2. FATTY ACID OXIDATION
• Fats (triacylglycerols) are the most important energy reserve in the animal
organism
• In many tissues, fatty acids are taken up from the blood plasma for the
synthesis of fats or to obtain energy by oxidizing them
• Fatty acids metabolism is intensive in the hepatocytes
• Fatty acid breakdown occurs in the cytosol of prokaryotes, in peroxisomes in
plants, in the mitochondrial matrix of all other eukaryotes
• β-oxidation—a metabolic pathway in the mitochondrial matrix, most
important process in the degradation of fatty acids
• Activation of Fatty Acids
– After uptake by the cell, fatty acids are activated by conversion into their
CoA derivatives—acyl CoA is formed
– Reaction catalyzed by acyl CoA synthase (fatty acid thiokinase) present on
outer mitochondrial membrane
– 2 energy-rich anhydride bonds of ATP per fatty acid are used up
3. • Transport into the Mitochondria
– Small- and medium-chain acyl CoA
CoA molecules (up to 10 carbon
atoms) readily cross the inner
mitochondrial membrane by
diffusion
– Longer chain acyl CoAs require a
specific transport mechanism,
– longer chain acyl CoAs conjugated
conjugated to the polar carnitine
molecule
– catalyzed by carnitine
acyltransferase I,on the outer face
of the inner mitochondrial
membrane
– acylcarnitine then transported
across the inner mitochondrial
membrane by a
carnitine/acylcarnitine translocase
– inside the mitochondrial matrix,
the acyl group is transferred back
on to CoA, releasing free carnitine,
carnitine, by the enzyme carnitine
acyltransferase II located on the
matrix side of the inner
mitochondrial membrane
4. β- Oxidation
• There are four individual reactions of beta-oxidation, each
catalyzed by a separate enzyme:
a) Dehydrogenation between the α and β carbons (C2
and C3) in a FAD-linked reaction, catalyzed by the
flavoenzyme acyl CoA dehydrogenase (AD),
leading to formation of a trans-α, β double bond.
Involvement of the β -carbon in this and subsequent
steps gives the pathway its name.
• There are three fatty acyl CoA
dehydrogenases. Each specific for a different
acyl chain length, so different enzymes are
involved in different stages of beta-oxidation.
Long chain fatty acyl CoA
dehydrogenase (LCAD) acts on chains
greater than C12.
Medium chain fatty acyl CoA
dehydrogenase (MCAD) acts on chains
of C6 to C12.
Short chain fatty acyl CoA
dehydrogenase (SCAD) acts on chains
of C4 to C6.
b) Hydration of the double bond by enoyl CoA
hydratase (EH). The product is an L-3-hydroxyacyl
CoA
c) A second dehydrogenation in a NAD-linked
reaction catalyzed by β-hydroxyacyl CoA
dehydrogenase to form the corresponding β-
ketoacyl-CoA, a ketone
d) Thiolytic cleavage of the thioester by β-ketoacyl
CoA thiolase (KT)
Reaction products are acetyl CoA and a new long
chain fatty acyl CoA that is two carbons shorter than
the original fatty acyl CoA.
The shortened fatty acyl group is now ready for
another round of β-oxidation.
• The process is repeated until the fatty acid is
completely broken down
• Even-numbered acyl chains will yield acetyl-CoA
only, whereas odd-numbered acyl chains will yield
one molecule of propionyl-CoA
β- Oxidation of fatty acids
5. Energy yield from fatty acid oxidation
• The energy yield from the β-oxidation pathway is high.
• Each round of degradation produces one FADH2, one NADH and one acetyl CoA
molecule
• Each NADH generates 3 ATP molecules, and each FADH2 generates 2 ATPs during
oxidative phosphorylation.
• In addition, each acetyl CoA yields 12 ATPs on oxidation by the citric acid cycle.
• The total yield for each round of fatty acid degradation is therefore 17 ATP
molecules.
