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 16-carbon chain of
palmitate to eight two-carbon acetyl-CoA molecules. Formation of each molecule
of acetyl-CoA 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. Acetyl-CoA 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 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.
The enzyme responsible for this first step, acyl-CoA 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.
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-hydroxyacyl-CoA (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-ketoacyl-CoA 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 β-ketoacyl-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.
![]()
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
The overall equation for the oxidation of palmitoyl-CoA to eight molecules of acetyl-CoA, including the electron transfers and oxidative phosphorylation, is
Palmitoyl-CoA + 7CoA + 7O2 + 35Pi + 35ADP![]()
8 acetyl-CoA + 35ATP +
42H2O
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. Acetyl-CoA 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 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-enoyl-CoA. 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 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.
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Chapter 18.
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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-hydroxyacyl-CoA (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-ketoacyl-CoA 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 β-ketoacyl-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 acyl-CoA, 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 +
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 acetyl-CoA 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 + .
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 The overall equation for the oxidation of palmitoyl-CoA to eight molecules of acetyl-CoA, including the electron transfers and oxidative phosphorylation, is
Palmitoyl-CoA + 7CoA + 7O2 + 35Pi + 35ADP
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 ............
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
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 the free-energy recovery is over 80%; the energy conservation is
remarkably efficient.
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
18-carbon 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, 12-carbon unsaturated fatty acid,
cis-Δ3dodecenoyl-CoA (Fig. 16-10). This product cannot
be acted upon by the next enzyme of the β-oxidation pathway, enoyl-CoA
hydratase, which acts only on trans double bonds. However, by the action of the
auxiliary enzyme, enoyl-CoA isomerase, the
cis-Δ3-enoyl-CoA is isomerized to yield the
trans-Δ3-enoyl-CoA, which is converted by enoyl-CoA
hydratase into the corresponding L-β-hydroxyacyl-CoA
(trans-Δ2dodecenoyl-CoA). 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 (decanoyl-CoA). 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,cis-Δ12
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 12-carbon unsaturated fatty acid with a
cis-Δ3,cis-Δ6 configuration.
This intermediate cannot be used by the enzymes of the β-oxidation 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 β-oxidation pathway and its degradation to six acetyl-CoAs. The
overall result is conversion of linoleate to nine molecules of acetyl-CoA.
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