Fatty Acid Oxidation

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 & unsaturated fatty acids are in the cis
configuration
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

gly c e ro l fa tty a c id tria c ylg lyc e ro l

Triacylglycerols (triglycerides) are the most abundant

dietary lipids.
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

g ly c e ro l fa tty a c id tria c y lg ly c e ro l

Lipases hydrolyze triacylglycerols, yielding glycerol and

three fatty acids
 O

  C
4
3 1 O
2

fatty acid with a cis- 9

double bond

Free fatty acids are transported in the blood bound to

albumin, a plasma protein produced by the liver.
Several proteins have been identified that facilitate
transport of long chain fatty acids into cells
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, is 
O P O P O P O CH2
O ATP
catalyzed by O 
O 
O  H H
Pyrophosphatase H
OH OH
H
NH2
2 ~P bonds of ATP
are cleaved. 2 Pi PPi
N
N

The acyl-CoA O O N N
product includes
R C O P O CH2
one "~" thioester O acyl-
O H H adenylate
linkage. CoA SH H H
OH OH
AMP
O

R C S CoA acyl-CoA
Summary of fatty aid activation:
fatty acid + ATP + HS-CoA  acyl-CoA + AMP + 2 Pi
 Mitochondrion

Fatty acid-oxidation -Oxidation

is considered to occur pathway in
in the mitochondrial matrix
matrix.

Fatty acids must enter

the matrix to be Fatty acyl-CoA formed in cytosol by enzymes
oxidized. of outer mitochondrial membrane & ER

Fatty acyl-CoA formed outside can pass through the

outer mitochondrial membrane, 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
across the inner R
mitochondrial
C O
membrane
involves CH3 O
carnitine. +
H3C N CH2 CH CH2 COO + HSCoA

CH3 fatty acyl carnitine

Carnitine Palmitoyl Transferases catalyze 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 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 thus inhibits fatty acid oxidation by
preventing its transport into mitochondria.
-Oxidation HHO
3 2
Pathway: H C
3 (CH ) C
2n   C C
1
SC oA
fattyac
yl-
C oA
Step 1. Acyl-CoA HH
Dehydrogenase FADA cyl-Co A Deh ydr
ogen a
se
catalyzes oxidation FAD H2 HO
of the fatty acid of
H C(C H n C C C SC
) oA
acyl-CoA to 3 2
trans-2
-enoyl-
C oA
produce a double H
bond between carbon atomsH 22O& 3.
There are different Acyl-CoA Dehydrogenases
H O for short
(4-6 C), medium (6-10H C),
C(C
3
long
H 2) and very
n CC H long (12-18
2C SC oA C)
chain fatty acids. OH

+
H+N
ADH
N
AD+ O 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
tr
ans
-2
-en
oyl-
C o
A
H

FAD His2O
the prosthetic group that functions as e acceptor
for Acyl-CoAHDehydrogenase.
O

The
HCreaction
3 (C
H
2)n CCis stereospecific,
H2C SC oA yielding a trans double
bond in enoyl-CoA.
OH

+
H+N
ADH
N
AD+ O O
 H H O
Step 2. H3C (C
3
H2)n C C
2
C SC
oA
1
 
Enoyl-CoA fattyacyl-CoA
H H
Hydratase FAD Acyl-CoADehydrogenase
catalyzes FAD
H2
H O
stereospecific
H3C (C
H2)n C C C SC
oA
hydration of the
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)
 O O
Step 4.
-Ketothiolase catalyzes H3C (CH2)n C CH2 C SCoA
thiolytic cleavage. -ketoacyl-CoA
HSCoA
Thiol sulfur of CoA
attacks the -keto carbon O O

H3C (CH2)n C SCoA + CH3 C SCoA

fatty acyl-CoA acetyl-CoA
(2 C shorter)
-Ketothiolase

Acetyl-CoA is released, leaving the fatty acyl in thioester

linkage to the CoA -fatty acyl-CoA (2 C less).
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 molecules of acetyl-CoA.
 FADH2 & NADH produced during fatty acid
oxidation are reoxidized by transfer of electrons to
respiratory chain.Transfer of electrons in the
respiratory chain leads to production of ATP

 Acetyl-CoA can enter Krebs cycle, yielding additional

NADH, FADH2, and ATP.

Fatty acid oxidation is a major source of cell ATP.

