Lec.

4 2nd term
Biochemistry II 3rd stage

OXIDATION OF FATTY ACIDS: KETOGENESIS

BIOMEDICAL IMPORTANCE

Although fatty acids are broken down by oxidation to acetyl-CoA and also
synthesized from acetyl-CoA, fatty acid oxidation is not the simple reverse of fatty
acid biosynthesis but an entirely different process taking place in a separate
compartment of the cell. The separation of fatty acid oxidation in mitochondria
from biosynthesis in the cytosol allows each process to be individually controlled
and integrated with tissue requirements. Each step in fatty acid oxidation involves
acyl-CoA derivatives, is catalyzed by separate enzymes, utilizes NAD+ and FAD as
coenzymes, and generates ATP. It is an aerobic process, requiring the presence of
oxygen.
Increased fatty acid oxidation is a characteristic of starvation and of diabetes
mellitus, and leads to ketone body production by the liver (ketosis). Ketone
bodies are acidic and when produced in excess over long periods, as in diabetes,
cause ketoacidosis, which is ultimately fatal. Because gluconeogenesis is
dependent upon fatty acid oxidation, any impairment in fatty acid oxidation leads
to hypoglycemia. This occurs in various states of carnitine deficiency or
deficiency of essential enzymes in fatty acid oxidation, for example, carnitine
palmitoyltransferase, or inhibition of fatty acid oxidation by poisons, for example,
hypoglycin.

OXIDATION OF FATTY ACIDS OCCURS IN MITOCHONDRIA

Fatty Acids Are Transported in the Blood as Free Fatty Acids

Free fatty acids (FFA)—also called unesterified (UFA) or nonesterified (NEFA) fatty
acids—are fatty acids that are in the unesterified state. In plasma, longer chain
FFA are combined with albumin, and in the cell they are attached to a fatty acid
binding protein, so that in fact they are never really "free." Shorter chain fatty
acids are more water-soluble and exist as the unionized acid or as a fatty acid
anion.
Fatty Acids Are Activated before Being Catabolized

Fatty acids must first be converted to an active intermediate before they can be
catabolized. This is the only step in the complete degradation of a fatty acid that
requires energy from ATP. In the presence of ATP and coenzyme A, the enzyme
acyl-CoA synthetase (thiokinase) catalyzes the conversion of a fatty acid (or FFA)
to an "active fatty acid" or acyl-CoA, which uses one high-energy phosphate with
the formation of AMP and PPi (Figure 22–1). The PPi is hydrolyzed by inorganic
pyrophosphatase with the loss of a further high-energy phosphate, ensuring that
the overall reaction goes to completion. Acyl-CoA synthetases are found in the
endoplasmic reticulum, peroxisomes, and inside and on the outer membrane of
mitochondria.

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Long-Chain Fatty Acids Penetrate the Inner Mitochondrial Membrane
as Carnitine Derivatives

Carnitine (β-hydroxy-γ-trimethylammonium butyrate), (CH3)3 N+—CH2—

CH(OH)—CH2—COO–, is widely distributed and is particularly abundant in
muscle. Long-chain acyl-CoA (or FFA) cannot penetrate the inner membrane of
mitochondria. In the presence of carnitine, however, carnitine
palmitoyltransferase-I, located in the outer mitochondrial membrane, converts
long-chain acyl-CoA to acylcarnitine, which is able to penetrate the inner
membrane and gain access to the β-oxidation system of enzymes (Figure 22–1).
Carnitineacylcarnitine translocase acts as an inner membrane exchange
transporter. Acylcarnitine is transported in, coupled with the transport out of one
molecule of carnitine. The acylcarnitine then reacts with CoA, catalyzed by
carnitine palmitoyltransferase-II, located on the inside of the inner membrane,
reforming acyl-CoA in the mitochondrial matrix, and carnitine is liberated.

β-OXIDATION OF FATTY ACIDS INVOLVES SUCCESSIVE CLEAVAGE WITH RELEASE

OF ACETYL-COA

In β-oxidation (Figure 22–2), two carbons at a time are cleaved from acyl-CoA
molecules, starting at the carboxyl end. The chain is broken between the α(2)-
and β(3)-carbon atoms—hence the name β-oxidation. The two-carbon units
formed are acetyl-CoA; thus, palmitoyl-CoA forms eight acetyl-CoA molecules.

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The Cyclic Reaction Sequence Generates FADH2 & NADH

Several enzymes, known collectively as "fatty acid oxidase," are found in the
mitochondrial matrix or inner membrane adjacent to the respiratory chain. These
catalyze the oxidation of acyl-CoA to acetyl-CoA, the system being coupled with
the phosphorylation of ADP to ATP (Figure 22–3).

