Oxidation of Fatty Acids

R. C. Gupta
Professor and Head Department of
Biochemistry
National Institute of Medical Sciences
Jaipur, India
Oxidation of fatty acids is an important
source of energy

Fatty acids are stored in tissues in the form

of triglycerides

Intestines, liver and adipose tissue release

triglycerides in blood
Triglycerides released by intestines and
liver are in the form of lipoproteins

VLDL and chylomicrons are

the lipo- proteins that
transport triglycerides in blood

VLDL is released by the liver Chylomicrons

are released by the intestine

Triglycerides are hydrolysed to free fatty
acids and glycerol by lipoprotein lipase

The hydrolysis occurs in the capillaries of

liver, adipose tissue and skeletal muscle

The fatty acids are taken up by the cells

Fatty acids released from adipose tissue
are bound to albumin in blood

They are transported to

different tissues
bound to albumin

Upon interaction with cell

surface, fatty
acids dissociate from albumin
Fatty acids are taken up by the cells with
the help of some proteins

These are membrane proteins having

high affinity for fatty acids

There are several such proteins

The proteins involved in cellular
fatty acid uptake include:

Fatty acid translocase (FAT)

Plasma membrane-associated fatty

acid-binding protein (FABPpm)

Fatty acid transport proteins (FATPs)

Mitochondria and peroxisomes are the
sites for oxidation of fatty acids

Short- and medium-chain fatty acids are

oxidized solely in mitochondria

Long-chain fatty acids are

oxidized both in
mitochondria and peroxisomes

Very-long-chain fatty acids are

oxidized in peroxisomes
Fatty acids have to be activated before
they are oxidized

Activation occurs in cytosol and

involves binding of fatty acid with
CoA

Two molar equivalents of ATP

are consumed in the reaction
(ATP→ AMP)

The reaction is catalysed by

 O O
|| Thiokinase ||
R−CH2−CH2−C−OH ++
R−CH2−CH2−C~S−CoA
Fatty acid Mg Acyl CoA
ATP AMP
+ +
CoA−SH PPi

Activation of fatty acid

The activation reaction is reversible

But immediate hydrolysis of PPi

prevents the backward reaction

Inorganic pyro- phosphatase

PPi 2 Pi
H2 O
There are several thiokinases in human
beings

They differ in substrate specificity and

intracellular localization

There are different thiokinases for short-,

medium- and long-chain fatty acids

Some of the thiokinases are also

involved in fatty acid uptake by the cells
A thiokinase is present in mitochondria
also

But the mitochondrial enzyme can act only

on short-chain fatty acids

It cannot activate medium- and long-chain

fatty acids
Thiokinases acting on medium- and long-
chain fatty acids are present on:

Outer mitochondrial membrane

Endoplasmic reticulum

They convert long- and medium-chain fatty

acids into acyl CoA
There are several pathways for oxidation of
fatty acids

The major pathway is -oxidation

-Oxidation occurs in mitochondrial matrix

Acyl CoA derivatives of long-and

medium-
The inner mitochondrial membrane is not
permeable to acyl CoA

A special transport system is required to

transport acyl CoA into mitochondria
The key component of acyl CoA transport
system is carnitine

Carnitine is  -hydroxy- -
ammonium butyrate trimethyl

Carnitine can react with acyl CoA to form

acyl-carnitine
 CH3
|
+
H 3C — N — CH 2— CH — CH 2— COOH
| |
CH3 OH
Carnitine
O
||
R—C~S—
CoA
Acyl CoA

CoA — SH
CoA
+
CH3
H 3C — N — CH
| 2— CH — CH 2— COOH
| |
CH3 O—C—R
|| O
Acylcarnitine
On the outer surface of inner mitochondrial
membrane, carnitine reacts with acyl CoA

Acyl group is transferred to

carnitine, forming acylcarnitine

This reaction is catalysed by

carnitine- palmitoyl transferase I

Acylcarnitine moves to the inner surface of

the membrane
Acylcarnitine reacts with the CoA present
in the matrix

The acyl group is transferred

to CoA This reaction is

catalysed by carnitine-
palmitoyl transferase II

Free carnitine moves back to the

Carnitine and acylcarnitine are transported
across the membrane by an enzyme

The enzyme is carnitine-acylcarnitine

translocase

It is present in the inner mitochondrial

membrane
 Outer side Inner mitochondrial Mitochondrial
membrane matrix
Acyl CoA Carnitine Acyl CoA
Carnitine- Carnitine- acylcarnitine Carnitine- palmitoyl
palmitoyl translocase transferase II
transferase I Acylcarnitine CoA
CoA
  -Oxidation
pathway
This major pathway for oxidation of fatty
acids was elucidated by Knoop

