This presentation provides the knowledge about Biosynthesis of Fatty acids & eicosanoids, Pathways, De novo fatty acid Synthesis, Advantages, Significance, Pharmacological Applications.
3. FATTY ACIDS
ďFatty acids are the class of compounds containing a long hydrophobic
hydrocarbon chain and a terminal carboxylate group.
ďExist free in the body as well as fatty acyl esters in more complex molecules
such as triglycerides or phospholipids.
ďOxidized in all tissues, particularly liver and muscle to provide energy.
ďPrecursors 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 â (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.
8. STAGES
ďProduction of acetyl CoA & NADPH.
ď Conversion of acetyl CoA to malonyl CoA.
ďReactions 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
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- â2-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 ι &
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.
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.
30. SIGNIFICANCE OF FAS COMPLEX
ďś FAS complex offers great efficiency that is free from
interference of other cellular reactions for the synthesis of
fatty acids.
ďśGood coordination in the synthesis of all enzymes of the
FAS complex.
31. REGULATIONOF FATTY ACIDSYNTHESIS
ďFatty acid production is controlled by enzymes, metabolites, end products,
hormones and dietary manipulations.
ďAcetyl CoA carboxylase: This enzyme controls a committed step in fatty acid.
synthesis. Inactive protomer (monomer) or an active polymer.
ďCitrate promotes polymer formation & increases fatty acid synthesis.
ďPalmitoyl CoA & malonyl CoA cause depolymerization of the enzyme, inhibits
the fatty acid synthesis.
32. HORMONAL INFLUENCE
ďHormones regulate acetyl CoA carboxylase by a separate mechanism-
phosphorylation (inactive form) & dephosphorylation (active form) of the
enzyme.
ď Glucagon, epinephrine & norepinephrine inactivate the enzyme by cAMP
dependent phosphorylation.
ďInsulin, dephosphorylates & activates the enzyme. Promotes fatty acid synthesis
while glucagon inhibits.
ďInsulin stimulates tissue uptake of glucose & conversion of pyruvate to acetyl
CoA. This also facilitates fatty acid formation
33. DIETARY REGULATION
ďConsumption of high carbohydrate or fat-free diet increases the
synthesis of acetyl CoA carboxylase & fatty acid synthase, which
promote fatty acid formation.
ďFasting or high fat diet decreases fatty acid production by reducing
the synthesis of acetyl CoA carboxylase & FAS.
35. INTRODUCTION
ďśEicosanoid" is derived from a Greek word âeicosaâ meaning "twentyâ.
ďśEicosanoids is the collective term for the signaling molecules made by oxidation.
ďśThey are oxygenated derivatives of 3 different 20-carbon fatty acids:
1. Eicosapentanoic acid
2. Arachidonic acid
3. Di-homo-gamma-linolenic acid
36. ďś Eicosapentaenoic acid (EPA), an Ď-3 fatty acid with 5 double bonds.
ďś Arachidonic acid (AA), an Ď-6 fatty acid, with 4 double bonds.
ďś Dihomo-gamma-linolenic acid (DGLA), an Ď-6, with 3 double bonds.
ďś Eicosanoids are derived from either omega-3 (Ď-3) or omega-6 (Ď-6) fatty
acids
37. CLASSIFICATION
ďśEicosanoids are classified into two main
groups:
1) Prostanoids
2) Leukotrienes and Lipoxins
ďśProstanoids are further sub-classified into
three groups
a) Prostaglandins(PGs)
b) Prostacyclins(PGIs)
c) Thromboxanes (TXs)
38. BIOSYNTHESIS
ďTwo 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.
ď 5-lipoxygenase (5-LO) generates the leukotrienes and via transcellular
biosynthesis is also involved in lipoxin generation.
ďEicosanoids are not stored within cells, but are synthesized as required.
ďDerive from the fatty acids that make up the cell membrane and nuclear
membrane.
39. ďEicosanoid 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).
ďTriggers the release of a phospholipase at the cell membrane.
ďThe phospholipase travels to the nuclear membrane.
ďThere, the phospholipase catalyzes ester hydrolysis of phospholipid or
diacylglycerol (by phospholipase C).
ďFrees a 20-carbon fatty acid.
ďThe fatty acids may be released by any of several phospholipases.
ď Type IV cytosolic phospholipase A2 (cPLA2 ) is the key actor, as cells lacking
cPLA2 are, in general, devoid of eicosanoid synthesis.
ďPhospholipase cPLA2 is specific for phospholipids that contain AA, EPA or
GPLA at the SN2 position.
ďcPLA2 may also release the lysophospholipid that becomes platelet-activating
factor
40. PROSTANOID PATHWAYS
ďCyclooxygenase (COX) catalyzes the conversion of the free fatty acids to
prostanoids by a two-step process.
ďFirst, 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).
ďOne of the peroxide linkages sheds a single oxygen, forming PGH.
41. ďAll three classes of prostanoids originate from PGH. All have distinctive rings in
the center of the molecule. They differ in their structures.
ďDerived prostaglandins contain a single, unsaturated 5-carbon ring.
ďIn prosta cyclins, this ring is conjoined to another oxygen-containing ring. In
thromboxanes the ring becomes a 6-member ring with one oxygen.
ďLeukotrienes do not have rings.
ďSeveral drugs lower inflammation by blocking prostanoid synthesis.
43. LEUKOTRIENE PATHWAYS
ďEnzyme 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).
ďEnzyme 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. ďEosinophils, 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.
ďLeukotriene LTD4 is then cleaved by dipeptidases to make LTE4 .
ďLeukotrienes LTC4 , LTD4 and LTE4 all contain cysteine and are
collectively known as the cysteinyl leukotrienes.
46. PHARMACOLOGICAL APPLICATIONS OF EICOSANOIDS
ďCardiovascular 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.
ďDigestive Uses- Indicated in the treatment of gastro duodenal ulcer and for the
prevention of NSAID-induced ulcers.
ďGynecological and obstetrical uses - Induce cervical dilatation and uterine
contractions, particularly in late pregnancy. Used for medical termination of
pregnancy and induction of labour.
47. ďOphthalmologic Use- lower intraocular pressure.
ďAnti- inflammatory use- Inhibitors of cyclooxygenases have anti-inflammatory
properties and include nonsteroidal anti-inflammatory drugs or NSAID.
ďThe 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. ďUlcerative 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.
ďBronchial Asthma- PGE2 agonists and leukotrienes receptor antagonists
are used for the treatment of bronchial asthma.