1. Chapter 22: Fatty Acid Metabolism Copyright © 2007 by W. H. Freeman and Company Berg Tymoczko Stryer Biochemistry Sixth Edition

2. An Adipocyte A fat cell, for storage of triacylglycerol.

3. A Triacylglycerol (TAG) TAG is primarily storage fat to be used for energy. Other important functions are thermal insulation and protection from mechanical shock.

4. Outline of Fatty Acid Metabolism Synthesis which occurs in the cytosol adds 2 C at a time to the carboxylate end. Degradation is mitochondial and removes 2 C at a time from the carboxylate end. R = CoA (catabolic) and ACP (anabolic)

5. Metabolic reaction steps Degradation is oxidative. Synthesis is reductive. Catabolic Anabolic

6. Triacylglycerol (TAG) Hydrolysis Lipases catalyze enzymatic hydrolysis of TAG to yield fatty acids and glycerol.

7. Glycocholic Acid Bile acids are emulsifiers that aid in intestinal digestion of triacylglycerols. (cholic acid + glycine)

8. Transport intestine to lymph TAG is hydrolyzed for membrane transport and reformed for chylomicron transport.

9. TAG Hydrolysis for energy Lipases in adipose tissue are hormone stimulated.

10. Hormonal effects TAGs are stored in adipocytes, and fatty acids are released to supply energy demands. Hormone-sensitive lipases convert TAGs to free fatty acids and glycerol. At low carbohydrate and low insulin concentrations, TAG hydrolysis is stimulated by increased epinephrine and glucagon which activate Protein kinase A through G- protein transduction.

11. Mobilization of TAG Stimulated by epinephrine and glucagon.

12. Chylomicrons TAGs, cholesterol and cholesterol esters are insoluble in water and cannot be transported in blood or lymph as free molecules so are transported as lipoproteins. Chylomicrons are the largest and most nonpolar lipoproteins. They deliver TAGs from the intestine (via lymph and blood) to tissues (muscle for energy, adipose for storage) and are present in blood only after feeding. Cholesterol-rich chylomicron remnants deliver cholesterol to the liver

13. Glycerol and Fatty Acid

14. Metabolism of Glycerol

15. Fatty Acid Oxidation Knoop proposed  -oxidation in 1904 based on studies with phenyl substituted fatty acids of odd and even chain lengths. Even chains -- > phenylacetic acid Odd chains -- > benzoic acid Later discoveries: (1) reactions occur in the mitochondrial matrix (2) reactions are enzyme catalyzed (3) Coenzyme A is required (4) ATP is required

16. Fatty Acid Oxidation FA Catabolism can be viewed in three stages: (1) Activation of fatty acids in the cytosol. A coenzyme A derivative is formed. (2) Transport into the mitochondria. Carnitine is used for FA transport. (3)  -oxidation degrades fatty acids to two- carbon fragments (as acetyl CoA) in the matrix where it goes to the Krebs cycle.

17. Fatty Acid (FA) Activation Occurs in the cytosol. Hydrolysis of pyrophosphate (PPi  2 Pi) enhances formation of the acylSCoA product. This is a two step process catalyzed by acylCoA synthetase. AcylCoA synthetase

18. Activation Steps There are several chain length specific acylCoA synthetases (short, medium, long).

19. Acyl Adenylate Precursor to Acyl Coenzyme A.

20. Formation of Acyl Carnitine Catalyzed by Carnitine acyltransferase. Carnitine acyltransferase I is in the intermembrane space and Carnitine acyltransferase II is in the mitochondrial matrix.

21. Transport of activated FA 1.Acylcarnitine formation 2.Translocation 3.AcylSCoA formation Maintains separate CoA pools.

22.  -Oxidation of Fatty Acids The pathway for degradation of fatty acids is in the mitochondrial matrix and is called the  -oxidation spiral. Oxidation occurs at the  -carbon of the fatty acid.

23.  -Oxidation Sequence 1.Oxidation (FAD) 2.Hydration (HOH) 3.Oxidation (NAD + ) 4.Cleavage (CoASH)

24. FAD Oxidation Double bond is trans. Compare to succinate dehydrogenase in the Kreb’s cycle. AcylCoA DH

25. Electron Flow/Proton Pumps

26. Electrons from FAD Oxidation

27. Hydration 1.Oxidation (FAD) 2.Hydration (HOH) 3.Oxidation (NAD + ) 4.Cleavage (CoASH) EnoylCoA hydratase

28. NAD + Oxidation 1.Oxidation (FAD) 2.Hydration (HOH) 3.Oxidation (NAD + ) 4.Cleavage (CoASH)  -hydroxyacylCoA dehydrogenase

29. Cleavage 1.Oxidation (FAD) 2.Hydration (HOH) 3.Oxidation (NAD + ) 4.Cleavage (CoASH)  -ketothiolase

30.  -Oxidation Summary The pathway for degradation of fatty acids is called the  -oxidation spiral.

