Oxidation of fatty acids and its energetics

1. Oxidation of Fatty Acids
R. C. Gupta
Professor and Head
Department of Biochemistry
National Institute of Medical Sciences
Jaipur, India

2. 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

3. 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

4. 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

5. 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

6. 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

7. 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)

8. 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

9. 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 thiokinase
(acyl CoA synthetase)

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

11. The activation reaction is reversible
PPi 2 Pi
H2O
Inorganic pyro-
phosphatase
But immediate hydrolysis of PPi
prevents the backward reaction

12. 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

13. 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

14. 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

15. There are several pathways for oxidation of
fatty acids
The major pathway is b-oxidation
b-Oxidation occurs in mitochondrial matrix
Acyl CoA derivatives of long- and medium-
chain fatty acids have to enter mitochondria

16. The inner mitochondrial membrane is not
permeable to acyl CoA
A special transport system is required to
transport acyl CoA into mitochondria

17. The key component of acyl CoA transport
system is carnitine
Carnitine is b-hydroxy-g-trimethyl
ammonium butyrate
Carnitine can react with acyl CoA to form
acyl-carnitine

18. CH3
|
H C — N — CH — CH — CH — COOH3
+
2 2
| |
CH3 O — C — R
||
O
Acylcarnitine
CH3
|
H C — N — CH — CH — CH — COOH3
+
2 2
| |
CH3 OH
CoA — SH
CoA
R — C ~ S — CoA
||
O
Acyl CoA
Carnitine

19. 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

20. 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
outer surface of the membrane

21. 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

22. Outer
side
Inner mitochondrial
membrane
Mitochondrial
matrix
Acyl CoA Carnitine Acyl CoA
Carnitine-
palmitoyl
transferase I
Carnitine-
acylcarnitine
translocase
Carnitine-
palmitoyl
transferase II
AcylcarnitineCoA CoA

23. 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
b-Oxidation pathway

24. 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

25. Knoop’s experiments
Phenylacetic acid
Benzoic acid
‒CH2‒(CH2)2n‒COOH ‒CH2‒COOH
‒CH2‒(CH2)2n+1‒COOH ‒COOH
Oxidation of a fatty acid having an even
number of carbon atoms
Oxidation of a fatty acid having an odd
number of carbon atoms

26. 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 b-oxidation as the b-
carbon (C3) is oxidized in each cycle

27. 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

28. 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

29. 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
Reactions of b-oxidation pathway

30. One hydrogen atom is removed from a-
carbon and one from b-carbon of acyl CoA
A double bond is formed between a- and
b-carbon atoms
The product is a, b-unsaturated acyl CoA

31. 
R ‒ CH2 ‒ CH2 ‒ C ~ S‒CoA
O
II
R ‒ CH = CH ‒ C ~ S‒CoA
Acyl CoA (Cn)
Acyl CoA
dehydrogenase
Fp
FpH2
a, b-Unsaturated acyl CoA
O
II

32. The second reaction is addition of H and
OH
Crotonase splits H2O into H and OH
It adds H to a-carbon and OH to b-carbon
a, b-Unsaturated acyl CoA is converted
into b-L-hydroxyacyl CoA

33. 
O
II
R ‒ CH ‒ CH2 ‒ C ~ S‒CoA
O
II
R ‒ CH = CH ‒ C ~ S‒CoA
b-L-Hydroxyacyl CoA
Crotonase
H2O
a, b-Unsaturated acyl CoA
I
OH

34. In the third reaction, two hydrogen atoms
are removed from the b-carbon
These are transferred to NAD+
b-L-Hydroxyacyl CoA is converted into b-
ketoacyl CoA

35. O
II
R ‒ CH ‒ CH2 ‒ C ~ S‒CoA
b-L-Hydroxyacyl CoA
b-Hydroxyacyl CoA
dehydrogenase
NADH + H+
O
II
R ‒ C ‒ CH2 ‒ C ~ S‒CoA
b-Ketoacyl CoA

NAD+
II
O
I
OH

36. The fourth (and final) reaction is catalysed
by thiolase
The last two carbon atoms and CoA are
removed from b-keto acyl CoA as acetyl
CoA

37. 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

38. II
CH3‒C~S‒CoA
Thiolase
O
II
R‒C‒CH2‒C~S‒CoA
II
b-Ketoacyl CoA

CoA‒SH
O
R‒C~S‒CoA
II
O
O
Acyl CoA (Cn‒2)

39. Thus, during one cycle of
b-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

40. 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

41. R ‒ CH2 ‒ CH2 ‒ C ~S ‒ CoA
O

Fp
FpH2
H2O


Acyl CoA (Cn)
R ‒ CH = CH ‒ C ~S ‒ CoA
O
R ‒ C ‒ CH2 ‒ C ~S ‒ CoA
O
R ‒ C ~S ‒ CoA
O
O
R ‒ CH ‒ CH2 ‒ C ~S ‒ CoA
OOH
CH3 ‒ C ~S ‒ CoA
O

