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Fatty acid synthesis

Dr. Sandeep Agrawal MD DNB
Assistant Professor
Dept. of Biochemistry
AIIMS, Raebareli
[email protected]
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Fatty acid synthesis

 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.

 In most mammals, glucose is the primary substrate for lipogenesis, but in

ruminants it is acetate, the main fuel molecule they obtain from the diet.

 Fatty acids are synthesized by an extra mitochondrial system (cytosol).

 Liver and lactating mammary glands are main organs for endogenous
synthesis, although adipose tissue, brain and kidney are involved to a lesser
extent.

 Acetyl-CoA is the immediate substrate, and free palmitate (16:0) is the end
product.

 Its cofactor requirements include NADPH, ATP, Mn2+, biotin, and HCO3– (as a
source of CO2).
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Subcellular localization of lipid metabolism

Fatty acid synthesis takes place in the compartment in which NADPH is available
for reductive synthesis (i.e., where the [NADPH]/[NADP+] ratio is high).
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Sources of NADPH for lipid biosynthesis

NADPH acts as a donor of reducing equivalents, is generated from various
sources:

1. Reactions of HMP shunt: Two of the reactions of this pathway yield an

NADPH each. In hepatocytes and in the mammary gland of lactating animals,
the NADPH required for fatty acid biosynthesis is supplied primarily by the
pentose phosphate pathway.
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Sources of NADPH for lipid biosynthesis

2. Malate to pyruvate conversion: Malic enzyme (pyruvate produced in
the reaction re-enters the mitochondrion for further utilization)

3. Cytosolic isocitrate dehydrogenase: This is a minor source of NADPH

(except in ruminants).
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Transport of acetyl CoA into cytosol

 Acetyl CoA is generated in mitochondrial matrix, which needs to be
transported into the cytosol, where fatty acids are synthesised. However, IMM
is impermeable to acetyl CoA. This problem is overcome in three steps:

 Mitochondrial acetyl CoA combines with oxaloacetate to form citrate

(enzyme: citrate synthase).

 The citrate readily crosses the IMM and moves into the cytosol aided by a
specific carrier protein (facilitated diffusion).

 In cytosol, the citrate is cleaved to regenerate acetyl CoA (enzyme: ATP

citrate lyase).
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Transport of acetyl CoA into cytosol

 Oxaloacetate is returned to the mitochondrial matrix through the malate
shuttle in the following steps:

 Oxaloacetate is first converted to malate (enzyme: malate dehydrogenase)

and then to pyruvate (enzyme: malic enzyme). The latter reaction
generates NADPH, which is used in fatty acid synthesis.

 Pyruvate crosses the IMM, aided by a specific carrier protein (facilitated

transport).

 In mitochondrial matrix, pyruvate is carboxylated to form oxaloacetate

(enzyme: pyruvate carboxylase)
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Enzymes and cofactors involved in FA synthesis

 Two main enzymes
 Acetyl CoA carboxylase
 Fatty acid Synthase

Both the enzymes are multienzyme complexes

 Coenzymes and cofactors are

 Biotin
 NADPH
 Mn++
 Mg++
 HCO3–
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Acetyl CoA carboxylase (ACC)

 Acetyl CoA carboxylase catalyses the initial and controlling step of fatty acid
synthesis.

 It is a multienzyme complex containing:

 Biotin
 Biotin Carboxylase
 Biotin carboxyl carrier protein
 Transcarboxylase
 A regulatory allosteric site
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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.

 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.
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First step: Acetyl CoA to malonyl CoA conversion

 Malonyl CoA is produced by carboxylation of acetyl CoA. The reaction is
catalysed by the enzyme acetyl CoA carboxylase, and requires free energy
which is provided by ATP hydrolysis.

 Biotin is covalently linked to the BCP (Biotin carrier protein). The reaction
proceeds in 2 steps.

 In step 1, catalysed by E1 (biotin carboxylase), biotin is carboxylated as

it accepts a COO− group from HCO3− and ATP is used.