He labeled the methyl end of fatty acids with

a phenyl group and fed them to animals

The end products of oxidation were

recovered from urine and were identified
It was seen that when fatty acids having
an even number of carbon atoms were
fed, phenylacetic acid was recovered from
urine

When fatty acids having an odd number of

carbon atoms were fed, benzoic acid was
recovered from urine
 Knoop’s experiments

‒CH2‒(CH2)2n‒COOH ‒CH2‒COOH

Phenylacetic acid
Oxidation of a fatty acid having an even
number of carbon atoms

‒CH2‒(CH2)2n+1‒COOH ‒COOH

Benzoic acid
Oxidation of a fatty acid having an odd
number of carbon atoms
Knoop concluded that oxidation of fatty
acids occurs at the carboxyl end

It involves removal of the last two carbon

atoms from the carboxyl end in one cycle

This was termed as -oxidation as the -

carbon (C3) is oxidized in each cycle
If the fatty acids has an even
number of carbon atoms:

The last two carbon atoms on the methyl

end remain tagged with the label

The final product is phenylacetic acid

If the fatty acids has an odd
number of carbon atoms:

The last carbon atom on the methyl end

remains tagged with the label

The final product is benzoic acid

 Reactions of  -oxidation
pathway
The first reaction that acyl CoA under-
goes is dehydrogenation

It is catalysed by acyl CoA dehydro-

genase, a flavoprotein

FAD, which is a prosthetic group of the

enzyme, accepts the hydrogen atoms
One hydrogen atom is removed from -
carbon and one from -carbon of acyl CoA

A double bond is formed

between - and
-carbon atoms

The product is , -unsaturated acyl CoA

 O
II
R ‒ CH2 ‒ CH2 ‒ C ~ S‒CoA
Acyl CoA (Cn)
Acyl CoA Fp
dehydrogenase
 FpH2
O
II
R ‒ CH = CH ‒ C ~ S‒CoA
 ,  -Unsaturated acyl
CoA
The second reaction is addition of H and
OH

Crotonase splits H2O into H and OH

It adds H to  -carbon and OH to  -

carbon
, -Unsaturated acyl CoA is converted
into -L-hydroxyacyl CoA
 O
II
R ‒ CH = CH ‒ C ~
S‒CoA
, -Unsaturated acyl
CoA
H2O
CrotonaseO
II
R ‒ CH
I OH‒ CH2 ‒ C ~ S‒CoA
-L-Hydroxyacyl
CoA
In the third reaction, two hydrogen atoms
are removed from the  -carbon

These are transferred to NAD+

 -L-Hydroxyacyl CoA is converted into

 -
ketoacyl CoA
 O
II
R ‒ CH ‒ CH2 ‒ C ~ S‒CoA
I OH
-L-Hydroxyacyl
CoA
NAD+
 -Hydroxyacyl
CoAdehydrogenase
  NADH + H+

O
II
R‒C ‒ CH2 ‒ C ~ S‒CoA
II O
-Ketoacyl CoA
The fourth (and final) reaction is catalysed
by thiolase

The last two carbon atoms and CoA

are removed from -keto acyl CoA as
acetyl CoA
A new CoA molecule is added to the acyl
chain

The product is an acyl CoA shorter by

two carbon atoms than the initial acyl
CoA
 O
II
II O 2‒C~S‒CoA
R‒C‒CH
-Ketoacyl
CoA
CoA‒SH
Thiolase O
 II
 CH3‒C~S‒CoA
R‒C~S‒CoA
II O
Acyl CoA
(Cn‒2)
Thus, during one cycle of
 -oxidation:

Two carbon atoms are removed from the

carboxyl end as acetyl CoA

An acyl CoA having two carbon

atoms less than the original acyl CoA
is formed
The new acyl CoA goes through the cycle
again

Two more carbon atoms are

removed in
the form of acetyl CoA

This continues until only a two-carbon

acyl CoA (acetyl CoA) is left
 Acyl CoA (Cn) R ‒ CH2 ‒ CH2 ‒ C ~S ‒ CoA
O
Fp

 FpH2
 ,  - R ‒ CH = CH ‒ C ~S ‒ CoA
Unsaturated O
acyl CoA H2O


-Hydroxy- R ‒ CH ‒ CH2 ‒ C ~S ‒ CoA
acyl CoA O
OH
NAD+

NADH +
-Keto- H+ R ‒ C ‒ CH2 ‒ C ~S ‒
acyl CoA CoAO O
CoA‒SH

  CH3 ‒ C ~S ‒ CoA
Acyl CoA (Cn‒2) R ‒ C ~S ‒ CoA O
O
Energetics

In eachcycle
of
-oxidation:

