This presentation provides the knowledge about Biosynthesis of Fatty acids & eicosanoids, Pathways, De novo fatty acid Synthesis, Advantages, Significance, Pharmacological Applications.

1. BIOSYNTHESIS OF FATTY ACIDS
& EICOSANOIDS
Presented by
K. Meghana M. Pharm 1st Year
Department of Pharmacology
K.K College of Pharmacy, Chennai

2. BIOSYNTHESIS OF FATTY ACIDS

3. FATTY ACIDS
Fatty acids are the class of compounds containing a long hydrophobic
hydrocarbon chain and a terminal carboxylate group.
Exist free in the body as well as fatty acyl esters in more complex molecules
such as triglycerides or phospholipids.
Oxidized in all tissues, particularly liver and muscle to provide energy.
Precursors of Eicosanoids..

4. DE NOVO FATTY ACID SYNTHESIS
• Fatty acids are synthesized by an Extra mitochondrial system or Cytoplasmic
Fatty Acid Synthase System.
• This system is present in many tissues, including liver, kidney, brain, lung,
mammary gland, and adipose tissue.
• Acetyl-CoA is the immediate substrate, and free palmitate is the end product.
• Cofactor requirements includes NADPH, ATP, Mn2+, biotin, and HCO3 – (as a
source of CO2 ).

5. SOURCES OF NADPH
• Involved as donor of Reducing equivalents.
• Produced from HMP shunt & Malic enzyme reaction.
• Every molecule of acetyl CoA delivered to cytoplasm, one molecule of NADPH
is formed.
• ATP supplies energy.

6. PENTOSE PHOSPHATE PATHWAY/HMP PATHWAY
In hepatocytes, adipose tissue and the lactating mammary
glands, the NADPH is supplied primarily by the pentose
phosphate pathway.

7. MALIC ENZYME
Reversible reaction, pyruvate produced in
the reaction reenters the mitochondrion
for further utilization.

8. STAGES
Production of acetyl CoA & NADPH.
 Conversion of acetyl CoA to malonyl CoA.
Reactions of fatty acid synthase complex.

9. I. PRODUCTION OF ACETYL COA &NADPH
• Acetyl CoA is the starting material for de novo synthesis of fatty acids.
• Produced in the mitochondria by the oxidation of pyruvate, fatty acids,
degradation of carbon skeleton of certain amino acids & from ketone bodies.
• Mitochondria are not permeable to acetyl CoA.
• An alternate or a bypass arrangement is made for the transfer of acetyl CoA to
cytosol.
• Acetyl CoA condenses with oxaloacetate in mitochondria to form citrate

10. • Citrate is freely transported to cytosol by tricarboxylic acid transporter.
• In cytosol it is cleaved by ATP citrate lyase to liberate acetyl CoA &
oxaloacetate.
• Oxaloacetate in the cytosol is converted to malate.
• Malic enzyme converts malate to pyruvate.
• NADPH & CO2 are generated in this reaction.
• Both of them are utilized for fatty acid synthesis

11. TRANSFER OF ACETYL COA FROM MITOCHONDRIA TO CYTOSOL

12. II. CONVERSION OF ACETYL COA-MALONYL COA
• Acetyl CoA is carboxylated to malonyl CoA by the enzyme acetyl CoA carboxylase.
• ATP-dependent reaction & requires biotin for CO2 fixation.
• Mechanism of action of acetyl CoA carboxylase is similar to that of pyruvate carboxylase.
• Acetyl CoA carboxylase is a regulatory enzyme.

13. III. REACTIONS OF FATTY ACID SYNTHASE COMPLEX
• Fatty acid synthase (FAS) - multifunctional enzyme.
• In eukaryotic cells, fatty acid synthase exists as a dimer with two
identical units.
• Each monomer possesses the activities of seven different enzymes
& an acyl carrier protein (ACP) bound to 4'-phosphopantetheine.
• Fatty acid synthase functions as a single unit catalyzing all the
seven reactions.