 In step 2, catalysed by E2 (carboxyl transferase), the COO− is

transferred to acetyl-CoA forming malonyl-CoA.

 This is the first committed step in fatty acid biosynthesis.

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First step: Acetyl CoA to malonyl CoA conversion

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Why is it necessary to convert acetyl CoA to malonyl CoA?

 The key reactions of fatty acid synthesis are the carbon-to-carbon

condensation.

 They require input of considerable energy and are, therefore,

thermodynamically unfavourable.

 To overcome this energy barrier, acetyl CoA needs to be converted to

activated form, which will serve as the donor of carbon units to the growing
fatty acid chain.

 Malonyl CoA, a three carbon compound, is one such activated form.

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Reactions of FAS complex

 FAS is a dimer of two identical polypeptide chains with a carrier protein called
acyl carrier protein (ACP). The two subunit polypeptide chains are arranged in
head-to-tail configuration, each of which has seven catalytic sites.

 Role of ACP: It attaches with the reaction intermediates and passes them
from one catalytic site to the next until the final product (palmitate) is released.
Thus, the whole reaction sequence is catalysed by FAS while the intermediates
are shuttled by ACP between successive catalytic sites.

 The subunits together form two functional divisions, generating two fatty acid
molecules (palmitate) simultaneously.

 The growing fatty acid chain is bound to two types of sulfhydryl groups:
 Central thiol
 Peripheral thiol
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Reactions of FAS complex

 Central thiol is contributed by ACP. The reactive unit of ACP is 4-

phosphopantetheine.

 Peripheral thiol is contributed by the condensing enzyme (ketoacyl

synthase) of FAS which contains cysteine with a terminal SH group.

 The two types of thiol groups lie in close proximity in the dimer.

 Each subunit (monomer) contains all the seven enzyme activities, but the
actual functional unit consists of one-half of a monomer interacting with the
complementary half of the other monomer. Thus, two fatty acid chains can be
synthesised simultaneously.

 Overall, seven reactions are catalysed by FAS:

 Two priming reactions and four elongation steps. The priming reactions
ensure that the two thiol groups are loaded with correct acyl groups, while
the elongation steps are involved in actual building of the fatty acid chain.
 The seventh reaction releases the newly synthesised fatty acid from FAS.
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Fatty acid synthase complex

Peripheral thiol

Central thiol
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Priming reactions of FAS complex

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Reactions of FAS complex

1. A molecule of acetate is transferred from acetyl CoA to the –SH group of the
ACP. Enzyme: Acetyl transacylase.

2. Next, this two-carbon fragment is transferred to a temporary holding site, the

thiol group of a cysteine residue on the enzyme.

3. The now-vacant ACP accepts a three-carbon malonate unit from malonyl CoA.
Enzyme: Malonyl transacylase.

4. The acetyl group on the cysteine residue condenses with the malonyl group on
ACP as the CO2 originally added by acetyl CoA carboxylase is released. The
result is a four-carbon unit attached to the ACP Enzyme. The loss of free
energy from the decarboxylation drives the reaction. Enzyme: 3-Ketoacyl
synthase.
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Reactions of FAS complex

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Reactions of FAS complex

The next three reactions convert the 3-ketoacyl group to the corresponding
saturated acyl group by a pair of reductions requiring NADPH and a dehydration
step.

5. The keto group is reduced to an alcohol. Enzyme: 3-Ketoacyl reductase.

6. A molecule of water is removed to introduce a double bond between carbons 2

and 3 (the α- and β-carbons). Enzyme: 3-Hydroxyacyl dehydratase.