One FAD is reduced

One NAD is

reduced One acetyl

If the fatty acid being oxidized
is palmitic acid (C16):

Seven cycles of -oxidation will form

seven molecules of acetyl CoA

A two-carbon acyl CoA i.e. acetyl CoA

will be left at the end of the last cycle
Thus, eight molecules of acetyl CoA
are formed

When oxidized in the citric acid cycle,

these will form 8 x 12 = 96
ATP equivalents
Seven molecules of FAD are reduced
in seven cycles

When oxidized in the citric acid cycle,

these will form 7x2 = 14 ATP equivalents
Seven molecules of NAD are reduced
in seven cycles

When oxidized in the citric acid cycle,

these will form 7x3 = 21 ATP equivalents
Therefore, the total number of ATP
equivalents formed is 96+14+21 = 131

Two ATP equivalents are used in the initial

activation reaction (ATP  AMP + PPi)

Hence, the net gain is

131–2 = 129 ATP
equivalents per molecule of palmitic acid
Hydrolysis of terminal phosphate group
of ATP yields 7.3 kcal/mol of ATP

Hence, oxidation of
palmitic acid yields
129 x 7.3 = 942 kcal/mol of
palmitic acid
Molecular weight of palmitic acid is 256

Hence, its potential energy is 256 x 9.1 =

2,330 kcal/mol

Therefore, efficiency of -oxidation

is
942  2,330 x 100 or ≈ 40%
If the fatty acid being oxidized
is stearic acid (C18):

Eight cycles of -oxidation will form

eight molecules of acetyl CoA

A two-carbon acyl CoA i.e. acetyl CoA

will be left at the end of the last cycle
Thus, nine molecules of acetyl CoA are
formed

When oxidized in the citric acid cycle,

these will form 9 x 12 = 108 ATP
equivalents
Eight molecules of FAD are reduced
in eight cycles

When oxidized in the citric acid cycle,

these will form 8x2 = 16 ATP equivalents
Eight molecules of NAD are reduced
in eight cycles

When oxidized in the citric acid cycle,

these will form 8x3 = 24 ATP equivalents
Therefore, the total number of ATP
equivalents formed is 108+16+24 = 148

Two ATP equivalents are used in the

initial
activation reaction (ATP  AMP + PPi)

Therefore, the net gain is 148–2 = 146 ATP

equivalents per molecule of palmitic acid
or 146x7.3 = 1,066 kcal/mol of palmitic acid
Molecular weight of stearic acid is 284

Its potential energy is 284 x 9.1 = 2,584

kcal/mol

So, efficiency of -oxidation is 1066 

2,584 x 100 or ≈ 41%
Fatty acids having an odd number of carbon
atoms are also oxidized by -oxidation

After the last cycle of -oxidation, a 3-carbon

acyl CoA is left which is propionyl CoA

This is converted by a series

of reactions into succinyl CoA

Succinyl CoA can enter the citric acid cycle

 O Propionyl CoA COOH
|| carboxylase, biotin |
CH3 − CH2 − C ~ S − H − C − CH3
CoA |
CO2 + ATP ADP + Pi C ~ S − CoA
Propionyl CoA ||
O
D-Methylmalonyl CoA
Methylmalonyl
CoA racemase
CH2 − COOH Methylmalonyl CoA COOH
| isomerase, cobamide |
CH2 − C ~ S − CoA H3C − C − H
|| |
O C ~ S − CoA
Succinyl CoA ||
O
L-Methylmalonyl CoA
 Defects in -oxidation

Inborn errors of -oxidation are uncommon

Rarely, defects have been reported in:

▪Acyl CoA dehydrogenase
▪-Hydroxyacyl CoA dehydrogenase

Clinical manifestation is unexplained hypo-

glycaemia with or without ketosis
Sometimes, transport of fatty acids into
mitochondria is defective

Carnitine-palmitoyl transferase I,
carnitine- palmitoyl transferase II or
carnitine-acyl- carnitine translocase may be
defective

Rarely, the defect may be due to

carnitine
deficiency
Dietary carnitine deficiency has not been
identified in healthy people

Carnitine deficiency can occur in patients

undergoing repeated haemodialysis

This happens because haemodialysis

removes carnitine from blood
 Regulation of fatty acid oxidation

Fatty acid metabolism is regulated

according to the availability of energy

When energy is needed, oxidation of fatty

acids in increased and their synthesis is
decreased

The reverse occurs when energy is

abundant
 Rate of fatty acid oxidation depends on
the availability of substrates i.e. fatty acids

