BIOSYNTHESIS

OF FATTY ACIDS
doc. Ing. Zenbia Chavkov, CSc.

The pathway for the of FAs is

not the reversal of the oxidation pathway

Both pathways are separated within

different cellular compartments

In humans
the pathway for FA synthesis occurs primarily

in the cytoplasm
of the liver and adipose tissue,

to a lesser extend in lactating mammary glands,

brain,
lungs,
and kidneys
whereas,

oxidation occurs in the mitochondria

The other major difference

is the use of nucleotide co-factors
Oxidation of fats
involves the reduction of FAD, NAD+
Synthesis of fats
involves the oxidation of NADPH

Both oxidation and synthesis of fats

utilize an activated 2C intermediate,

acetyl-CoA

Acetyl-CoA
must be first transported
out of mitochondria
using

citrate shuttle transport system

The total energy requirement
for converting mitochondrial acetyl-CoA
into cytoplasmic acetyl-CoA is

1 ATP

Origin of Cytoplasmic Acetyl-CoA

Acetyl-CoA
is generated in the mitochondria primarily from the sources:

The pyruvate dehydrogenase (PDH)

reaction
(glycolysis

glucose

pyruvic acid

acetyl-CoA)

Fatty acid oxidation

AAs degradation and ketone bodies
In order to be utilized for fatty acid synthesis

they must be present in the cytoplasm

The shift from fatty acid oxidation and glycolytic oxidation
occurs when the need for energy diminishes

Source of reducing equivalents

The origin of NADPH for FA biosynthesis
depends on cell type
In liver, the 2 NADPHs come from
the pentose phosphate pathway

In adipose tissue,
NADPH is generated by malic enzyme
-OOC-CH

2-CH-COO

|
OH

CH3-C-COO||
O

The pentose phosphate pathway

Another source for NADPH

for these reactions is
the isocitrate shuttle

The synthesis of malonyl-CoA

is the first committed step of FAs synthesis

Acetyl-CoA carboxylase (ACC),

is the major site of regulation of FAs synthesis

ACC requires a biotin co-factor

First, CO2 is covalently bound to biotin

using energy from hydrolysis of ATP
Then, the CO2 is transferred to acetyl-CoA
producing malonyl-CoA
The biotinyl group serves as a temporary carrier of CO2

The carboxylation of
acetyl-CoA
to form
malonyl-CoA
catalyzed by
acetyl-CoA
carboxylase

Enzyme-biotin
HCO3 + ATP

ADP + Pi
Enzyme-biotin-CO2
O
ll

CH3-C-SCoA
acetyl-CoA

2
Enzyme-biotin
O

ll

O2C-CH2-C-SCoA
malonyl-CoA

is the rate-limiting step of FA biosynthesis

The overall reaction may be summarized as:

Acetyl-CoA Carboxylase activity,

in the mammals
is regulated by

phosphorylation
allosteric regulation by local metabolites
The active conformation of the enzyme
associates in

multimeric filamentous complexes

The inactive conformation of the enzyme
exists as
individual protomers

The rate of fatty acid synthesis

is controlled by the equilibrium between
monomeric and polymeric acetyl-CoA carboxylase
(ACC)
The activity of ACC requires polymerization
This conformational change
is controlled

by local metabolites
(citrate, palmitoyl-CoA
and other long-chain
fatty acyl-CoAs)

Regulation by local metabolites

The equilibrium between monomeric and polymeric
acetyl-CoA carboxylase is
inhibited by
palmitoyl- CoA
(product of FA synthase)
other long-chain
fatty acyl-CoAs
enhanced by
citrate
(promoting enzyme
polymerization)

Regulation of Acetyl-CoA carboxylase activity

through

hormone mediated phosphorylation

Glucagon and epinephrine
promote phosphorylation
and
decrease the enzymatic
activity ( )
Insulin
promotes dephosphorylation
and increases the activity ( )

With Acetyl-CoA Carboxylase inhibited,

acetyl-CoA
remains available
for ketone bodies synthesis
the alternative metabolic fuel
used when blood glucose is low