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 &
propionyl-CoA.
Propionyl-CoA is converted to the Krebs cycle
intermediate succinyl-CoA, by a pathway
involving vitamin B12.
 Most double bonds of naturally occurring fatty acids
have the cis configuration.
They are not correct substrates for Enoyl-CoA
hydratase, which acts only on trans compounds.
Additional enzymes, isomerase and reductase , are
required for oxidation of unsaturated fatty acids.
-Oxidation of very long-chain fatty acids also occurs
within peroxisomes.
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 H2 O2  2 H2 O + O 2
These reactions produce no ATP.
Once fatty acids are reduced in length within the
peroxisomes they may shift to the mitochondria to be
catabolized to acetylCoA.
 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 (from 
H2 H2
O C C C C C SCoA
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
-Hydroxybutyrate
CH3 CH3
Dehydrogenase H 
+
+
catalyzes reversible C O NADH NAD HO CH
interconversion of CH2 CH2
the ketone bodies
COO COO
acetoacetate &
acetoacetate D -  -hydroxybutyrate
-hydroxybutyrate.
Ketone bodies are transported in the blood to other cells,
where they are converted back to acetyl-CoA for
catabolism in Krebs cycle, to generate ATP.
Ketone bodies thus function as an alternative fuel.
Ketoacidosis is caused by excess of ketone bodies.
Fatty Acid Synthesis
 O

H 3C C SC o A
acetyl-C oA
The input to fatty acid O
synthesis is acetyl-CoA,

which is carboxylated to OOC CH2 C SC o A
malonyl-CoA. m alonyl-C oA

ATP-dependent carboxylation provides energy input.

The CO2 is lost later during condensation with the
growing fatty acid.
 Enzyme-biotin
-
Acetyl-CoA HCO3 + ATP
1
Carboxylase ADP + Pi
-
catalyzes the Enzyme-biotin-CO2
2-step reaction O
ll 2
by which CH3-C-SCoA
Enzyme-biotin
acetyl-CoA is acetyl-CoA
carboxylated O
-
ll
to form O2C-CH2-C-SCoA
malonyl-CoA. malonyl-CoA

As with other carboxylation reactions, the enzyme

prosthetic group is biotin.
ATP-dependent carboxylation of the biotin, carried out at
one active site 1 , is followed by transfer of the carboxyl
group to acetyl-CoA at a second active site 2 .
 Enzyme-biotin
-
HCO3 + ATP
1
ADP + Pi
-
Enzyme-biotin-CO2
O
ll 2
CH3-C-SCoA
Enzyme-biotin
acetyl-CoA
O
-
ll
O2C-CH2-C-SCoA
malonyl-CoA

The overall reaction may be summarized as:

HCO3 + ATP + acetyl-CoA  ADP + Pi + malonyl-CoA
 O

O C
C N NH
O
CH CH
O O C
H2 C CH
S (CH 2)4 C NH (CH 2)4 CH

Carboxybiotin lysine NH
residue

Biotin is linked to the enzyme by an amide bond between

the terminal carboxyl of the biotin side chain and the
-amino group of a lysine residue.
Fatty acid synthesis from acetyl-CoA & malonyl-CoA
occurs by a series of reactions that are:
 catalyzed by individual domains of a very large
polypeptide that includes an ACP domain. This
multienzyme complex is called Fatty Acid Synthase
NADPH serves as electron donor in the two reactions
involving substrate reduction.
The NADPH is produced mainly by the Pentose Phosphate
Pathway.
 H SH
Coenzyme A
H3N+ C COO CH2

CH2 CH2  -mercaptoethylamine

SH NH

Fatty Acid cysteine C O

Synthase CH2

prosthetic groups: CH2

pantothenate
 the thiol of the side- NH
NH2

chain of a cysteine C O
N
ADP-3'- N
residue. HO C H
phosphate
 the thiol of H3C C CH3 O O N N

phosphopantetheine, H2C O P O P O CH2

O
H H
equivalent in structure O O
H H
to part of coenzyme A. phosphopantetheine
O OH


O P O

O
 SH
phosphopantetheine
CH2 of acyl carrier protein
Phosphopantetheine
CH2
(Pant) is covalently -mercaptoethylamine
linked via a phosphate NH

ester to a serine OH of C O
the acyl carrier protein CH2
(ACP) of Fatty Acid
CH2
Synthase. pantothenate
NH

C O

HO C H

H3C C CH3 O NH

H2C O P O CH2 CH
serine
residue
O C O
phosphate
 acetyl-S-CoA HS-CoA malonyl-S-CoA HS-CoA CO2

Pant Cys Pant Cys Pant Cys Pant Cys

1 2 3
SH SH SH S S S S SH

C O C O C O C O

CH3 CH2 CH3 CH2

1 Malonyl/acetyl-CoA-ACP Transacylase COO
Acetyl-CoA-ACP Transacylase C O
2 Malonyl/acetyl-CoA-ACP Transacylase
Malonyl-CoA-ACP Transacylase
CH3
3 Condensing Enzyme (-Ketoacyl Synthase)

