Molecular Biochemistry II

Fatty Acid Oxidation

Copyright © 1999-2005 by Joyce J. Diwan.

All rights reserved.
 O

  C
4
3 1 O
2

fatty acid with a cis- 9

double bond

A 16-C fatty acid with numbering conventions is shown.

Most naturally occurring fatty acids have an even number of
carbon atoms.
The pathway for catabolism of fatty acids is referred to as
the -oxidation pathway, because oxidation occurs at the
-carbon (C-3).
 O

H 2C OH H 2C O C R
O
O
HC OH HC O C R
HO C R O
H 2C OH H 2C O C R

glyc ero l fatty a c id triac ylgly ce ro l

Triacylglycerols (triglycerides) are the most abundant
dietary lipids. They are the form in which we store reduced
C for energy.
Each triacylglycerol has a glycerol backbone to which are
esterified 3 fatty acids
Most triacylglycerols are “mixed.” The 3 fatty acids differ
in chain length & number of double bonds.
 O

H 2C OH H 2C O C R
O
O
HC OH HC O C R
HO C R O
H 2C OH H 2C O C R

glyc ero l fatty a c id triac ylgly ce ro l

Lipid digestion, absorption, transport will be covered

separately.
Lipases hydrolyze triacylglycerols, releasing 1 fatty acid
at a time, yielding diacylglycerols, & eventually glycerol.
 CH2 OH CH2 OH +
H+ + CH2 OH
ATP ADP NAD NADH
HO CH HO CH C O
1 2 

CH2 OH CH2 O PO 3 CH2 O PO 3

g ly c e ro l g ly c e ro l-3 -P d ih y d r o x y a c e to n e -P

Glycerol, arising from hydrolysis of triacylglycerols, is

converted to the Glycolysis intermediate
dihydroxyacetone phosphate, by reactions catalyzed by:
1 Glycerol Kinase
2 Glycerol Phosphate Dehydrogenase.
 O

  C
4
3 1 O
2

fatty acid with a cis- 9

double bond

Free fatty acids, which in solution have detergent

properties, are transported in the blood bound to
albumin, a serum protein produced by the liver.
Several proteins have been identified that facilitate
transport of long chain fatty acids into cells, including
the plasma membrane protein CD36.
Fatty acid activation:
Acyl-CoA Synthases (Thiokinases) of ER & outer
mitochondrial membranes catalyze activation of long
chain fatty acids, esterifying them to coenzyme A.
This process is ATP-dependent, & occurs in 2 steps.
There are different Acyl-CoA Synthases for fatty
acids of different chain lengths. 
 NH2
Fatty acid activation
Acyl-CoA O N
Synthases fatty acid R C
N
O
Exergonic PPi O O O N N
(P~P) hydrolysis, 
O P O P O P O CH2
O ATP
catalyzed by O 
O 
O  H H
Pyrophosphatase, H
OH OH
H

makes the coupled NH2

reaction 2 Pi PPi
N
N
spontaneous.
O O N N
2 ~P bonds of ATP
are cleaved. R C O P O CH2
O acyl-
O H H adenylate
The acyl-CoA CoA SH H H
OH OH
product includes AMP
one "~" thioester O
linkage. R C S CoA acyl-CoA
Summary of fatty aid activation:
 fatty acid + ATP  acyladenylate + PPi
PPi  2 Pi
 acyladenylate + HS-CoA  acyl-CoA + AMP
Overall:
fatty acid + ATP + HS-CoA  acyl-CoA + AMP + 2 Pi

-Oxidation pathway:
For most steps of the -oxidation pathway, there are
multiple enzymes specific for particular fatty acid chain
lengths.
 Mitochondrion
Fatty acid-oxidation
is considered to occur -Oxidation
in the mitochondrial pathway in
matrix. matrix