Changes in diet
affect the amount of fatty acid biosynthesis

by affecting
the amount of acetyl-CoA carboxylase
A diet
rich in carbohydrate or low in fat
increases the biosynthesis of the enzyme
by affecting the rate of transcription

Starvation or diet high in fat

has the opposite effect and

reduces the rate

of synthesis of acetyl-CoA carboxylase

Synthesis of the Acyl chain

The reactions of FA biosynthesis take place on

a multifunctional protein,
called fatty acid synthase
(fatty acid synthase complex)
A polyprotein is a single protein with more then 1 activity,

and fatty acid synthase is formed

from 2 chains of this protein
The active enzyme
is a dimer of identical subunits

There is some evidence

that the 2 copies of the multi-domain enzyme
are aligned antiparallel, as below

Pant-SH HS-Cys

Cys-SH HS-Pant

Fatty Acid Synthase dimer

H
H3N+

COO

CH2
SH
Fatty Acid
cysteine
Synthase
prosthetic groups:

The thiol of the sidechain of a cysteine

residue of condensing
enzyme domain
The thiol
of phosphopantetheine,
equivalent in structure
to part of coenzyme A

The fatty acid synthase complex

contains 2 types thiol groups
The central thiol,
made up of

4phosphopantetheine

a derivative of coenzyme A, covalently linked

by a phosphodiester bond to

serine residue of acyl carrier protein, or ACP

The peripheral thiol,
belongs to a cysteinyl residue

on ketoacyl-ACP synthase

Like fat oxidation,

fat synthesis involves 4 enzymatic activities
-keto-ACP synthase,
-keto-ACP reductase,
3-OH acyl-ACP dehydratase
Enoyl-CoA reductase
The two reduction reactions
require NADPH oxidation to NADP+

The acetyl-CoA
+ malonyl CoA

are transferred to ACP

by the action of:

Acetyl-CoA transacylase
Malonyl-CoA transacylase

The attachment of these carbon atoms

to ACP

allows them to enter the fatty acid synthesis cycle

During the sequence of reaction, the growing FA takes

the form of a thioester attached to the:
Peripheral SH group of a cysteine residue
of the protein
or to the central SH group
of a protein-bound phosphopantetheine

The biosynthetic intermediates

do not diffuse away
from the polyprotein
but are passed

from one enzyme active site

to the next active site
by acyl carrier protein (ACP)

Individual steps
of the Fatty Acid Synthase
reaction pathway
In the first reaction,
(to initiate biosynthesis)

acetyl-CoA is transferred:
From CoA
To the central SH (thiol) group
of phosphopantetheine
to form a covalent bond with release of CoA

 Then,

Central thiol

the acetyl group

is transferred

Peripheral thiol

to the peripheral SH
(thiol) group
of a cysteine

Next,

the malonyl group

is transferred to the pantetheine central SH group
of ACP, just vacated by the acetyl group
Now the reactants are poised
for the first condensation reaction

 In the first step,

the acetyl group
and
malonyl groups
are condensed,
with the release of CO2

This forms
CONDENSATION
acetoacetate attached
to the pantetheine (central) SH group

The condensation reaction of fatty acid biosynthesis is

catalyzed by -ketoacyl-ACP synthase

REDUCTION, step 2.
Using NADPH,
acetoacetyl-ACP undergoes
a reduction,

yielding -hydroxybutyryl-ACP
and NADP+ in reaction
The ketone is reduced
to a hydroxyl group,
mediated
by -ketoacyl-ACP reductase

DEHYDRATION, step 3.
Then the compound is dehydrated

to 2,3-trans-butenoyl-ACP
(crotonyl-ACP)
catalyzed by
-hydroxyacyl-ACP-dehydratase

REDUCTION, step 4.
The double bond is reduced
by NADPH + H+
in reaction
catalyzed by

2,3 trans-enoyl-ACP reductase

to form butyryl-S-ACP

Lengthened fatty acid chain is then translocated

to the peripheral SH (thiol) group of a cysteine
ketoacyl ACP synthase

Another malonyl group is added

to the central SH (thiol) group
of ACP

This series of reactions form

a 4-carbon acyl group still attached

to the phosphopantetheine (central -SH)