CoA‒SH
NADH + H+
NAD+

a,b-Unsaturated
acyl CoA
b-Hydroxy-
acyl CoA
b-Keto-
acyl CoA
Acyl CoA (Cn‒2)

42. Energetics
In each cycle of
b-oxidation:
One FAD is reduced
One NAD is reduced
One acetyl CoA is formed

43. If the fatty acid being oxidized
is palmitic acid (C16):
Seven cycles of b-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

44. Thus, eight molecules of acetyl CoA are
formed
When oxidized in the citric acid cycle,
these will form 8 x 12 = 96 ATP
equivalents

45. Seven molecules of FAD are reduced
in seven cycles
When oxidized in the citric acid cycle,
these will form 7x2 = 14 ATP equivalents

46. Seven molecules of NAD are reduced
in seven cycles
When oxidized in the citric acid cycle,
these will form 7x3 = 21 ATP equivalents

47. 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

48. 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

49. Molecular weight of palmitic acid is 256
Hence, its potential energy is 256 x 9.1 =
2,330 kcal/mol
Therefore, efficiency of b-oxidation is
942  2,330 x 100 or ≈ 40%

50. If the fatty acid being oxidized
is stearic acid (C18):
Eight cycles of b-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

51. Thus, nine molecules of acetyl CoA are
formed
When oxidized in the citric acid cycle,
these will form 9 x 12 = 108 ATP
equivalents

52. Eight molecules of FAD are reduced
in eight cycles
When oxidized in the citric acid cycle,
these will form 8x2 = 16 ATP equivalents

53. Eight molecules of NAD are reduced
in eight cycles
When oxidized in the citric acid cycle,
these will form 8x3 = 24 ATP equivalents

54. 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

55. Molecular weight of stearic acid is 284
Its potential energy is 284 x 9.1 = 2,584
kcal/mol
So, efficiency of b-oxidation is 1066 
2,584 x 100 or ≈ 41%

56. Fatty acids having an odd number of carbon
atoms are also oxidized by b-oxidation
After the last cycle of b-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

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

58. Inborn errors of b-oxidation are uncommon
Rarely, defects have been reported in:
▪ Acyl CoA dehydrogenase
▪ b-Hydroxyacyl CoA dehydrogenase
Clinical manifestation is unexplained hypo-
glycaemia with or without ketosis
Defects in b-oxidation

59. 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

60. 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

61. 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
Regulation of fatty acid oxidation

62. 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

63. 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

64. When energy is abundant, insulin
secretion increases
Insulin inhibits lipolysis and availability of
fatty acids
Therefore, oxidation of fatty acids is
decreased

65. 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

66. NADH inhibits b-hydroxyacyl CoA
dehydrogenase
Acetyl CoA inhibits thiolase
This decreases the oxidation of fatty acids

67. Unsaturated fatty acids are also oxidized
by b-oxidation
Two additional enzymes are needed to
deal with the double bonds
Double bonds in naturally occurring fatty
acids have a cis conformation
Oxidation of unsaturated fatty acids

68. On hydration, cis double bonds form the
D-isomers of hydroxyacyl CoA
b-Hydroxyacyl CoA dehydrogenase can
act only on b-L-hydroxyacyl CoA
Therefore, the D-isomers have to be
racemised to L-isomers

69. When a double bond occurs between b-
and g-carbon, acyl CoA dehydrogenase
cannot act on it
The reason is that the single hydrogen
atom attached to b-carbon atom
cannot be removed

70. Hence, the double bond is shifted
between the a- and b-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

71. CH3‒(CH2 )4‒CH = CH‒CH2‒CH =CH‒(CH2 )7‒C~S‒CoA
O
3 Cycles of b-oxidation
3
H2O



Linoleyl CoA
CH3 ‒ C ~S ‒ CoA
O
Crotonase
D3-cis, D6-cis-
Dienoyl CoA
CH3‒(CH2 )4‒CH = CH‒CH2‒CH =CH‒CH2 ‒C~S‒CoA
O
CH3‒(CH2 )4‒CH = CH‒CH2‒CH2 ‒ C = C ‒ C~S‒CoA
O
H
H
CH3‒(CH2 )4‒CH = CH‒CH2‒CH2‒CH2‒CH‒ C~S‒CoA
OOH
D2-trans, D6-cis-
Dienoyl CoA
b-L-Hydroxy-D6-
cis-enoyl CoA
D3-cis → D2-trans-Dienoyl CoA isomerase

9101213
7 6 4 3
6 2
6

72. 
2 Cycles of b-oxidation
2
H2O

CH3 ‒ C ~S ‒ CoA
O
Crotonase
a, b-Unsaturated
acyl CoA
CH3‒(CH2 )4‒CH = CH‒C~S‒CoA
O
CH3‒(CH2 )4‒CH ‒ CH2‒C~S‒CoA
O
OH
CH3‒(CH2 )4‒CH = CH‒CH2‒CH2‒CH2‒CH‒C~S‒CoA
OOH
b-L-Hydroxy-D6-
cis-enoyl CoA
b-D-Hydroxyacyl
CoA

