Download Fatty Acid Oxid

Molecular Biochemistry II
Fatty Acid Oxidation
Copyright © 1999-2007 by Joyce J. Diwan.
All rights reserved.

4
b

3
2
O
C
1
O
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 b-oxidation pathway, because oxidation occurs at
the b-carbon (C-3).
O
H2C
OH
HC
OH
O
HO
H2C
C
OH
glycerol
fatty acid
H2C
O
C
O
R
HC
O
C
O
R
H2C
O
C
R
R
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
C
OH
glycerol
fatty acid
H2C
O
C
O
R
HC
O
C
O
R
H2C
O
C
R
R
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
HO
ATP
CH
CH2 OH
glycerol
CH2 OH
ADP
HO
1
+
NAD
H + CH OH
2
NADH
C
CH
CH2 O
PO3
glycerol-3-P

O
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.

4
b

3
2
O
C
1
O
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

O
O
P
O
R
O
O
O
N
P

O
O
CH2

H
2 Pi
R
O
C
H
OH
H
OH
P
N
N
O
CH2
O
CoA
SH
H
O
H
H
OH
H
OH
N
acyladenylate
AMP
O
R
NH2
N
O
O
N
ATP
H
PPi
O
N
O
O
P

C
N
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
Mitochondrion
b-Oxidation
pathway:
Fatty acids are
degraded in the
mitochondrial matrix
via the b-Oxidation
Pathway.
b-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
b-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
R
CH CH2 COO +
C
carnitine
O
SCoA
Carnitine Palmitoyl
Transferase
Transfer of the fatty
acid moiety across
the mitochondrial
inner membrane
involves carnitine.
R
C
CH3
H3C
+
N
CH3
O
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
O
O
R-C-SCoA HO-carnitine
1
HO-carnitine R-C-SCoA
3
2
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
O
O
R-C-SCoA HO-carnitine
1
HO-carnitine R-C-SCoA
3
2
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
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
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.
SCoA
Acetyl-CoA
Carboxylase
(inhibited by
AMP-Activated
Kinase)
O
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
b-Oxidation
3
2
1
Pathway:
H3C (CH2)n C C C SCoA
b
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 atomsH22O& 3.
H
O
There are different Acyl-CoA Dehydrogenases
for short
(4-6 C), medium (6-10H3C),
long
and very long
(12-18 C)
C (CH
SCoA
2)n C CH2 C
chain fatty acids.
OH
Very Long Chain Acyl-CoA Dehydrogenase is bound to
+
H
+ NADH
the inner mitochondrial membrane.
The others are soluble
O
O
NAD +
enzymes located in the mitochondrial
matrix.
H
3
H3C (CH2)n C
b
H
FAD
H
O
2
C
H
C
1
SCoA
fatty acyl-CoA
glutamate
H
H3N+
Acyl-CoA Dehydrogenase
FADH2
H
O
H3C (CH2)n C
C
C
H
COO
C
CH2
CH2
SCoA
2
trans- -enoyl-CoA
C
O
O
H2O
FAD is the prosthetic group that functions as e acceptor
H Dehydrogenase.
O
for Acyl-CoA
Proposed mechanism:
H
C SCoA
(CH
2)n C CH2carboxyl
A3CGlu
side-chain
extracts
a proton from the
-carbon ofOH
the substrate, facilitating transfer of 2 e
with H+ (a hydride) from the b position to FAD.
H+ + NADH
nd H+, yielding FADH .
The reduced
FAD
accepts
a
2
2
+
NAD
O
O
H
3
H3C (CH2)n C
b
H
FAD
H
O
2
C
H
C
1
SCoA
fatty acyl-CoA
Acyl-CoA Dehydrogenase
FADH2
H
O
H3C (CH2)n C
C
C
H
SCoA
trans-2-enoyl-CoA
H2O
The carbonyl O of theH thioester
O substrate is hydrogen
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
O
H
C
C
N
O

