β Oxidation
Mitochondrial oxidation of fatty acids
takes place in three stages (Fig. 16-7).
In the first stage-β oxidation-the fatty
acids undergo oxidative removal of
successive two-carbon units in the
form of acetyl-CoA, starting from the
carboxyl end of the fatty acyl chain.
For example, the 16-carbon fatty acid
palmitic acid (palmitate at pH 7)
undergoes seven passes through this
oxidative sequence, in each pass losing
two carbons as acetyl-CoA. At the end
of seven cycles the last two carbons of
palmitate (originally C-15 and C-16)
are left as acetyl-CoA. The overall
result is the conversion of the 16carbon chain of palmitate to eight twocarbon acetyl-CoA molecules.
Formation of each molecule of acetylCoA requires removal of four
hydrogen atoms (two pairs of electrons
and four H+) from the fatty acyl moiety
by the action of dehydrogenases.
In the second stage of fatty acid
oxidation the acetyl residues of
acetyl-CoA are oxidized to CO2
via the citric acid cycle, which
also takes place in the
mitochondrial matrix. AcetylCoA derived from fatty acid
oxidation thus enters a final
common pathway of oxidation
along with acetyl-CoA derived
from glucose via glycolysis and
pyruvate oxidation (see Fig.
15-1).
The first two stages of fatty
acid oxidation produce the
reduced electron carriers
NADH and FADH2, which in
the third stage donate electrons
to the mitochondrial respiratory
chain, through which the
electrons are carried to oxygen
(Fig. 16-7). Coupled to this
flow of electrons is the
Figure 16-7 Stages of fatty acid oxidation. Stage 1:
A long-chain fatty acid is oxidized to yield acetyl
residues in the form of acetyl-CoA. Stage 2: The acetyl
residues are oxidized to CO2 via the citric acid cycle.
Stage 3: Electrons derived from the oxidations of
stages 1 and 2 are passed to O2 via the mitochondrial
respiratory chain, providing the energy for ATP
synthesis by oxidative phosphorylation.
phosphorylation of ADP to
ATP, to be described in
Chapter 18. Thus energy
released by fatty acid oxidation
is conserved as ATP.
We will now look in more
detail at the first stage of fatty
acid oxidation, for the simple
case of a saturated chain with
an even number of carbons,
and for the slightly more
complicated cases of
unsaturated and odd-number
chains. We then consider the
regulation of fatty acid
oxidation, and the β-oxidative
processes occurring in
organelles other than
mitochondria.
β Oxidation of Saturated Fatty
Acids Has Four Basic Steps
Four enzyme-catalyzed
reactions are involved in the
first stage of fatty acid
oxidation (Fig. 16-Sa). First,
dehydrogenation produces a
double bond between the α and
β carbon atoms (C-2 and C-3),
yielding a trans-Δ2-enoylCoA. The symbol Δ2
designates the position of the
double bond. (It may be helpful
to review fatty acid
nomenclature, described on p.
240.) The new double bond has
the trans configuration; recall
that naturally occurring
unsaturated fatty acids
normally have their double
bonds in the cis configuration.
We shall consider the
significance of this difference
later.
The enzyme responsible for
this first step, acyl-CoA
Figure 16-8 The fatty acid oxidation (β-oxidation)
pathway. (a) In each pass through this sequence, one
acetyl residue (shaded in red) is removed in the form of
acetyl-CoA from the carboxyl end of palmitate (C16),
which enters as palmitoyl-CoA. (b) Six more passes
through the pathway yield seven more molecules of
acetyl-CoA, the seventh arising from the last two
carbon atoms of the 16-carbon chain. Eight molecules
dehydrogenase, includes FAD
as a prosthetic group. The
electrons removed from the
fatty acyl-CoA are transferred
to the FAD, and the reduced
form of the dehydrogenase then
immediately donates its
electrons to an electron carrier,
the electron-transferring
flavoprotein (ETFP). ETFP, an
integral protein of the inner
mitochondrial membrane, is
one of the electron carriers of
the mitochondrial respiratory
chain (Fig. 16-9). The transfer
of a pair of electrons from the
FADH2 of acyl-CoA
dehydrogenase to O2 via the
respiratory chain provides the
energy for the synthesis of two
ATP molecules.
