Fatty Acid Biosynthesis

March 26, 2003

Bryant Miles

Fatty acid biosynthesis is different from the β-oxidation degradation pathway.

1. Fatty acid biosynthesis occurs in the cytosol, β-oxidation occurs in the mitochondrial matrix.
2. All of the carbon atoms of fatty acids come from acetyl CoA.
3. Intermediates of fatty acid biosynthesis are covalently linked to an acyl carrier protein. In β-
oxidation the fatty acids are attached to Coenzyme A.
4. In animals, the enzymes involved in fatty acid biosynthesis are contained in one long polypeptide
chain, whereas the enzymes of β-oxidation are independent enzymes found in the matrix.
5. The oxidation/reduction reagents of fatty acid biosynthesis are NADP+/NADPH whereas the redox
reagents of β-oxidation are NAD+/NADH and FAD/FADH2.

The Design Strategy for Fatty Acid Biosynthesis.

• Fatty acids are constructed by the addition of two carbon units derived from acetyl-CoA.
• The acetate units are activated by the formation of malonyl-CoA at the expense of ATP.
• The driving force for the addition of two carbon units to the growing chain is the
decarboxylation of malonyl-CoA.
• The chain elongation stops at palmitoyl-CoA.
• Other enzymes add double bonds or additional carbon atom to the carbon chain.

Fatty acid biosynthesis in the cytosol requires a sufficient concentration of NADPH and acetyl-CoA.
NADPH is generated in the cytosol by the pentose phosphate pathway, and by the malic enzyme which
oxidizes malate into pyruvate and CO2, generating NADPH.
There are 3 principle ways of producing acetyl-CoA in the cytosol of the cell.
1. Amino acid degradation produces acetyl-CoA.
2. Fatty acid oxidation in the matrix of the mitochondria produces acetyl-CoA which is converted
into citrate which is transported into the cytosol by the tricarboxylate transporter. ATP-citrate
lyase convertes citrate in the cytosol into acetyl-CoA.
3. Glycolysis generates pyruvate which can be carboxylated in the mitochondria into oxaloacetate
and then converted into citrate which is transported into the cytosol by the translocase. ATP-citrate
lyase converts citrate in the cytosol into acetyl-CoA.
The acetyl-CoA formed by amino acid degradation is insufficient for fatty acid biosynthesis.
 I. Acetyl-CoA Carboxylase (ACC)
O
Acetyl CoA molecules are the building blocks
O of fatty acid synthesis.
HO C O-
CoA S C C H :B
ATP H2
The acetyl CoA molecules need to be activated
ADP for fatty acid biosynthesis.
O-
CoA S C CH2
O O Acetyl CoA is carboxylated to form malonyl
HO C O P O CoA by acetyl CoA carboxylase which is a
O - O
-
biotin containing enzyme.
O CoA S C CH2

C The carboxylation reaction is irreversible and is

HN NH O B the first committed step of fatty acid
H H
H
CoA S C CH O
H
O biosynthesis.
H2C C C
-
S R O N NH
H H The mechanism of this carboxylase is the same
H2C
H as pyruvate carboxylase and propionyl CoA
Pi S R carboxylase.
ATP is used to activate bicarbonate in the form
O O
of carboxyphosphate which leads to the
C C
-
O N NH
O O carboxylation of biotin.
H H CoA C C
O- The activated CO2 group is transferred to acetyl-
S C
H2C
H
H2 CoA to form malonyl CoA.
S R Malonyl CoA
Acetyl CoA carboxylase has three domains:
1. A biotin carboxyl group carrier protein.
2. Biotin carboxylase which adds CO2 to biotin.
3. A transcarboxylase which transfers the CO2 group from biotin to acetyl CoA to form malonyl
CoA.