Fatty acids are released from fat depots

by lipolysis

When lipolysis increases, so does the

oxidation of fatty acids

When lipolysis decreases, oxidation of

fatty acids also decreases
When energy is scarce, secretion of
glucagon and epinephrine increases

These two activate hormone-sensitive

lipase through cAMP

Lipolysis increases; fatty acids are

released from stored triglycerides

Increased availability of fatty acids

increases their oxidation
 When energy is abundant, insulin
secretion increases

Insulin inhibits lipolysis and

availability of fatty acids

Therefore, oxidation of fatty acids is

decreased
 In times of energy abundance,
concentrations of acetyl CoA, malonyl
CoA and NADH are also high

Malonyl CoA inhibits carnitine-

palmitoyl transferase I

This decreases the entry of fatty acids

into mitochondria
 NADH inhibits -hydroxyacyl CoA
dehydrogenase

Acetyl CoA inhibits thiolase

This decreases the oxidation of fatty acids

 Oxidation of unsaturated fatty acids

Unsaturated fatty acids are also

oxidized by -oxidation

Two additional enzymes are needed to

deal with the double bonds

Double bonds in naturally occurring fatty

acids have a cis conformation
On hydration, cis double bonds form the
D-isomers of hydroxyacyl CoA

-Hydroxyacyl CoA dehydrogenase

can act only on -L-hydroxyacyl CoA

Therefore, the D-isomers have to be

racemised to L-isomers
When a double bond occurs between
and  -carbon, CoA dehydrogenase
 -
acyl
cannot act on it

The reason is that the single hydrogen

atom attached to -carbon
atom cannot be removed
Hence, the double bond is shifted
between the - and -carbon atoms by
an isomerase

This isomerase also converts the cis

double bond into a trans double bond

These reactions are illustrated by

oxidation of linoleic acid
 13 12 10 9
Linoleyl CoA CH3‒(CH2 )4‒CH = CH‒CH2‒CH =CH‒(CH2 )7‒C~S‒CoA
O
3 Cycles of -oxidation

 3 CH3 ‒ C ~S ‒ CoA
O
7 6 4 3
-cis, -cis- CH3‒(CH2 )4‒CH = CH‒CH2‒CH =CH‒CH2 ‒C~S‒CoA
Dienoyl CoA


O
-cis → -trans-Dienoyl CoA isomerase
 H
6 2
 - CH3‒(CH2 )4‒CH = CH‒CH2‒CH2 ‒ C = C ‒ C~S‒CoA
t r a ns,
H O
 -cis-
Dienoyl CoA H2O
Crotonase
6
 -L-Hydroxy- CH3‒(CH2 )4‒CH = CH‒CH2‒CH2‒CH2‒CH‒ C~S‒CoA
 -
OH O
cis-enoyl CoA
 -L-Hydroxy- CH3‒(CH2 )4‒CH = CH‒CH2‒CH2‒CH2‒CH‒C~S‒CoA
 -
OH O
cis-enoyl CoA
2 Cycles of -oxidation

 2 CH3 ‒ C ~S ‒ CoA
O
 -Unsaturated CH3‒(CH2 )4‒CH =
acyl CoA CH‒C~S‒CoA
O
H2O
 Crotonase
OH
 -D- CH3‒(CH2 )4‒CH ‒ CH2‒C~S‒CoA
Hydroxyacyl
O
CoA
 OH
-D-Hydroxyacyl CH3‒(CH2 )4‒CH ‒ CH2‒C~S‒CoA
CoA
O

-Hydroxyacyl CoA racemase


-L-Hydroxyacyl CH3‒(CH2 )4‒CH ‒
CoA CH ‒C~S‒CoA
OH O 2
3 Cycles of  -oxidation

 3 CH3 ‒ C ~S ‒ CoA
Acetyl CoA CH3 ‒ C ~S ‒ CoA O
O
 Other pathways for oxidation of fatty
acids
There are two other pathways for oxidation
of fatty acids

These are quantitatively insignificant

Of these, -oxidation pathway is

present in brain

The other pathway is -oxidation

  -Oxidation

 -Oxidation is a pathway for the

oxidation
of 3-methyl-branched chain fatty acids

Phytanic acid is the most important

3-methyl-branched chain fatty acid
Phytanic acid is 3,7,11,15-tetramethyl-
hexadecanoic acid
 CH3 CH3 CH3 CH3
CH3‒ CH‒ (CH2)3‒ CH‒ (CH2)3‒ CH‒ (CH2)3‒ CH‒ CH2‒ COOH
Phytanic acid
Phytanic acid is oxidized by  -
oxidation