In the next reaction:

The growing fatty acyl chain is transferred to the
cysteine (peripheral thiol),

Another malonyl group is added to the pantetheine

-SH (central thiol) and cycle begins again

This cycle of condensation,

reduction,
dehydration,
reduction
and transfer of the acyl group continues

until chain of 16 carbons has been created

The resulting palmitoyl group - palmitate

is released from

the fatty acid synthase complex

by an exergonic hydrolysis reaction

Palmitate,
a 16-C saturated fatty acid,
is the final product of the FA synthase reactions

Therefore:
Acetate group is added at the beginning
Then need 1 malonate to extend the chain by 2 carbons

3C

= malonate

2C

= acetate

1C

= CO2

2C
2C

2C

3C

2C

FAS
1C

2C

3C

2C

2C

FAS

FAS
1C

3C

1C

That fatty acid synthesis by multienzyme complex

stops at palmitate is probably

due to limitation in the size of an active site

of fatty acid synthase
Palmitate
can then undergo separate

elongation and/or unsaturation

to yield other fatty acid molecules

Fatty acid biosynthesis

is energetically expensive
however, occurs when is

abundant precursor
to provide both

the mass
the energy

BIOENERGETICS OF FA BIOSYNTHESIS
1 mole ATP is required for the generation of 1 mole of acetyl-CoA
from citrate

7 moles of ATP are required for the transport of acetyl-CoA

from mitochondria into cytosol, as a substrate
for the synthesis of malonyl-CoA

7 additional moles of ATP are required for the synthesis

of 7 moles of malonyl-CoA
from acetyl-CoA and CO2

A total of 15 ATP
equivalents are required
for the synthesis of palmitate from citrate

14 moles of NADPH
are required for the biosynthesis of 1 mole of palmitate

THE REGULATION OF FAT METABOLISM

Occurs via two distinct mechanisms

One is short term regulation
which is regulation effected by events such as

substrate availability,
allosteric effectors
and/or enzyme modification
Control of a given pathways' regulatory enzymes can
also occur by

alteration of enzyme synthesis

and turn-over rates of synthesis
These changes are long term regulatory effects

Insulin stimulates lipogenesis

by several mechanisms
It increases the transport of glucose into cell
(in adipose tissue)
and thereby increases the availability of both:
pyruvate for FAs synthesis
glycerol-3-P for esterification of the newly formed FAs
Insulis converts the inactive form of pyruvate
dehydrogenase to the active form
(in adipose tissue but not in liver!)
Insulin activates acetylCo-A carboxylase.
It involves dephosphorylation by a protein phosphatase
accompanied by change in aggregation
of monomers to a more polymeric state

Insulin by its ability

to depress the level of intracellular cAMP,
inhibits lipolysis in adipose tissue
and thereby reduces the concentration
of plasma free FAs and long-chain acyl-CoA,
an inhibitor of lipogenesis

By this same mechanism

insulin antagonizes the action of
glucagon and epinephrine,
which inhibit acetyl-CoA carboxylase
and therefore lipogenesis,
by increasing cAMP, allowing cAMP dependent protein
kinase to inactivate the enzyme by phosphorylation

Regulation of fat metabolism also occurs through

malonyl-CoA induced inhibition
of carnitine acyltransferase I.
This functions
to prevent the newly synthesized FAs
from entering the mitochondria
and being oxidized

ELONGATION AND DESATURATION

Stearic
are major constituent of FAs
Oleic acids
found in human cells
The fatty acid product released
from fatty acid synthase (FAS)
is palmitate
which is a 16:0 fatty acid,
(16 carbons and no sites of unsaturation)

Although

the FA synthase complex stops

at 16 C atoms,
human cells:
Can extend the length of the FA chain
Posseses the machinery for converting
saturated to unsaturated FAs

2-carbon units can be added:

To endogenously synthesized
or dietary fatty acids
by elongation reactions

ELONGATION AND UNSATURATION

of fatty acids occurs after palmitate (16C)

in both the mitochondria

and endoplasmic reticulum
(microsomal membranes)
The endoplasmic reticulum pathway

is quantitatively more important

This strategy agrees with the role of mitochondria
functioning as a catabolic organell

