FATTY ACID METABOLISM

1. FATTY ACID METABOLISM

2. FATTY ACID METABOLISM A fatty acid is a carboxylic acid. often with a long unbranched aliphatic chain, which is either saturated or unsaturated. Carboxylic acids as short as butyric acid (4 carbon atoms) are considered to be fatty acids, Fatty acids are produced by the hydrolysis of the ester linkages in a fat or biological oil (both of which are triglycerides), with the removal of glycerol. The most abundant natural fatty acids have an even number of carbon atoms.

3. Major Physiological Roles oF Fatty Acids • Fatty Acids (FAs) are building blocks of phospholipids and glycolipids. • Many proteins are modified by the covalent attachment of FAs, which targets them to membrane locations. • FAs are fuel molecules that are stored as triacylglycerols (or triglycerides) (TGs). • FAs derivatives serve as hormones and intracellular messengers.

4. In mammals, the major site of accumulation of triacylglycerols is the cytoplasm of adipose cells (fat cells)

5. Fatty acid degradation and synthesis are relatively simple processes that are essentially the reverse of each other.

6. TGs Are Highly Concentrated Energy Stores • 9 Kcal/g VS 4 Kcal/g for protein/carbohydrates. • Anhydrous storage in adipose VS hydrated glycogen in liver/muscle • A typical 70kg man would have fuel reserves: 100,000 kcal in TGs, 25,000 kcal in protein (mostly in muscle), 600 kcal in glycogen 40 kcal in glucose. • TGs constitute about 11 kg of his total body weight. • If the 11kg TGs to be stored in Glycogen form, his total body weight would be 55kg greater (11+55 kg) + 59. • Storing energy in TGs saves 55Kg of body weight. • Glycogen & Glucose provide energy for short-term (a day or so), while TGs support the body with energy for several weeks.

7. Dietary Lipids Are Digested by Pancreatic Lipases • TGs in the intestinal lumen are incorporated into micelles formed with the aid of bile salts (e.g. glycocholate) • Amphipathic molecules synthesized from cholesterol in the liver and secreted from the gall bladder.

8. Incorporation of TGs into micelles orients the ester bonds of the TGs toward the surface of the micelle. • This renders the bonds more susceptible to digestion by pancreatic lipases that are in aqueous solution. • The digestion products are carried in micelles to the intestinal epithelium where they are absorbed across the plasma membrane.

9. Dietary Lipids Are Transported in Chylomicrons • In the intestinal mucosal cells, the TGs are re-synthesized from FAs and monoacylglycerols. • Then packaged into chylomicrons.

10. Dietary TGs Bile salt Other lipids & proteins micelles H2O Pancreatic Lipase Chylomicrons Lymph system FAs TGs Monoglyceroles Intestinal lumen Intestinal mucosa Monoglyceroles FAs lipoprotein lipases Storage in adipose tissue TGs b-oxidation in muscle

11. The Utilization of Fatty Acids as Fuel Requires Three Stages of Processing • The lipids must be mobilized: • TGs are degraded to FAs and glycerol • They are released from the adipose tissue and transported to the energy-requiring tissues. • At these tissues, the fatty acids must be activated and transported into mitochondria for degradation. • The fatty acids are broken down step-by-step into acetyl CoA, which is then processed in the citric acid cycle.

12. Triacylglycerols Are Hydrolyzed by Cyclic AMP-Regulated Lipases epinephrine, norepinephrine, glucagon, adrenocorticotropic hormone

13. Glycerol formed by lipolysis is absorbed by the liver where it enters glycolysis or gluconeogenesis. phosphatase

14. Fatty Acids Are Linked to CoA Before They Are Oxidized • Fatty acids are oxidized in mitochondria. • They are activated before they enter the mitochondrial matrix. • ATP drives the formation of a thioester linkage between the carboxyl group of a FA and the sulfhydryl group of CoA. • This activation reaction takes place on the outer mitochondrial membrane, where it is catalyzed by acyl CoA synthase (also called fatty acid thiokinase).

15. The activation of a fatty acid is accomplished in two steps: • The FA reacts with ATP to form an acyl adenylate. • The sulfhydryl group of CoA then attacks the acyl adenylate, which is tightly bound to the enzyme, to form acyl CoA and AMP.