14. ADVANTAGES OF MULTI-ENZYME COMPLEX
• Intermediates of the reaction can easily interact with the active sites of the
enzymes.
• One gene codes all the enzymes.
• All enzymes are in equimolecular concentrations.
• Efficiency of the process is enhanced.

15. FAS COMPLEX
• First domain or Condensing unit:
• It is initial substrate binding site.
• The enzymes involved are β-keto acyl synthase or condensing
enzyme (CE), acetyl transferase (AT) & malonyl transacylase (MT).

16. SECOND DOMAIN OR REDUCING UNIT
• It contains the dehydratase (DH), enoyl reductase
(ER), β-keto acyl reductase (KR) & acyl carrier
protein (ACP).
• Acyl carrier protein is a polypeptide chain having a
phospho-pantotheine group, to which acyl groups are
attached in thioester linkage.
• ACP acts like CoA carrying fatty acyl groups.

17. DEHYDRATION
• Dehydration yields a double bond in the product,
trans- ∆2-butenoyl-ACP.
• Reaction is catalyzed by β-hydroxybutyrylACP
dehydratase.

18. REACTIONS
• Two carbon fragment of acetyl CoA is transferred to ACP of fatty acid synthase,
catalyzed by the enzyme - acetyl CoA-ACP transacylase.
• Acetyl unit is then transferred from ACP to cysteine residue of the enzyme.
• ACP site falls vacant.
• Enzyme malonyl CoA-ACP transacylase transfers malonate from malonyl CoA
to bind to ACP.
• Acetyl unit attached to cysteine is transferred to malonyl group (bound to ACP).

19. • Malonyl moiety loses CO2 which was added by acetyl CoA carboxylase.
• CO2 is never incorporated into fatty acid carbon chain.
• Decarboxylation is accompanied by loss of free energy which allows the
reaction to proceed forward.
• Catalyzed by β-ketoacyl ACP synthase.
• β -Ketoacyl ACP reductase reduces ketoacyl group to hydroxyacyl group.
• Reducing equivalents are supplied by NADPH.
• β -Hydroxyacyl ACP undergoes dehydration.
• A molecule of water is eliminated & a double bond is introduced between α &
β carbons.
• A second NADPH-dependent reduction, catalysed by enoyl-ACP reductase
occurs to produce acyl-ACP.
• Four-carbon unit attached to ACP is butyryl group.

20. • Carbon chain attached to ACP is transferred to cysteine residue & the
reactions of malonyl CoA-ACP transacylase & enoyl-ACP reductase are
repeated 6 more times.
• Each time, the fatty acid chain is lengthened by a two-carbon unit
(obtained from malonyl CoA).
• At the end of 7 cycles, the fatty acid synthesis is complete & a 16-carbon
fully saturated fatty acid-namely palmitate-bound to ACP is produced.
• Enzyme palmitoyl thioesterase separates palmitate from fatty acid
synthase.
• This completes the synthesis of palmitate.

21. DE NOVO FATTY ACID SYNTHESIS: REACTIONS

27. FATTY ACID SYNTHASE COMPLEX
• Multienzyme complex.
• Fatty acid synthase is a dimer composed of two identical subunits (monomers).
Each with a molecular weight of 240,000.
• Each subunit contains the activities of 7 enzymes of FAS & an ACP with 4'-
phosphopantetheine SH group.
• The two subunits lie in antiparallel (head to tail) orientation.

28. • The -SH group of phosphopantetheine of one subunit is in close
proximity to the -SH of cysteine residue (of the enzyme ketoacyl
synthase) of the other subunit.
• Each monomer of FAS contains all the enzyme activities of fatty acid
synthesis.
• Only the dimer is functionally active.
• Functional unit consists of half of each subunit interacting with the
complementary half of the other.
• FAS structure has both functional division & subunit division.
• The two functional subunits of FAS independently operate &
synthesize two fatty acids simultaneously.

