Chapter 23 Fatty Acid Catabolism

1. Chapter 23Fatty Acid Catabolism

2. Outline • How are fats mobilized from dietary intake and adipose tissue ? • How are fatty acids broken down ? • How are odd-carbon fatty acids oxidized ? • How are unsaturated fatty acids oxidized ? • Are there other ways to oxidize fatty acids ? • What are ketone bodies, and what role do they play in metabolism ?

3. Why Are Fatty Acids an Efficient Form of Energy Storage ? • Two reasons: • The carbon in fatty acids (mostly -CH2-) is in the most reduced form found in biomolecules (so its oxidation yields the maximum energy possible). • Fatty acids are not hydrated (as mono- and polysaccharides are), as a result they can pack more closely in storage tissues. Saving space in energy storage.

4. 23.1 How Are Fats Mobilized from Dietary Intake and Adipose Tissue ? • Most of the fatty material in diet and stored in adipose tissue are triacylglycerols (TAG). • Triglycerides represent the major dietary energy source for modern Americans. • Triglycerides are the major form of stored energy in the body, see Table 23.1. • Release of fatty acids from adipose tissue is under hormonal control triggered by glucagon, epinephrine or ACTH.

5. Energy Storage in Biomolecules Triacylglycerols = Triglycerides = Fats (saturated fatty acids) = Oils (unsaturated fatty acids).

6. 23.1 Mobilization of TAG: Dietary Intake Figure 23.3 (a) Digesting triacylglycerols (fats and oils). The pancreatic duct secretes digestive fluids (bile acids) into the duodenum, the first portion of the small intestine in order to emulsify fats. Glycocholic acid and taurocholic acid are the most abundant.

7. 23.1 Mobilization of TAG: Dietary Intake Figure 23.3 (b) Hydrolysis of triacylglycerols by pancreatic and intestinal lipases. Pancreatic lipases cleave fatty acids at the C-1 and C-3 positions. Resulting monoacylglycerols with fatty acids at C-2 are hydrolyzed by intestinal lipases. Fatty acids and monoacylglycerols are absorbed through the intestinal wall and assembled into lipoprotein aggregates termed chylomicrons (see Chapter 24).

8. 23.1 Mobilization of TAG: Dietary Intake Figure 23.3(b) Fatty acids and monoacylglycerols are absorbed through the intestinal wall and assembled into lipoprotein aggregates termed chylomicrons (see Chapter 24) for transport to adipose and other tissue.

9. 23.1 Mobilization of TAG: Adipose Tissue Figure 23.2 Hormonal initiated release of fatty acids from triacylglycerols (TAGs) in adipose tissue. Fatty acids are transported to other cell sites via albumin.

10. 23.2 How Are Fatty Acids Broken Down ? Knoop (1904) showed that fatty acids are degraded by removal of 2-C units • Knoop fed odd and even chain length, phenyl substituted fatty acids to dogs. • Urinalysis showed that odd chain lengths gave benzoic acid and even chain lengths gave phenylacetate. • The process begins with oxidation of the carbon that is "β" to the carboxyl carbon, so the process is called "β-oxidation".

11. 23.2 How Are Fatty Acids Broken Down ? Later discoveries: Albert Lehninger showed that this occurred inside the mitochondria. F. Lynen and E. Reichart showed that the 2-C unit released is acetyl-CoA, not free acetate. Also it was determined that β-oxidation is enzyme catalyzed and ATP is required. The process involves: 1. Activation of the fatty acids. 2. Transport into the mitochondria. 3. The reactions of β-oxidation.

12. 23.2 How Are Fatty Acids Broken Down ? Figure 23.5 Fatty acids are degraded by repeated cycles of oxidation at the β-carbon and cleavage of the Cα-Cβ bond to yield acetate units, in the form of acetyl-CoA.

13. 23.2 Activation of Fatty Acids Acyl-CoA synthetase (FA thiokinase) catalyzes activation of fatty acid. • Formation of a CoA ester is energetically expensive. There are several chain length specific enzymes. • Reaction just barely breaks even with ATP hydrolysis to AMP and PPi. • But subsequent hydrolysis of PPi drives the reaction strongly forward. • Note the acyl-adenylate intermediate in the mechanism.

14. 23.2 Activation of Fatty Acids Figure 23.6 The acyl-CoA synthetase activates fatty acids for β-oxidation. The reaction is driven by hydrolysis of ATP to AMP and pyrophosphate and by the subsequent hydrolysis of pyrophosphate. Even though only one ATP is used in this conversion, the cost of regeneration of ATP from the AMP level is equivalent to 2 ATP.

