Molecular Biochemistry II

Fatty Acid Oxidation

Copyright 1999-2007 by Joyce J. Diwan.

All rights reserved.

C
1

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
H2C

OH

HC

OH

O
HO

H2C

OH

glycerol

fatty acid

H2C

C
O

HC

C
O

H2C

triacylglycerol

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
H2C

OH

HC

OH

O
HO

H2C

OH

glycerol

fatty acid

H2C

C
O

HC

C
O

H2C

triacylglycerol

Lipid digestion, absorption, transport will be covered

separately.
Lipases hydrolyze triacylglycerols, releasing 1 fatty acid
at a time, yielding diacylglycerols, & eventually glycerol.

CH2 OH ATP
HO

CH
CH2 OH

glycerol

CH2 OH

ADP
1

HO

NAD+

CH
CH2 O

PO3

glycerol-3-P

H+ +
NADH CH2 OH
C

CH2 O

PO3

dihydroxyacetone-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.

C
1

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:

Fatty acids must be esterified to Coenzyme A before they
can undergo oxidative degradation, be utilized for
synthesis of complex lipids, or be attached to proteins as
lipid anchors.
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.

Acyl-CoA
Synthases
Exergonic PPi
(P~P) hydrolysis,
catalyzed by
Pyrophosphatase,
makes the coupled
reaction
spontaneous.
2 ~P bonds of ATP
are cleaved.
The acyl-CoA
product includes
one "~" thioester
linkage.

NH2

Fatty acid activation

O

fatty acid
O

P
O

CH2

2 Pi

OH

H
OH

N
O

CH2

O
CoA

SH

OH

H
OH

acyladenylate

AMP
O
R

NH2

O
O

ATP

PPi
O

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

Mitochondrion

-Oxidation
pathway:
Fatty acids are
degraded in the
mitochondrial matrix
via the -Oxidation
Pathway.

-Oxidation
pathway in
matrix

Fatty acyl-CoA formed in cytosol by enzymes

of outer mitochondrial membrane & ER

For most steps of the pathway there are multiple enzymes

specific for particular fatty acid chain lengths.

Many of the constituent Mitochondrion

enzymes are soluble
proteins located in the
-Oxidation
mitochondrial matrix.
pathway in
But enzymes specific
for very long chain
fatty acids are
associated with the
inner membrane,
facing the matrix.

matrix

Fatty acyl-CoA formed in cytosol by enzymes

of outer mitochondrial membrane & ER

Fatty acyl-CoA formed outside can pass through the

outer mitochondrial membrane (which has large VDAC
channels), but cannot penetrate the inner membrane.

CH3
H3C

N
CH3

CH2

OH

CH CH2 COO +

carnitine

SCoA

Carnitine Palmitoyl
Transferase

Transfer of the fatty

acid moiety across
the mitochondrial
inner membrane
involves carnitine.

R
C
CH3
H3C

N
CH3

O
CH2

CH CH2 COO

+ HSCoA

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

R-C-SCoA HO-carnitine
1

HO-carnitine R-C-SCoA
3

HSCoA R-C-O-carnitine
O

R-C-O-carnitine HSCoA
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

R-C-SCoA HO-carnitine
1

HO-carnitine R-C-SCoA
3

HSCoA R-C-O-carnitine
O

R-C-O-carnitine HSCoA
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

SCoA

acetyl-CoA
O

OOC

CH2

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.

AMP-Activated Kinase, a
H3C C
sensor of cellular energy
acetyl-CoA
levels, is allosterically

ATP
+
HCO
3
activated by AMP, which is
high in concentration when
ADP + Pi
[ATP] is low.

SCoA

Acetyl-CoA
Carboxylase
(inhibited by
AMP-Activated
Kinase)

Acetyl-CoA Carboxylase

OOC CH2 C SCoA

is inhibited when
malonyl-CoA
phosphorylated by AMPActivated Kinase, leading to decreased malonyl-CoA.

The decrease in malonyl-CoA concentration leads to

increased activity of Carnitine Palmitoyl Transferase I.
Increased fatty acid oxidation then generates acetyl-CoA,
for entry into Krebs cycle with associated ATP production.

AMP-Activated Kinase functions under a variety of

conditions that lead to depletion of cellular ATP
(reflected as increased AMP), including:
glucose deprivation, exercise, hypoxia & ischaemia.
AMP-Activated Kinase regulates various metabolic
pathways to:
promote catabolism leading to ATP synthesis
(e.g., stimulation of fatty acid oxidation)
inhibit energy-utilizing anabolic pathways
(e.g., fatty acid synthesis).
AMP-Activated Kinase in the hypothalamus of the
brain is involved also in regulation of food intake.

