Fatty acid synthesis revised version

Fatty Acid Synthesis
Namrata Chhabra
15-May-20 Namrata Chhabra 1

Fatty acids
❑Fatty acids are a class of compounds containing a long hydrophobic hydrocarbon
chain and a terminal carboxylate group
❑They exist free in the body as well as fatty acyl esters in more complex molecules
such as triglycerides or phospholipids.
❑Fatty acids can be oxidized in all tissues, particularly liver and muscle to provide
energy
❑They are also structural components of membrane lipids such as phospholipids
and glycolipids.
❑Esterified fatty acids, in the form of triglycerides are stored in adipose cells
❑Fatty acids are also precursors of Eicosanoids

Sources of Fatty acids
• Diet
• Adipolysis
• De novo synthesis(from precursors)- Carbohydrates, protein, and
other molecules obtained from diet in excess of the body’s need can
be converted to fatty acids, which are stored as triglycerides

Introduction
❑Fatty acids are synthesized by an extra mitochondrial 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.
❑Its cofactor requirements include NADPH, ATP, Mn2+, biotin, and
HCO3
– (as a source of CO2).

Site of fatty acid synthesis
• FA synthase complex is
found exclusively in the
cytosol.
• The location segregates
synthetic processes from
degradative reactions.

Substances required for fatty acid biosynthesis
• Acetyl CoA
• NADPH
• Enzymes
• Co-factors
• Carbon-dioxide
• Energy

Acetyl Co A- Sources
❑Acetyl co A, the precursor of fatty
acid synthesis is produced from
pyruvate, ketogenic amino acids,
fatty acid oxidation and by alcohol
metabolism
❑It is a substrate for TCA cycle and a
precursor for fatty acids, ketone
bodies and sterols.

NADPH- Sources
❑NADPH is involved as donor of reducing equivalents
❑The oxidative reactions of the pentose phosphate pathway are the
chief source of the hydrogen required for the reductive synthesis of
fatty acids.
❑Tissues specializing in active lipogenesis—ie, liver, adipose tissue, and
the lactating mammary gland—possess an active pentose phosphate
pathway.

NADPH- Sources
• Other sources of NADPH include the reaction that converts malate to
pyruvate catalyzed by the "Malic enzyme" (NADP malate
dehydrogenase) and
• the extra mitochondrial isocitrate dehydrogenase reaction (probably
not a substantial source, except in ruminants).

The pentose phosphate /HMP Pathway
• In hepatocytes, adipose tissue and the lactating mammary glands, the
NADPH is supplied primarily by the pentose phosphate pathway.

Malic enzyme- the alternative source of NADPH
• Reversible reaction, pyruvate produced in the reaction reenters the
mitochondrion for further utilization

Cytosolic Isocitrate Dehydrogenase- Source of
NADPH
• There are three isoenzymes of isocitrate dehydrogenase.
• One, which uses NAD+, is found only in mitochondria.
• The other two use NADP+ and are found in mitochondria and the cytosol.
• Respiratory chain-linked oxidation of isocitrate proceeds almost completely through the
NAD+-dependent enzyme.

Transportation of Acetyl co A
❑Fatty acid synthesis requires considerable amounts of acetyl-CoA
❑Nearly all acetyl-CoA used in fatty acid synthesis is formed in
mitochondria
❑ Acetyl co A has to move out from the mitochondria to the cytosol.
❑The mitochondrial inner membrane is impermeable to acetyl-CoA

❑Acetate is shuttled out of mitochondria as citrate
❑Intra-mitochondrial acetyl-CoA first reacts with oxaloacetate to form
citrate, in the TCA cycle catalyzed by citrate synthase
❑Citrate then passes into the cytosol through the mitochondrial inner
membrane on the citrate transporter.
❑In the cytosol, citrate is cleaved by citrate lyase regenerating acetyl-
CoA.

Fate of Oxaloacetate
• The other product of Citrate cleavage, oxaloacetate can be-
❑Channeled towards glucose production
❑Converted to malate by malate dehydrogenase
❑Converted to Pyruvate by Malic enzyme, producing more NADPH,
that can be used for fatty acid synthesis
❑Pyruvate and Malate pass through special transporters present in the
inner mitochondrial membrane

Fate of Oxaloacetate

Enzymes and cofactors involved in the
process of Fatty acid synthesis
• Two main enzymes-
❑Acetyl co A carboxylase
❑Fatty acid Synthase
• Both the enzymes are multienzyme complexes
• Coenzymes and cofactors are-
❑Biotin
❑NADPH
❑Mn++
❑Mg++