 -Oxidation occurs in peroxisomes

Phytanic acid is first activated to
phytanoyl CoA

Phytanoyl CoA is hydroxylated to 2-

hydroxyphytanoyl CoA
 CH3 CH3 CH3 CH3
CH3‒ CH‒ (CH2)3‒ CH‒ (CH2)3‒ CH‒ (CH2)3‒ CH‒ CH2‒ COOH
Phytanic acid
ATP + CoA‒SH
Acyl CoA synthetase
AMP + PPi CH3


CH3 CH3 CH3 O
CH3‒ CH‒ (CH2)3‒ CH‒ (CH2)3‒ CH‒ (CH2)3‒ CH‒ CH2‒ C~S‒CoA
Phytanoyl CoA
O2 +  -
Ketoglutarate  Phytanoyl CoA hydroxylase
CO2 + Succinate CH3 CH3 OH O
CH3 CH3
CH3‒ CH‒ (CH2)3‒ CH‒ (CH2)3‒ CH‒ (CH2)3‒ CH‒ CH‒ C~S‒CoA
2-Hydroxyphytanoyl CoA
Hydroxyphytanoyl CoA is cleaved into
pristanal and formyl CoA

Formyl CoA is broken down into formate

and eventually CO2

Pristanal is oxidized to pristanic acid

 CH3 CH3 CH3 CH3 OH O
CH3‒ CH‒ (CH2)3‒ CH‒ (CH2)3‒ CH‒ (CH2)3‒ CH‒ CH‒ C~S‒CoA
2-Hydroxyphytanoyl CoA
O
2-Hydroxyphytanoyl CoA lyase
H‒ C~S‒CoA


CH3 CH3 CH3 CH3
CH3‒ CH‒ (CH2)3‒ CH‒ (CH2)3‒ CH‒ (CH2)3‒ CH‒ CHO
Pristanal

H2O + NADH+
 Aldehyde dehydrogenase
NADH + H+
CH3 CH3 CH3 CH3
CH3‒ CH‒ (CH2)3‒ CH‒ (CH2)3‒ CH‒ (CH2)3‒ CH‒ COOH
Pristanic acid
Pristanic acid is activated to pristanoyl
CoA, which then undergoes -oxidation

Six cycles of -oxidation produce

iso- butyryl CoA and three molecules each
of acetyl CoA and propionyl CoA
 CH3 CH3 CH3 CH3
CH3‒ CH‒ (CH2)3‒ CH‒ (CH2)3‒ CH‒ (CH2)3‒ CH‒ COOH
Pristanic acid
ATP + CoA‒SH
Acyl CoA synthetase
AMP + PPi


CH3 CH3 CH3 CH3 O
CH3‒ CH‒ (CH2)3‒ CH‒ (CH2)3‒ CH‒ (CH2)3‒ CH‒ C~S‒CoA
Pristanoyl CoA

Six cycles of  -oxidation



CH3 O O O
CH3‒ CH ‒C~S‒CoA + 3 CH3‒ CH2‒C~S‒CoA + 3 CH3‒ C~S‒CoA
Isobutyryl CoA Propionyl CoA Acetyl CoA
An inherited defect in -oxidation results
in Refsum's disease

This is most
commonly due to
deficiency of phytanoyl CoA
hydroxylase

Some times, it is due

to deficiency of
peroxin-7, a peroxisomal receptor
Large amounts of phytanic acid
accumulate in brain in Refsum's disease

This causes neurological damage,

cerebellar degeneration, and peripheral
neuropathy

Patients are advised to take a phytanic

acid-restricted diet
  -
Oxidation
This is another minor pathway for
oxidation of fatty acids

It becomes important when  -oxidation

is
defective

It is located in endoplasmic reticulum of

liver and kidney cells
The first reaction is introduction of a
hydroxyl group onto the -carbon

The reaction is catalysed by

the
microsomal hydroxylase system

Alcohol dehydrogenase then oxidizes the

hydroxyl group to an aldehyde group

Aldehyde dehydrogenase
oxidizes the
CH3 –(CH2)n –COOH

CH2 –(CH2)n –COOH

I OH

CH –(CH2)n –COOH
II O

HOOC–(CH2)n –
COOH
The fatty acid now has a carboxyl group
at each end

The dicarboxylic acid is activated and

enters the mitochondria

 -Oxidation starts from both the

ends
Two carbon atoms are removed in one cycle
from both the ends

This continues until a 6-carbon or

8-carbon
dicarboxylic acid is left

The 6-carbon product is adipic acid,

and the 8-carbon product is suberic
acid

These are excreted in urine