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De Novo Synthesis Of Fatty Acids
Dr. N.Sivaranjani, MD
Asst. Prof.
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Beta-oxidation Fatty acid synthesis
Site Mitochondria Cytoplasm
Intermediates Present as CoA derivatives Covalently linked to SH gr of ACP
Enzymes Present as independent proteins Multienzyme complex
Sequential units 2C units released as Acetyl CoA 2C added as Malonyl CoA(3C)
Co-enzymes NAD and FAD NADPH
Transport Carnitine Citrate
End product Acetyl CoA Palmitate
Lynen's spiral / LipogenesisIt is not a reversal of oxidation.Difference b/w synthesis and breakdown of fatty acids are :-
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Subcellular organelle - Cytoplasm (extra-mitochondrial)
Source of carbon atoms - Acetyl CoA
Source of reducing equivalent - NADPH
Source of energy - ATP
Site :-
Liver, adipose tissue, kidney, brain and mammary glands
Source of fatty acids :-
Exogenous - Diet (major)
Denovo / Endogenous - Pathway operates – excess of caloric in the diet – fatty acids are synthesized – and stored as Triacylglycerol (TAG)
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• Stages of fatty acids synthesis
Transport of Acetyl CoA and NADPH into cytoplasm.
Conversion of Acetyl CoA to Malonyl CoA.
Reactions of Fatty acid synthase complex.
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Transport of Acetyl CoA to cytoplasm
Acetyl CoA is produced in the mitochondria by the oxidation of pyruvate
and fatty acids, degradation of carbon skeleton of ketogenic amino acids.
Because it is impermeable, Acetyl CoA is converted to citrate and
transported to cytoplasm.
This transport is coupled with the cytosomal production of NADPH and
CO2 which is also required for FA synthesis.
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Acetyl-CoA
Pyruvate
AAs
Fatty acids
PDH
Mitochondria Cytoplasm
Oxaloacetate
Citrate
ATP Citrate synthase
Malate
NAD+
NADH+H
Malate DH
Pyruvate
NADP+
NADPH+H
Malicenzyme
Tricarboxylicacid transporter
Citrate
Acetyl-CoA
Oxaloacetate
ATP Citrate lyase
Malate
NAD+
NADH+HMalate DH
Pyruvate
NADP+
NADPH+HCO2
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Conversion of Acetyl CoA to Malonyl CoA / Carboxylation of Acetyl CoA
(3C) Malonyl-CoA
CO2
ADP+Pi
CH3-C-SCoA
O=
(2C) Acetyl-CoA
-OOC-CH2-C-SCoA
O=
Biotin
Acetyl CoA carboxylase
+
Acetyl CoA carboxylase is the rate limiting enzyme of this pathway.
ATP
The elongation of the fatty acid occurs by addition of 2C at a time. But the 2-carbon units are added as 3-carbon, Malonyl units
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Fatty Acid Synthase (FAS) Complex
• exists as a multi-enzyme complex• The enzymes form a dimer with identical subunits• Each subunit is organized into 3 domains with 7 enzymes• Subunits independently operate & both synthesize FA simultaneously
subunits lie in Antiparallel (head to tail) orientation
1st Domain or Condensing Unit - initial substrate binding siteBeta-keto acyl synthase or Condensing enzyme (CE); Acetyl transferase (AT)
and Malonyl trans acylase (MT)
2nd Domain or Reduction Unit - Dehydratase (DH); Enoyl reductase(ER); Beta-keto acyl reductase (KR) and Acyl carrier protein (ACP) 3rd Domain or Releasing Unit - release the FA synthesised.
Thio-esterase (TE) or Deacylase
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• ACP - polypeptide chain having a phospho-pantotheine gr, to which the
acyl groups are attached in thioester linkage.
• ACP acts like the CoA carrying fatty acyl groups
• Eukaryotes - ACP is a part of FAS complex
• Prokaryotes – FAS complex + separate acyl carrier protein
•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; so all the enzymes are in equimolecular concentrations.
