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Lecture 32

Lecture 32. Last lecture!! Fatty acid biosynthesis. b -oxidation. 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.

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Lecture 32

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  1. Lecture 32 • Last lecture!! • Fatty acid biosynthesis

  2. b-oxidation • 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

  3. Acyl-CoA Dehydrogenase • Oxidation of the C-Cbond • Mechanism involves proton abstraction, followed by double bond formation and hydride removal by FAD • Electrons are passed to an electron transfer flavoprotein (ETF), and then to the electron transport chain.

  4. Acyl-CoA Dehydrogenase Net: 2 ATP/2 e- transferred

  5. Formation of a trans  double bond by dehydrogenation by acyl-CoA dehydrogenase (AD). • Hydration of the double bond by enoyl-CoA hydratase (EH) to form 3-L-hydroxyacyl-CoA • NAD+-dependent dehydrogenation of b-hydroxyacyl-CoA by 3-L-hydroxyacyl-CoA dehydrogense (HAD) to form -ketoacyl-CoA. • C-C bond cleavage by -ketoacyl-CoA thiolase (KT) Page 917

  6. Enoyl-CoA Hydratase • aka crotonases • Adds water across the double bond • Uses substrates with trans-D2-and cisD2double bonds (impt in b-oxidation of unsaturated FAs) • With trans-D2 substrate forms L-isomer, withcisD2 substrate forms D-isomer. • Normal reaction converts trans-enoyl-CoA to L--hydroxyacyl-CoA

  7. Formation of a trans  double bond by dehydrogenation by acyl-CoA dehydrogenase (AD). • Hydration of the double bond by enoyl-CoA hydratase (EH) to form 3-L-hydroxyacyl-CoA • NAD+-dependent dehydrogenation of b-hydroxyacyl-CoA by 3-L-hydroxyacyl-CoA dehydrogense (HAD) to form -ketoacyl-CoA. • C-C bond cleavage by -ketoacyl-CoA thiolase (KT) Page 917

  8. Hydroxyacyl-CoA Dehydrogenase • Oxidizes the-Hydroxyl Group to keto group • This enzyme is completely specific for L-hydroxyacyl-CoA • D-hydroxylacyl-isomers are handled differently • Produces one NADH

  9. Formation of a trans  double bond by dehydrogenation by acyl-CoA dehydrogenase (AD). • Hydration of the double bond by enoyl-CoA hydratase (EH) to form 3-L-hydroxyacyl-CoA • NAD+-dependent dehydrogenation of -hydroxyacyl-CoA by 3-L-hydroxyacyl-CoA dehydrogense (HAD) to form -ketoacyl-CoA. • C-C bond cleavage by -ketoacyl-CoA thiolase (KT) Page 917

  10. Thiolase • Nucleophillic sulfhydryl group of CoA-SH attacks the -carbonyl carbon of the 3-keto-acyl-CoA. • Results in the cleavage of the C-Cbond. • Acetyl-CoA and an acyl-CoA (-) 2 carbons are formed

  11. Figure 25-15 Mechanism of action of b-ketoacyl-CoA thiolase. • An active site thiol is added to the substrate -keto group. • C-C bond cleavage forms an acetyl-CoA carbanion intermediate (Claisen ester cleavage) • The acetyl-CoA intermediate is protonated by an enzyme acid group (acetyl-CoA released) • CoA binds to the enzyme-thioester intermediate • Acyl-CoA is released. Net reaction reduces fatty acid by 2C and acyl-CoA group is free to pass through the cyle again. Page 919

  12. Formation of a trans  double bond by dehydrogenation by acyl-CoA dehydrogenase (AD). • Hydration of the double bond by enoyl-CoA hydratase (EH) to form 3-L-hydroxyacyl-CoA • NAD+-dependent dehydrogenation of -hydroxyacyl-CoA by 3-L-hydroxyacyl-CoA dehydrogense (HAD) to form -ketoacyl-CoA. • C-C bond cleavage by -ketoacyl-CoA thiolase (KT) Page 917

