Lecture 5

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 Fatty acid β-oxidation

• The major pathway for catabolism of fatty acids is a

mitochondrial pathway called β-oxidation, in which two-

carbon fragments are successively removed from the

carboxyl end of the fatty acyl CoA, producing acetyl CoA,

NADH, and FADH2.

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1. Long-chain fatty acid transport into cytosol and mitochondria:

Carnitine shuttle.
The net effect is that a long-chain (LC) fatty acyl coenzyme A (CoA) is transported
from the outside to the inside of mitochondria.
AMP = adenosine monophosphate; 3
PPi = pyrophosphate.
Long-chain fatty acid transport into cytosol and mitochondria:
• The uptake of fatty acids may occur using several different mechanisms. It may
involve passive diffusion or one of several lipid transportation proteins, such as
fatty acid translocase (FAT), fatty acid–binding protein (FABP) and fatty acid
transport protein (FATP).

• The long chain fatty acids (LCFA) are taken up by FATP. After a LCFA enters a
cell, it is converted in the cytosol to its CoA derivative by long-chain fatty acyl
CoA synthetase (thiokinase), an enzyme of the outer mitochondrial membrane.

• Because β-oxidation occurs in the mitochondrial matrix, the fatty acid must be
transported across the inner mitochondrial membrane that is impermeable to
CoA. Therefore, a specialized carrier transports the long chain acyl group from
the cytosol into the mitochondrial matrix. This carrier is carnitine, and this rate-
limiting transport process is called the carnitine shuttle.

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Carnitine sources:

• Carnitine can be obtained from the diet, where it is found primarily

in meat products. It can also be synthesized from the amino acids
lysine and methionine by an enzymatic pathway found in the liver
and kidneys but not in skeletal or cardiac muscle. Therefore, these
latter tissues are totally dependent on uptake of carnitine provided
by endogenous synthesis or the diet and distributed by the blood.
(Note: Skeletal muscle contains ∼97% of all carnitine in the body.)

• Carnitine enters into cells through carnitine transporters. In heart,

muscle and kidney, the high-affinity transporter is organic cation
transporter novel 2 (OCTN2). The liver has a different, low-affinity,
high-capacity carnitine transporter.
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Translocation steps:

• First, the acyl group is transferred from CoA to carnitine by

carnitine palmitoyl transferase I (CPT-I), an enzyme of the outer
mitochondrial membrane. (Note: CPT-I is also known as CAT-I for
carnitine acyltransferase I.) This reaction forms an acylcarnitine
and regenerates free CoA.

• Second, the acylcarnitine is transported into the mitochondrial

matrix. Carnitine palmitoyl transferase 2 (CPT-II, or CAT-II), an
enzyme of the inner mitochondrial membrane, catalyzes the
transfer of the acyl group from carnitine to CoA in the mitochondrial
matrix, thus regenerating free carnitine.

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2. Shorter-chain fatty acid entry into mitochondria:

• Fatty acids ≤12 carbons can cross the inner

mitochondrial membrane without the aid of carnitine or

the CPT system.

• Once inside the mitochondria, they are activated to their

CoA derivatives by matrix enzymes and are oxidized.

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 β-Oxidation reactions
• The first cycle of β-oxidation consists of a sequence
of four reactions involving the β-carbon (carbon 3)
that results in shortening the fatty acid by two carbons
at the carboxylate end.
• The steps include an oxidation that produces FADH2,
a hydration, a second oxidation that produces NADH,
and a CoA-dependent thiolytic cleavage that releases
a molecule of acetyl CoA.
• Each step is catalyzed by enzymes with chain-length
specificity.
• These four steps are repeated for saturated fatty acids
of even-numbered carbon chains (n/2) − 1 times
(where n is the number of carbons), each cycle
producing one acetyl CoA plus one NADH and one
FADH2. The final cycle produces two acetyl CoA.
• The acetyl CoA can be oxidized or used in hepatic
ketogenesis. The reduced coenzymes are oxidized by
the electron transport chain, NADH by Complex I,
and FADH2 by coenzyme Q.
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β-Oxidation energy yield:

• The energy yield from fatty acid β-oxidation is high. For

example, the oxidation of a molecule of palmitoyl CoA to CO2

and H2O produces eight acetyl CoA, seven NADH, and

seven FADH2, from which 131 ATP can be generated.

However, activation of the fatty acid requires two ATP.

Therefore, the net yield from palmitate is 129 ATP.

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 Summary of the energy yield from the oxidation of palmitoyl coenzyme A (CoA) (16 carbons).
(Note: *Activation of palmitate to palmitoyl CoA requires the equivalent of 2 ATP [ATP → AMP + PPi].)
FADH2 = flavin adenine dinucleotide; NADH = nicotinamide adenine dinucleotide; TCA = tricarboxylic acid; CoQ =
coenzyme Q; CO2 = carbon dioxide. 10
 Oxidation of fatty acids with an odd number of
carbons:

• This process proceeds by the same reaction steps as that of

fatty acids with an even number of carbons, until the final three
carbons are reached. This product, propionyl CoA, is
metabolized by a three-step pathway.

a. D-Methylmalonyl CoA synthesis: First, propionyl CoA is

carboxylated, forming D-methylmalonyl CoA. The enzyme
propionyl CoA carboxylase has an absolute requirement for the
coenzymes biotin and ATP.