15. 23.2 Mechanism of Activation Figure 23.7 The mechanism of the acyl-CoA synthetase reaction involves fatty acid carboxylate attack on ATP to form an acyl-adenylate intermediate. The fatty acyl-CoA thioester product is formed by CoA attack on this intermediate.

16. Transport: The Carnitine Shuttle Carnitine carries fatty acyl groups across the inner mitochondrial membrane • Short chain fatty acids are carried directly into the mitochondrial matrix. • Medium and long-chain fatty acids cannot be directly transported into the matrix. • These FAs are converted to acyl carnitines and are then transported into the mitochondria. • Acyl-CoA esters are formed inside the inner membrane in this way. • CoASH pools inside and outside are separate.

17. Carnitine Acyl Transferase I - outside CAT I Figure 23.8 The formation of acylcarnitines and their transport across the inner mitochondrial membrane. The process involves the coordinated actions of two carnitine acyltransferases. CATI on the outer mitochondrial membrane is the regulatory site of fatty acid catabolism. Access ??

18. Carnitine Acyl Transferase II - inside An antiport CAT II Figure 23.8 The formation of acylcarnitines and their transport across the inner mitochondrial membrane. And CATII on the inner mitochondrial membranes plus a translocase that shuttles O-acylcarnitines across the inner membrane.

19. -Oxidation of Fatty Acids The Repeated Sequence of 4 Reactions is a spiral. • Strategy: Create a carbonyl group on the -C. • First 3 reactions do that; fourth cleaves the "-keto ester" in a reverse Claisen condensation. • Products: An acetyl-CoA and a fatty acid two carbons shorter. • The first three reactions are crucial and classic - we will see them again and again in other pathways.

20. -Oxidation of Fatty Acids Figure 23.9 The β-oxidation of saturated fatty acids involves a cycle (spiral) of four enzyme catalyzed reactions. 1. AcylCoA dehydrogenase 2. EnoylCoA hydratase 3. HydroxyacylCoA dehydrogenase 4. Thiolase or β-thiolase

21. A Family of Acyl-CoA Dehydrogenases Carry Out the First Reaction of β-Oxidation • The acylCoA dehydrogenases are a family of membrane-bound and soluble matrix enzymes. • As a fatty acyl chain is shortened in successive cycles of β-oxidation, it moves from the membrane-bound complex to the family of soluble matrix enzymes (Figure 23.10). • Mechanism involves proton abstraction, followed by double bond formation and hydride removal by FAD. • Electrons are passed to an electron transfer flavoprotein, and on to the electron transport chain.

22. 23.2 How Are Fatty Acids Broken Down ? Figure 23.12 The first step of β-oxidation is the acyl-CoA dehydrogenase reaction. The two electrons removed in this oxidation reaction are delivered to the electron-transport chain in the form of reduced coenzyme Q (UQH2).

23. 23.2 How Are Fatty Acids Broken Down ? Figure 23.13 The mechanism of acyl-CoA dehydrogenase. Removal of a proton from the α-carbon is followed by hydride transfer from the β-carbon to FAD.

24. 23.2 How Are Fatty Acids Broken Down ? Enoyl-CoA Hydratase Adds Water Across the Double Bond The second step is the addition of the elements of H2O across the new double bond in a stereospecific manner, yielding the corresponding hydoxyacyl-CoA. The reaction is catalyzed by enoyl-CoA hydratase, also called crotonase. These enzymes convert trans-enoyl-CoA derivatives to L-β-hydroxyacyl-CoA.

25. 23.2 How Are Fatty Acids Broken Down ? L-Hydroxyacyl-CoA Dehydrogenase Oxidizes the β-Hydroxyl Group The third reaction of this spiral is the oxidation of the hydroxyl group at the β-position to produce a β-ketoacyl-CoA derivative. This second oxidation reaction is catalyzed by L-hydroxyacyl-CoA dehydrogenase, an enzyme that requires NAD+ as a coenzyme. NADH produced in this reaction represents metabolic energy. Each NADH produced by this reaction drives the synthesis of 2.5 ATP.

26. The Fourth Reaction of β-Oxidation: Thiolase Thiolase or -ketothiolase • A cysteine thiolate on the thiolase enzyme attacks the -carbonyl group. • Thiol group of a new CoA attacks the shortened chain, forming a new, shorter acyl-CoA. • This is the reverse of a Claisen condensation: attack of the enolate of acetyl-CoA on a thioester. • Even though it forms a new thioester, the reaction is favorable and drives other three.