16. These partial reactions are freely reversible • One high-transfer-potential compound is cleaved (between PPi and AMP) and one is formed (the thioester acyl CoA). • How is the overall reaction driven forward? • The answer is that PPi is rapidly hydrolyzed by a pyrophosphatase • The equivalent of two molecules of ATP are consumed.

17. Carnitine Carries Long-Chain Activated Fatty Acids into the Mitochondrial Matrix • Activated long-chain FAs are transported across the membrane by conjugating them to carnitine, a zwitterionic alcohol. • This reaction is catalyzed by carnitine acyltransferase I (also called carnitine palmitoyl transferase I), bound to the outer mitochondrial membrane.

18. Acyl carnitine is shuttled across the inner mitochondrial membrane by a translocase • The acyl group is transferred back to CoA on the matrix side of the membrane. • This reaction, which is catalyzed by carnitine acyltransferase II (carnitine palmitoyl transferase II), is simply the reverse of the reaction that takes place in the cytosol.

19. Normally, the transfer of an acyl group from an alcohol to a sulfhydryl group is thermodynamically unfavorable. • However equilibrium constant for this reaction for carnitine is near 1. • Apparently because carnatine and its esters are solvated differently from most other alcohols and their esters due to the zwitterionic nature of carnitine. • As a result, the O-acyl link in carnitine has a high group-transfer potential. • Finally, the translocase returns carnitine to the cytosolic side in exchange for an incoming acyl carnitine.

20. Fatty Acid Oxidation • Through a sequence of 4 reactions: • Oxidation by FAD • Hydration by H2O • Oxidation by NAD+ • Thiolysis by CoA

21. oxidation of acyl CoA by an acyl CoA dehydrogenase to give an enoyl CoA with a transC2=C3. electron-transferring flavoprotein ETF:ubiquinone reductase, an iron-sulfur protein

22. Stereospecific hydrationof the C2=C3by enoyl CoA hydratase.

23. A second oxidation reaction converts the hydroxyl group at C3 into a keto group and generates NADH. This oxidation is catalyzed by L-3-hydroxyacyl CoA dehydrogenase, which is specific for the L isomer of the hydroxyacyl substrate

24. Thiolysis of 3-ketoacyl CoA by the thiol group of a second molecule of CoA yields acetyl CoA and an acyl CoAshortened by two carbon atoms. This thiolytic cleavage is catalyzed by b-ketothiolase.

25. The shortened acyl CoA then undergoes another cycle of oxidation, starting with the reaction catalyzed by acyl CoA dehydrogenase

26. Step Reaction Enzyme 1 Fatty acid + CoA + ATP acyl CoA + AMP + PPi Acyl CoA synthetase [also called fatty acid thiokinase and fatty acid:CoA ligase (AMP)] 2 Carnitine + acyl CoA acyl carnitine + CoA Carnitine acyltransferase (also called carnitine palmitoyl transferase) 3 Acyl CoA + E-FAD trans- D2 -enoyl CoA + E-FADH2 Acyl CoA dehydrogenases (several isozymes having different chain-length specificity) 4 trans-D2 -Enoyl CoA +H2O L-3-hydroxyacyl CoA Enoyl CoA hydratase (also called crotonase or 3-hydroxyacyl CoA hydrolyase) 5 L-3-Hydroxyacyl CoA + NAD+ 3-ketoacyl CoA + NADH + H+ L-3-Hydroxyacyl CoA dehydrogenase 6 3-ketoacyl CoA + CoA acetyl CoA + acyl CoA (shortened by C2) b-Ketothiolase (also called thiolase)

27. The Complete Oxidation of Palmitate Yields 106 Molecules of ATP • The degradation of palmitoyl CoA (C16-acyl CoA) requires seven reaction cycles. • In seventh cycle, C4-ketoacyl CoA is thiolyzed to two molecules of acetyl CoA. • Hence, the stoichiometry of oxidation of palmitoyl CoA is:

28. Approximately 2.5 of ATP are generated per each of the 7 molecules of NADH. • 1.5 of ATP are formed for each of the 7 molecules of FADH2. • Recall that the oxidation of acetyl CoA by the citric acid cycle yields 10 molecules of ATP. • Hence, the number of ATP molecules formed in the oxidation of palmitoyl CoA is: • 10.5 from the 7 molecules of FADH2 • 17.5 from the 7 molecules of NADH • 80 from the 8 molecules of acetyl CoA • ~2 ATP are consumed in the activation of palmitate • The total is 106 ATP.

