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Fatty Acid Biosynthesis

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 Fatty Acid Biosynthesis
Prof. Dr. Fadhil Jawad Al-Tu’ma . Ph.D.
College of Medicine – University of Kerbala

Objective:
1. Describe the reaction catalyzed by acetyl-CoA carboxylase and understand the
mechanisms by which its activity is regulated to control the rate of fatty acid
synthesis.
2. Outline the structure of the fatty acid synthase multienzyme complex, indicating the
sequence of enzymes in the two peptide chains of the homodimer.
3. Explain how long-chain fatty acids are synthesized by the repeated condensation of
two carbon units, with formation of the 16-carbon palmitate being favored in most
tissues, and identify the cofactors required.
4. Indicate the sources of reducing equivalents (NADPH) for fatty acid synthesis.
5. Understand how fatty acid synthesis is regulated by nutritional status and identify
other control mechanisms that operate in addition to modulation of the activity of
acetyl-CoA carboxylase.
6. Explain how polyunsaturated fatty acids are synthesized by desaturase and elongation
enzymes.

Fatty acid synthesis is not simply a reversal of the degradative pathway. Rather, it consists
of a new set of reactions, again exemplifying the principle that synthetic and degradative
pathways are almost always distinct. Some important differences between the pathways are:
1. Fatty acid biosynthesis takes place in the cytosol, in contrast with degradation, which
takes place primarily in the mitochondrial matrix.
2. Intermediates in fatty acid biosynthesis are covalently linked to the sulfhydryl groups of
an acyl carrier protein (ACP), whereas intermediates in fatty acid breakdown are
covalently attached to the sulfhydryl group of coenzyme A.
3. The enzymes of fatty acid biosynthesis in higher organisms are joined in a single
polypeptide chain called fatty acid synthase. In contrast, the degradative enzymes do not
seem to be associated.
4. The growing fatty acid chain is elongated by the sequential addition of two-carbon units
derived from acetyl CoA. The activated donor of two carbon units in the elongation step
is malonyl ACP. The elongation reaction is driven by the release of CO2.
5. The reductant in fatty acid biosynthesis is NADPH, whereas the oxidants in fatty acid
degradation are NAD+ and FAD.
6. Elongation by the fatty acid synthase complex stops on formation of palmitate (C16).
Further elongation and the insertion of double bonds are carried out by other enzyme
systems.
Biomedical Importance:
Fatty acids are synthesized by an extra-mitochondrial system, which is responsible for
the complete synthesisof palmitate from acetyl-CoA in the cytosol. In most mammals,
glucose is the primary substrate for lipogenesis, but in ruminants it is acetate, the main fuel
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molecule produced by the diet. Critical diseases of the pathway have not been reported in
humans. However, inhibition of lipogenesis occurs in type 1 (insulin-dependent) diabetes
mellitus, and variations in its activity may affect the nature and extent of obesity.
De novo Biosynthesis of Fatty Acids (lipogenesis) :
A large proportion of the fatty acids used by the body is supplied by the diet.
Carbohydrates, protein, and other molecules obtained from the diet in excess of the body's
needs for these compounds can be converted to fatty acids, which are stored as
triacylglycerols. This system is present in many tissues, including liver,kidney, brain, lung,
mammary gland, and adipose [Link] cofactor requirements include NADPH, ATP, Mn2+,
biotin, and HCO3− (as a source of CO2).
Acetyl-CoA is the immediate substrate, and free palmitate is the end product.
Source of NADPH for Fatty Acid Biosynthesis:
NADPH is involved as donor of reducing equivalents in both the reduction of the 3-
ketoacyl and of the 2,3- unsaturated acyl derivatives. The oxidative reactions of the pentose
phosphate pathway are the chief source of the hydrogen required for the reductive synthesis
of fatty acids. Significantly, tissues specializing in active lipogenesis i.e, liver, adipose
tissue, and the lactating mammary gland—also possess an active pentose phosphate
pathway. Moreover, both metabolic pathways are found in the cytosol of the cell, so there
are no membranes or permeability barriers against the transfer of NADPH.
Other sources of NADPH include the reaction that converts malate to pyruvate catalyzed
by the “malic enzyme” (NADP malate dehydrogenase) (see below) and the extra-
mitochondrial isocitrate dehydrogenase reaction (probably not a substantial source, except
in ruminants).
Production of Cytosolic Acetyl-CoA :
The following figure indicate the Shuttle required for transfer of acetyl groups from
mitochondria to the cytosol. The mitochondrial outer membrane is freely permeable to all
these compounds. Pyruvate derived from amino acid catabolism in the mitochondrial
matrix, or from glucose by glycolysis in the cytosol, is converted to acetyl-CoA in the
matrix. Acetyl groups pass out of the mitochondrion as citrate; in the cytosol they are
delivered as acetyl-CoA for fatty acid synthesis. Oxaloacetate is reduced to malate, which
returns to the mitochondrial matrix and is converted to oxaloacetate. An alternative fate for
cytosolic malate is oxidation by malic enzyme to generate cytosolic NADPH; the pyruvate
produced returns to the mitochondrial matrix.