73. 3 Cycles of b-oxidation
3 CH3 ‒ C ~S ‒ CoA
O
CH3‒(CH2 )4‒CH ‒ CH2‒C~S‒CoA
O
OH
Acetyl CoA
b-D-Hydroxyacyl
CoA


CH3‒(CH2 )4‒CH ‒ CH2‒C~S‒CoA
OOH

CH3 ‒ C ~S ‒ CoA
O
b-L-Hydroxyacyl
CoA
b-Hydroxyacyl CoA racemase

74. There are two other pathways for oxidation
of fatty acids
These are quantitatively insignificant
Of these, a-oxidation pathway is present in
brain
The other pathway is w-oxidation
Other pathways for oxidation of fatty
acids

75. a-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
a-Oxidation

76. CH3‒ CH‒ (CH2)3‒ CH‒ (CH2)3‒ CH‒ (CH2)3‒ CH‒ CH2‒ COOH
CH3CH3 CH3 CH3
Phytanic acid

77. Phytanic acid is oxidized by a-oxidation
a-Oxidation occurs in peroxisomes
Phytanic acid is first activated to
phytanoyl CoA
Phytanoyl CoA is hydroxylated to 2-
hydroxyphytanoyl CoA

78. CH3CH3 CH3 CH3
CH3‒ CH‒ (CH2)3‒ CH‒ (CH2)3‒ CH‒ (CH2)3‒ CH‒ CH2‒ COOH
Acyl CoA synthetase

CH3CH3 CH3 CH3
CH3‒ CH‒ (CH2)3‒ CH‒ (CH2)3‒ CH‒ (CH2)3‒ CH‒ CH2‒ C~S‒CoA
O
 
Phytanoyl CoA hydroxylase
CH3CH3 CH3 CH3
CH3‒ CH‒ (CH2)3‒ CH‒ (CH2)3‒ CH‒ (CH2)3‒ CH‒ CH‒ C~S‒CoA
OOH
ATP + CoA‒SH
AMP + PPi
O2 + a-Ketoglutarate
CO2 + Succinate
Phytanic acid
Phytanoyl CoA
2-Hydroxyphytanoyl CoA

79. 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

80. CH3CH3 CH3 CH3
CH3‒ CH‒ (CH2)3‒ CH‒ (CH2)3‒ CH‒ (CH2)3‒ CH‒ CHO
Pristanic acid
 
2-Hydroxyphytanoyl CoA lyase
CH3CH3 CH3 CH3
CH3‒ CH‒ (CH2)3‒ CH‒ (CH2)3‒ CH‒ (CH2)3‒ CH‒ CH‒ C~S‒CoA
OOH
NADH + H+
Pristanal
2-Hydroxyphytanoyl CoA
H‒ C~S‒CoA
O


H2O + NADH+
Aldehyde dehydrogenase
CH3CH3 CH3 CH3
CH3‒ CH‒ (CH2)3‒ CH‒ (CH2)3‒ CH‒ (CH2)3‒ CH‒ COOH

81. Pristanic acid is activated to pristanoyl
CoA, which then undergoes b-oxidation
Six cycles of b-oxidation produce iso-
butyryl CoA and three molecules each of
acetyl CoA and propionyl CoA

82. CH3CH3 CH3 CH3
CH3‒ CH‒ (CH2)3‒ CH‒ (CH2)3‒ CH‒ (CH2)3‒ CH‒ C~S‒CoA
Pristanic acid


O
Pristanoyl CoA
Acetyl CoA

Six cycles of b-oxidation
CH3CH3 CH3 CH3
CH3‒ CH‒ (CH2)3‒ CH‒ (CH2)3‒ CH‒ (CH2)3‒ CH‒ COOH
Acyl CoA synthetase
ATP + CoA‒SH
AMP + PPi
3 CH3‒ C~S‒CoA
O
Propionyl CoAIsobutyryl CoA
3 CH3‒ CH2‒C~S‒CoA +
OCH3
CH3‒ CH ‒C~S‒CoA +
O

83. An inherited defect in a-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
Peroxisomal enzymes fail to reach
peroxisomes in peroxin-7 deficiency

84. 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

85. This is another minor pathway for
oxidation of fatty acids
It becomes important when b-oxidation is
defective
It is located in endoplasmic reticulum of
liver and kidney cells
w-Oxidation

86. The first reaction is introduction of a
hydroxyl group onto the w-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
aldehyde group to a carboxyl group

87. CH3 –(CH2)n –COOH
CH2 –(CH2)n –COOH
OH
I
HOOC–(CH2)n –COOH
CH –(CH2)n –COOH
O
II

88. The fatty acid now has a carboxyl group
at each end
The dicarboxylic acid is activated and
enters the mitochondria
b-Oxidation starts from both the ends

89. 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