Fatty acids must enter

the matrix to be
oxidized.
However enzymes of Fatty acyl-CoA formed in cytosol by enzymes
of outer mitochondrial membrane & ER
the pathway specific
for very long chain fatty acids are associated with the
inner membrane, facing the matrix.
Fatty acyl-CoA formed outside can pass through the outer
mitochondrial membrane (which has large VDAC
channels), but cannot penetrate the inner membrane.
 CH3 OH R
+
H3C N CH2 CH CH2 COO + C O

CH3 carnitine SCoA

Transfer of the
Carnitine Palmitoyl
fatty acid Transferase
moiety across R
the inner
C O
mitochondrial
membrane CH3 O
involves +
H3C N CH2 CH CH2 COO + HSCoA
carnitine. 
CH3 fatty acyl carnitine

Carnitine Palmitoyl Transferases catalyzes transfer of a fatty

acid between the thiol of Coenzyme A and the hydroxyl on
carnitine.
 cytosol mitochondrial matrix

O O
R-C-SCoA HO-carnitine HO-carnitine R-C-SCoA
1 3
2
HSCoA R-C-O-carnitine R-C-O-carnitine HSCoA
O O

Carnitine-mediated transfer of the fatty acyl moiety into

the mitochondrial matrix is a 3-step process:
1. Carnitine Palmitoyl Transferase I, an enzyme on the
cytosolic surface of the outer mitochondrial membrane,
transfers a fatty acid from CoA to the OH on carnitine.
2. An antiporter in the inner mitochondrial membrane
mediates exchange of carnitine for acylcarnitine.
 cytosol mitochondrial matrix

O O
R-C-SCoA HO-carnitine HO-carnitine R-C-SCoA
1 3
2
HSCoA R-C-O-carnitine R-C-O-carnitine HSCoA
O O

3. Carnitine Palmitoyl Transferase II, an enzyme

within the matrix, transfers the fatty acid from carnitine
to CoA. (Carnitine exits the matrix in step 2.)
The fatty acid is now esterified to CoA in the matrix.
 O

H3C C SCoA
acetyl-CoA
O

OOC CH2 C SCoA
malonyl-CoA

Control of fatty acid oxidation is exerted mainly at the

step of fatty acid entry into mitochondria.
Malonyl-CoA, which is also a precursor for fatty acid
synthesis, inhibits Carnitine Palmitoyl Transferase I.
Malonyl-CoA is produced from acetyl-CoA by the
enzyme Acetyl-CoA Carboxylase.
 O
AMP-Activated Kinase, H3C C SCoA
a sensor of cellular energy acetyl-CoA
levels, is allosterically 
Acetyl-CoA
activated by AMP, which ATP + HCO3 Carboxylase
is high in concentration (inhibited by
ADP + Pi AMP-Activated
when [ATP] is low. Kinase)
O
Phosphorylation via AMP- 
OOC CH2 C SCoA
Activated Kinase inhibits
malonyl-CoA
Acetyl-CoA Carboxylase,
thereby decreasing malonyl-CoA production.
The decrease in [malonyl-CoA] releases Carnitine
Palmitoyl Transferase I from inhibition.
The increase in fatty acid oxidation generates acetyl-CoA,
for entry into Krebs cycle with associated ATP production.
-Oxidation HHO
3 2
Pathway: H3C (C H ) C
2n   C C
1
SC oA
fattyac
yl-
C oA
Step 1. Acyl-CoA HH
Dehydrogenase FADA cyl-Co A Dehydrogenas
e
catalyzes oxidation FAD H2 HO
of the fatty acid
H C(C H n C C C SC
) oA
moiety of acyl-CoA 3 2
tr
a ns-2
-enoy
l-CoA
to produce a double H
bond between carbon atoms H 22O
& 3.
There are different Acyl-CoA Dehydrogenases
H O for short
(4-6 C), medium (6-10H C), long
3C(C H2)and very
n CC H long (12-18
2C SC oA C)
chain fatty acids.
OH
Very Long Chain Acyl-CoA Dehydrogenase is bound to
the inner mitochondrialHmembrane.
+
+ NADH The others are soluble
enzymes located in the mitochondrial
NAD+ O matrix.
O
 HHO
3 2 1 glutamate
H
3C(C
H
2nC C
) 
C SC
o
A H