7. The double bond is reduced. Enzyme: Enoyl reductase

 The result of these seven steps is production of a four-carbon compound

(butyryl) whose three terminal carbons are fully saturated, and which remains
attached to the ACP.
 These seven steps are repeated, beginning with the transfer of the butyryl chain
from the ACP to the Cys residue [2*], the attachment of a molecule of malonate
to the ACP [3*], and the condensation of the two molecules liberating CO2 [4*].
 The carbonyl group at the β-carbon (carbon 3—the third carbon from the
sulphur) is then reduced [5*], dehydrated [6*], and reduced [7*], generating
hexanoyl-ACP.
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Reactions of FAS complex

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Reactions of FAS complex

 This cycle of reactions is repeated five more times, each time incorporating a
two-carbon unit (derived from malonyl CoA) into the growing fatty acid chain at
the carboxyl end. When the fatty acid reaches a length of 16 carbons, the
synthetic process is terminated with palmitoyl-S-ACP.

 Shorter-length fatty acids are important end products in the lactating mammary
gland.

 Palmitoyl thioesterase cleaves the thioester bond, releasing a fully saturated

molecule of palmitate (16:0).

 All the carbons in palmitic acid have passed through malonyl CoA except the
two donated by the original acetyl CoA, which are found at the methyl-group (ω)
end of the fatty acid. This underscores the rate-limiting nature of the acetyl CoA
carboxylase reaction.
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Summary of palmitate synthesis from acetyl CoA

Part-1

First, the formation of seven malonyl-CoA molecules:

7 Acetyl-CoA + 7 CO2 + 7 ATP  7 malonyl CoA + 7 ADP + 7 Pi

Part-2

Then the seven cycles of condensation and reduction:

Acetyl-CoA + 7 malonyl-CoA + 14 NADPH + 14 H+

Palmitate + 7 CO2 + 8 CoA + 14 NADP+ + 6 H2O

 Finally, only two carbons in palmitic acid come directly from acetyl CoA. The
remaining 14 C are obtained from malonyl CoA.
 In general NADPH is associated with synthetic pathways and NAD+ is
associated with degradative pathways.
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Regulation of fatty acid biosynthesis

Availability of citrate in cytoplasm
 Availability of citrate in cytoplasm is an important factor for short-term
regulation of fatty acid synthesis. The citrate is formed in mitochondrial
matrix, and has a choice between:

 Progressing further in the TCA cycle

 Diffusing into cytoplasm, where it can supply the acetyl CoA precursor
for fatty acid synthesis.

 In high energy states, when NADH:NAD+ ratio is high and ADP low, the
pathway for progression of citrate into TCA cycle is blocked and hence,
concentration of citrate builds up in the mitochondrial matrix.

 The excess of citrate is then passed into the cytoplasm, where it supplies the
acetyl CoA precursor for fatty acid synthesis.

 Citrate also allosterically stimulates the key enzyme of fatty acid synthesis,
i.e., acetyl CoA carboxylase (ACC).
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Regulation of fatty acid biosynthesis

Nutritional state
 Excess carbohydrate 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.
 Lipogenesis converts surplus glucose and intermediates such as pyruvate,
lactate, and acetyl-CoA to fat, assisting the anabolic phase of this feeding
cycle.
 The nutritional state of the organism is the main factor regulating the rate of
lipogenesis. Thus, the rate is high in the well-fed animal whose diet contains a
high proportion of carbohydrate.
 It is depressed by restricted caloric intake, high-fat diet, or a deficiency of
insulin, as in diabetes mellitus. These latter conditions are associated with
increased concentrations of plasma-free fatty acids, and an inverse
relationship has been demonstrated between hepatic lipogenesis and the
concentration of serum-free fatty acids.
 Lipogenesis is increased when sucrose is fed instead of glucose because
fructose bypasses the phosphofructokinase control point in glycolysis and
floods the lipogenic pathway.
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Regulation of fatty acid biosynthesis

Regulation of ACC activity

a) Allosteric modulation

 Most important allosteric modulators of ACC activity are citrate and

ATP, which stimulate the enzyme, and palmitoyl CoA, which inhibits the
activity of enzyme.

 When the substrate citrate is available in plenty, fatty acid synthesis is

desirable. Conversely, decreased synthesis of fatty acids is appropriate
when the product (palmitoyl CoA) accumulates.