31. Successive 2 carbon cleavages Each comes off as acetylSCoA and the four  -oxidation steps are required.

32. Unsaturated FA metabolism Normal  -oxidation occurs until the cis double bond is in the  position. Isomerization with migration moves the bond to a trans  An FADH 2 is lost due to the already present double bond.

33. Multiple Unsaturations For multiple double bonds,  9,12,  -oxidation proceeds as before until the second db is in the  position.  -oxidation inserts an  unsaturation giving two conjugated double bonds. The NADPH enzyme, 2,4-dienoyl reductase, acts with migration to yield a trans  unsaturation. An isomerase catalyzes migration to  and retains the trans structure &  -ox. continues. For the second db an FADH 2 is not lost but an NADPH is used negating the NADH produced.

34. Multiple Unsat. 3 cycles of  -ox

35. Odd Chain Fatty Acids  -oxidation of a FA with an odd number of carbon atoms produces propionic acid.

36. Odd Chain Fatty Acids Odd-chain fatty acids occur in bacteria and microorganisms and is a minor metabolic route in mammals (occurs in liver). Three enzymes are needed to convert propionyl CoA to succinyl CoA (so the last three carbons do not yield acetylCoA but do give a citric acid cycle intermediate). Propionyl CoA carboxylase (requires biotin) Malonyl CoA racemase Malonyl CoA mutase(requires B-12)

37. Odd Chain FA Pathway Propionyl CoA carboxylase Malonyl CoA racemase Malonyl CoA mutase

38. B-12, a coenzyme for mutase

39. Methylmalonyl SCoA mutase Co +3 -- > Co +2

40. Deoxyadenosyl Radical Homolytic bond cleavage between the deoxyadenosyl 5'CH 2 and Cobalt.

41. Radical Mechanism Methyl malonylSCoA mutase catalyzes a radical migration of the thioester carbonyl.

42. Peroxisome oxidation  -oxidation occurs in liver peroxisomes as well as the mitochondrial matrix. All  - oxidation in plants occurs in the peroxisomes(glyoxysomes) and none in the matrix. Peroxisomes use oxygen and produce peroxide. Peroxisomes do well with very long chain fatty acids and can do branched chains. The peroxisomal thiolase does not do well with small chains which are moved to the matrix for further oxidation.

43. Peroxisome oxidation

44. Ketone Body Pathway Ketone bodies are synthesized in the liver. The pathway is active under conditions that cause elevated levels of acetylCoA. Ketone bodies are:acetoacetate  -hydroxybutyrate and acetone The pathway requires the enzymes: 1.  -ketothiolase 2. hydroxymethylglutarylCoA synthase 3. hydroxymethylglutarylCoA lyase 4.  -hydroxybutyrate dehydrogenase

45. Ketone Body Pathway  -ketothiolase In presence of excess acetyl CoA, the last reaction of  -oxidation reverses to form acetoacetylCoA (not a ketone body).

46. Ketone Body Pathway 2. Hydroxymethyl glutarylCoA synthase AcetoacetylCoA combines with another acetylCoA with loss of Coenzyme A.

47. Ketone Body Pathway 3. Hydroxymethyl glutarylCoA lyase To get the ketone body, acetoacetate, acetylCoA is cleaved from HMGCoA.

48. Ketone Body Pathway 4.  -hydroxybutyrate dehydrogenase Acetoacetate is reduced with NADH to  -hydroxybutyrate Decarboxylation of the  -ketoacid to acetone is slow but Is spontaneous and irreversible.

49. Ketone Body Pathway 1.  -ketothiolase 2. hydroxymethylglutarylCoA synthase 3. hydroxymethylglutarylCoA lyase 4.  -hydroxybutyrate dehydrogenase

50. Ketone Body Use

51. Activating Acetoacetate Acetoacetate is converted to acetylCoA for energy. First it must be converted back to the CoA form. The acylCoA synthetase used to activate fatty acids is not used for acetoacetate.