The substrate for the elongation reactions

is fatty acyl-CoA
and not fatty acyl-ACP

The endoplasmic reticulum

contains the enzyme activities found in the FA synthase
complex, that succesively

reduce,
dehydrate,
and reduce
the compound

to produce fatty acyl-CoA

containing 2 additional carbon atoms
The reactions are analogous to the condensation reactions
that occurs during conventional FA biosynthesis

The resultant product is 2C longer

The fatty acyl-CoA substrate

for the elongation reaction

is malonyl-CoA
More then 1 elongation reaction can occur,

and fatty acids up to 26 C atoms

can be synthesized
The reduction reactions of elongation

require NADPH as co-factor

just as for the similar reactions

catalyzed by FAS

Mitochondrial elongation
Involves acetyl-CoA units
is a reversal of oxidation

Except that the final reduction

utilizes NADPH instead of FADH2
as co-factor
Acetyl-CoA, not malonyl-CoA
donates the 2C units in mitochondria

x,y..)

n:=(
18

14

11

C-C-C-C-C-C=C-C-C=C-C-C=C-C-C=C-C-C-C-COOH
18

12

C-C-C-C-C-C=C-C-C=C-C-C-C-C-C-C-C-COOH

Animals cannot
put double
bonds in this
part of the
molecule,
plants can!

Essential fatty acids:

Linoleate

18:2(9,12)

Arachidonate
20:4(5,8,11,14)

Since these enzymes

cannot introduce sites of unsaturation beyond C9
they cannot synthesize either

linoleate (18:2D9, 12 )
linolenate (18:3D9, 12, 15)
17

15

12

COOH
CH3

Linoleic acid (cis 9,12 octadecadienoic acid)

These fatty acids must be acquired from the diet

and are, therefore, referred to as

essential fatty acids

These essential FAs are

necessary for normal membrane structure

Linoleic acid
especially important, serves as a precursor
for the synthesis of arachidonic acid,
from which

the eicosanoids
(the prostaglandins, thromboxanes)
are formed
is also a constituent of epidermal cell

sphingolipids
that function as

the skins water permeability barrier

It is this role of FAs in eicosanoid synthesis that leads to

poor growth,
wound healing
dermatitis
in persons on fat free diets

Desaturation
occurs in the ER membranes
in mammalian cells involves

4 broad specificity fatty acyl-CoA

desaturases
(non-heme iron containing enzymes)
These mixed-function oxidase require
NADPH and molecular oxygen
to add a hydroxyl group to the fatty acid

Arachidonic acid
is produced by
elongation and the addition of 2 double bonds
as shown in Fig.
Common in
healthy diet
desaturation

Some available
from meat and eggs

-6 Pathway
Linoleic acid (18:2)

-Linolenic acid (18:3)

Anti-inflammatory
metabolites

Dihomo- -linolenic acid (20:3)

(Slow)

Pro-inflammatory
metabolites

Arachidonic acid (20:4)

Released from stores

Meat and
eggs

Acetyl CoA carboxylase

CO2

Biotin
carboxylase

O
-O

BIOTIN

Irreversible
two-step reaction

C=O
H-N

Lys

Biotin carrier
protein

H-N Lys
C=O

BIOTIN
O=C
-O

Transcarboxylase

AMP-Activated
Kinase
catalyzes
phosphorylation of
Acetyl-CoA
Carboxylase
causing
inhibition ( )

Phosphorylated protomer of
Acetyl-CoA Carboxylase (inactive)
Citrate
Dephosphorylated,
e.g., by insulinactivated Protein
Phosphatase

Palmitoyl-CoA
Phosphorylated, e.g., via
AMP-activated Kinase
when cellular stress or
exercise depletes ATP.

Dephosphorylated Polymer of
Acetyl-CoA Carboxylase (active)
Regulation of Acetyl-CoA Carboxylase

The primary phosphorylation of ACC occurs through the action

of AMP-activated protein kinase, AMPK
This is not the same as cAMP- dependent protein kinase, PKA!

Phosphorylation
causes the filamentous enzyme to dissociate
into inactive mononomers