 ACC can exist in an inactive monomeric form (MW 400 kD) and an
active polymeric form (MW 6000-8000 kD). Citrate stabilizes the
polymeric form, and palmitoyl CoA dissociates into monomers.
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Regulation of fatty acid biosynthesis

Regulation of ACC activity
b) Covalent modulation

 ACC is inactivated by phosphorylation by an AMP dependent protein

kinase (AMPK). It is activated by dephosphorylation.

 These effects are under the influence of hormones- insulin promotes

dephosphorylation and thereby stimulates the ACC activity.

 Glucagon or epinephrine, on the other hand, promotes the inhibitory

phosphorylation (via AMPK), thereby inactivating the enzyme.

 Thus insulin promotes fatty acid synthesis while glucagon and

epinephrine inhibit it.

 In severe illness, cellular energy charge is dangerously low, as reflected

by high AMP level. The latter activates AMPK. This helps the cell to
survive the energy shortage by switching off nonessential biosynthetic
pathways including fatty acid synthesis.
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Regulation of fatty acid biosynthesis

Regulation of ACC activity
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Regulation of fatty acid biosynthesis

Role of insulin
 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 (e.g., in adipose tissue),
increasing the availability of both pyruvate for fatty acid synthesis and
glycerol-3-phosphate for triacylglycerol synthesis via esterification of the
newly formed fatty acids, and also converts the inactive form of pyruvate
dehydrogenase to the active form in adipose tissue, but not in liver.

 Insulin also—by its ability to depress the level of intracellular cAMP—

inhibits lipolysis in adipose tissue and reducing the concentration of plasma-
free fatty acids and, therefore, long-chain acyl-CoA, which are inhibitors of
lipogenesis.
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Regulation of fatty acid biosynthesis

Regulation of FAS activity
 In common with ACC system, FAS activity also is similarly affected by
regulatory mechanisms, such as allosteric effect, and induction-repression of
the enzyme.

 For instance a high-carbohydrate/low-fat diet increases synthesis of FAS

and conversely a low-carbohydrate/high-fat diet or fasting decreases the
synthesis.

 These regulatory mechanisms for FAS ensure that the excess dietary
carbohydrates are converted to fatty acids in liver. The latter are esterified to
TAGs, which are released as constituents of VLDL.

 This carbohydrate to fat pathway is important only on a high carbohydrate

diet, as the regulatory enzymes are stimulated by insulin.

 This explains why people with carbohydrate based diets are prone to develop
obesity, even when they restrict fat intake.
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Coordinated regulation of fatty acid synthesis and breakdown

Two enzymes are key to the coordination of fatty acid metabolism: acetyl-CoA
carboxylase (ACC), the first enzyme in the synthesis of fatty acids, and carnitine
acyltransferase I, which governs the transport of fatty acids into the mitochondrial
matrix for beta oxidation.
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Chain elongation system

 Chain elongation beyond C-16 palmitate is possible in endoplasmic

reticulum (microsomes) and in the mitochondrion.

 The microsomal system elongates palmitate by the addition of two carbon

fragments derived from malonyl CoA.

 The system of reactions during the chain elongation is similar to that

involved in fatty acid synthesis, except that here the fatty acid is attached to
CoA, instead of the ACP.

 Mitochondrial elongation system is less active and uses acetyl CoA as a

donor of carbons. It is NADH dependent and the substrate for chain
elongation are short and medium chain fatty acids.
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The desaturase system

 Monounsaturated fatty acids can be obtained by introduction of double bond
in the corresponding saturated fatty acids.

 A membrane bound desaturase system in the ER of liver and some other

organs can do so.

 The bond is most commonly introduced at position ∆9 of palmitic or stearic

acid producing palmitoleic or oleic acid respectively.

 The system for desaturation requires molecular oxygen, NADH and

Cytochrome B5.

 PUFA may be formed by introduction of additional double bonds. But these

double bonds can be introduced only between the existing double bond and
the carboxyl end of the fatty acid (but not between the methyl end and the
existing double bond).
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Chain elongation and desaturase system

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