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Malonyl-Co A Biosynthesis Is the Committed Step:
Fatty acid biosynthesis starts with the carboxylation of acetyl-CoA to malonyl-CoA. This
irreversible reaction is the committed step in fatty acid biosynthesis.

The synthesis of malonyl-CoA is catalyzed by acetyl-CoA carboxylase. Acetyl-CoA

carboxylase has a requirement for the vitamin biotin. The enzyme is a multienzyme
protein containing a variable number of identical subunits, each containing biotin, biotin
carboxylase, biotin carboxyl carrier protein, and transcarboxylase, as well as a regulatory
allosteric site. The reaction takes place in two steps:
1. Carboxylation of biotin involving ATP.
2. Transfer of the carboxyl to acetyl-CoA to form malonyl-CoA.

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 Bicarbonate as a source of CO2 is required in the initial reaction for the carboxylation of
acetyl-CoA to malonyl-CoA in the presence of ATP and acetyl-CoA carboxylase.

Regulation of Acetyl-CoA Carboxylase:

1. Short-term regulation of acetyl CoA carboxylase:This carboxylation is both the rate-
limiting and the regulated step in fatty acid biosynthesis. The inactive form of acetyl–
CoA carboxylase is a protomer (dimer). The enzyme undergoes allosteric activation by
citrate, which causes dimers to polymerize. The enzyme can be allosterically inactivated
by long-chain fatty acyl-CoA (the end product of the pathway), which causes its
depolymerization.
A second mechanism of short-term regulation is by reversible phosphorylation. In the
presence of counterregulatory hormones, such as epinephrine and glucagon, acetyl-CoA
carboxylase is phosphorylated and, thereby, inactivated (see below). In the presence of
insulin, acetyl-CoA carboxylase is dephosphorylated and, thereby, activated.

2. Long-term regulation of acetyl CoA carboxylase: Prolonged consumption of a diet

containing excess calories such as high-carbohydrate diets causes an increase in acetyl-
CoA carboxylase synthesis, thus increasing fatty acid synthesis. Conversely, a low-
calorie diet or fasting causes a reduction in fatty acid biosynthesis by decreasing the
synthesis of acetyl-CoA carboxylase.

The Fatty Acid Synthase Complex Has Seven Different Active Sites
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 (see below). Initially, a priming molecule of acetyl-CoA combines with a
cysteine-SH group catalyzed by acetyl transacylase.

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 Fatty acid synthase multienzyme complex. The sequence of the enzymes in each
monomer is based on Salih J. Wakil / Baylor Medical College.

Fatty Acid Synthesis Proceeds in a Repeating Reaction Sequence:

The long carbon chains of fatty acids are assembled in a repeating four-step sequence (see
below).

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 Addition of two carbons to a growing fatty acyl chain: a four-step sequence. Each malonyl group
and acetyl (or longer acyl) group is activated by a thioester that links it to fatty acid synthase, a
multienzyme.
1. Condensation of an activated acyl group (an acetyl group from acetyl-CoA is the first acyl
group) and two carbons derived from malonyl-CoA, with elimination of CO2 from the
malonyl group, extends the acyl chain by two carbons. The mechanism of the first step of this
reaction is given to illustrate the role of decarboxylation in facilitating condensation. The β-
keto product of this condensation is then reduced in three more steps nearly identical to the
reactions of β-oxidation, but in the reverse sequence.
2. The β-keto group is reduced to an alcohol.
3. Elimination of H2O creates a double bond.
4. The double bond is reduced to form the corresponding saturated fatty acyl group.

The equation for the overall synthesis of palmitate from acetyl-CoA and malonyl-CoA is:
CH3 CO-SCoA + 7HOOC-CH2CO-SCoA +14 NADPH + 14H+→
CH3(CH2)14COOH + 7CO2 + 6H2O + 8CoA-SH + 14NADP+

The acetyl-CoA used as a primer forms carbon atoms 15 and 16 of palmitate. The
addition of all the subsequent C2 units is via malonyl-CoA. Propionyl-CoA acts as primer
for the synthesis of long-chain fatty acids having an odd number of carbon atoms, found
particularly in ruminant fat and milk.
The following figure indicates the overall process of palmitate synthesis. The fatty acyl
chain grows by two-carbon units donated by activated malonate, with loss of CO2 at each
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step. The initial acetyl group is shaded yellow, C-1 and C-2 of malonate are shaded pink,
and the carbon released as CO2 is shaded green. After each two-carbon addition, reductions
convert the growing chain to a saturated fatty acid of four, then six, then eight carbons, and
so on. The final product is palmitate (16:0).