Because this is the first committed step of fatty acid biosynthesis, this acetyl CoA carboxylase (ACC) is
allosterically regulated. In animals, ACC is a filamentous polymer composed of 230 kD protomers. Each
protomer contains the biotin carboxyl carrier protein, the carboxylase and the transcarboxylase domains as
well as allosteric regulatory sites. The polymeric form of this enzyme is active, the individual protomers
are not. The activity of ACC is dependent of the equilibrium between the two forms of this enzyme.

Inactive protamers Active polymers

The final product of fatty acid biosynthesis is palmitoyl CoA. There is an allosteric binding site for
palmitoyl-CoA which shifts the equilibrium toward the inactive protamers.

Citrate which is a precursor for acetyl CoA formation in the cytosol is an allosteric activator of this
enzyme. It binds to an allosteric binding site shifting the equilibrium towards the active polymers.

The regulatory effects of citrate and palmitoyl CoA are modulated by the phosphorylation state of acetyl-
CoA carboxylase. The animal ACC enzyme has 8-10 potential phosphorylation sites on each protomer.
Some of these phosphorylation sites are regulatory others are silent and have no effect on enzyme activity.
Acetyl CoA carboxylase is phosphorylated by protein kinases and dephosphorylated by protein
phosphatases.
 The unphosphorylated ACC binds citrate with
high affinity and thus becomes fully active a
very low citrate concentration.

The phosphorylated ACC enzyme has a greatly

reduced affinity for citrate, so high
concentrations of citrate are required to activate
the enzyme.

Palmitoyl CoA binds preferably to the

phosphorylated form of the enzyme, so when
the ACC enzyme is phosphorylated it takes a
small concentration of palmitoyl CoA to
inactivate it.

When the enzyme is dephosphorylated, the

dephosphorylated enzyme has a low affinity for
palmitoyl CoA and thus it takes a large
concentration of palmitoyl CoA to inactivate it.

II Acyl Carrier Protein (ACP)

The intermediates of fatty acid biosynthesis are not linked to Coenzyme A. Rather the intermediates of
fatty acid biosynthesis are linked to an acyl carrier protein. The acyl carrier protein is very similar to
Coenzyme A in that it contains a phosphopantetheine prosthetic group. Coenzyme contains the
phosphopantetheine group attached to an adenosine nucleotide. In the acyl carrier protein, the
phosphopantetheine group is attached to a serine residue.
S H
S H

CH2
CH2

CH2
CH2

NH
NH

C O
C O

CH2
CH2

CH2
CH2

NH
NH NH2

C O
C O
N
N
HO C H
HO C H

N H3 C C CH 3 O
H3 C C CH 3 O O N

H2 C O P O Serine
H2 C O P O P O
O
O-
O- O- H H

H H
O OH

O P O-

O-

III. Elongation Phase of Fatty Acid Biosynthesis

Before fatty acid biosynthesis begins, fatty acid synthetase must be primed with acetyl CoA. The
elongation phase of fatty acid biosynthesis begins with the formation of acetyl ACP and malonyl ACP.
Acetyl transacylase and malonyl transacylase catalyzes the reactions.
 Acetyl CoA + ACP Acetyl ACP + CoA
Malonyl CoA + ACP Malonyl ACP + CoA
Acetyl transacylase will also catalyze the following reaction at a much slower rate:
Propionyl CoA + ACP Propionyl ACP + CoA
This reaction allows for the synthesis of fatty acid chains of odd length. Malonyl transacylase is specific
for malonyl CoA.
O

C CoA Elongation Cycle of Fatty Acid Biosynthesis.