Details of enzymes
• Acetyl co A carboxylase -Is the Initial & Controlling Step in Fatty Acid
Synthesis
❑Multienzyme complex containing-
❑Biotin
❑Biotin Carboxylase
❑Biotin carboxyl carrier protein
❑Transcarboxylase
❑A regulatory allosteric site

Acetyl co A carboxylase
• The input to fatty acid synthesis is acetyl-CoA, which is carboxylated
to malonyl-CoA.
• The reaction is catalyzed by Acetyl co A carboxylase

Role of Biotin
• Biotin is linked to the
enzyme by an amide
bond between the
terminal carboxyl of the
biotin side chain and
the e-amino group of a
lysine residue.
CHCH
H2C
S
CH
NH
C
N
O
(CH2)4 C NH (CH2)4 CH
CO
NH
O
C
O
O−
Carboxybiotin lysine
residue

Fatty acid synthase complex
❑The Fatty Acid Synthase Complex is a polypeptide containing seven
enzyme activities
❑In bacteria and plants, the individual enzymes of the fatty acid
synthase system are separate, and the acyl radicals are found in
combination with a protein called the acyl carrier protein (ACP).
❑In yeast, mammals, and birds, the synthase system is a multienzyme
polypeptide complex that incorporates ACP, which takes over the role
of CoA.

Fatty acid Synthase complex
❑In mammals, the fatty acid synthase complex is a dimer comprising
two identical monomers, each containing all seven enzyme activities
of fatty acid synthase on one polypeptide chain
❑The use of one multienzyme functional unit has the advantages of
achieving the effect of compartmentalization of the process within
the cell without the erection of permeability barriers,
❑Synthesis of all enzymes in the complex is coordinated since it is
encoded by a single gene.

Fatty acid Synthase complex
• Domain-1-Condensation unit- The substrate entry and condensation
unit, contains acetyl transferase, malonyl transferase, and β-ketoacyl
synthase (condensing enzyme).
• Domain-2-Reduction unit- The reduction unit, contains the acyl carrier
protein, β-ketoacyl reductase, dehydratase, and enoyl reductase.
• Domain-3-Releasing unit- the palmitate release unit, contains the
thioesterase.

• The —SH of the 4'-phosphopantetheine of one monomer is in close
proximity to the —SH of the cysteine residue of the ketoacyl synthase
of the other monomer, suggesting a "head-to-tail" arrangement of
the two monomers.
• Though each monomer contains all the partial activities of the
reaction sequence, the actual functional unit consists of one-half of
one monomer interacting with the complementary half of the other.
• Thus, two acyl chains are produced simultaneously.

❑Each segment of the disk
represents one of the six enzymatic
activities of the complex
❑(Thioesterase not shown)
❑At the center is the ACP – acyl
carrier protein - with its
phosphopantethein-e arm ending
in –SH.

The function of the prosthetic group of the ACP
❑Serve as a flexible arm,
tethering the growing fatty acyl
chain to the surface of the
synthase complex
❑Carrying the reaction
intermediates from one
enzyme active site to the next.

Structure of Phosphopantetheine
❑Phosphopantetheine (Pant) is
covalently inked via a phosphate ester
to a serine OH of the acyl carrier
protein domain of Fatty Acid Synthase.
❑The long flexible arm of
phosphopantetheine helps its thiol to
move from one active site to another
within the complex.
OPOH2C
O−
OC
C
C
NH
CH2
CH2
C
NH
CH3H3C
HHO
O
CH2
CH2
SH
O
CH2 CH
NH
C O
-mercaptoethylamine
pantothenate
serine
residue
phosphopantetheine
of acyl carrier protein
phosphate

Steps of fatty acid synthesis
• Fatty acid synthesis is a cyclic process.
• The initial step of carboxylation of acetyl co A is catalyzed by Acetyl co
A carboxylase, the remaining steps are catalyzed by fatty acid
synthase complex.

The first round of FA biosynthesis
• a) Formation of Malonyl co A- Step-1
Fatty acid synthesis starts with the carboxylation of acetyl CoA
to malonyl CoA.
• This irreversible reaction is the committed step in fatty acid synthesis.
❑As with other carboxylation reactions, the enzyme prosthetic group is
biotin.
❑The reaction takes place in two steps: carboxylation of biotin
(involving ATP) and transfer of the carboxyl to acetyl-CoA to form
malonyl-CoA.

Formation of Malonyl co A- Step-1
❑ATP-dependent carboxylation provides energy input.
❑The CO2 is lost later during condensation with the growing fatty acid.
❑The spontaneous decarboxylation drives the condensation reaction.