b. L-Methylmalonyl CoA formation: Next, the D-isomer is

converted to the L-form by the enzyme methylmalonyl CoA
racemase.

c. Succinyl CoA synthesis: Finally, the carbons of L-

methylmalonyl CoA are rearranged, forming succinyl CoA, which
can enter the TCA cycle. The enzyme methylmalonyl CoA

Metabolism of propionyl CoA. mutase requires a coenzyme form of vitamin B12

ADP = adenosine diphosphate; (HCO3−) = 11
bicarbonate; Pi = inorganic phosphate.
(deoxyadenosylcobalamin).
Unsaturated fatty acid β-oxidation:

• The oxidation of unsaturated fatty acids generates intermediates

that cannot serve as substrates for 2,3-enoyl CoA hydratase.
Consequently, additional enzymes are required.

• Oxidation of a double bond at an odd-numbered carbon, such as

18:1(9) (oleic acid), requires one additional enzyme, 3,2- enoyl CoA
isomerase, which converts the 3-cis derivative obtained after three
rounds of β-oxidation to the 2-trans derivative required by the
hydratase.

• Oxidation of a double bond at an even-numbered carbon, such as

18:2(9,12) (linoleic acid), requires an NADPH-dependent 2,4-dienoyl
CoA reductase in addition to the isomerase. 12
Peroxisomal β-oxidation:
• VLCFA ≥22 carbons in length undergo a preliminary β-oxidation in
peroxisomes, because peroxisomes and not mitochondria are the primary
site of the synthetase that activates fatty acids of this length.

• In contrast to mitochondrial β-oxidation, the initial dehydrogenation in

peroxisomes is catalyzed by a FAD-containing acyl CoA oxidase. The
FADH2 produced is oxidized by O2, which is reduced to hydrogen peroxide
(H2O2). The H2O2 is reduced to H2O by catalase. Therefore, no ATP is
generated from this step.

• Genetic defects in the ability either to target matrix proteins to peroxisomes

[resulting in Zellweger syndrome, a peroxisomal biogenesis disorder] or to
transport VLCFA across the peroxisomal membrane [resulting in X-linked
adrenoleukodystrophy] lead to accumulation of VLCFA in the blood and
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tissues.
 Peroxisomal α-oxidation
• Branched-chain phytanic acid, a product of
chlorophyll metabolism, is not a substrate for
acyl CoA dehydrogenase because of the
methyl group on its β-carbon. Instead, it is
hydroxylated at the α-carbon by phytanoyl
CoA α- hydroxylase (PhyH); carbon 1 is
released as CO2; and the product, 15-
carbon-long pristanal, is oxidized to pristanic
acid, which is activated to its CoA derivative
and undergoes β- oxidation.
Phytanic acid, a branched-chain
fatty acid 16 carbons in length.

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 KETONE BODIES: ALTERNATIVE FUEL FOR CELLS
• Liver mitochondria have the capacity to convert acetyl CoA derived from fatty
acid oxidation into ketone bodies. The compounds categorized as ketone bodies
are acetoacetate, 3-hydroxybutyrate (also called β-hydroxybutyrate), and
acetone.

• Ketone bodies are important sources of energy for the peripheral tissues
because they are soluble in aqueous solution and, therefore, not need to be
incorporated into lipoproteins or carried by albumin as do the other lipids; Thus,
ketone bodies spare glucose, which is particularly important during prolonged
periods of fasting. Acetoacetate and 3-hydroxybutyrate are transported in the
blood to the peripheral tissues. There they can be reconverted to acetyl CoA,
which can be oxidized by the TCA cycle.

• Ketone body synthesis by the liver is ketogenesis.

• Ketone body use by the peripheral tissues is ketolysis. 15
Ketone body synthesis in the liver and use in peripheral tissues.
Extrahepatic tissues, including the brain but excluding cells lacking mitochondria (e.g., RBCs), efficiently
oxidize acetoacetate and 3-hydroxybutyrate in this manner. In contrast, although the liver actively produces
ketone bodies, it lacks thiophorase and, therefore, is unable to use ketone bodies as fuel.
(Note: Thiophorase is also known as succinyl CoA:acetoacetate CoA transferase.)
CoA = coenzyme A; NAD(H) = nicotinamide adenine dinucleotide; TCA = tricarboxylic acid; CO2 = carbon
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dioxide.
Excessive ketone body production in diabetes mellitus
• When the rate of formation of ketone bodies is greater than the
rate of their use, their levels begin to rise in the blood (ketonemia)
and, eventually, in the urine (ketonuria). This is seen most often in
cases of uncontrolled type 1 diabetes mellitus (T1D), where the
blood concentration of ketone bodies may reach 90 mg/dl (versus
<3 mg/dl in normal individuals), and urinary excretion of ketone
bodies may be as high as 5,000 mg/24 hrs.

• The elevation of the ketone body concentration in the blood can

result in acidemia. A frequent symptom of diabetic ketoacidosis
[DKA] is a fruity odor on the breath, which results from increased
production of acetone. Ketoacidosis may also be seen in cases of
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prolonged fasting and excessive ethanol consumption.