  13. b-oxidation • Each round of -oxidation produces 1 NADH, 1 FADH2 and 1 acetyl-CoA. • -oxidation of palmitate (C16:0) yields 129 molecules of ATP • C 16:0-CoA + 7 FAD + 7 NAD+ + 7 H2O + 7 CoA  8 acetyl-CoA + 7 FADH2 + 7 NADH + 7 H+ • Acetyl-CoA = 8 GTP, 24 NADH, 8 FADH2 • Total = 31 NADH = 93 ATPs + 15 FADH2 = 30 ATPs • 2 ATP equivalents (ATP  AMP + PPi, PPi  2 Pi) consumed during activation of palmitate to acyl-CoA • Net yield = 129 ATPs

  14. Beta-oxidation of unsaturated fatty acids • Nearly all fatty acids of biological origin have cisdouble bonds between C9 and C10 (9 or 9-double bond). • Additional double bonds occur at 3-carbon intervals (never conjugated). • Examples: oleic acid and linoleic acid. • In linoleic acid one of the double bonds is at an even-numbered carbon and the other double bond is at an odd-numbered carbon atom. • 4 additional enzymes are necessary to deal with these problems. • Need to make cis into trans double bonds

  15. Figure 25-17 Problems in the oxidation of unsaturated fatty acids and their solutions. Page 920

  16. -oxidation of unsaturated fatty acids • -oxidation occurs normally for 3 rounds until a cis-3-enoyl-CoA is formed. • Acyl-CoA dehydrogenase can not add double bond between the  and carbons. • Enoyl-CoA isomerase converts this to trans- 2 enoyl-CoA • Now the -oxidation can continue on w/ the hydration of the trans-2-enoyl-CoA • Odd numbered double bonds handled by isomerase

  17. -oxidation of fatty acids with even numbered double bonds

  18. -oxidation of odd chain fatty acids • Odd chain fatty acids are less common • Formed by some bacteria in the stomachs of ruminants and the human colon. • -oxidation occurs pretty much as w/ even chain fatty acids until the final thiolase cleavage which results in a3 carbon acyl-CoA (propionyl-CoA) • Special set of 3 enzymes are required to further oxidize propionyl-CoA • Final Product succinyl-CoA enters TCA cycle

  19. Propionyl-CoA Carboxylase • The first reaction • Tetrameric enzyme that has a biotin prosthetic group • Reactions occur at 2 sites in the enzyme. • Carboxylation of biotin at the N1’ by bicarbonate ion (same as pyruvate carboxylase). Driven by hydrolysis of ATP to ADP and Pi-activates carboxyl group for transfer • Stereospecific transfer of the activated carboxyl group from carboxybiotin to propionyl-CoA to form (S)-methylmalonyl-CoA. Occurs via nucleophillic attack on the carboxybiotin by a carbanion at C2 of propionyl-CoA

  20. Page 922

  21. Methylmalonyl-CoA Racemase • 2nd reaction for odd chain fatty acid oxidation • Transforms (S)-methylmalonyl-CoA to (R)-methylmalonyl-CoA • Takes place through a resonance stablized carbanion intermediate (p. 923)

  22. H X X H -C1-C2- -C1-C2- Methylmalonyl-CoA mutase • 3rd reaction of the pathway: converts (R)-methylmalonyl-CoA to succinyl-CoA • Utilizes 5’-deoxyadenosylcobalamin (AdoCbl) - coenzyme B12. • AdoCbl has a reactive C-Co bond that is used for 2 types of reactions: • Rearrangements in which a hydrogen atom is directly transferred between 2 adjacent C atoms. • Methyl group transfers between molecules.