29. FATTY ACID SYNTHASE –MULTIENZYME COMPLEX

30. SIGNIFICANCE OF FAS COMPLEX
 FAS complex offers great efficiency that is free from
interference of other cellular reactions for the synthesis of
fatty acids.
Good coordination in the synthesis of all enzymes of the
FAS complex.

31. REGULATIONOF FATTY ACIDSYNTHESIS
Fatty acid production is controlled by enzymes, metabolites, end products,
hormones and dietary manipulations.
Acetyl CoA carboxylase: This enzyme controls a committed step in fatty acid.
synthesis. Inactive protomer (monomer) or an active polymer.
Citrate promotes polymer formation & increases fatty acid synthesis.
Palmitoyl CoA & malonyl CoA cause depolymerization of the enzyme, inhibits
the fatty acid synthesis.

32. HORMONAL INFLUENCE
Hormones regulate acetyl CoA carboxylase by a separate mechanism-
phosphorylation (inactive form) & dephosphorylation (active form) of the
enzyme.
 Glucagon, epinephrine & norepinephrine inactivate the enzyme by cAMP
dependent phosphorylation.
Insulin, dephosphorylates & activates the enzyme. Promotes fatty acid synthesis
while glucagon inhibits.
Insulin stimulates tissue uptake of glucose & conversion of pyruvate to acetyl
CoA. This also facilitates fatty acid formation

33. DIETARY REGULATION
Consumption of high carbohydrate or fat-free diet increases the
synthesis of acetyl CoA carboxylase & fatty acid synthase, which
promote fatty acid formation.
Fasting or high fat diet decreases fatty acid production by reducing
the synthesis of acetyl CoA carboxylase & FAS.

34. BIOSYNTHESIS OF EICOSANOIDS

35. INTRODUCTION
Eicosanoid" is derived from a Greek word “eicosa” meaning "twenty”.
Eicosanoids is the collective term for the signaling molecules made by oxidation.
They are oxygenated derivatives of 3 different 20-carbon fatty acids:
1. Eicosapentanoic acid
2. Arachidonic acid
3. Di-homo-gamma-linolenic acid

36.  Eicosapentaenoic acid (EPA), an ω-3 fatty acid with 5 double bonds.
 Arachidonic acid (AA), an ω-6 fatty acid, with 4 double bonds.
 Dihomo-gamma-linolenic acid (DGLA), an ω-6, with 3 double bonds.
 Eicosanoids are derived from either omega-3 (ω-3) or omega-6 (ω-6) fatty
acids

37. CLASSIFICATION
Eicosanoids are classified into two main
groups:
1) Prostanoids
2) Leukotrienes and Lipoxins
Prostanoids are further sub-classified into
three groups
a) Prostaglandins(PGs)
b) Prostacyclins(PGIs)
c) Thromboxanes (TXs)

38. BIOSYNTHESIS
Two families of enzyme catalyze fatty acid oxygenation to produce the
eicosanoids:
- Cyclooxygenase (Suicide Enzyme), or COX, generates the
prostanoids.
- Lipoxygenase, or LOX, in several forms.
 5-lipoxygenase (5-LO) generates the leukotrienes and via transcellular
biosynthesis is also involved in lipoxin generation.
Eicosanoids are not stored within cells, but are synthesized as required.
Derive from the fatty acids that make up the cell membrane and nuclear
membrane.

