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Molecular Biochemistry II

Fatty Acid Oxidation

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
2

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

ATP
+
HCO
3
activated by AMP, which
is high in concentration
ADP + Pi
when [ATP] is low.
Acetyl-CoA Carboxylase
is inhibited when
phosphorylated by AMP-

OOC

CH2

SCoA

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

O
C

SCoA

malonyl-CoA

Activated 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.

-Oxidation
Pathway:
Step 1. Acyl-CoA
Dehydrogenase
catalyzes oxidation
of the fatty acid
moiety of acyl-CoA
to produce a double

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
bond between carbon atoms
2 & 3.
O
There are different Acyl-CoA HDehydrogenases
for
short (4-6 C), medium (6-10 C),long and very
H3C (CH2)n C CH2 C SCoA
long (12-18 C) chain
fatty acids.
OH
Very Long Chain Acyl-CoA Dehydrogenase
is
bound to the inner mitochondrial
membrane. The
+
H + NADH located in the
others are soluble enzymes
O
O
mitochondrial matrix.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


eacceptor Hfor Acyl-CoA
Dehydrogenase. Proposed
O
mechanism:
H3C (CH2)n C CH2 C SCoA
A Glu side-chain carboxyl extracts a proton from the
-carbon of
OHthe substrate, facilitating transfer of 2
e with H+ (a hydride) from the position to FAD.
H+ + NADH
The
reduced FAD accepts a 2nd H+, yielding FADH2.
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
H the thioester
O
The carbonyl O of
substrate is
hydrogen
bonded
to
the
2'-OH
of
the
ribityl
H3C (CH2)n C CH2 C SCoA
moiety of FAD, giving this part of FAD a role
in positioning theOHsubstrate and increasing
acidity of+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

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.

Ribose

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
The reactive GluHand FAD
are on opposite
sides of
at SCoA
the active site.
H3Cthe
(CHsubstrate
2)n C CH2 C
Thus the reaction
OHis stereospecific, yielding a
trans 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
LhydroxyacylCoenzyme 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

-ketoacyl-CoA

HSCoA

Step 4.
-Ketothiolase
catalyzes thiolytic
cleavage.

SCoA

H3C (CH2)n C

SCoA + CH3 C

fatty acyl-CoA
(2 C shorter)

SCoA

acetyl-CoA

-Ketothiolase

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 & P i.

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
2 A? ()________
How many times is the -oxidation pathway repeated during oxidation
of a 12-C fatty acid? _________

How many each of NADH______, FADH2______, and Acetyl CoA______


5
6
are produced, per 12-carbon fatty acid,5 in the -oxidation pathway?
Oxidation of each acetyl CoA in Krebs cycle yields 3 NADH and one
FADH2 (from succinate), resulting in additional production of
_______NADH and _______FADH2.
18

6 the yield is a total of _______NADH and _______FADH2.


Thus

23
11 of ATP are produced per
In the respiratory chain, approx.
2.5 ~ bonds
NADH and 1.5 ~ 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 1274
C fatty acid.
Add to this the ~P bonds of GTP produced in Krebs Cycle (one GTP per
acetyl-CoA) for a total of _______ ~P bonds produced.
Summing input and80output yields a total of _______ ~P bonds per 12-C
fatty acid oxidized. Does fat yield more78
energy than carbohydrate?
_______

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
H 2O 2:
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 CO 2.
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

-Hydroxybutyrate
CH3
+
H
Dehydrogenase
C O NADH
catalyzes reversible
interconversion of
CH2
the ketone bodies
COO
acetoacetate &
acetoacetate
-hydroxybutyrate.

CH3
+

NAD HO

CH
CH2
COO

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.