Initiation of fatty acid synthesis
❑To initiate FA biosynthesis, malonyl and acetyl groups are activated
on to the enzyme fatty acid synthase.
❑Initially, a priming molecule of acetyl-CoA combines with a cysteine
—SH group catalyzed by acetyl transacylase
❑Malonyl-CoA combines with the adjacent —SH on the 4'-
phosphopantetheine of ACP of the other monomer, catalyzed by
malonyl transacylase (to form acetyl (acyl)-malonyl enzyme.

The activation of the acetyl group
❑The acetyl group from acetyl-CoA is
transferred to the Cys-SH group of the -
ketoacyl ACP synthase
❑This reaction is catalyzed by acetyl-CoA
transacetylase.

The activation of the malonyl group
❑Transfer of the malonyl group to the –SH
group of the ACP is catalyzed by malonyl-
CoA ACP transferase.
❑The charged acetyl and malonyl groups
are now in close proximity to each other

2) Elongation cycle in fatty acid synthesis
• After activation, the processes involved are-
1. Condensation
2. Reduction
3. Dehydration
4. Reduction
•These steps are repeated till a fatty acid with 16 carbon atoms is
synthesized

Step-1-Condensation
Condensation –
❑Condensation of the activated
acetyl and malonyl groups takes
place to form Acetoacetyl-ACP
❑The reaction is
catalyzed by β-ketoacyl-ACP
synthase.

Condensation
❑The acetyl group attacks the methylene group of the malonyl residue,
catalyzed by 3-ketoacyl synthase, and liberates CO2, forming 3-
ketoacyl enzyme (Acetoacetyl enzyme),freeing the cysteine —SH
group.
❑Decarboxylation allows the reaction to go to completion, pulling the
whole sequence of reactions in the forward direction.

Step-2 Reduction)
Reduction-
❑The Acetoacetyl-ACP is reduced
to b-hydroxybutyryl-ACP, catalyzed
by b-ketoacyl-ACP reductase
❑NADPH + H+ are required for
reduction

Step-3 Dehydration
Dehydration –
❑Dehydration yields a double bond in
the product, trans-∆2-butenoyl-ACP,
❑Reaction is catalyzed by β-
hydroxybutyryl-ACP dehydratase.

Step-4 Reduction
Reduction
❑Reduction of the double bond takes
place to form butyryl-ACP,
❑ Reaction is catalyzed by enoyl-
reductase.
❑Another NADPH dependent
reaction.

The growing chain is transferred from the acyl carrier protein
❑This reaction makes way for the
next incoming malonyl group.
❑The enzyme involved is acetyl-
CoA transacetylase

Beginning of the second round of the FA synthesis cycle
❑The butyryl group is on the Cys-SH group
❑The incoming malonyl group is first attached to
ACP.
❑In the condensation step, the entire butyryl
group is exchanged for the carboxyl group on
the malonyl residue

Repetition of these four steps leads to fatty
acid synthesis
❑The 3-ketoacyl group is reduced, dehydrated, and reduced again (reactions 2, 3,
4) to form the corresponding saturated acyl-S-enzyme.
❑A new malonyl-CoA molecule combines with the —SH of 4'-
phosphopantetheine, displacing the saturated acyl residue onto the free cysteine
—SH group.
❑The sequence of reactions are repeated until a saturated 16-carbon acyl radical
(Palmityl) has been assembled.
❑ It is liberated from the enzyme complex by the activity of a seventh enzyme in
the complex, Thioesterase (deacylase).

Repetition of these four steps leads to fatty acid
synthesis

The result of fatty acyl synthase activity
❑Seven cycles of condensation and reduction produce the 16-carbon
saturated palmitoyl group, still bound to ACP.
❑Chain elongation usually stops at this point, and free palmitate is
released from the ACP molecule by hydrolytic activity in the synthase
complex.
❑Smaller amounts of longer fatty acids such as stearate (18:0) are also
formed
❑In mammary gland, there is a separate Thioesterase specific for acyl
residues of C8, C10, or C12, which are subsequently found in milk lipids.

The overall reaction for the synthesis of
palmitate from acetyl-CoA can be
considered in two parts.

Part 1
First, the formation of seven malonyl-CoA molecules:
7Acetyl-CoA + 7CO2 + 7ATP
7malonyl CoA + 7ADP + 7Pi

Part 2
Then the seven cycles of condensation and reduction
Acetyl-CoA + 7malonyl-CoA + 14NADPH + 14H+
palmitate + 7CO2 + 8CoA +
14NADP+ + 6H2O
The biosynthesis of FAs requires acetyl-CoA and the input of
energy in the form of ATP and reducing power of NADPH.