f
attya
cyl-
C o
A
HH H3N+ C COO
FADA
cy
l-
C o
A D
eh
ydr
oge
nas
e CH2
FAD
H2 HO CH2
H
3C(C
H
2n C C C SC
) o
A C
tr
ans
--
eno
y2
l-
C o
A O O
H
H
2O
FAD is the prosthetic group that functions as e acceptor
HDehydrogenase.
for Acyl-CoA O Proposed mechanism:
H
A3C
Glu
(C n CC H
side-chain
H2) C SC o
carboxyl
2 A
extracts a proton from the -
carbon of the
O substrate, facilitating transfer of 2 e  with
H
H+ (a hydride) from the  position to FAD.
+
H+N
ADH
The reduced FAD accepts a 2 nd H+, yielding FADH .
2
AD O
N + O
 HHO
3 2 1
H
3C(C
H
2nC C
) 
C SC
o
A

f
attya
cyl-
C o
A
HH
FADA
cy
l-
C o
A D
eh
ydr
oge
nas
e
FAD
H2 HO

H
3C(C
H
2n C C C SC
) o
A
2
tr
ans
--en
oyl-
C o
A
H
H
2O
The carbonyl O of theH thioester
Osubstrate is hydrogen
bonded toH the
C
2'-OH
(CH )
of the
CC H
ribityl
C SC
moiety
oA
of FAD, giving
3 2n 2
this part of FAD a role in positioning the substrate and
increasing acidity of OH
the substrate -proton.
+
H+N
ADH
dimethylisoalloxazineO O
H H H
C N C  + C N C
H3C C C C NH 2e +2H H C C C C NH
3

H3C C C C C O H3C C C C C O
C N N C N N
H H H
CH2 CH2

HC OH HC OH

HC OH HC OH
FAD Adenine
FADH2
HC OHO O HC OHO O Adenine

H2C O P O P O Ribose H2C O P O P O Ribose

O- O- O- O-

The carbonyl O of the thioester substrate is hydrogen

bonded to the 2'-OH of the ribitol moiety of FAD, giving
the sugar alcohol a role in positioning the substrate and
increasing acidity of the substrate -proton.
 HHO
3 2 1
H
3C(C
H
2nC C
) 
C SC
o
A

f
attya
cyl-
C o
A
HH
FADA
cy
l-
C o
A D
eh
ydr
oge
nas
e
FAD
H2 HO

H
3C(C
H
2n C C C SC
) o
A
2
tr
ans
--en
oyl-
C o
A
H
H
2O

The reactive Glu andH FAD are

Oon opposite sides of the
substrateH
at
3Cthe
(Cactive
H2)n CC
site.
H2C SC o A
Thus the reaction is stereospecific,
O H yielding a trans
double bond in enoyl-CoA.
+
H+N
ADH
 Matrix
H+ + NADH NAD+ + 2H+ 2H+ + ½ O2 H2O

2 e ––
I Q III IV

++
+ + cyt c
4H 4H 2H+
Intermembrane Space

FADH2 is reoxidized by transfer of 2 electrons to

an electron transfer flavoprotein (ETF), which in
turn passes the electrons to coenzyme Q of the
respiratory chain.
 H H O
Step 2. H3C (C
3
H2)n C C
2
C SC
oA
1
 
Enoyl-CoA H H
fattyacyl-CoA
Hydratase FAD Acyl-CoADehydrogenase
catalyzes FAD
H2
H O
stereospecific
hydration of the H3C (C
H2)n C C C SC
oA
trans-2-enoyl-CoA
trans double bond H
produced in the H2O Enoyl-CoAHydratase
1st step, yielding H O
L-hydroxyacyl- H3C (C
H2)n C CH2 C SC
oA
Coenzyme A. 3-L-hydroxyacyl-CoA
OH