H H O
-Oxidation
3
2
1
H3C (CH2)n C C C SCoA
Pathway:

fatty acyl-CoA
Step 1. Acyl-CoA
H H
FAD
Dehydrogenase
Acyl-CoA Dehydrogenase
FADH2
catalyzes oxidation
H O
of the fatty acid
H3C (CH2)n C C C SCoA
moiety of acyl-CoA
trans-2-enoyl-CoA
H
to produce a double
bond between carbon atoms H22O
& 3.
H
O
There are different Acyl-CoA Dehydrogenases
for short
(4-6 C), medium (6-10HC),
long and very longSCoA
(12-18 C)
3C (CH2)n C CH2 C
chain fatty acids.
OH
Very Long Chain Acyl-CoA Dehydrogenase is bound to the
H+ + NADH The others are soluble
inner mitochondrial membrane.
O matrix.
O
enzymes located in the mitochondrial
NAD+

H
3

H3C (CH2)n C

H
FAD

O
2

C
H

SCoA

fatty acyl-CoA

Acyl-CoA Dehydrogenase

FADH2
H3C (CH2)n C
H

SCoA

trans-2-enoyl-CoA

H2O

FAD is the prosthetic group that functions as e acceptor

H Dehydrogenase.
O
for Acyl-CoA
Proposed mechanism:
a proton from the carbon of theOHsubstrate, facilitating transfer of 2 e with
H+ (a hydride) from the position to FAD.

H
C SCoA
2)n C CH2
A3CGlu(CH
side-chain
carboxyl
extracts

H+ + NADH

nd H+, yielding FADH .

The reduced
FAD
accepts
a
2
2
+

NAD

H
3

H3C (CH2)n C

H
FAD

O
2

C
H

SCoA

fatty acyl-CoA

Acyl-CoA Dehydrogenase

FADH2
H3C (CH2)n C
H

SCoA

trans-2-enoyl-CoA

H2O
O substrate is hydrogen
The carbonyl O of theH thioester
bonded toHthe
2'-OH
of the
ribityl
moiety of FAD, giving
C
C
CH
C
SCoA
(CH
)
3
2
2 n
this part of FAD a role in positioning the substrate and
increasing acidity of OH
the substrate -proton.
H+ + NADH

dimethylisoalloxazine
H3 C

H3 C

H
C

C
H

FAD

C
C

C
C

O
C

2e +2H

NH
C

CH2
HC

OH

HC

OH

HC

OH O

H2C

P
O-

Adenine

O
O

P
O-

Ribose

H3C

H3C

H
C

C
H

C
C

FADH2

H
N

O
C
C

CH2

N
H

HC

OH

HC

OH

HC

OH O

H2C

P
O-

NH
C

Adenine

O
O

Ribose

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.

H
3

H3C (CH2)n C

H
FAD

O
2

C
H

SCoA

fatty acyl-CoA

Acyl-CoA Dehydrogenase

FADH2
H3C (CH2)n C
H

SCoA

trans-2-enoyl-CoA

H2O
O on opposite sides of the
The reactive Glu andHFAD are
substrateHat3Cthe(CH
active
site.
SCoA
2)n C CH2 C
Thus the reaction is stereospecific,
yielding a trans
OH
double bond in enoyl-CoA.
H+ + NADH

Matrix
H+ + NADH NAD+ + 2H+

2 e
Q

2H+ + O2 H2O

III

IV

++
4H

4H

cyt c

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.

Step 2.
Enoyl-CoA
Hydratase
catalyzes
stereospecific
hydration of the
trans double bond
produced in the 1st
step, yielding
L-hydroxyacylCoenzyme A.

H3C (CH2)n C

H
FAD

O
2

C
H

fatty acyl-CoA

Acyl-CoA Dehydrogenase

FADH2
H3C (CH2)n C
H

SCoA

trans-2-enoyl-CoA

Enoyl-CoA Hydratase

H2O
H

H3C (CH2)n C CH2 C

OH
H+ + NADH

SCoA

SCoA

3-L-hydroxyacyl-CoA

H
H2O
H

H3C (CH2)n C CH2 C

Step 3.
Hydroxyacyl-CoA
Dehydrogenase
catalyzes oxidation
of the hydroxyl in
the position (C3)
to a ketone.
NAD+ is the
electron acceptor.

NAD+
H+ + NADH

OH

SCoA

3-L-hydroxyacyl-CoA

Hydroxyacyl-CoA
Dehydrogenase
O

H3C (CH2)n C CH2 C

SCoA

-ketoacyl-CoA

HSCoA

-Ketothiolase
O

H3C (CH2)n C

SCoA + CH3 C

fatty acyl-CoA
(2 C shorter)

SCoA

acetyl-CoA

H3C (CH2)n C CH2 C

SCoA

-ketoacyl-CoA

HSCoA
O

H3C (CH2)n C

SCoA + CH3 C

SCoA

fatty acyl-CoA
acetyl-CoA
Step 4.
(2 C shorter)
-Ketothiolase
-Ketothiolase
catalyzes thiolytic
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.

ADP + Pi ATP

Matrix
H+ + NADH NAD+ + 2H+

2 e
Q

2H+ + O2 H2O

III

IV

Fo
++

4H

F1

4H

cyt c

2H

3H+

Intermembrane Space

NADH produced during fatty acid oxidation is reoxidized

by transfer of 2e to respiratory chain complex I.
Transfer of 2e from complex I to oxygen causes sufficient
proton ejection to yield approximately 2.5 ATP.
Recall that 4H+ enter the matrix per ATP synthesized,
taking into account transmembrane flux of ADP, ATP & Pi.