52. Hydroxybutyrate Hydroxybutyrate is converted back to acetoacetate to get acetylCoA and acetone is lost.  -hydroxybutyrate DH

53. Fatty Acid Synthesis (FAS) Synthesis occurs in the cytosol of a liver cell or an adipocyte in mammals. It starts with acetyl CoA and requires NADPH. The NADPH required comes from the HMS or malic enzyme in the citrate shuttle. Note the cytosolic NADH/NADPH exchange. The initial product is palmitoyl CoA (no intermediate size fatty acids are observed). As one might expect,  -oxidation and fatty acid synthesis (FAS) occur by different pathways. When glucose is plentiful, acetylCoA can be used for FAS.

54. Oxidation vs Synthesis Oxidation Synthesis Localizationmitochondria/cytosol peroxisomes TransportCarnitine shuttleCitrate Shuttle Acyl carrierCoenzymeAAcylCarrierProtein Carbon unitsC2C2 Acceptor/donorAcetylSCoA, C2MalonylSCoA, C3 Redox CofactorsFAD, NAD + NADPH EnzymesSeparateMultifunctional enzymesdimer

55. Acetyl CoA into the Cytosol  -Oxidation produces acetylCoA in the mitochondria. Transport to the cytosol is via the Citrate Shuttle.

56. NADPH and Acetyl CoA Synthesis is in the cytosol starting with acetyl CoA.

57. The Citrate Shuttle Enzymes: Pyruvate Carboxylase (ATP, Biotin, CO 2 ) Citrate Synthase Citrate Lyase (ATP) Malate Dehydrogenase (NADH) Malic enzyme (NADP + ) Two ATPs used per transport cycle. Transport: Pyruvate into mito. uses a Pyr/H + symport Citrate out of mito. one of several antiports

58. Acetyl CoA Carboxylase Fatty acid synthesis starts in the cytosol with the addition of CO 2 to acetyl CoA. biotin A bifunctional enzyme in animals. A multienzyme complex in bacteria: Biotin carrier protein Biotin carboxylase Carboxyl transferase

59. Acetyl CoA Carboxylase (ACC) This the regulatory enzyme for fatty acid anabolism in animals. Under influence of epinephrine or glucagon it is phosphorylated and dissociates into monomeric units. ACC monomer-P ACC polymer less active more active Allosteric control (enzyme has no covalent P): polymerization activated by citrate (+) polymerization inhibited by palmitoylSCoA (-) Also, increased levels of malonylCoA inhibits CAT I.

60. Reactions of Fatty Acid Synthesis Synthesis uses a small protein, 77 amino acid residues, called acyl carrier protein (ACP) to hold the growing fatty acid chain. It resembles CoASH. Acyl-malonyl ACP condensing enzyme =  -ketoacyl ACP synthase

61. Acyl Carrier Protein(ACP) & CoA CoASH ACP

62. ACP and Coenzyme A

63. Reactions of Fatty Acid Synthesis FA are synthesized by the repetitive condensation of two-carbon units derived from malonyl CoA This occurs in separate steps: (1) Synthesis of malonylCoA (2) Loading of acetyl & malonyl using thioesters (3) Condensation of the precursors (4) Reduction (5) Dehydration (6) Reduction (7) Cleavage (at the end)

66. Detail of steps in FAS 1. AcetylCoA ACP transacylase loads ACP,  -ketoacyl ACP synthase transfers acetyl to Cys, 2. MalonylCoA ACP transacylase loads ACP, 3.  -KAS transfers acetyl to malonyl (loss of CO 2 ), 4.  -KAS now on ACP goes to  -ketoacyl reductase (NADPH) and forms a hydroxy group, 5. Then  -hydroxyacyl ACP dehydrase forms a trans double bond, 6. Enoyl ACP reductase (NADPH) reduces the db, 7.  -KAS transfers acyl to Cys & steps 2-6 repeat, 8. Thioesterase cleaves palmitate from ACP.  -ketoacyl ACP synthase = condensing enzyme

67. Fatty Acid Synthase A dimer and multifunctional enzyme in mammals, a multienzyme complex in yeast and separate enzymes in Ecoli and plants. Three domains with flexible connections

68. Regulation Active is polymeric and inactive monomeric.

69. Regulation ACC-P (less active) can be allosterically activated with citrate (+) and inhibited by palmitoylCoA (-).

71. Elongation and Unsaturation Palmitate can be elongated either in the mito or the cytosol but do not use FASase. Unsaturation occurs in the microsomes. Mammals can insert double bonds at carbons 4, 5, 6, & 9 but cannot insert beyond carbon 9. Unsaturation uses both O 2 and NADPH or NADH (two enzymes).

72. Eicosanoid Outline

73. Eicosanoid Structures

74. End of Chapter 22 Copyright © 2007 by W. H. Freeman and Company Berg Tymoczko Stryer Biochemistry Sixth Edition