H3C S
ACP
Acetyl
transferase
CoA
O
O O O

C ACP H2 H2
C C CoA C C C ACP
H3C S
-
O C S H3C C C S
Ksase H2 H2
H2 ACP
β-Ketoacyl-ACP
Malonyl
Synthase
Transferase
ACP
CoA
O O O

C KSase C C ACP
-
H3C S O C S
H2

O O
H2
β-Ketoacyl-ACP C C C A
Synthase H3C C S C
C P
CO2 H2 H2

O O

C C ACP CO2
H3C H2C S
β-Ketoacyl-ACP
Synthase
NADPH + H+
β-ketoacyl-ACP
Reductase

NADP+ O
H
HO O H2
C C KSase
C C ACP H3C C S
H3C H2
H2C S

ACP
β-Ketoacyl-ACP
D−β-hydroxyacyl-ACP Synthase
H 2O
Dehydratase Ksase
NADPH + H+ NADP+
O
O
H
H2
C C ACP C C ACP
H3 C C S H3C C S
H
H2
2,3-Trans-Enoyl-ACP
Reductase
Once acetyl KSase and malonyl ACP have been formed, elongation can begin. First the acetyl group of
acetyl ACP is transferred to a sulfhydryl residue of ketoacyl-ACP synthase also known as acyl-malonyl
ACP condensing enzyme. The decarboxylation of malonyl ACP generates an enolate anion which is a
good nucleophile that attacks the carbonyl of thioester of acetyl-S-KSase to form acetoacetyl ACP. The
exergonic decarboxylation reaction drives the condensation reaction. The CO2 group added to acetyl CoA
by acetyl CoA carboxylase is given up in this reaction. In effect this reaction is driven by ATP. ATP was
used to activate bicarbonate in the acetyl CoA carboxylase reaction. The free energy was conserved in the
malonyl CoA and released upon decarboxylation to drive the synthesis of acetoacetyl CoA.

The next three reactions are the reverse of β-oxidation. First the ketone is reduced to the alcohol by the
enzyme β-ketoacyl ACP reductase. This reaction differs from the reverse of the β-oxidation reaction in
that the D-isomer rather than the L-isomer is formed and NADPH is the reducing agent rather than NADH.
The alcohol is dehydrated D-β-hydroxyacyl ACP dehydratase to form crotonyl ACP which is reduced by
enoyl-ACP reductase to form butyryl ACP. This step is also different than the mere reversal of β-
oxidation in that NADPH is the reducing agent instead of FADH2.

In the next round of fatty acid biosynthesis, butyryl ACP it transferred to the sulfhydryl group of β-
ketoacyl-ACP synthase. The decarboxylation of a second molecule of malonyl CoA generates another
nucleophile that attacks the carbonyl of the thioester of butyryl-S-KSase to form a C6-β-ketoacyl ACP
which is then reduced the β-hydoxyacyl ACP, dehydrated to from the C6-∆2-enoyl-ACP which is then
reduced to form the C6-acyl-ACP which is then transferred to the sulfhydryl group of β-ketoacyl-ACP
synthase, condensed with a third maloyl CoA ect. Ect. Ect., unitil palmitoyl ACP is formed. A
thioesterase hydrolyzes the thioester bond of palmitoyl ACP to yield palmitate and ACP.

The overall reaction is shown below:

Acetyl CoA + 7Malonyl CoA + 14 NADPH + 14 H+  Palmitate + 7CO2 + 14 NADP+ + 8CoA + 6H2O

The formation of 7 malonyl CoA requires:

7Acetyl CoA + 7CO2 + 7ATP  7Malonyl CoA + 7ADP + 7Pi + 7H+

Combining these two steps:

8Acetyl CoA + 7 ATP + 14 NADPH + 7H+  Palmitate + 14NADP+ + 7ADP + 7Pi + 8CoA+ 6H2O

IV. The Fatty Acid Synthase Multienzyme Complex of Eukaryotes.

In contrast to bacterial fatty acid biosynthesis, Eukaryotes fatty acid synthase is a multienzyme complex
contained in 2 different polypeptide chains. The α subunit is 213 kD, the β subunit is 203 kD. Animal
fatty acid synthase complexes are dimers of αβ subunits. The separate activities of each dimer of αβ
subunits are shown in the figure below. The α subunits contain the β-ketoacyl-ACP synthase (KSase)
domain and the β-ketoacyl reductase domain. The β subunit contains the acetyl transferase domain, the
malonyl transferase domain, the β-hydroxyacyl dehydrogenase domain and the enoyl reductase domain.

