1. Oxidation and biosynthesis of fatty acids1

2. Stages of fatty acid oxidation
(1) Activation of fatty acids takes place on the outer mitochondrial membrane
(2) Transport into the mitochondria
(3) Degradation to two-carbon fragments (as acetyl CoA) in the mitochondrial matrix (b-oxidation pathway) 2

3. (1) Activation of Fatty Acids
Fatty acids are converted to CoA thioesters by acyl-CoA synthetase (ATP dependent)
The PPi released is hydrolyzed by a pyrophosphatase to 2 Pi
Two phosphoanhydride bonds (two ATP equivalents) are consumed to activate one fatty acid to a thioester 3

4. (2) Transport of Fatty Acyl CoA into Mitochondria
The carnitine shuttle system.
Fatty acyl CoA is first converted to acylcarnitine (enzyme carnitine acyltransferase I (bound to the outer mitochondrial membrane).
Acylcarnitine enters the mitochondria by a translocase.
The acyl group is transferred back to CoA (enzyme - carnitine acyltransferase II). 4

5. Carnitine shuttle system
Path of acyl group in red 5

6. (3) The Reactions of b oxidation
The b-oxidation pathway (b-carbon atom (C3) is oxidized) degrades fatty acids two carbons at a time   6

7. 1. Oxidation of acyl CoA by an acyl CoA dehydrogenase to give an enoyl CoA
Coenzyme - FAD 7

8. 2. Hydration of the double bond between C-2 and C-3 by enoyl CoA hydratase with the 3-hydroxyacyl CoA (b-hydroxyacyl CoA) formation 8

9. 3. Oxidation of 3-hydroxyacyl CoA to 3-ketoacyl CoA by 3-hydroxyacyl CoA dehydrogenase
Coenzyme – NAD+ 9

10. Enzyme - b-ketothiolase.
4. Cleavage of ketoacyl CoA by the thiol group of a second molecule of CoA with the formation of acetyl CoA and an acyl CoA shortened by two carbon atoms.
Enzyme b-ketothiolase. 10

11. The shortened acyl CoA then undergoes another cycle of oxidation
The number of cycles: n/2-1, where n – the number of carbon atoms 11

12. b-Oxidation of saturated fatty acids
Fatty acyl CoA 12

13. One round of b oxidation: 4 enzyme steps produce acetyl CoA from fatty acyl CoA
Each round generates one molecule each of: FADH2 NADH Acetyl CoA Fatty acyl CoA (2 carbons shorter each round)
Fates of the products of b-oxidation: NADH and FADH2 - are used in ETC acetyl CoA - enters the citric acid cycle acyl CoA – undergoes the next cycle of oxidation 13

14. ATP Generation from Fatty Acid Oxidation
Net yield of ATP per one oxidized palmitate
Palmitate (C15H31COOH) - 7 cycles – n/2-1
The balanced equation for oxidizing one palmitoyl CoA by seven cycles of b oxidation
Palmitoyl CoA + 7 HS-CoA + 7 FAD+ + 7 NAD+ + 7 H2O Acetyl CoA + 7FADH2 + 7 NADH + 7 H+
ATP generated
8 acetyl CoA 10x8=80 7 FADH x1.5= NADH 7x2.5= ATP
ATP expended to activate palmitate
Net yield: ATP 14

15. LIPID METABOLISM: FATTY ACID OXIDATION15

16. b-OXIDATION OF ODD-CHAIN FATTY ACIDS
Odd-chain fatty acids occur in bacteria and microorganisms
Final cleavage product is propionyl CoA rather than acetyl CoA
Three enzymes convert propionyl CoA to succinyl CoA (citric acid cycle intermediate) 16

17. Propionyl CoA Is Converted into Succinyl CoA
1. Propionyl CoA is carboxylated to yield the D isomer of methylmalonyl CoA.
The hydrolysis of an ATP is required.
Enzyme: propionyl CoA carboxylase
Coenzyme: biotin 17

18. 2. The D isomer of methylmalonyl CoA is racemized to the L isomer
Enzyme: methylmalonyl-CoA racemase 18

19. Enzyme: methylmalonyl CoA mutase Coenzyme: vitamin B12 (cobalamin)
3. L isomer of methylmalonyl CoA is converted into succinyl CoA by an intramolecular rearrangement
Enzyme: methylmalonyl CoA mutase
Coenzyme: vitamin B12 (cobalamin) 19

20. OXIDATION OF FATTY ACIDS IN PEROXISOMES
Peroxisomes - organelles containing enzyme catalase, which catalyzes the dismutation of hydrogen peroxide into water and molecular oxygen
Acyl CoA dehydrogenase transfers electrons to O2 to yield H2O2 instead of capturing the high-energy electrons by ETC, as occurs in mitochondrial b-oxidation. 20

21. METABOLISM OF LIPIDS: SYNTHESIS OF FATTY ACIDS21

22. Fatty Acid Synthesis
Occurs mainly in liver and adipocytes, in mammary glands during lactation
Occurs in cytoplasm
FA synthesis and degradation occur by two completely separate pathways
When glucose is plentiful, large amounts of acetyl CoA are produced by glycolysis and can be used for fatty acid synthesis 22

23. Three stages of fatty acid synthesis:
A. Transport of acetyl CoA into cytosol
B. Carboxylation of acetyl CoA
C. Assembly of fatty acid chain 23

24. A. Transport of Acetyl CoA to the Cytosol
Acetyl CoA from catabolism of carbohydrates and amino acids is exported from mitochondria via the citrate transport system
Cytosolic NADH also converted to NADPH
Two molecules of ATP are expended for each round of this cyclic pathway 24