29. Certain Fatty Acids Require Additional Steps for Degradation 1. The oxidation of unsaturated fatty acids requires additional steps. 2. Fatty acids containing an odd number of carbon atoms yield a propionyl CoA at the final thiolysis step. 3. Must be converted into an easily usable form by additional enzyme reactions.

30. Unsaturated fatty acids • Palmitoleate, C16 unsaturated fatty acid has one double bond between C-9 and C-10, (D9). • It is activated and transported across the inner mitochondrial membrane in the same way as saturated fatty acids. • Palmitoleoyl CoA then undergoes 3 cycles of degradation; carried out by the same enzymes as in saturated ones.

31. The cis-D3-enoyl CoA formed in the third round is not a substrate for acyl CoA dehydrogenase. • The presence of a C3=C4 prevents the formation of another double bond between C2 and C3. • An isomerase changes the position and configuration of this double bond.

32. The polyunsaturated fatty acid: (linoleate, C18) with cis-D9 and cis-D12 double bonds. The cis-D3 formed after three rounds of b-oxidation is converted into a trans-D2 by the isomerase. The acyl CoA produced by another round of b-oxidation contains a cis-D4 double bond.

33. Dehydrogenation of this species by acyl CoA dehydrogenase yields a 2,4-dienoyl intermediate, which is not a substrate for the next enzyme in the b-oxidation pathway. 2,4-dienoyl CoA reductase, uses NADPH to reduce the 2,4-dienoyl intermediate to trans-D3-enoyl CoA. Cis-D3-Enoyl CoA isomerase then converts trans-D3-enoyl CoA into the trans-D2 form, a customary intermediate in the b-oxidation pathway

34. Only two extra enzymes needed for oxidation of any polyunsaturated fatty acid: • Odd-numbered double bonds are handled by the isomerase. • Even-numbered ones by both reductase and isomerase.

35. Odd-Chain Fatty Acids Yield Propionyl Coenzyme A in the Final Thiolysis Step • Fatty acids having an odd number of carbon atoms are minor species. • They’re oxidized in same way as those having an even number of C. • Propionyl CoA and acetyl CoA, rather than two molecules of acetyl CoA, are produced in the final round of degradation.

36. Propionyl CoA is carboxylated by propionyl CoA carboxylase yielding D-isomer of methylmalonyl CoA at the expense of ATP hydrolysis. • This enzyme is a biotin enzyme that is homologous to and has a catalytic mechanism like that of pyruvate carboxylase. • The D-isomer is racemized into the L-isomer. • The L-isomer of methylmalonyl CoAis converted by a mutase into succinyl CoA • The (-CO-S-CoA) group migrates from C2 to C3 in exchange for a hydrogen atom.

37. Fatty Acids Are Also Oxidized in Peroxisomes • Some oxidation takes place in cellular organelles called peroxisomes that have high concentrations of catalase. • Fatty acid oxidation in these organelles, stops at octanyl CoA. • It may serve to shorten long chains to make them better substrates of b-oxidation in mitochondria.

38. Peroxisomal oxidation differs from b-oxidation in the initial dehydrogenation reaction. • In peroxisomes, a flavoprotein dehydrogenase transfers electrons to O2 to yield H2O2 • Catalase is needed to convert the H2O2 produced in the initial reaction into H2Oand O2 . • Subsequent steps are identical with their mitochondrial counterparts, although they are carried out by different isoforms of the enzymes.

39. Fats burn in the flame of carbohydrates • The entry of acetyl CoA into the citric acid cycle depends on the availability of oxaloacetate for the formation of citrate. • Oxaloacetate is normally formed from pyruvate, the product of glycolysis, by pyruvate carboxylase • The concentration of oxaloacetate is lowered if carbohydrate is unavailable or improperly utilized.