The oxidation catalyzed by
acyl-CoA dehydrogenase is
analogous to succinate
dehydrogenation in the citric
acid cycle (p. 457); in both
reactions the enzyme is bound
to the inner membrane, a
double bond is introduced into
a carboxylic acid between the α
and β carbons, FAD is the
electron acceptor, and electrons
from the reaction ultimately
enter the respiratory chain and
are carried to O2 with the
concomitant synthesis of two
ATP molecules per electron
pair.
of acetyl-CoA are formed in all.
Figure 16-9
Electrons
removed from
fatty acids
during β
oxidation pass
into the
mitochondrial
respiratory
chain and
eventually to
O2. The
structures I
through IV are
enzyme
complexes that
catalyze
portions of the
electron
transfer to
oxygen. Fatty
acyl-CoA
dehydrogenase
feeds electrons
into an
electrontransferring
flavoprotein
(ETFP)
containing an
iron-sulfur
center, which
in turn reduces
a lipid-soluble
electron
carrier,
ubiquinone
(UQ, or
coenzyme Q).
βHydroxyacylCoA
dehydrogenase
transfers
electrons to
NAD+, and the
resulting
NADH is
reoxidized by
NADH
dehydrogenase
(Complex I of
the respiratory
chain).
Propionate
produced from
odd-chain fatty
acids is
converted to
succinate.
Succinate
dehydrogenase,
which acts in
the citric acid
cycle (p. 457),
feeds electrons
into the
respiratory
chain at
Complex II.
Cytochrome c
(cyt c) is a
soluble
electron carrier
that transfers
electrons
between
Complexes III
and IV. All of
these transfers
are described
in detail in
Chapter 18.
In the second step of the fatty acid oxidation cycle (Fig. 16-8a), water is added to the
double bond of the trans-Δ2-enoyl-CoA to form the L stereoisomer of β-hydroxyacylCoA (also designated β-hydroxyacyl-CoA). This reaction, catalyzed by enoyl-CoA
hydratase, is formally analogous to the fumarase reaction in the citric acid cycle, in
which H2O adds across an α-β double bond (p. 458).
In the third step, the L-β-hydroxyacyl-CoA is dehydrogenated to form β-ketoacylCoA by the action of β-hydroxyacyl-CoA dehydrogenase (Fig. 16-8a); NAD+ is the
electron acceptor. This enzyme is absolutely specific for the r. stereoisomer. The
NADH formed in this reaction donates its electrons to NADH dehydrogenase
(Complex I), an electron carrier of the respiratory chain (Fig. 16-9). Three ATP
molecules are generated from ADP per pair of electrons passing from NADH to O2
via the respiratory chain. The reaction catalyzed by β-hydroxyacyl-CoA
dehydrogenase is closely analogous to the malate dehydrogenase reaction of the citric
acid cycle (p. 459).
The fourth and last step of the fatty acid oxidation cycle is catalyzed by acyl-CoA
acetyltransferase (more commonly called thiolase), which promotes reaction of βketoacyl-CoA with a molecule of free coenzyme A to split off the carboxyl-terminal
two-carbon fragment of the original fatty acid as acetyl-CoA. The other product is the
coenzyme A thioester of the original fatty acid, now shortened by two carbon atoms
(Fig. 16-8a). This reaction is called thiolysis, by analogy with the process of
hydrolysis, because the β-ketoacyl-CoA is cleaved by reaction with the thiol group of
coenzyme A.