• Example, complete degradation of palmitoyl CoA (C16:0) requires seven rounds
of degradation
Palmitoyl CoA + 7 CoA + 7 FAD + 7 H2O 8 Acetyl CoA + 7 FADH2 + 7 NADH + 7 H+
– 8 acetyl CoA, 7 NADH, and 7 FADH2, from which 131 ATP can be generated
– however, activation of the fatty acid requires 2 ATP
– thus, the net yield from palmitate is 129 ATP
6. Propionic Acid Pathway
• Fatty acids with an odd number of carbon atoms
yield one acetyl CoA and one propionyl CoA from
the 5-carbon fragment remaining after β-
oxidation
• The propionyl-CoA is converted, by a three-step
ATP-dependent pathway, to succinyl-CoA
– Propionyl Co-A is carboxylated to the S form
of methylmalonyl CoA by biotin-dependent
propionyl CoA carboxylase.
– The S form of methylmalonyl CoA is
racemized to the R form by methylmalonyl
CoA racemase.
– The R form of methlmalonyl CoA is then
converted to succinyl CoA by methylmalonyl
CoA mutase, which requires a cofactor
derived from vitamin B12, 5’-
deoxyadenosyl cobalamin.
• The succinyl- CoA can then enter the TCA cycle, of
which it is an intermediate for further oxidation
Metabolism of Propionyl CoA
Note: Succinyl-CoA is a citric acid cycle intermediate and can be converted to glucose. Propionyl- CoA is
therefore, an exception to the rule that carbon from fatty acids cannot be used for gluconeogenesis.
However, fatty acids with odd numbers of carbons account only for a small fraction of all fatty acids.
7. Oxidation of unsaturated fatty acids
• Oxidation of unsaturated fatty acids essentially
the same process as for saturated fats, except
when a double bond is encountered.
• The double bond is isomerized by a specific
enoyl-CoA isomerase and oxidation continues.
• The double bonds in the naturally occurring fatty
acids are cis, and the β-oxidation pathway can
only deal with trans double bonds as at the enoyl
CoA hydratase step.
• ∆3 – cis – ∆2 trans enoyl CoA isomerase shifts the
double bond to the preferred ∆2 – trans
configuration.
• Polyunsaturated fatty acids require another
enzyme for complete oxidation.
• Hydration of a cis – ∆2 double bond yields the D –
isomer of β-hydroxyacyl CoAs, which are not
substrates for L- β-hydroxyacyl CoA
dehydrogenase.
• An epimerase acts on the D isomer of a β-
hydroxyacyl CoA to yield the required L isomer.
• In the case of linoleate, the presence of the D-12
unsaturation results in the formation of a dienoyl-
CoA during oxidation. This molecule is the
substrate for an additional oxidizing enzyme, the
NADPH requiring 2,4-dienoyl-CoA reductase.
8. Alternative/Minor Oxidation Pathways
• ω-Oxidation involves oxidation of the terminal methyl group to form an ω-hydroxy fatty
acid. This is a minor pathway observed with liver microsomal preparation
• α-Oxidation pathway involves the oxidation of long-chain fatty acids to 2-hydroxy fatty
acids, which are constituents of brain lipids, followed by oxidation to fatty acid with one
less carbon e.g. oxidation of phytanic acid
– Phytanic acid is a fatty acid present in the tissues of ruminants and in dairy products and is,
therefore, an important dietary component of fatty acid intake.
– Phytanic acid is methylated, and cannot act as a substrate for the first enzyme of the β-oxidation
pathway (acyl-CoA dehydrogenase).
– An additional mitochondrial enzyme, a-hydroxylase, adds a hydroxyl group to the α-carbon of
phytanic acid, which then serves as a substrate for the remainder of the normal oxidative
enzymes. This process is termed a-oxidation.
9. KETONE BODIES
• During high rates of fatty acid oxidation when large amounts of acetyl-CoA are generated
e.g. in the fasting state, liver mitochondria converts acetyl CoA into ketone bodies.