  23. Figure 25-21 Structure of 5¢-deoxyadenosylcobalamin (coenzyme B12). Co is coordinated by the corrin ring’s 4 pyrrole N atoms, a N from the dimethylbenzimadazole (DMB), and C5’ from the deoxyribose unit. One of only 2 known C-metal bonds in biology. Page 923

  24. Figure 25-20 The rearrangement catalyzed by methylmalonyl-CoA mutase. Page 923

  25. Methylmalonyl-CoA mutase • Mechanism begins with homolytic cleavage of the C-Co(III) bond. • The AdoCbl is a free radical generator • C-Co(III) bond is weak and it is broken and the radical is stabilized favoring the formation of the adenosyl radical. • Rearrangement to form succinyl-CoA from a cyclopropyloxy radical • Abstraction of a hydrogen atom from 5’deoxyadenosine to regenerate the adenosyl radical • Release of succinyl-CoA

  26. Page 926

  27. Odd chain fatty acids • Transform odd chain length FAs to succinyl-CoA • 3 enzymes • Propionyl-CoA carboxylase (biotin cofactor): activates bicarbonate and transfers to propionyl-CoA to form S-methylmalonyl-CoA. • Methylmalonyl-CoA racemase: Transforms (S)-methylmalonyl-CoA to (R)-methylmalonyl-CoA through a resonance-stabilized intermediate. • Methylmalonyl-CoA mutase (B12 cofactor(AdoCbl)): Transforms (R)-methylmalonyl-CoA to succinyl-CoA by generating a radical. • Succinyl-CoA enters TCA cycle

  28. Combination of fatty acid activation, transport into mitochondrial matrix and b oxidation • Resulting acetyl CoA enters citric acid cycle. • Production of NADH, FADH2, oxidized by respiratory chain.

  29. Fatty Acid Breakdown Summary • Even numbered fatty acids are broken down into acetyl-CoA by 4 enzymes: acyl-CoA dehydrogenase (AD), enoyl-CoA hydratase (EH), 3-L-hydroxyacyl-CoA dehydrogenase (HAD) and -ketoacyl-CoA thiolase (KT). • The breakdown of unsaturated fatty acids (cis double bonds) requires 4 additional enzymes in mammals: enoyl-CoA isomerase, 2,4 dienoyl-CoA reductase, 3,2-enoyl-CoA isomerase, and 3,5-2,4-dienoyl-CoA isomerase. In bacteria, they only need enoyl-CoA isomerase and 2,4-dienoyl-CoA reductase. • Have to convert cis double bonds to trans double bonds. • Unsaturated fatty acids -oxidation results in the production of acetyl-CoA.

  30. Fatty Acid Breakdown Summary • Odd numbered fatty acids are broken down into propionyl-CoA. • Propionyl-CoA is converted to S-Methylmalonyl-CoA by propionyl-CoA carboxylase with ATP and CO2. Uses a carboxybiotynyl cofactor for the mechanism. • S-Methylmalonyl-CoA is converted to R-Methylmalonyl-CoA by methylmalonyl-CoA racemase. • R-Methylmalonyl-CoA is converted to Succinyl-CoA by methylmalonyl-CoA mutase. Uses a 5’-deoxyadenosylcobalimin (AdoCbl aka coenzyme B12) cofactor for the mechanism.

  31. Fatty Acid Synthesis • Fatty acid biosynthesis occurs through condensation of C2 units (reverse of -oxidation) • Acetyl-CoA is the precursor molecule; converted to malonyl-CoA • In mammals fatty acid synthesis occurs primarily in the liver and adipose tissues • Also occurs in mammary glands during lactation. • Fatty acid synthesis and degradation go by different routes • There are four major differences between fatty acid breakdown and biosynthesis

  32. The differences between fatty acid biosynthesis and breakdown • Intermediates in synthesis are linked to -SH groups of acyl carrier proteins (as compared to -SH groups of CoA) • Synthesis in cytosol; breakdown in mitochondria • Enzymes of synthesis are one polypeptide in eukaryotes. • Dissociated in bacteria • Biosynthesis uses NADPH/NADP+; breakdown uses NADH/NAD+