The carbon-carbon single bond that connects methylene (-CH2-) groups in fatty acids
is relatively stable. The β-oxidation sequence represents an elegant solution to the
problem of breaking these bonds. The first three reactions of β oxidation have the
effect of creating a much less stable C-C bond, in which one of the carbon atoms (the
a carbon, C-2) is bonded to two carbonyl carbons. The ketone function on the β
carbon (C-3) makes it a good point for nucleophilic attack by -SH of coenzyme A,
catalyzed by thiolase. The acidity of the a carbon makes the terminal -CH2-CO-S-CoA
a good leaving group, facilitating breakage of the α-β bond.
The Four Steps Are Repeated to Yield Acetyl-CoA and ATP
In one pass through the fatty acid oxidation sequence, one molecule of acetyl-CoA,
two pairs of electrons, and four H+ ions are removed from the long-chain fatty acylCoA, to shorten it by two carbon atoms. The equation for one pass, beginning with the
coenzyme A ester of our example, palmitate, is
Palmitoyl-CoA + CoA + FAD + NAD+ + H2O
myristoyl-CoA + acetyl-CoA +
FADH2 + NADH + H+
............ (16-2)
Following removal of one acetyl-CoA unit from palmitoyl-CoA, the coenzyme A
thioester of the shortened fatty acid remains, in this case the 14-carbon myristate. The
myristoyl-CoA can now enter the β-oxidation sequence and go through another set of
four reactions, exactly analogous to the first, to yield a second molecule of acetylCoA and lauroylCoA, the coenzyme A thioester of the 12-carbon laurate. Altogether,
seven passes through the β-oxidation sequence are required to oxidize one molecule
of palmitoyl-CoA to eight molecules of acetyl-CoA (Fig. 16-8b). The overall equation
is
Palmitoyl-CoA + 7CoA + 7FAD + 7NAD+ + 7H2O
8 acetyl-CoA + 7FADH2 +
7NADH + 7H+
............(16-3)
Each molecule of FADH2 formed during oxidation of the fatty acid donates a pair of
electrons to ETFP of the respiratory chain (Fig. 16-9); two molecules of ATP are
generated during the ensuing transfer of the electron pair to O2 and the coupled
oxidative phosphorylations. Similarly, each molecule of NADH formed delivers a pair
of electrons to the mitochondrial NADH dehydrogenase; the subsequent transfer of
each pair of electrons to O2 results in formation of three molecules of ATP. Thus five
molecules of ATP are formed for each two-carbon unit removed in one pass through
the sequence as it occurs in animal tissues, such as the liver or heart. Note that water
is also produced in this process. Condensation of ADP and Pi releases one H2O for
each ATP formed, and transfer of electrons from NADH or FADH2 to O2 yields one
H2O per electron pair. R,eduction of O2 by NADH also consumes one H+ per NADH:
NADH + H+ + 2O2
NAD+ + H2O. In hibernating animals, fatty acid oxidation
provides metabolic energy, heat, and water-all essential for survival of an animal that
neither eats nor drinks for long periods (Box 16-1).
The overall equation for the oxidation of palmitoyl-CoA to eight molecules of acetylCoA, including the electron transfers and oxidative phosphorylation, is
Palmitoyl-CoA + 7CoA + 7O2 + 35Pi + 35ADP
42H2O
............ (16-4)
8 acetyl-CoA + 35ATP +
Acetyl-CoA Can Be Further Oxidized via the Citric Acid Cycle
The acetyl-CoA produced from the oxidation of fatty acids can be oxidized to CO2
and H2O by the citric acid cycle. The following equation represents the balance sheet
for the second stage in the oxidation of our example, palmitoyl-CoA, together with
the coupled phosphorylations of the third stage:
8 Acetyl-CoA + 16O2 + 96Pi + 96ADP
8CoA + 96ATP + 104H2O +
16CO2
............ (16-5)
Combining Equations 16-4 and 16-5, we obtain the overall equation for the complete
oxidation of palmitoyl-CoA to carbon dioxide and water:
Palmitoyl-CoA + 23O2 + 131Pi + 13lADP
CoA + 13lATP + l6CO2 +
146H2O
............ (16-6)
Because the activation of palmitate to palmitoyl-CoA consumes two ATP equivalents
(p. 484), the net gain per molecule of palmitate is 129 ATP. Table 16-1 summarizes
the yields of NADH, FADH2, and ATP in the successive steps of fatty acid oxidation.