ADP + Pi ATP

Matrix
H+ + NADH NAD+ + 2H+

2 e
Q

2H+ + O2 H2O

III

IV

Fo
++

4H+

F1

4H+

cyt c

2H+

3H+

Intermembrane Space

FADH2 of Acyl-CoA Dehydrogenase is reoxidized by

transfer of 2e via ETF to CoQ of the respiratory chain.
H+ ejection from the matrix that accompanies transfer of
2e from coenzyme Q to oxygen, leads to production of
approximately 1.5 ATP.

 Acetyl-CoA can enter Krebs cycle, yielding

additional NADH, FADH2, and ATP.
Fatty acid oxidation is a major source of cell ATP.

ADP + Pi ATP

Matrix
H+ + NADH NAD+ + 2H+

Problem
(See web
handout,
tutorial)

2 e
Q

2H+ + O2 H2O

III

IV

Fo
++

4H

F1

4H

cyt c

2H

3H+

Intermembrane Space

Catabolism of two 6-C glucose through Glycolysis, Krebs,

& ox phos yields about 60 ~P bonds of ATP (30/glucose).
Compare energy yield oxidizing a 12-C fatty acid. Assume:
1.5 ATP produced per FADH2 reoxidized in the
respiratory chain (via coenzyme Q).
2.5 ATP produced per NADH reoxidized in the
respiratory chain.

How many "high energy" (~) bonds are utilized in activating the fatty acid, by
esterifying it to coenzyme A? ()________
2
How many times is the -oxidation pathway repeated during oxidation of a 12-C fatty
acid? _________5
How many each of NADH______,
5 FADH2______,
5 and Acetyl CoA______6 are
produced, per 12-carbon fatty acid, in the -oxidation pathway?
Oxidation of each acetyl CoA in Krebs cycle yields 3 NADH and one FADH 2 (from
succinate), resulting in additional production of _______NADH
18 and _______FADH 2.
Thus6 the yield is a total of _______NADH and _______FADH .
2

11 produced per NADH and 1.5

In the respiratory chain, approx.232.5 ~ bonds of ATP are
~ bonds of ATP per FADH2 (electrons entering the respiratory chain via coenzyme Q).
Thus from reoxidation of NADH and FADH2 a total of _______
~ bonds of ATP
are produced per 12-C fatty acid.
74
Add to this the ~P bonds of GTP produced in Krebs Cycle (one GTP per acetyl-CoA)
for a total of _______ ~P bonds produced.
80 yields a total of _______ ~P bonds per 12-C fatty acid
Summing input and output
oxidized. Does fat yield more energy than carbohydrate?
_______
78

YES

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
Hydroxyacyl-CoA Dehydrogenases for medium &
short chain length fatty acids
Medium Chain -Ketothiolase
the trifunctional protein complex
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
sudden infant death (SIDS).
Hereditary deficiency of Medium Chain Acyl-CoA
Dehydrogenase (MCAD), the most common genetic
disease relating to fatty acid catabolism, has been linked
to 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 &
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 to be the correct substrate for EnoylCoA 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 very 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 involved in transfer of fatty acids into and
out of peroxisomes.

Serious genetic diseases are associated with defects in or

deficiency of enzymes of the peroxisomal -oxidation
system.
Peroxisomes also contain enzymes for an essential
-oxidation pathway that degrades fatty acids having
methyl branches, such as phytanic acid, a breakdown
product of chlorophyll.

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.

Ketone body
synthesis:
-Ketothiolase. The
final step of the oxidation pathway
runs backward.
HMG-CoA
Synthase catalyzes
condensation with a
3rd acetate moiety
(from acetyl-CoA).
HMG-CoA Lyase
cleaves HMG-CoA to
yield acetoacetate &
acetyl-CoA.

O
H3C

acetyl-CoA

SCoA + H3C

Thiolase

HSCoA

O
H3C

O
H3C

SCoA

acetyl-CoA HSCoA
O

SCoA

acetyl-CoA

H2
C C

SCoA

acetoacetyl-CoA

HMG-CoA Synthase
OH

H2
C C

H2
C C

CH3

SCoA

HMG-CoA

HMG-CoA Lyase
O

H2
C C

acetoacetate

O
CH3 + H3C

SCoA

acetyl-CoA

-Hydroxybutyrate
Dehydrogenase
catalyzes reversible
interconversion of
the ketone bodies
acetoacetate &
-hydroxybutyrate.

-Hydroxybutyrate Dehydrogenase
CH3
C

H
+
O NADH NAD HO

CH3
CH

CH2

CH2

COO

COO

acetoacetate

D--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.
While ketone bodies thus function as an alternative fuel,
amino acids must be degraded to supply input to
gluconeogenesis when hypoglycemia occurs, since acetate
cannot be converted to glucose.