Figure 22-3.

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The first step is the removal of two hydrogen atoms from the 2(α)- and 3(β)-
carbon atoms, catalyzed by acyl-CoA dehydrogenase and requiring FAD. This
results in the formation of Δ2-trans-enoyl-CoA and FADH2. The reoxidation of
FADH2 by the respiratory chain requires the mediation of another flavoprotein,
termed electron-transferring flavoprotein. Water is added to saturate the double
bond and form 3-hydroxyacyl-CoA, catalyzed by Δ2-enoyl-CoA hydratase. The 3-
hydroxy derivative undergoes further dehydrogenation on the 3-carbon catalyzed
by L(+)-3-hydroxyacyl-CoA dehydrogenase to form the corresponding 3-ketoacyl-
CoA compound. In this case, NAD+ is the coenzyme involved. Finally, 3-ketoacyl-
CoA is split at the 2,3-position by thiolase (3-ketoacyl-CoA-thiolase), forming
acetyl-CoA and a new acyl-CoA two carbons shorter than the original acyl-CoA
molecule. The acyl-CoA formed in the cleavage reaction reenters the oxidative
pathway at reaction 2 (Figure 22–3). In this way, a long-chain fatty acid may be
degraded completely to acetyl-CoA (C2 units). Since acetyl-CoA can be oxidized to
CO2 and water via the citric acid cycle (which is also found within the
mitochondria), the complete oxidation of fatty acids is achieved.

Oxidation of a Fatty Acid with an Odd Number of Carbon Atoms Yields

Acetyl-CoA Plus a Molecule of Propionyl-CoA

Fatty acids with an odd number of carbon atoms are oxidized by the pathway of
β-oxidation, producing acetyl-CoA, until a three-carbon (propionyl-CoA) residue
remains. This compound is converted to succinyl-CoA, a constituent of the citric
acid cycle . Hence, the propionyl residue from an odd-chain fatty acid is the only
part of a fatty acid that is glucogenic.

Oxidation of Fatty Acids Produces a Large Quantity of ATP

Transport of electrons from FADH2 and NADH via the respiratory chain leads to
the synthesis of four high-energy phosphates for each of the seven cycles needed
for the breakdown of the C16 fatty acid, palmitate, to acetyl-CoA (7 x 4 = 28). A
total of 8 mol of acetyl-CoA is formed, and each gives rise to 10 mol of ATP on
oxidation in the citric acid cycle, making 8 x 10 = 80 mol. Two must be subtracted
for the initial activation of the fatty acid, yielding a net gain of 106 mol of ATP per
mole of palmitate, or 106 x 51.6*= 5470 kJ. This represents 68% of the free
energy of combustion of palmitic acid.

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Peroxisomes Oxidize Very Long Chain Fatty Acids

A modified form of β-oxidation is found in peroxisomes and leads to the

formation of acetyl-CoA and H2O2 (from the flavoprotein-linked dehydrogenase
step), which is broken down by catalase . Thus, this dehydrogenation in
peroxisomes is not linked directly to phosphorylation and the generation of ATP.
The system facilitates the oxidation of very long chain fatty acids (eg, C20, C22).
These enzymes are induced by high-fat diets and in some species by
hypolipidemic drugs such as clofibrate.
The enzymes in peroxisomes do not attack shorter chain fatty acids; the β-
oxidation sequence ends at octanoyl-CoA. Octanoyl and acetyl groups are both
further oxidized in mitochondria. Another role of peroxisomal β-oxidation is to
shorten the side chain of cholesterol in bile acid formation . Peroxisomes also take
part in the synthesis of ether glycerolipids, cholesterol, and dolichol .

OXIDATION OF UNSATURATED FATTY ACIDS OCCURS BY A MODIFIED β-

OXIDATION PATHWAY

The CoA esters of unsaturated fatty acids are degraded by the enzymes normally
responsible for β-oxidation until either a Δ3-cis-acyl-CoA compound or a Δ4-cis-
acyl-CoA compound is formed, depending upon the position of the double bonds
(Figure 22–4). The former compound is isomerized ( Δ3cis or trans Δ2-trans-
enoyl-CoA isomerase) to the corresponding Δ2-trans-CoA stage of β-oxidation for
subsequent hydration and oxidation. Any Δ4-cis-acyl-CoA either remaining, as in
the case of linoleic acid, or entering the pathway at this point after conversion by
acyl-CoA dehydrogenase to Δ2-trans- Δ4-cis-dienoyl-CoA, is then metabolized as
indicated in Figure 22–4.

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Figure 22–4.