27. 23.2 How Are Fatty Acids Broken Down ? Figure 23.15 The mechanism of the thiolase reaction. Attack by an enzyme Cys thiolate group at the β-carbonyl carbon produces a tetrahedral intermediate, which decomposes with departure of acetyl-CoA, leaving an enzyme thioester intermediate. Attack by the thiol group of a second CoA yields a new acyl-CoA.

28. Summary of -Oxidation Repetition of the spiral yields a succession of acetate units • Thus, palmitic acid yields eight acetyl-CoAs. • Complete -oxidation of one palmitic acid yields 106 molecules of ATP. • Large energy yield is a consequence of the highly reduced state of the carbon in fatty acids. • This makes fatty acids the fuel of choice for migratory birds and many other animals.

29. Complete β-Oxidation of One Palmitic Acid Yields 106 Molecules of ATP Figure 23.16 Reduced coenzymes produced by β-oxidation and TCA cycle activity provide electrons that drive synthesis of ATP in oxidative phosphorylation. Complete oxidation of palmitoyl-CoA yields a net of 106 ATP.

30. 23.2 How Are Fatty Acids Broken Down ?

31. Summary – Enzymes Invoved Activation: AcylSCoA synthetase (ATP dependent) Transport: Carnitine acyltransferase I (CAT-I is regulatory site and is inhibited by malonylSCoA) Carnitine acyltransferase II b-Oxidation: AcylSCoA dehydrogenase EnoylSCoA hydratase b-hydroxyacylSCoA dehydrogenase b-ketothiolase

32. Fatty Acid Oxidation is an Important Source of Metabolic Water for Some Animals • Large amounts of metabolic water are generated by β-oxidation. • For certain animals – including desert animals (such as gerbils), killer whales (which do not drink seawater), and camels (whose hump is a large fat deposit) – the oxidation of stored fatty acids can be a significant source of dietary water. • Metabolism of fatty acids from such stores provides needed water, as well as metabolic energy, during periods when drinking water is not available.

33. Exercise Can Reverse the Consequences of Metabolic Syndrome • Metabolic syndrome is a combination of disorders that increase the risk of diabetes and cardiovascular disease. • Insights into how the body deals with high fat and sugar diets are emerging from a variety of studies. • Endurance training increases the mass of slow-twitch muscle, whereas resistance-training builds fast-twitch muscle fibers. • Slow-twitch muscles depend on -oxidation and TCA cycle activity and are “oxidative”. • Fast-twitch muscles are adapted for short bursts of energy from glycolysis and are “glycolytic”.

34. 23.3 How Are Odd-Carbon Fatty Acids Oxidized ? -Oxidation of odd-carbon fatty acids yields propionyl-CoA • This is a minor pathway (not many odd chains) and occurs in the microsomes. • Odd-carbon fatty acids are metabolized normally, until the last three-C fragment (propionyl-CoA) is reached. • Three reactions convert propionyl-CoA to succinyl-CoA. • Note the involvement of biotin and B12. • Note pathway for net oxidation of succinyl-CoA.

35. 23.3 How Are Odd-Carbon Fatty Acids Oxidized ? Figure 23.18 The conversion of propionyl-CoA (formed from β-oxidation of odd-carbon fatty acids) to succinyl-CoA is carried out by a trio of enzymes, as shown. Succinyl-CoA can be used as a net precursor to carbohydrate or enter the TCA cycle after conversion to pyruvate and acetylCoA.

36. A B12-Catalyzed Rearrangement Yields Succinyl-CoA from L-Methylmalonyl-CoA Figure 23.19 The methylmalonyl-CoA epimerase mechanism involves a resonance-stabilized carbanion at the α-position.

37. Net Oxidation of Succinyl-CoA Requires Conversion to Acetyl-CoA • Succinyl-CoA derived from propionyl-CoA can enter the TCA cycle. • But TCA cycle intermediates are catalytic and net consumption of succinyl-CoA does not occur directly in TCA. • However, succinyl-CoA from β-oxidation CAN be converted to malate and then transported from the mitochondrial matrix to the cytosol, where it can be oxidatively decarboxylated to pyruvate and CO2 by malic enzyme. • Pyruvate can move back to the matrix where it can enter the TCA cycle via AcetylCoA.