40. In fasting or diabetes • Oxaloacetate is consumed to form glucose by the gluconeogenesis. • Under these conditions, acetyl CoA is diverted to the formation of acetoacetate and D-3-hydroxybutyrate. • Acetoacetate, D-3-hydroxybutyrate, and acetone are often referred to as ketone bodies.

41. D-3-Hydroxybutyrate dehydrogenase Thiolase Hydroxymethylglutaryl CoA synthase Hydroxymethylglutaryl CoA cleavage enzyme

42. Ketone Bodies Are a Major Fuel in Some Tissues • Acetoacetate and 3-hydroxybutyrate are produced mainly in the liver mitochondria. • Acetoacetate and 3-hydroxybutyrate are normal fuels of respiration and are quantitatively important as sources of energy. • Heart muscle and the renal cortex use acetoacetate in preference to glucose. • In contrast, glucose is the major fuel for the brain and red blood cells in normal conditions. • However, the brain adapts to the utilization of acetoacetate during starvation and diabetes

43. Acetoacetate can be activated by the transfer of CoA from succinyl CoA in a reaction catalyzed by a specific CoA transferase. • Acetoacetyl CoA is then cleaved by thiolase to yield two molecules of acetyl CoA, which can then enter the citric acid cycle. • The liver has acetoacetate available to supply other organs because it lacks this particular CoA transferase. • Acetoacetate also has a regulatory role: • High levels of acetoacetate in the blood lead to a decrease in the rate of lipolysis in adipose tissue.

44. Animals Cannot Convert Fatty Acids into Glucose • Acetyl CoAcannot be converted intopyruvate or oxaloacetate in animals. • Oxaloacetate is regenerated, but it is not formed de novo when the acetyl unit of acetyl CoA is oxidized by the citric acid cycle. • In contrast, plants have two additional enzymes enabling them to convert the carbon atoms of acetyl CoA into oxaloacetate.

45. Fatty Acids Synthesis

46. Fatty Acids Are Synthesized and Degraded by Different Pathways • Some important differences between the pathways are: • Synthesis takes place in the cytosol, in contrast with degradation, which takes place primarily in the mitochondrial matrix. • Intermediates in fatty acid synthesis are covalently linked to the sulfhydryl groups of an acyl carrier protein (ACP), whereas intermediates in fatty acid breakdown are covalently attached to the sulfhydryl group of coenzyme A. • The enzymes of fatty acid synthesis in higher organisms are joined in a single polypeptide chain calledfatty acid synthase. In contrast, the degradative enzymes do not seem to be associated.

47. The growing fatty acid chain is elongated by the sequential addition of two-carbon units derived from acetyl CoA. • The activated donor of two carbon units in the elongation step is malonyl ACP. The elongation reaction is driven by the release of CO2. • The reductant in fatty acid synthesis is NADPH, whereas the oxidants in fatty acid degradation are NAD+ and FAD. • Elongation by the fatty acid synthase complex stops on formation of palmitate (C16). • Further elongation and the insertion of double bonds are carried out by other enzyme systems.

48. The Formation of Malonyl Coenzyme A Is the Committed Step in Fatty Acid Synthesis • Fatty acid synthesis starts with the carboxylation of acetyl CoA to malonyl CoA by acetyl CoA carboxylase, which contains a biotin prosthetic group.

49. The carboxyl group of biotin is covalently attached to the e-amino group of a lysine residue, as in pyruvate carboxylase. • A carboxybiotin intermediate is formed at the expense of the hydrolysis of a molecule of ATP. • The activated CO2 group in this intermediate is then transferred to acetyl CoA to form malonyl CoA. • Acetyl CoA carboxylase is also the essential regulatory enzyme for fatty acid metabolism.

50. Intermediates in Fatty Acid Synthesis Are Attached to an Acyl Carrier Protein • They are linked to the sulfhydryl terminus of a phosphopantetheine group, which is, in turn, attached to a serine residue of the acyl carrier protein. • Acyl carrier protein (ACP) a single polypeptide chain of 77 residues, can be regarded as a giant prosthetic group, a "macro CoA."