H++NADH
 H
H2O

H O

H3C (C
H2)n C CH2 C SC
oA
3-L-hydroxyacyl-CoA
Step 3. OH
+
NAD Hydroxyacyl-CoA
Hydroxyacyl-CoA H++NADH Dehydrogenase
Dehydrogenase O O
catalyzes oxidation
H3C (C
H2)n C CH2 C SC
oA
of the hydroxyl in -ketoacyl-CoA
the  position (C3) HSCoA -Ketothiolase
to a ketone. O O

NAD+ is the H3C (C

H2)n C SC
oA + CH3 C SC
oA
electron acceptor. fattyacyl-CoA acetyl-CoA
(2Cshorter)
 H
O O
H3N+ C COO
H3C (CH2)n C CH2 C SCoA
CH2 -ketoacyl-CoA
HSCoA
SH
O O
cysteine
H3C (CH2)n C SCoA + CH3 C SCoA
Step 4. fatty acyl-CoA acetyl-CoA
(2 C shorter)
-Ketothiolase
catalyzes thiolytic -Ketothiolase
cleavage.
A cysteine S attacks the -keto C.
Acetyl-CoA is released, leaving the fatty acyl moiety in
thioester linkage to the cysteine thiol.
The thiol of HSCoA displaces the cysteine thiol, yielding
fatty acyl-CoA (2 C less).
A membrane-bound trifunctional protein complex
with two subunit types expresses the enzyme
activities for steps 2-4 of the -oxidation pathway for
long chain fatty acids.
Equivalent enzymes for shorter chain fatty acids are
soluble proteins of the mitochondrial matrix.
Summary of one round of the -oxidation pathway:
fatty acyl-CoA + FAD + NAD+ + HS-CoA 
fatty acyl-CoA (2 C less) + FADH2 + NADH + H+
+ acetyl-CoA
The -oxidation pathway is cyclic.
The product, 2 carbons shorter, is the input to another
round of the pathway.
If, as is usually the case, the fatty acid contains an
even number of C atoms, in the final reaction cycle
butyryl-CoA is converted to 2 copies of acetyl-CoA. 
 Matrix
H+ + NADH NAD+ + 2H+ 2H+ + ½ O2 H2O

2 e ––
I Q III IV

++
cyt c
ATP 4H+ 4H+ 2H+
Production: Intermembrane Space

 FADH2 of Acyl-CoA Dehydrogenase is reoxidized by

transfer of 2 e via ETF to CoQ of the respiratory chain.
H+ ejection from the matrix that accompanies transfer
of 2 e from CoQ to oxygen, leads via chemiosmotic
coupling to production of approximately 1.5 ATP.
(Approx. 4 H+ enter the matrix per ATP synthesized.)
 Matrix
H+ + NADH NAD+ + 2H+ 2H+ + ½ O2 H2O