Comparison of β-Oxidation & Fatty Acid Synthesis
Βeta Oxidation pathway Fatty acid Synthesis
Location Mitochondrial Cytoplasmic
Acyl Carriers(Thiols) Coenzyme A 4’ Phosphopantetheine and
Cysteine
Electron acceptors and donors FAD/NAD NADPH
OH Intermediates L D
2 Carbon product/donor Acetyl co A Acetyl co A/ Malonyl co A

Regulation of fatty acid synthesis
When a cell has more energy, the
excess is generally converted to
Fatty Acids and stored as lipids such
as triacylglycerol.
Glycerol-P
Triacylglycerol
Fatty acyl CoA
Malonyl CoA
Acetyl CoA
Glucose
Pyruvate
TCA cycle

Regulation of fatty acid synthesis
The reaction catalyzed by acetyl-CoA
carboxylase is the rate limiting step in
the biosynthesis of fatty acids.
CH3-C-S-CoA
=
O
HCO3
-
-OOC-CH2-C-S-CoA
=
O
Acetyl-CoA
Malonyl-CoA

Regulation of Acetyl-coA carboxylase
The mammalian enzyme is regulated, by
 Allosteric control by local metabolites
 Phosphorylation
 Conformational changes associated with regulation:
 In the active conformation, Acetyl-CoA Carboxylase associates to form
multimeric filamentous complexes.
 Transition to the inactive conformation is associated with dissociation
to yield the monomeric form of the enzyme (protomer).

Allosteric control
❑Palmitoyl-CoA acts as a feedback
inhibitor of the enzyme, and citrate is
an activator.
❑When there is an increase in
mitochondrial acetyl-CoA and ATP,
citrate is transported out of
mitochondria,
❑Citrate becomes both the precursor
of cytosolic acetyl-CoA and a signal for
the activation of acetyl-CoA
carboxylase.

Acetyl co A carboxylase

Phosphorylation
Acetyl-CoA carboxylase is also regulated
by hormones such as glucagon,
epinephrine, and insulin via changes in
its phosphorylation state

❑Additionally, these pathways are regulated at the level of gene
expression
❑Long-chain fatty acid synthesis is controlled in the short term by
allosteric and covalent modification of enzymes and in the long term by
changes in gene expression governing rates of synthesis of enzymes.

Nutritional state regulates lipogenesis
❑Excess carbohydrates is stored as fat in many animals in anticipation of
periods of caloric deficiency such as starvation, hibernation, etc, and to
provide energy for use between meals in animals, including humans,
that take their food at spaced intervals.
❑The nutritional state of the organism is the main factor regulating the
rate of lipogenesis.

Fatty acid synthesis during Fed state
❑The rate is higher in the well-fed state if the diet contains
a high proportion of carbohydrate
❑Lipogenesis converts surplus glucose and intermediates
such as pyruvate, lactate, and acetyl-CoA to fat, assisting the
anabolic phase of this feeding cycle
❑Lipogenesis is increased when sucrose is fed instead of
glucose because fructose bypasses the phosphofructokinase
control point in glycolysis and floods the lipogenic pathway

Fatty acid synthesis during Fasting
❑It is depressed by restricted caloric intake, high fat diet, or a
deficiency of insulin, as in diabetes mellitus
❑These conditions are associated with increased concentrations of
plasma free fatty acids
❑An inverse relationship has been demonstrated between hepatic
lipogenesis and the concentration of serum-free fatty acids.

Role of Insulin in fatty acid synthesis
❑Insulin stimulates lipogenesis by several other mechanisms as
well as by increasing acetyl-CoA carboxylase activity.
❑It increases the transport of glucose into the cell (eg, in adipose
tissue),
❑Increases the availability of both pyruvate for fatty acid synthesis
and glycerol 3-phosphate for esterification of the newly formed
fatty acids,

Fatty acid elongation
❑Palmitate in animal cells is the precursor of other
long-chained FAs.
❑By further additions of acetyl groups, fatty acid
chain length is elongated through the action of FA
elongation systems present in the smooth
endoplasmic reticulum and the mitochondria.

Fatty acid elongation

The desaturation of Fatty Acids

Essential fatty acids
❑Mammalian hepatocytes readily introduce double bonds at the D9
position of FAs but cannot between C-10 and the methyl-terminal
end.
❑Linoleate, 18:2D9,12 and linolenate 18:3D9,12,15 cannot be
synthesized by mammals, but plants can synthesize both.
❑Arachidonic acid is semi essential, since it can be synthesized from
Linoleic acid

The fate of fatty acids
Most of the FAs synthesized or ingested by an organism have one
of two fates:
❑Incorporated into triacylglycerols for the storage of metabolic
energy
❑Incorporated into the phospholipid components of membranes

Thank you

Fatty acid synthesis revised version

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