The subunits are arranged in a head to tail fashion that allows the first domain of one subunit of fatty acid
synthase to interact with the second and third domains of the other subunits.
 O O
O- O
O O
O O-
H 3C C S CoA O -
- O C C C S CoA
H 3C C S CoA H3C C S CoA O C C C S CoA
H -
O C C C S CoA H2
H2 H
O :B H2 H B O
O H :B O H B O :B
SER O H :B SER
SER SER SER
ENZ SER
ENZ
ENZ ENZ ENZ
ENZ

Acetyl Transferase CoA

Malonyl Transferase CoA

O- O
O O- O O
O O O
-
H 3C C S ACP S ACP O C C C S ACP -
O C C C S ACP
H 3C C -
O C C C S ACP
H 3C C S ACP H2 H2
H H2 H
O O H B O
H B O O H :B
O H :B :B
:B SER SER
SER SER SER
SER ENZ
ENZ ENZ
ENZ ENZ
ENZ

The first step of the fatty acid synthase reaction is the formation of acetyl-O-enzyme intermediate
between an acetyl group of acetyl CoA and an active site serine residue of the acetyl transferase domain
as shown above on the left. In a similar manner, a malonyl-O-enzyme intermediate is formed between
malonyl CoA and an active site serine residue of the malonyl transferase domain as shown above on the
right.
The next step is the transfer of the acetyl group to the sulfhydryl of the acyl carrier protein (ACP). This
acyl group is then transferred one more time to a cysteine residue of β-ketoacyl-ACP synthase as shown
below. This frees the acyl carrier protein to acquire the malonyl group from the malonyl transferase. The
next step is the condensation reaction in which decarboxylation of the malonyl-ACP generates a highly
reactive nucleophile that attacks the carbonyl of acetyl-S-KSase.

The next three steps are the reduction of the carbonyl to the alcohol, the dehydration and the reduction of
the alkene to form saturated butyryl-ACP. A second malonyl group is transferred from malonyl CoA to
the active site serine of malonyl transferase. The butyryl-ACP is then transferred to the cysteine residue
of KSase. This frees the acyl carrier protein to acquire the malonyl group from the malonyl transferase.
The next step is the condensation reaction in which decarboxylation of the malonyl-ACP generates a
highly reactive nucleophile that attacks the carbonyl of butyryl-S-KSase. This cycle continues until
palmitoyl ACP is formed. The thioester bond is hydrolyzed by thioesterase to form palmitate.

V. Elongation of Palmitate

Palmitate is the primary product of fatty acid synthase. Fatty acids with shorter chains can be generated
by releasing the fatty acid before reaching 16 carbons of length. Fatty acids longer that 16 carbon atoms
require special elongation reactions which occur either in the mitochondria or at the surface of the
endoplasmic reticulum.
The elongation reaction that occurs in the endoplasmic reticulum is similar to fatty acid synthesis. As
shown on the next page.
Elongation in the ER
O
H B

S CoA

-
O S CoA

O O
CoA + CO2

O O

S CoA

S CoA

The mitochondrial elongation reaction involves the addition and subsequent reduction of acetyl groups.
The condensation reaction is catalyzed by thiolase. The reaction is the reverse of the thiolase reaction in
β-oxidation.
Mitochondrial Elongation
O H B

C S CoA

S H :B

ENZ

HS CoA

O
H B
C
S

ENZ
H
H :B
CoA S C
H
C

O O

C C
C S CoA
H2

NADH + H+
β-Hydroxyacyl-CoA dehydrogenase

NAD+ OH O

CH C
C S CoA
H2

Enoyl-CoA hydratase

H
C C
C S CoA
H

NADPH + H+

Trans-enoyl-CoA reductase

NADP+
O

H2
C C
C S CoA
H2