Acyl carrier protein (ACP) is a small protein (Mr 8,860) containing the prosthetic
group 4'-phosphopantetheine (see below). Hydrolysis of thioesters is highly exergonic,
and the energy released helps to make two different steps (1 and 5) in fatty acid synthesis
(condensation) thermodynamically favorable. The 4'-phosphopantetheine prosthetic group
of ACP is believed to serve as a flexible arm, tethering the growing fatty acyl chain to the
surface of the fatty acid synthase complex while carrying the reaction intermediates from
one enzyme active site to the next. Fatty Acid Synthase Receives the Acetyl and Malonyl
Groups

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 Before the condensation reactions that build up the fatty acid chain can begin, the two
thiol groups on the enzyme complex must be charged with the correct acyl groups. First, the
acetyl group of acetyl-CoA is transferred to the Cys-SH group of the β-ketoacyl-ACP
synthase. This reaction is catalyzed by acetyl-CoA–ACP transacetylase (AT).
The second reaction, transfer of the malonyl group from malonyl-CoA to the OSH group
of ACP, is catalyzed by malonyl-CoA–ACP transferase (MT), also part of the complex. In
the charged synthase complex, the acetyl and malonyl groups are very close to each other
and are activated for the chain-lengthening process. The first four steps of this process are
now considered in some details:
1. Condensation.
2. Reduction of the Carbonyl Group.
3. Dehydration.
4. Reduction of the Double Bond.

The Fatty Acid Synthase Reactions Are Repeated again to Form

Palmitate:

Butyryl group is now transferred from thephosphopantetheine-SH group of ACP to the

Cys-SH group of β-ketoacyl-ACP synthase, which initially borethe acetyl group. To start
the next cycle of four reactions that lengthens the chain by two more carbons, another
malonyl group is linked to the now unoccupied phosphopantetheine-SH group of ACP (see
below). Condensation occurs as the butyryl group, acting like the acetyl group in the first
cycle, is linked to two carbons of the malonyl-ACP group with concurrent loss of CO2. The
product of this condensation is a six carbon acyl group, covalently bound to the
phosphopantetheine -SH group. Its β-keto group is reduced inthe next three steps of the
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synthase cycle to yield the saturated acyl group, exactly as in the first round of reactions, in
this case forming the six-carbon product.
Seven cycles of condensation and reduction produce the 16-carbon saturated palmitoyl
group, still bound to ACP. For reasons not well understood, chain elongation by the
synthase complex generally stops at this point and free palmitate is released from the ACP
by a hydrolytic activity in the complex.
Small amounts of longer fatty acids such as stearate (18:0) are also formed. In certain
plants (coconut and palm, for example) chain termination occurs earlier; up to 90% of the
fatty acids in the oils of these plants are between 8 and 14 carbons long.
Long-Chain Saturated Fatty Acids Are Synthesized from Palmitate:

This pathway (the “microsomal system”) elongates saturated and unsaturated fatty acyl-
CoAs (from C10 upward) by two carbons, using malonyl-CoA as acetyl donor and NADPH
as reductant, and is catalyzed by the microsomal fatty acid elongase system of enzymes.
Elongation of stearyl-CoA in brain increases rapidly during myelination in order to provide
C22 and C24 fatty acids for sphingolipids. Palmitate is the precursor of other long-chain
fatty acids (see below). It may be lengthened to form stearate (18:0) or even longer
saturated fatty acids by further additions of acetyl groups, through the action of fatty acid
elongation systems present in the smooth endoplasmic reticulum and in mitochondria.
Although different enzyme systems are involved, and coenzyme A rather than ACP is the
acyl carrier in the reaction, the mechanism of elongation in the ER is otherwise identical to
that in palmitate synthesis: donation of two carbons by malonyl-CoA, followed by
reduction, dehydration, and reduction to the saturated 18-carbon product, stearoyl-CoA.

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 Desaturation of Fatty Acids Requires a Mixed-Function Oxidase
Palmitate and stearate serve as precursors of the two most common monounsaturated
fatty acids of animal tissues: palmitoleate, 16:1(∆9), and oleate, 18:1(∆9); both of these fatty
acids have a single cis double bond between C-9 and C-10. The double bond is introduced
into the fatty acid chain by an oxidative reaction catalyzed by fatty acyl-CoA desaturase (a
mixed-function oxidase) (see below).
Mammalian hepatocytes can readily introduce double bonds at the ∆9 position of fatty
acids but cannot introduce additional double bonds between C-10 and the methyl-terminal
end. Thus mammals cannot synthesize linoleate, 18:2(∆9,12), or α-linolenate, 18:3(∆9,12,15).
Plants, however, can synthesize both; the desaturases that introduce double bonds at the
∆ and ∆15 positions are located in the ER and the chloroplast. The ER enzymes act not on
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free fatty acids but on a phospholipid, phosphatidylcholine, that contains at least one oleate
linked to the glycerol. Both plants and bacteria must synthesize polyunsaturated fatty acids
to ensure membrane fluidity at reduced temperatures. Because they are necessary precursors
for the synthesis of other products, linoleate and linolenate are essentialfatty acids for
mammals; they must be obtained from dietary plant material. Once ingested, linoleate may
be converted to certain other polyunsaturated acids, particularly γ-linolenate,
eicosatrienoate, and arachidonate (eicosatetraenoate), all of which can be made only from
linoleate. Arachidonate, 20:4(∆5,8,11,14), is an essential precursor of regulatory lipids, the
eicosanoids. The 20-carbon fatty acids are synthesized from linoleate (and linolenate) by
fatty acid elongation reactions.

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