• So the efficiency of the process is enhanced.sivaranjani
FAS complex
Cys
Cys
4’-phospho-pantetheine
4’-phospho-pantetheine
SH
SH
SH
SHSubunitdivision
Thioesterase
ACP
-SH group of phosphopantetheineof one subunit is in close proximityto the -SH of cysteine residue of CE of the other subunit
1
2
ThioesteraseCE
AT
MT
Acetyltransacylase
Malonyltransacylase
Ketoacylsynthase
DH
ER
KR
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Reactions of fatty acid synthase complex
CH3-C-SCoA
O=
Acetyl-CoA
CoA-SH
ACP SH
Cys SH
ACP S
Cys SH
-C-CH3
O=
ACP SH
Cys S-C-CH3
O=
Acetyl S-enzyme
Acetyl S-ACP
FAS complex
Acetyl CoAtransacylase
Transfer of acetyl to cys
2C of acetyl CoA is transferred to ACP of FAS by Acetyl CoA-ACP transacylase.
The acetyl unit is then transferred from ACP to cysteine residue of the E
Thus ACP site falls vacant
1
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Malonyl transacylase transfer malonatefrom malonyl CoA to ACP to form acetyl-malonyl enzyme
ACP SH
Cys S-C-CH3
O=
Acetyl S-enzyme
Malonyl-CoA
-OOC-CH2-C-SCoA
O=
CoA-SH Malonyl trasacylase
ACP S
Cys S-C-CH3
O=
-C-CH2-COO
O=
β-Ketoacyl-ACP
CO2
β-Ketoacyl synthase / CE
ACP S
Cys S
-C-CH2
O=
Acetyl-Malonyl E
condensing enzyme or keto acyl synthase condenses Acetyl-S-Cys and malonyl-S-ACP -C-CH3
O=
Condensation reaction
2
3
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β-Ketoacyl-ACPACP S
Cys SH
-C-CH2
O=
-C-CH3
O=
NADP+
NADPH+H+
β-Ketoacyl reductase
ACP S
Cys SH
-C-CH2
O=
-C-CH3
OH
β-Hydroxyacyl-ACP
H2O
Trans-enoyl-ACP
β-hydroxyacyl dehydratase
ACP S
Cys SH
-C-CH
O=
CH-CH3
ketoacyl ACP is reduced by NADPH dependent beta-keto acyl reductase to form beta-hydroxyfatty acyl ACP
β-Hydroxyacy ACP undergoes dehydration.A molecule of water is eliminated & a doublebond is introduced b/w α and β carbons.
Reduction
Dehydration
4
5
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NADP+
NADPH+H+
Trans-enoyl-ACPACP S
Cys SH
-C-CH
O=
CH-CH3
Acyl-ACP / butyrylACP
ACP S
Cys SH
-C-CH
O=
CH2-CH3
Enoyl reductase
Transfer of C chain from ACP to cys-SH
ACP SH
Cys S-C-CH
O=
CH2-CH3
Acyl-S-enzyme
4C unit attached to ACP is butyryl group
Reduction
6
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Palmitate (16C)Palmitoyl Thioesterase
reactions of 2-6 are repeated 6 times
ACP SH
Cys S-C-CH
O=
CH2-CH3
Acyl-S-enzyme
ACP S -
Cys SH
ACP SH
Cys SHCH3-CH2
-(CH2)13-COO-
+
7
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Summary of palmitate synthesis
• End product –(16C) Palmitate
• 2C - Acetyl CoA directly
• 14C - Malonyl CoA
• Over all reaction :
Palmitoyl-coA
CO-S-coA
CH3
1
2
3
4
5
6
7
8
9
10121416
15 13 11
8 Acetyl-coA = Acetyl-CoA + 7 malonyl-CoA
CH3-CO-SCoA
14 NADPH+H+
7 Cycles of Fatty acid synthesis
7 ATP
7 ADP+Pi
14 NADP+6 H2O
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Short-term control – rapid, with in min
Allosteric regulation
Covalent modification
Long-term control – slow, takes hr to manifest
Induction
Repression
Regulation of fatty acid synthesis
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Malonyl-CoA
Acetyl-CoA
Glucose
Citrate
Palmitoyl CoA
NADPH HMP shunt
-
G6PD
Allosteric regulation
Acetyl CoA Carboxylase
+
-
Rate limiting enzyme –Acetyl CoA Carboxylase
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Insulin
Protein phosphatase
Proteinkinase
Glucagon & Epinephrine
ATPADP
-
+
Covalent modification
Acetyl CoA Carboxylase
(inactive)
P
Acetyl CoA Carboxylase
(active)
P
Acetyl CoA
Malonyl CoA
Dephosphorylated E – Active Phosphorylated E - Inactive
Acyl CoA
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• Involves change in the gene expression which controls the rate of synthesis of these enzymes.