The standard free-energy change for the oxidation of palmitate to CO2 + H2O is about
9,800 kJ/ mol. Under standard conditions, 30.5 × 129 = 3,940 kJ/mol (about 40% of
the theoretical maximum) is recovered as the phosphate bond energy of ATP.
However, when the free-energy changes are calculated from actual concentrations of
reactants and products under intracellular conditions (see Box 13-2), the free-energy
recovery is over 80%; the energy conservation is remarkably efficient.
Table 16-1 Yield of ATP during oxidation of one molecule of palmitoyl-CoA to
CO2 and H2O
Enzyme catalyzing
oxidation step
Number of NADH or
FADH2 formed
Number of ATP
ultimately formed
Acyl-CoA dehydrogenase
7 FADH2
14
β-Hydroxyacyl-CoA
dehydrogenase
7 NADH
21
Isocitrate dehydrogenase
8 NADH
24
α-Ketoglutarate
dehydrogenase
8 NADH
24
Succinyl-CoA synthetase
8*
Succinate dehydrogenase
8 FADH2
16
Malate dehydrogenase
8 NADH
24
Total
131
GTP produced directly in this step yields ATP in the reaction catalyzed by nucleoside diphos- phate
kinase (p. 457).
*
BOX 16-1
Fat Bears Carry On β Oxidation in Their Sleep
Many animals depend on fat stores for energy during hibernation or dormancy, during
migratory periods, and in other situations involving radical metabolic adjustments (as
in the case of the camel, which can obtain its water supply from the oxidation of fat).
One of the most pronounced adjustments of fat metabolism occurs in the hibernation
of the grizzly bear (Fig. 1). Bears go into a continuous state of dormancy for periods
as long as seven months without arousal. Unlike most other hibernating species, the
bear maintains its body temperature between 32 and 35 °C, nearly the normal level.
Although the bear in this state expends about 6,000 kcal/day (25,000 kJ/day), it does
not eat, drink, urinate, or defecate for months at a time. When accidentally aroused,
the bear is almost immediately alert and ready to defend itsel?
Experimental studies have shown that the bear uses body fat as its sole fuel during
hibernation. The oxidation of fat yields sufficient energy for maintaining body
temperature, for active synthesis of amino acids and proteins, and for other energyrequiring activities, such as membrane transport. Fat oxidation also releases large
amounts of water (p. 488), which replenishes water loss during
breathing. In addition, degradation of triacylglycerols yields glycerol, which,
following its enzymatic phosphorylation to glycerol-3-phosphate and oxidation to
dihydroxyacetone phosphate, is converted into blood glucose. Urea formed during the
degradation of amino acids is reabsorbed and recycled by the bear, the amino groups
being used to make new amino acids for maintaining body proteins.
Bears store an
enormous
amount of
body fat in
preparation for
their long
hibernation
periods.
Normally, an
adult grizzly
bear consumes
about 9,000
kcal/day
during the late
spring and
summer. But
as winter
approaches
bears will feed
20 hours a day
Figure 1 A grizzly bear prepares its hibernation nest, near the McNeil
and consume
up to 20,000
kcal, in
response to
seasonal
changes in
hormone
secretion.
Large amounts
of body
triacylglycerols
are formed
from the huge
amounts of
carbohydrate
consumed
during the
fattening-up
period. Other
hibernating
species,
including the
tiny dormouse,
also
accumulate
large amounts
of body fat.
The camel,
although not a
hibernator, can
synthesize and
store
triacylglycerols
in large
amounts in its
hump, a
metabolic
source of both
energy and
water under
desert
conditions.
River in Canada.