38. Net Oxidation of Succinyl-CoA Requires Conversion to Acetyl-CoA Figure 23.20 Route to the Kreb’s cycle. The malic enzyme reaction proceeds by oxidation of malate to oxaloacetate, followed by decarboxylation to yield pyruvate. Pyruvate goes to the matrix to PDH, etc.

39. The Activation of Vitamin B12 • Conversion of inactive vitamin B12 to active 5'-deoxyadenosylcobalamin involves three steps. • Two flavoprotein reductases convert Co3+ to Co2+ and then to Co+. • Co+ is a powerful nucleophile, which can attack the C-5’ of ATP to form 5-deoxyadenosylcobalamin. • This is one of only two known adenosyl transfers in biological systems (the other is the formation of S-adenosylmethionine – see Chapter 25).

40. The Activation of Vitamin B12 Formation of the active coenzyme form of B12 is initiated by the action of flavoprotein reductases.

41. The Activation of Vitamin B12 Formation of the active coenzyme form of B12 is initiated by the action of flavoprotein reductases. The resulting Co+ species, dubbed a supernucleophile, attacks the 5'-carbon of ATP in an unusual adenosyl transfer.

42. The Activation of Vitamin B12 Homolytic cleavage of the Co3+-C bond produces a reactive free radical that facilitates rearrangements such as that in the methylmalonyl-CoA mutase reaction.

43. The Activation of Vitamin B12 The reactive free radical form of vitamin B12 that facilitates rearrangements such as in the methylmalonyl-CoA mutase.

44. 23.4 How Are Unsaturated Fatty Acids Oxidized ? Consider monounsaturated fatty acids: • Oleic and palmitoleic acids are 9 acids. • Normal -oxidation proceeds for three cycles. • cis-3 acyl-CoA cannot be utilized by acyl-CoA dehydrogenase. • Enoyl-CoA isomerase converts this to trans- 2 acyl CoA. • β-oxidation continues from this point. • An FADH2 is lost since the double bond was already present, so less ATP results.

45. 23.4 How Are Unsaturated Fatty Acids Oxidized ? γβ Figure 23.21 β-Oxidation of unsaturated fatty acids. In the case of oleoyl-CoA, three β-oxidation cycles produce three molecules of acetyl-CoA and leave cis-Δ3-dodecenoyl-CoA. Rearrangement of enoyl-CoA isomerase gives the trans-Δ2 species, which then proceeds normally through the β-oxidation pathway.

46. 23.4 How Are Unsaturated Fatty Acids Oxidized ? βα Figure 23.21 β-Oxidation of unsaturated fatty acids. In the case of oleoyl-CoA, three β-oxidation cycles produce three molecules of acetyl-CoA and leave cis-Δ3-dodecenoyl-CoA. Rearrangement of enoyl-CoA isomerase gives the trans-Δ2 species, which then proceeds through the β-oxidation pathway.

47. Degradation of Polyunsaturated Fatty Acids Requires 2,4-Dienoyl-CoA Reductase • Degradation of polyunsaturated fatty acids is slightly more complicated. • As in the case of oleic acid, -oxidation proceeds through 3 cycles, then enoyl-CoA isomerase converts the cis-Δ3 double bond to a trans-Δ2 double bond and the 4th cycle is completed. • AcylCoA dehydrogenase inserts a double bond. • This results in a trans-2, cis-4 conjugated double bond structure which is the problem. • 2,4-Dienoyl-CoA reductase solves this problem.

48. Degradation of Polyunsaturated Fatty Acids Degradation of Polyunsaturated Fatty Acids Figure 23.22 The oxidation pathway for polyunsaturated fatty acids in mammals. The 9 double bond is treated the same as oleic acid.

49. Degradation of Polyunsaturated Fatty Acids Degradation of Polyunsaturated Fatty Acids Requires 2,4-Dienoyl-CoA Reductase Figure 23.22 The oxidation pathway for polyunsaturated fatty acids in mammals. There is an NADH equivalent lost here due to the NADPH used in the reductase reaction as well as an FADH2 for the first double bond.

50. 23.5 Are There Other Ways to Oxidize Fatty Acids ? • β-Oxidation also occurs in liver peroxisomes with a loss of ATP production. • This is the primary site of β-oxidation in plants. • Peroxisomal β-oxidationrequires FAD-dependent acyl-CoA oxidase. • Branched-chain fatty acids are degraded via -oxidation. • -Oxidation of fatty acids yields small amounts of dicarboxylic acids.