• Insulin - induces
• Glucagon - represses
Long-term control mechanism
+High fat dietStarvationDM – dec. Insulin
-All the enzymes of fatty acid synthesis
High carbohydrate diet
Low fat diet
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Elongation of FA
Microsomal elongation system Mitochondrial elongation system
Endoplasmic reticulumLIVER
Mitochondria LESS ACTIVE
Malonyl-CoA – the donor of the C2 units AcetylCoA – the donor of the C2 unit
It elongates FA having C10 to C22 , C24
LCFA - present in sphingomyelin –important for myelination of Nerves.
Only MCFA and SCFA
FA > C16 elongases (chain elongation)
CO-S-coA
CH3
1
2
3
4
5
6
7
8
9
10121416
15 13 1117
18
19
20CH3
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Desaturation system
Smooth endoplasmic reticulum
There are 4 fatty acyl desaturase enzymes in mammals designated 9 , 6, 5 and 4 fatty acyl-CoA desaturase
Palmitoyl CoA 16C– Palmitoleic acid 16:1(9) Stearyl CoA 18C – Oleic acid 18:1(9)
Mammals cannot incorporate a double bond beyond 9 – PUFA cannot be synthesized so supplied in diet, but plants contains 12,15 fatty acyl CoA desaturase.
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Desaturase
Cyt b5
reductase
Cyt b5
C18 Stearoly-CoA + O2 + 2H+ C18 9-oleyl-CoA + 2H2O
2 cyt b5 Fe2+ 2 cyt b5 Fe3+
2H++ 2 cyt b5 reductaseFAD+
2 cyt b5 reductaseFADH2
NADH + H+ NAD+
9Fattyl acyl CoA Desaturase
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Palmitate
Stearate
Oleate
Linoleate
-Linolenate
-Linolenate
Homo -Linolenate
Arachidonate
18:3(9,12,15)
18:2(9,12) 18:3(6,9,12)
16 C
18 C
Elongase
18:1(9)
Palmitoleate 16:1(9)
9 Desaturase
12 Desaturase
9 Desaturase
15 Desaturase
6 Desaturase
5 Desaturase
Elongase
20:3(8,11,14)
20:4(5,8,11,14)
Mammals
Essential fatty acid
Plants
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Summary of FA synthesis
Site: Liver, Adipose tissue, Mammary gland during lactation
Localization:Cytoplasm (up to C16)
Enzymes: Acetyl-CoA-carboxylase (HCO3- - source of CO2, biotin, ATP)
Fatty acid synthase (NADPH ,CoA)
Primary substrate: Acetyl-CoA
Final product: Palmitate
(always in excess calories)
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2-6 Rn are repeated by 2C in each cycle to form chain length C16
(palmitate)
Palmitate,is a precursor of saturated and unsaturated FA:
Saturated FA (> C16) elongation systems
Unsaturated FA (=) desaturation systems
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