  33. ACP vs. Coenzyme A • Intermediates in synthesis are linked to -SH groups of acyl carrier proteins (ACP) as compared to -SH groups of CoA

  34. Figure 25-28 A comparison of fatty acid b oxidation and fatty acid biosynthesis. Page 931

  35. Citrate Lyase Citrate synthase Malate dehydrogenase Pyruvate carboxylase Malate Enzyme Fatty Acid Synthesis Occurs in the Cytosol • Must have source of acetyl-CoA • Most acetyl-CoA in mitochondria • Citrate-malate-pyruvate shuttle provides cytosolic acetate units and reducing equivalents for fatty acid synthesis

  36. Fatty Acid Synthesis • Fatty acids are built from 2-C units derived from acetyl-CoA • Acetate units are activated for transfer to growing FA chain by conversion to malonyl-CoA • Decarboxylation of malonyl-CoA and reducing power of NADPH drive chain growth • Chain grows to 16-carbons (eight acetyl-CoAs) • Other enzymes add double bonds and more carbons

  37. Acetyl-CoA Carboxylase Acetyl-CoA + HCO3- + ATP  malonyl-CoA + ADP • The "ACC enzyme" commits acetate to fatty acid synthesis • Carboxylation of acetyl-CoA to form malonyl-CoA is the irreversible, committed step in fatty acid biosynthesis

  38. Acetyl-CoACarboxylase

  39. Regulation of Acetyl-CoA Carboxylase (ACCase) • ACCase forms long, active filamentous polymers from inactive protomers • Accumulation of palmitoyl-CoA (product) leads to the formation of inactive polymers • Accumulation of citrate leads to the formation of the active polymeric form • Phosphorylation modulates citrate activation and palmitoyl-CoA inhibition

  40. Figure 25-30 Association of acetyl-CoA carboxylase protomers. Page 932

  41. Regulation of Acetyl-CoA Carboxylase (ACCase) • Unphosphorylated ACCase has low Km for citrate and is active at low citrate • Unphosphorylated ACCase has high Ki for palmitoyl-CoA and needs high palmitoyl-CoA to inhibit • Phosphorylated E has high Km for citrate and needs high citrate to activate • Phosphorylated E has low Ki for palmitoyl-CoA and is inhibited at low palmitoyl-CoA

  42. Fatty acid biosynthesis Acetyl-CoA is converted by MAT to Acetyl ACP Acetyl-ACP is attached to KS (condensation reaction). Malonyl ACP is formed by MAT. Acetyl-group is coupled to beta carbon of malonyl-ACP with release of CO2 to form acetoacetyl-ACP(2b) by KS. Reduction of acetoacetyl-ACP with NADPH to form D--hydroxybutyrl-ACP by DH Dehydration of D--hydroxybutyrl-ACP by ER to form a,b-trans-butenoyl-ACP Reduction of the double bond to form butyryl-ACP Repeat until Palmitoyl-ACP (C16) is formed. ACP is cleaved by TE releasing free fatty acid. Page 933

  43. Fatty Acid Synthesis • Step 1: Loading – transferring acetyl- and malonyl- groups from CoA to ACP • Step 2: Condensation – transferring 2 carbon unit from malonyl-ACP to acetyl-ACP to form 2 carbon keto-acyl-ACP • Step 3: Reduction – conversion of keto-acyl-ACP to hydroxyacyl-ACP (uses NADPH) • Step 4: Dehydration – Elimination of H2O to form Enoyl-ACP • Step 5: Reduction – Reduce double bond to form 4 carbon fully saturated acyl-ACP

  44. Step 1: Loading Reactions MAT

  45. Step 2: Condensation Rxn -ketoacyl-ACP synthase (KS)

  46. Step 3: Reduction KR

  47. Step 4: Dehydration DH

  48. Step 5: Reduction ER

  49. Step 6: next condensation KS

  50. Termination of Fatty Acid Synthesis Acyl-CoA synthetase

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