The condensation reaction (step 3) involves

decarboxylation of the malonyl, followed by attack of
the acetyl (or acyl).
 NADPH NADP+ H2O NADPH NADP+
Pant Cys Pant Cys Pant Cys Pant Cys

S SH 4 S SH 5 S SH 6 S SH

C O C O C O C O

CH2 CH2 CH CH2

C O HC OH HC CH2

CH3 CH3 CH3 CH3

4 -Ketoacyl-ACP Reductase
5 -Hydroxyacyl-ACP Dehydratase
6 Enoyl-ACP Reductase

4. The -ketone is reduced to an alcohol by e transfer

from NADPH.
5. Dehydration yields a trans double bond.
6. Reduction by NADPH yields a saturated chain.
 Malonyl-S-CoA HS-CoA

Pant Cys Pant Cys Pant Cys

S SH 7 SH S 2 S S

C O C O C O C O

CH2 CH2 CH2 CH2

CH2 CH2 COO CH2

CH3 CH3 CH3

7 Condensing Enzyme
Malonyl-CoA-ACP Transacylase
2 Malonyl/acetyl-CoA-ACP (repeat)(repeat).
Transacylase

Following transfer of the growing fatty acid from

phosphopantetheine to cysteine sulfhydryl, the cycle
begins again, with another malonyl-CoA.
Product release:
When the fatty acid is 16 carbon atoms long, a
Thioesterase catalyzes hydrolysis of the thioester
linking the fatty acid to phosphopantetheine.
The 16-C saturated fatty acid palmitate is the final
product of the Fatty Acid Synthase complex .
Palmitate, a 16-C saturated fatty acid, is the final product
of the Fatty Acid Synthase reactions.
Summary (ignoring H+ & water):
acetyl-CoA + 7 malonyl-CoA + 14 NADPH 
palmitate + 7 CO2 + 14 NADP+ + 8 CoA
Accounting for ATP-dependent synthesis of malonate:
8 acetyl-CoA + 14 NADPH + 7 ATP 
palmitate + 14 NADP+ + 8 CoA + 7 ADP + 7 Pi

Fatty acid synthesis occurs in the cytosol. Acetyl-CoA

generated in mitochondria is transported to the cytosol
via a shuttle mechanism involving citrate.
 -Oxidation & Fatty Acid Synthesis
Compared
 Oxidation Pathway Fatty Acid Synthesis

pathway location mitochondrial matrix cytosol

acyl carriers phosphopantetheine

Coenzyme-A
(thiols) (ACP) & cysteine

e acceptors/donor FAD & NAD+ NADPH

-OH intermediate L D
malonyl-CoA
2-C product/donor acetyl-CoA
(& acetyl-CoA)
Elongation beyond the 16-C length of the palmitate
product of Fatty Acid Synthase occurs in mitochondria and
endoplasmic reticulum (ER).
 Fatty acid elongation within mitochondria involves the
-oxidation pathway running in reverse, but NADPH
serves as electron donor for the final reduction step.
 Fatty acids esterified to CoA are substrates for the ER
elongation machinery, which uses malonyl-CoA as
donor of 2-carbon units.
The reaction sequence is similar to Fatty Acid Synthase
but individual steps are catalyzed by separate proteins.
A family of enzymes designated Fatty Acid Elongases
catalyze the initial condensation step for elongation of
saturated or polyunsaturated fatty acids.
 O
10 9
C
OH

oleate 18:1 cis 9

Desaturases introduce double bonds at specific

positions in a fatty acid chain.
Mammalian cells are unable to produce double bonds
at certain locations, e.g., 12.
Thus some polyunsaturated fatty acids are dietary
essentials, e.g., linoleic acid, 18:2 cis 9,12 (18 C atoms
long, with cis double bonds at carbons 9-10 & 12-13).
 O
10 9
C
OH

oleate 18:1 cis 9

Formation of a double bond in a fatty acid involves the

following endoplasmic reticulum membrane proteins in
mammalian cells:
 NADH-cyt b5 Reductase, a flavoprotein with FAD
as prosthetic group.
 Cytochrome b5
 Desaturase
The overall reaction for desaturation of stearate (18:0) to
form oleate (18:1 cis 9) is:

stearate + NADH + H+ + O2  oleate + NAD+ + 2H2O

There is a 4-electron reduction of O2  2 H2O as a fatty

acid is oxidized to form a double bond.
 2e pass from NADH to the desaturase via the
FAD-containing reductase & cytochrome b5, the
order of electron transfer being:
NADH  FAD  cyt b5  desaturase
 2e are extracted from the fatty acid as the double
 Control of fatty acid synthesis is exerted
mainly at acetyl-CoA carboxylase step

• Citrate & Insulin activate the enzyme

• Acyl-CoA & Glucagon & Epinephrine

inhibite the enzyme
Thank you for your attention