– Ketone bodies: acetoacetate, 3-hydroxybutyrate (also called β-hydroxybutyrate), and acetone (a
nonmetabolized side product
• Acetoacetate and 3-hydroxybutyrate are interconverted by the mitochondrial enzyme
d(–)-3-hydroxybutyrate dehydrogenase
– the equilibrium is controlled by the mitochondrial [NAD+]/[NADH] ratio, i.e. the redox state.
• Active synthesis but little utilization of ketone bodies in the liver, and high utilization
with no production in extrahepatic tissues in extrahepatic tissues, result in a net flow of
the compounds to the extrahepatic tissues
• Acetoacetate and 3-hydroxybutyrate are transported in the blood to the peripheral
tissues where they can be reconverted to acetyl CoA, which can be oxidized by the TCA
cycle.
• Ketone bodies are important sources of energy for the peripheral tissues because
– they are soluble in aqueous solution and, therefore, do not need to be incorporated into
lipoproteins or carried by albumin as do the other lipids
– they are produced in the liver during periods when the amount of acetyl CoA present exceeds
the oxidative capacity of the liver; and
– they are used in proportion to their concentration in the blood by extrahepatic tissues, such as
the skeletal and cardiac muscle and renal cortex.
• Extrahepatic tissues utilize acetoacetate and β-hydroxybutyrate as respiratory
substrates.
– E.g. the brain can use ketone bodies to help meet its energy needs if the blood levels rise
sufficiently
• Ketone bodies thus, spare glucose; important during prolonged periods of fasting
10. Ketogenesis
• Ketone body synthesis occurs in the mitochondrion and
involves the following reactions:
– Formation of acetoacetyl-CoA from two molecules of
acetyl-CoA by thiolase, the reversal of the final step
in β-oxidation
– Formation of hydroxymethylglutaryl-CoA (HMG-
CoA) by HMGCoA synthase
– Release of acetoacetate by HMG-CoA lyase
– Reduction of acetoacetate to β-hydroxybutyrate by
β-hydroxybutyrate dehydrogenase
• Acetoacetate can spontaneously (yet slowly)
decarboxylate to acetone (CH3-CO-CH3).The latter cannot
be metabolized but is volatile and is exhaled
• A characteristic acetone smell can be detected in persons
that utilize fat at a high rate e.g. diabetic ketoacidosis, flu
or fever
• Utilization of ketone bodies in the brain, muscle and
other tissues is straightforward:
– b-Hydroxybutyrate is dehydrogenated to acetoacetate,
– Acetoacetate receives Co-A from succinyl-CoA to become
acetoacetyl-CoA
– Thiolase cleaves acetoacetyl-CoA to acetyl-CoA.
11. Overview of ketone bodies metabolism.
a: Structures of acetoacetate and b-hydroxybutyrate ( β-HB).
b: Overview of metabolic pathways. Triacylglycerol is cleaved to fatty acids and glycerol in the
in the fat tissue. Fatty acids undergo β-oxidation in the liver. Acetyl- CoA is converted to
acetoacetate, which is released into the blood. A fraction of the NADH generated in β-
oxidation is used to convert acetoacetate to β-hydroxybutyrate. In the brain and other
tissues, the two substrates are converted back again to acetyl-CoA and utilized in the citric
citric acid cycle.
12. DE NOVO SYNTHESIS OF FATTY ACIDS
• Fatty acids are synthesized by an extramitochondrial
system, which is responsible for the complete synthesis of
palmitate from acetyl-CoA in the cytosol.
• Present in many tissues, e.g. liver, kidney, brain, lung,
mammary gland, and adipose tissue.
• Its cofactor requirements include NADPH, ATP, Mn2+,
biotin, and HCO3
− (as a source of CO2).
• Acetyl- CoA is the immediate substrate, and free palmitate
is the end product.