39. Eicosanoid biosynthesis begins when a cell is activated by mechanical trauma,
cytokines, growth factors or other stimuli. (The stimulus may even be an
eicosanoid from a neighboring cell).
Triggers the release of a phospholipase at the cell membrane.
The phospholipase travels to the nuclear membrane.
There, the phospholipase catalyzes ester hydrolysis of phospholipid or
diacylglycerol (by phospholipase C).
Frees a 20-carbon fatty acid.
The fatty acids may be released by any of several phospholipases.
 Type IV cytosolic phospholipase A2 (cPLA2 ) is the key actor, as cells lacking
cPLA2 are, in general, devoid of eicosanoid synthesis.
Phospholipase cPLA2 is specific for phospholipids that contain AA, EPA or
GPLA at the SN2 position.
cPLA2 may also release the lysophospholipid that becomes platelet-activating
factor

40. PROSTANOID PATHWAYS
Cyclooxygenase (COX) catalyzes the conversion of the free fatty acids to
prostanoids by a two-step process.
First, two molecules of O2 are added as two peroxide linkages, and a 5-member
carbon ring is forged near the middle of the fatty acid chain. This forms the
short-lived, unstable intermediate Prostaglandin G (PGG).
One of the peroxide linkages sheds a single oxygen, forming PGH.

41. All three classes of prostanoids originate from PGH. All have distinctive rings in
the center of the molecule. They differ in their structures.
Derived prostaglandins contain a single, unsaturated 5-carbon ring.
In prosta cyclins, this ring is conjoined to another oxygen-containing ring. In
thromboxanes the ring becomes a 6-member ring with one oxygen.
Leukotrienes do not have rings.
Several drugs lower inflammation by blocking prostanoid synthesis.

42. SYNTHESIS OF EICOSANOIDS

43. LEUKOTRIENE PATHWAYS
Enzyme 5-lipoxygenase (5-LO) uses 5- lipoxygenase activating protein (FLAP) to
convert arachidonic acid into 5- Hydroperoxyeicosatetraenoic acid (5 HPETE)which
spontaneously reduces to 5- hydroxyeicosatetraenoic acid (5-HETE).
Enzyme LTA synthase acts on 5-HPETE to convert it into leukotriene A4 (LTA4) which
may be converted into LTB4 by the enzyme leukotriene A4 epoxide hydrolase.

44. Eosinophils, mast cells, and alveolar macrophages use the enzyme
leukotriene C4 synthase to conjugate glutathione with LTA4 to make
LTC4, transported outside the cell, where a glutamic acid moiety is
removed from it to make LTD4.
Leukotriene LTD4 is then cleaved by dipeptidases to make LTE4 .
Leukotrienes LTC4 , LTD4 and LTE4 all contain cysteine and are
collectively known as the cysteinyl leukotrienes.

45. BIOSYNTHESIS OF LEUOTRIENE

46. PHARMACOLOGICAL APPLICATIONS OF EICOSANOIDS
Cardiovascular uses- Pulmonary arterial hypertension, peripheral vascular
disease. For keeping the ductus arteriosus open until surgery in neonates carrying
certain cardiac malformations and platelet anti-aggregating agents.
Digestive Uses- Indicated in the treatment of gastro duodenal ulcer and for the
prevention of NSAID-induced ulcers.
Gynecological and obstetrical uses - Induce cervical dilatation and uterine
contractions, particularly in late pregnancy. Used for medical termination of
pregnancy and induction of labour.

47. Ophthalmologic Use- lower intraocular pressure.
Anti- inflammatory use- Inhibitors of cyclooxygenases have anti-inflammatory
properties and include nonsteroidal anti-inflammatory drugs or NSAID.
The useful effects in therapeutics are anti-inflammatory effect analgesic effect
antipyretic effect inhibition of platelet aggregation and decrease of thromboembolic
risk (well-known with aspirin at low doses).

48. Ulcerative Colitis- Mesalamine also called mesalazine or 5 aminosalicyclic
acid has antiinflammatory properties in the colon and is used in the
treatment of ulcerative colitis (Crohn's disease). Its mechanism of action is
complex and as yet incompletely known: in addition to cyclo-oxygenases,
it also inhibits lipoxygenases.
Bronchial Asthma- PGE2 agonists and leukotrienes receptor antagonists
are used for the treatment of bronchial asthma.

49. REFERENCES
Harper’s Illustrated Biochemistry Twenty-Eighth
Edition.
Biochemistry by Satyanarayana Five Edition.