2 e ––
I Q III IV

++
+ + cyt c
4H 4H 2H+
Intermembrane Space

 NADH is reoxidized by transfer of 2 e to the

respiratory chain complex I.
H+ pumping associated with transfer of 2 e from
complex I to oxygen yields approx. 2.5 ATP.
 Acetyl-CoA can enter Krebs cycle, yielding
additional NADH, FADH2, and ATP.
Fatty acid oxidation is a major source of cell ATP.
Problem (See web page handout and tutorial):
Catabolism of two 6-C glucose molecules through
glycolysis, Krebs cycle, and oxidative phosphorylation
yields approx. 60 ~P bonds of ATP (30/glucose).
Compare the energy yield for oxidizing a 12-C fatty acid.
Assume:
 1.5 ATP produced per FADH2 reoxidized via the
respiratory chain.
 2.5 ATP produced per NADH is reoxidized via the
respiratory chain.
Human genetic diseases have been identified that
involve mutations in:
 the plasma membrane fatty acid transporter CD36
 Carnitine Palmitoyltransferases I & II (required for
transfer of fatty acids into mitochondria)
 Acyl-CoA Dehydrogenases for various chain lengths
of fatty acids
 the trifunctional protein complex
 Medium Chain -Ketothiolase
 Electron Transfer Flavoprotein (ETF).
Human genetic diseases:
Symptoms vary depending on the specific genetic
defect but may include:
 hypoglycemia and failure to increase ketone body
production during fasting
 fatty degeneration of the liver
 heart and/or skeletal muscle defects
 maternal complications of pregnancy.
Hereditary deficiency of Medium Chain Acyl-CoA
Dehydrogenase (MCAD), the most common genetic
disease relating to fatty acid catabolism, has been
linked to sudden death in infants (SIDS). 
The reactions presented accomplish catabolism of a
fatty acid with an even number of C atoms & no
double bonds.
Additional enzymes deal with catabolism of fatty acids
with an odd number of C atoms or with double bonds.
 The final round of -oxidation of a fatty acid with
an odd number of C atoms yields acetyl-CoA and
propionyl-CoA.
Propionyl-CoA is converted to the Krebs cycle
intermediate succinyl-CoA, by a pathway involving
vitamin B12 (to be presented later).
 Most double bonds of naturally occurring fatty
acids have the cis configuration.
As C atoms are removed two at a time, a double
bond may end up in the wrong position or wrong
configuration for the enoyl-CoA to be a substrate
for Enoyl-CoA Hydratase.
The reactions that allow unsaturated fatty acids to
be fully catabolized by the -oxidation pathway are
summarized in the textbook.
 Peroxisome
Single membrane

Crystalline inclusion
often present

Enzymes, some of which produce H2O2 , &

always including Catalase, that degrades H2O2.

-Oxidation of long-chain fatty acids also occurs within

peroxisomes.
FAD is e acceptor for peroxisomal Acyl-CoA Oxidase,
which catalyzes the 1st oxidative step of the pathway.
Within the peroxisome, FADH2 generated by fatty acid
oxidation is reoxidized producing hydrogen peroxide:
FADH2 + O2  FAD + H2O2
The peroxisomal enzyme Catalase degrades H2O2:
2 H2O2  2 H2O + O2
These reactions produce no ATP.
Once fatty acids are reduced in length within the
peroxisomes they may shift to the mitochondria to be
catabolized all the way to CO2.
Carnitine is also involved in transfer of fatty acids into
and out of peroxisomes.
 Glucose-6-phosphatase
glucose-6-P glucose
Gluconeogenesis Glycolysis
pyruvate
fatty acids
During fasting acetyl CoA ketone bodies
or carbohydrate cholesterol
starvation, oxaloacetate citrate
oxaloacetate is
depleted in Krebs Cycle
liver due to
gluconeogenesis.
This impedes entry of acetyl-CoA into Krebs cycle.
Acetyl-CoA in liver mitochondria is converted then to
ketone bodies, acetoacetate & -hydroxybutyrate.
 O O
Ketone body
synthesis: H3C C SCoA + H3C C SCoA
acetyl-CoA acetyl-CoA
-Ketothiolase. The HSCoA Thiolase
final step of the - O O
oxidation pathway H2
H3C C C C SCoA
runs backward. O acetoacetyl-CoA
HMG-CoA H3C C SCoA
HMG-CoA Synthase
Synthase catalyzes acetyl-CoA HSCoA
condensation with a O OH O
3rd acetate moiety 
H2 H2
O C C C C C SCoA
(from acetyl-CoA).
CH3 HMG-CoA
HMG-CoA Lyase
HMG-CoA Lyase
cleaves HMG-CoA to O O O
yield acetoacetate & 
H2
O C C C CH3 + H3C C SCoA
acetyl-CoA. acetoacetate acetyl-CoA
  -H ydroxybutyrate D ehydrogenase
CH3 + CH3
H 
+
C O N A D H N A D HO CH

CH2 CH2

COO COO
acetoacetate D - -hydroxybutyrate

-Hydroxybutyrate Dehydrogenase catalyzes

reversible inter-conversion of the ketone bodies
acetoacetate and -hydroxybutyrate.
Ketone bodies are transported in the blood to
other tissue cells, where they are converted back
to acetyl-CoA for catabolism in Krebs cycle.