25. Citrate transport system25

26. Sources of NADPH for Fatty Acid Synthesis
1. One molecule of NADPH is generated for each molecule of acetyl CoA that is transferred from mitochondria to the cytosol (malic enzyme).
2. NADPH molecules come from the pentose phosphate pathway. 26

27. B. Carboxylation of Acetyl CoA
Enzyme: acetyl CoA carboxylase Prosthetic group - biotin
A carboxybiotin intermediate is formed.
ATP is hydrolyzed.
The CO2 group in carboxybiotin is transferred to acetyl CoA to form malonyl CoA.
Acetyl CoA carboxylase is the regulatory enzyme. 27

28. C. The Reactions of Fatty Acid Synthesis
Five separate stages: (1) Loading of precursors via thioester derivatives (2) Condensation of the precursors (3) Reduction (4) Dehydration (5) Reduction 28

29. During the fatty acid synthesis all intermediates are linked to the protein called acyl carrier protein (ACP-SH), which is the component of fatty acyl synthase complex.
The pantothenic acid is a component of ACP.
Intermediates in the biosynthetic pathway are attached to the sulfhydryl terminus of phosphopantotheine group. 29

30. Acetyl transacylase and malonyl transacylase catalyze these reactions.
The elongation phase of fatty acid synthesis starts with the formation of acetyl ACP and malonyl ACP.
Acetyl transacylase and malonyl transacylase catalyze these reactions.
Acetyl CoA + ACP  acetyl ACP + CoA Malonyl CoA + ACP  malonyl ACP + CoA 30

31. Condensation reaction.
Acetyl ACP and malonyl ACP react to form acetoacetyl ACP.
Enzyme acyl-malonyl ACP condensing enzyme. 31

32. Acetoacetyl ACP is reduced to D-3-hydroxybutyryl ACP.
Reduction.
Acetoacetyl ACP is reduced to D-3-hydroxybutyryl ACP.
NADPH is the reducing agent
Enzyme: -ketoacyl ACP reductase 32

33. D-3-hydroxybutyryl ACP is dehydrated to form crotonyl ACP
Dehydration.
D-3-hydroxybutyryl ACP is dehydrated to form crotonyl ACP
(trans-2-enoyl ACP).
Enzyme: hydroxyacyl ACP dehydratase 33

34. The final step in the cycle reduces crotonyl ACP to butyryl ACP.
Reduction.
The final step in the cycle reduces crotonyl ACP to butyryl ACP.
NADPH is reductant.
Enzyme - enoyl ACP reductase.
This is the end of first elongation cycle (first round). 34

35. In the second round butyryl ACP condenses with malonyl ACP to form a C6--ketoacyl ACP.
Reduction, dehydration, and a second reduction convert the C6--ketoacyl ACP into a C6-acyl ACP, which is ready for a third round of elongation. 35

36. Final reaction of FA synthesis
Rounds of synthesis continue until a C16 palmitoyl group is formed
Palmitoyl-ACP is hydrolyzed by a thioesterase
Overall reaction of palmitate synthesis from acetyl CoA and malonyl CoA
Acetyl CoA + 7 Malonyl CoA + 14 NADPH + 14 H+
Palmitate + 7 CO NADP+ + 8 HS-CoA + 6 H2O 36

37. Organization of Multifunctional Enzyme Complex in Eukaryotes
The synthase is dimer with antiparallel subunits.
Each subunit has three domains.
ACP is located in domain 2.
Domain 1 contains transacylases, ketoacyl-ACP synthase (condensing enzyme)
Domain 2 contains acyl carrier protein, -ketoacyl reductase, dehydratase, and enoyl reductase.
Domain 3 contains thioesterase activity. 37

38. 38

39. Fatty Acid Elongation and Desaturation
The common product of fatty acid synthesis is palmitate (16:0).
Cells contain longer fatty acids and unsaturated fatty acids they are synthesized in the endoplasmic reticulum.
The reactions of elongation are similar to the ones seen with fatty acid synthase (new carbons are added in the form of malonyl CoA).
For the formation of unsaturated fatty acids there are various desaturases catalizing the formation of double bonds. 39

40. THE CONTROL OF FATTY ACID METABOLISM
Acetyl CoA carboxylase plays an essential role in regulating fatty acid synthesis and degradation.
The carboxylase is controlled by hormones:
glucagon,
epinephrine, and
insulin.
Another regulatory factors:
citrate,
palmitoyl CoA, and
AMP 40

41. Global Regulation
is carried out by means of reversible phosphorylation
Acetyl CoA carboxylase is switched off by phosphorylation and activated by dephosphorylation
Insulin stimulates fatty acid synthesis causing dephosphorylation of carboxylase.
Glucagon and epinephrine have the reverse effect (keep the carboxylase in the inactive phosphorylated state).
Protein kinase is activated by AMP and inhibited by ATP.
Carboxylase is inactivated when the energy charge is low. 41

42. Local Regulation
Acetyl CoA carboxylase is allosterically stimulated by citrate.
The level of citrate is high when both acetyl CoA and ATP are abundant (isocitrate dehydrogenase is inhibited by ATP).
Palmitoyl CoA inhibits carboxylase. 42

43. Response to Diet Fed state: Starvation: Insulin level is increased
Inhibits hydrolysis of stored TGs
Stimulates formation of malonyl CoA, which inhibits carnitine acyltransferase I
FA remain in cytosol (FA oxidation enzymes are in the mitochondria)
Starvation:
Epinephrine and glucagon are produced and stimulate adipose cell lipase and the level of free fatty acids rises
Inactivate carboxylase, so decrease formation of malonyl CoA (lead to increased transport of FA into mitochondria and activate the b-oxidation pathway) 43