Oxidation of Unsaturated Fatty Acids Requires Two Additional Reactions
The fatty acid oxidation sequence just described is typical when the incoming fatty
acid is saturated (having only single bonds in its carbon chain). However, most of the
fatty acids in the triacylglycerols and phospholipids of animals and plants are
unsaturated, having one or more double bonds. These bonds are in the cis
configuration and cannot be acted upon by enoyl-CoA hydratase, the enzyme
catalyzing the addition of H2O to the trans double bond of the Δ2-enoyl-CoA
generated during β oxidation. However, by the action of two auxiliary enzymes, the
fatty acid oxidation sequence described above can also break down the common
unsaturated fatty acids. The action of these two enzymes, one an isomerase and the
other a reductase, will be illustrated by two examples.
First, let us
follow the
oxidation of
oleate, an
abundant 18carbon
monounsaturated
fatty acid with a
cis double bond
between C-9 and
C-10 (denoted
Δ9). Oleate is
converted into
oleoyl-CoA
(Fig. 16-10),
which is
transported
through the
mitochondrial
membrane as
oleoylcarnitine
and then
converted back
into oleoyl-CoA
in the matrix
(Fig. 16-6).
Oleoyl-CoA
then undergoes
three passes
through the fatty
acid oxidation
cycle to yield
three molecules
of acetyl-CoA
and the
coenzyme A
ester of a Δ3, 12carbon
unsaturated fatty
acid, cisΔ3dodecenoylCoA (Fig. 1610). This
Figure 16-10 The oxidation of a monounsaturated fatty acyl-CoA, such
as oleoyl-CoA (Δ9), requires an additional enzyme, enoyl-CoA isomerase.
This enzyme repositions the double bond, converting the cis isomer to a
trans isomer, a normal intermediate in β oxidation.
product cannot
be acted upon by
the next enzyme
of the βoxidation
pathway, enoylCoA hydratase,
which acts only
on trans double
bonds. However,
by the action of
the auxiliary
enzyme, enoylCoA isomerase,
the cis-Δ3-enoylCoA is
isomerized to
yield the transΔ3-enoyl-CoA,
which is
converted by
enoyl-CoA
hydratase into
the
corresponding Lβ-hydroxyacylCoA (transΔ2dodecenoylCoA). This
intermediate is
now acted upon
by the remaining
enzymes of β
oxidation to
yield acetyl-CoA
and a 10-carbon
saturated fatty
acid as its
coenzyme A
ester (decanoylCoA). The latter
undergoes four
more passes
through the
pathway to yield
altogether nine
acetyl-CoAs
from one
molecule of the
18-carbon
oleate.
The other
auxiliary enzyme
(a reductase) is
required for
oxidation of
polyunsaturated
fatty acids. As an
example, we take
the 18-carbon
linoleate, which
has a cis-Δ9,cisΔ12 configuration
(Fig. 16-11).
Linoleoyl-CoA
undergoes three
passes through
the standard
βoxidation
sequence to yield
three molecules
of acetyl-CoA
and the
coenzyme A
ester of a 12carbon
unsaturated fatty
acid with a cisΔ3,cis-Δ6
configuration.
This intermediate
cannot be used
by the enzymes
of the βoxidation
pathway; its
double bonds are
in the wrong
position and have
the wrong
configuration
(cis, not trans).
However, the
combined action
of enoyl-CoA
isomerase and
2,4-dienoyl-CoA
reductase (Fig.
16-11) allows
reentry of this
intermediate into
the normal βoxidation
pathway and its
degradation to
six acetyl-CoAs.
The overall result
is conversion of
linoleate to nine
molecules of
acetyl-CoA.
Figure 16-11
Oxidation of
polyunsaturated fatty
acids requires a
second auxiliary
enzyme in addition
to enoyl-CoA
isomerase: NADPHdependent 2,4dienoyl-CoA
reductase. The
combined action of
these two enzymes
converts a transΔ2,cis-Δ4dienoylCoA intermediate
into the trans-Δ2enoylCoA substrate
necessary for β
oxidation.