• Fatty acid synthesis takes plac in 3 stages:
– Production of cytoslic acetyl CoA
– Carboxylation of acetyl CoA to form malonyl CoA
– Assembly of fatty acid chain via fatty acid synthase
13. – Involves the transfer of acetate
units from mitochondrial acetyl
CoA to the cytosol
– Mitochondrial acetyl CoA is
produced by the oxidation of
pyruvate, and by the catabolism
of fatty acids, ketone bodies, and
certain amino acids
– Mitochondrial acetyl CoA is
transported into cytosol via citrate
transport system
– Acetyl CoA is condensed with
oxaloacetate to form citrate which
is antiported into the cytosol
– Citrate is then cleaved to
regenerate acetyl CoA and
oxaloacetate by ATP-citrate lyase
Production of cytoslic acetyl CoA
14. Carboxylation of acetyl CoA to form malonyl CoA
• Production of malonyl-CoA is the
initial and controlling step in fatty
acid synthesis.
• Carboxylation of acetyl-CoA to
malonyl- CoA is catalyzed by acetyl-
CoA carboxylase, the key regulatory
enzyme and occurs in the presence
of ATP requiring bicarbonate as a
source of CO2.
• Acetyl-CoA carboxylase is a
multienzyme protein with a
requirement for the vitamin biotin
• The reaction takes place in two
steps: (i) carboxylation of biotin
involving ATP and (ii) transfer of
the carboxyl to acetyl-CoA to form
malonyl-CoA
15. Assembly of fatty acid chain via fatty acid synthase
• Consists of five separate stages:
1. Loading - acetyl CoA and malonyl CoA are
attached to acyl carrier protein.
2. Condensation - Condensation of acetyl-ACP
and malonyl-ACP to form acetoacetyl-ACP,
releasing free ACP and CO2; catalyzed by acyl-
malonyl-ACP condensing enzyme.
3. Reduction - Reduction of acetoacetyl-ACP to
form D-3-hydroxybutyryl-ACP, using NADPH as
reductant; catalyzed by β-ketoacyl-ACP
reductase.
4. Dehydration - Dehydration of D-3-
hydroxybutyryl-ACP to produce crotonyl-ACP;
catalyzed by 3-hydroxyacyl-ACP dehydratase.
5. Reduction - Reduction of crotonyl-ACP by a
second NADPH molecule to give butyryl- ACP;
catalyzed by enoyl-ACP reductase.
• The cycle repeats with malonyl-ACP adding two-
carbon units in each cycle to the lengthening acyl-ACP
chain.
• This continues until the 16-carbon palmitoyl- ACP is
formed.
• Palmitoyl- ACP is then hydrolyzed by a thioesterase to
give palmitate and ACP.
16. • Elongation of fatty acid chains
The elongation of fatty acid chains occurs in the endoplasmic reticulum.
This pathway (the “microsomal system”) elongates saturated and unsaturated fatty acyl-CoAs
CoAs (from C10 upward) by two carbons, using malonyl-CoA as acetyl donor and NADPH as
as reductant,
Elongation is catalyzed by the microsomal fatty acid elongase system of enzymes
• Formation of double bonds
– In eukaryotes the SER has enzymes able to introduce double bonds into fatty acyl CoA molecules in an
an oxidation reaction that uses molecular oxygen.
– This reaction is catalyzed by a membrane-bound complex of three enzymes: NADH cytochrome b5
reductase, cytochrome b5 and a desaturase. The overall reaction is:
saturated fatty acyl CoA + NADH + H+ + O2 monounsaturated acyl CoA + NAD+ + 2H2O
– Reaction may be repeated to introduce more than one double bond into a fatty acid.
– Mammals lack the enzymes to insert double bonds at carbon atoms beyond C-9 in the fatty acid chain.
chain.
– Thus cannot synthesize linoleate and linolenate, both of which have double bonds later in the chain
than C-9 (linoleate has cis, cis Δ9, Δ12 double bonds, and linolenate has all-cis Δ9, Δ12, Δ15 double bonds).