H3C
C
C
C
NH
H3C
C
C
C
C
C
H
N
H
C
+
2e +2H
O
N
H
N
H3C
C
C
C
NH
H3C
C
C
C
C
C
H
CH2
FAD
N
O
N
H
CH2
HC
OH
HC
OH
HC
OH O
H2C
C
O
P
O-
Adenine
O
O
P
O-
O
Ribose
FADH2
HC
OH
HC
OH
HC
OH O
H2C
O
P
O-
Adenine
O
O
P
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
b
H
FAD
H
O
2
C
H
C
1
SCoA
fatty acyl-CoA
Acyl-CoA Dehydrogenase
FADH2
H
O
H3C (CH2)n C
C
C
H
SCoA
trans-2-enoyl-CoA
H2O
O on opposite sides of the
The reactive Glu andH FAD are
substrateHat
the active site.
SCoA
3C (CH2)n C CH2 C
Thus the reaction is OH
stereospecific, yielding a trans
double bond in enoyl-CoA.
H+ + NADH
Matrix
H+ + NADH NAD+ + 2H+
2 e
Q
I
2H+ + ½ 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.
H
Step 2.
Enoyl-CoA
Hydratase
catalyzes
stereospecific
hydration of the
trans double bond
produced in the
1st step, yielding
L-hydroxyacylCoenzyme A.
3
H3C (CH2)n C
b
H
FAD
H
O
2
C
H
C
1
fatty acyl-CoA
Acyl-CoA Dehydrogenase
FADH2
H
O
H3C (CH2)n C
C
C
H
H2O
SCoA
trans-2-enoyl-CoA
Enoyl-CoA Hydratase
H
O
H3C (CH2)n C CH2 C
OH
H+ + NADH
SCoA
SCoA
3-L-hydroxyacyl-CoA
H
H2O
H
O
H3C (CH2)n C CH2 C
Step 3.
Hydroxyacyl-CoA
Dehydrogenase
catalyzes oxidation
of the hydroxyl in
the b position (C3)
to a ketone.
NAD+ is the
electron acceptor.
NAD +
H+ + NADH
OH
SCoA
3-L-hydroxyacyl-CoA
Hydroxyacyl-CoA
Dehydrogenase
O
O
H3C (CH2)n C CH2 C
SCoA
b-ketoacyl-CoA
b-Ketothiolase
HSCoA
O
O
H3C (CH2)n C
SCoA + CH3 C
fatty acyl-CoA
(2 C shorter)
SCoA
acetyl-CoA
H
H3N+
C
COO
CH2
SH
cysteine
O
O
H3C (CH2)n C CH2 C
SCoA
b-ketoacyl-CoA
HSCoA
O
O
H3C (CH2)n C
SCoA + CH3 C
SCoA
Step 4.
fatty acyl-CoA
acetyl-CoA
(2 C shorter)
b-Ketothiolase
b-Ketothiolase
catalyzes thiolytic
cleavage.
A cysteine S attacks the b-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 b-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 b-oxidation pathway:
fatty acyl-CoA + FAD + NAD+ + HS-CoA 
fatty acyl-CoA (2 C less) + FADH2 + NADH + H+
+ acetyl-CoA
The b-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
I
2H+ + ½ 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
I
2H+ + ½ 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
I
2H+ + ½ 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
2
esterifying it to coenzyme A? ()________
How many times is the b-oxidation pathway repeated during oxidation of a 12-C
5
fatty acid? _________
5
6 are
5
How many each of NADH______,
FADH2______,
and Acetyl CoA______
produced, per 12-carbon fatty acid, in the b-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
18
6
_______FADH
2.
23
11
Thus the yield is a total of _______NADH
and _______FADH
2.
In the respiratory chain, approx. 2.5 ~ bonds of ATP are produced per NADH and
1.5 ~ bonds of ATP per FADH2 (electrons entering the respiratory chain via
74
coenzyme Q). Thus from reoxidation of NADH and FADH2 a total of _______
~ bonds of ATP are produced per 12-C fatty acid.
Add to this the ~P bonds of GTP produced in Krebs Cycle (one GTP per acetyl80 ~P bonds produced.
CoA) for a total of _______
78
Summing input and output yields a total of _______
~P bonds per 12-C fatty acid
YES
oxidized. Does fat yield more energy than carbohydrate? _______
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 b-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 b-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 b-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.
b-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 b-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 & b-hydroxybutyrate.
Ketone body
synthesis:
b-Ketothiolase. The
final step of the boxidation 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
O
C
acetyl-CoA
SCoA + H3C
HSCoA
H3C
O
H3C
C
SCoA
O
O
Thiolase
O
O
C
H2
C C
SCoA
acetyl-CoA
SCoA
acetoacetyl-CoA
acetyl-CoA HSCoA

C
C
HMG-CoA Synthase
OH
H2
C C
O
H2
C
CH3
C
SCoA
HMG-CoA
HMG-CoA Lyase
O

O
C
O
H2
C
acetoacetate
C
O
CH3 + H3C
C
SCoA
acetyl-CoA
b-Hydroxybutyrate Dehydrogenase
b-Hydroxybutyrate
CH3
+
H 
Dehydrogenase
C O NADH
catalyzes reversible
interconversion of
CH2
the ketone bodies
COO
acetoacetate &
acetoacetate
b-hydroxybutyrate.
CH3
+
NAD HO
CH
CH2
COO
D-b-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.