH yields a saturated chain.

12. Continue.. 8- Following transfer of the growing fatty acid from Pant to the Condensing Enzyme's cysteine sulfhydryl, the cycle begins again, with another malonyl-CoA. Note: Acetyl residue successively added is derived from the 2C atoms of malonyl CoA with the release of the third C as CO2 EXCEPT the 2 donated by the original acetyl CoA which are found at the methyl group end of the fatty acid.

13. Product Release • When the fatty acid is 16 carbon atoms long, a Thioesterase domain catalyzes hydrolysis of the thioester linking the fatty acid to phosphopantetheine. • The16-C saturated fatty acid palmitate is the final product of the Fatty Acid Synthase complex (but it may produce short chain FAs) • Further elongation and insertion of double bonds are carried out by other enzyme system.

14. Palmitate, a 16-C saturated fatty acid, is the final product of the Fatty Acid Synthase reactions. 1- a. How many acetyl-CoA used for initial priming of enzyme? 1 b. How many acetyl-CoA used for synthesis of each malonate? 1 c. How many malonate used (how many reaction cycles) per synthesis of one 16-C palminate? 7 d. Total acetyl-CoA used for priming & for syntheisis of malonate, a + b(c): 8 2- a. How many ~P bonds of ATP used for synthesis of each malonate? 1 b. Total ~P bonds of ATP used for synthesis of one 16-C palmitate,2a(1c): 7 3- a. How many NADPH used per reaction cycle? 2 b. Total NADPH used per synthesis of one 16-C palmitate, 3a(1c): 14 No. of cycles = (C/2) – 1 No. of Malonate molecules = (C/2) – 1 No. of Acetyl CoA molecules= [(C/2) – 1] +1 No. of NADPH molecules = [(C/2) – 1] x2

15. Regulation of FA Synthesis • Allosteric regulation • Acetyl CoA carboxylase, which catalyzes the committed step in fatty acid synthesis, is a key control site. • End-product fatty acid is a feedback inhibitor (palmitoyl-CoA) • Activatedby citrate, which increases in well-fed state and is an indicator of a plentiful supply of acetyl-CoA • Inhibitedby long-chain acyl-CoA

16. Regulation of FA Synthesis • Glucagon inhibits fatty acid synthesis while increasing lipid breakdown and fatty acid β-oxidation. • Acetyl CoA cayboxylase is inactivated by phosphorylation • Insulin prevents action of glucagonInhibits lipases/activates acetyl Co A cayboxylase

17. Further Processing of C16 Fatty Acids Additional Elongation In mammalian systems FA elongation can occur either in : • Microsomes • Mitochondria

18. Chain Elongation in Microsomes • The reactions are similar to that which occurs in the cytosolic FA synthase in that: a) The source of the 2 carbon units is malonyl CoA. b) NADPH is used as reducing power. • In contrast to denovo synthesis of Fatty Acids, the intermediates in the subsequent reactions are CoA esters, indicating that the process is carried out by separate enzymes rather than a complex of FA synthase type. (uses CoA instead of ACP as the acyl carrier) • It is the main site for elongation of existing long chain FAs molecules.

19. Chain Elongation in Mitochondria • It differs from the microsomal system in that acetyl CoA is the source of the added 2C atoms (instead of malonyl CoA) • NADH and NADPH are sources of reducing agents • This system operate by simple reversal of the pathway of FA oxidation with the exception that, NADPH-linked α,β-unsaturated acyl CoA reductase replaces FAD linked acyl CoA dehydrogenase. • The mitochondrial system serves in the elongation of shorter chain fatty acids to long chin FAs.

20. Biosynthesis of Unsaturated Fatty Acids • Desaturases introduce double bonds at specific positions in a fatty acid chain. • Mammalian cells are unable to produce double bonds at certain locations, e.g., ∆ 12. • Thus some polyunsaturated fatty acids are dietary essentials, e.g., linoleic acid, 18:2 cis ∆ 9,12 (18 C atoms long, with cis double bonds at carbons 9-10 & 12-13)

21. Continue.. • Formation of a double bond in a fatty acid involves the following endoplasmic reticulum membrane proteins in mammalian cells: • NADH-cyt b5 Reductase, a flavoprotein with FAD as prosthetic group. • Cytochrome b5, which may be a separate protein or a domain at one end of the desaturase. • Desaturase, with an active site that contains two iron atoms complexed by histidine residues

22. Continue.. • The ∆9 desaturase in the endoplasmic reticulum catalyzes the conversion of stearate (18:0) to oleate (18:1 cis ∆ 9) . • the overall reaction is: stearate + NADH + H+ + O2 oleate + NAD+ + 2H2O • Synthesis of polyunsaturated fatty acids involves desaturase and elongase systems

23. Differences in the oxidation and synthesis of FAs