Lecture III

Peroxisomal β-oxidation

• Shortens very long chain fatty acids (> 22 carbon atoms)

in animals.

• Enzymes of β-oxidation in peroxisomal and mitochondrial

pathways are different.

• Carnitine is not required for the transport of fatty

acyl-CoA into the peroxisomes.
• Very long chain fatty acids diffuse into the peroxisomal
compartment.

• These are activated by a peroxisomal very long chain

acyl-CoA synthetase to form their CoA-esters.

• These are oxidised directly to yield shorter chain acyl-CoA

products.

• These are then linked to carnitine for transport to the

mitochondria for further oxidation.
X-adrenoleukodystrophy (X-ALD)

• Rare X-linked inherited disease.

• Causes very long chain saturated fatty acids to

accumulate in the blood.

• Destroys myelin, the insulating sheath surrounding the

axons of many neurons.

• May be caused by a deficiency of peroxisomal very long

chain acyl-CoA synthetase enzyme.
 Bacterial β-oxidation system

• In bacteria grown in the absence of fatty acids, the β-

oxidation system is practically absent.

• It is readily induced by the presence of fatty acids in the

growth medium.

• The bacterial β-oxidation system is completely soluble,

being cytosolic in its location.

• It is never membrane-bound.
 β-oxidation system in seeds

• In germinating seeds, where there is a high lipid content,

the β-oxidation system is exclusively located in microbodies
called glyoxysomes.

• In seeds with low lipid content, the enzymes of the β-

oxidation system are associated with the mitochondria as
usual.
 Metabolic water

• An important biological significance of fatty acid oxidation

is the production of metabolic water.

• 1 mole of palmitic acid (C16) upon β-oxidation produces 16

moles of water.

• This metabolic production of water is important to many

organisms.
Examples

a. In case of camel, lipids stored in its humps serve as

both a source of energy and a source of water for its
sustenance for extended periods in deserts.

b. Also the kangaroo rat has been observed to live

indefinitely without drinking water, as they are supplied
with metabolic water in times of need.
 Minor pathways of fatty acid
oxidation

1. α-oxidation (for long-chain fatty acids)

Definition: It is the removal of one C-atom

(i.e. α-C or C-2) at a time from the
carboxyl-end of a molecule.

Discovery: This pathway was first observed in seeds

and leaf-tissues of plants.
Occurrence

• in seeds and leaf tissues of plants

• microsomes of brain and other tissues of animals

Sub-cellular occurrence

• hydroxylation reaction occurs in mitochondria

• the oxidative decarboxylation reaction occurs in

the endoplasmic reticulum of a cell
Salient features of α-oxidation

• Long-chain fatty acids are substrates; e.g. lignoceric,

nervonic and branched-chain phytanic acids.

• Molecular O2 is indirectly involved.

• Does not require Coenzyme A intermediates.

• Does not generate high-energy phosphates.

• Here, first there is a hydroxylation at the fatty acid Cα.

• Resulting product is subsequently oxidatively decarboxylated

to yield a new fatty acid with an unsubstituted Cβ.

• The resulting fatty acid is then further degraded via cycles of

the normal β-oxidation pathway.
Mechanism
Hydroxylation

α-hydroxylase
RCH2CH2CH2COOH
RCH2CH2CHOHCOOH
long-chain fatty acid or, monooxygenase α-hydroxy fatty acid
(n C) (O2, Mg2+, NADPH + H+,
heat-stable cofactor)

Oxidative decarboxylation
RCH2CH2COOH
RCH2CH2COCOOH
long-chain fatty acid (O2, Fe2+, ascorbate) α-keto fatty acid
(n-1 C)
+ CO2

Further degradation then continues via β-oxidation.

Physiological significance of α-oxidation –
an example

• The α-oxidation system plays a key role in the capacity of

mammalian tissues to oxidise the branched-chain fatty acid
phytanic acid (3,7,11,15-tetramethylhexadecanate) to
pristanic acid.

• Phytanic acid is a metabolic breakdown (oxidation) product

of chlorophyll’s phytol side chain.
‘Refsum’s Disease’ / ‘Phytanic acid
storage syndrome’

• Large amounts of phytanic acid accumulates (as much as

20% of the serum fatty acids and 50% of the hepatic fatty
acids) in the tissues and serum of such individuals.

• Rare inheritable autosomal recessive genetic disorder.

• Affects the nervous system of a person because of an

inability to oxidise phytanic acid.
• These individuals lack the enzyme phytanate-α-
hydroxylase that converts phytanic acid to α-hydroxy
phytanic acid.

• Disease symptoms - progressive neurological difficulties,

such as tremors, defective or unsteady gait, and poor night-
vision.

• Diets low in phytanic-acid containing animal fat and milk

products appear to relieve some of the symptoms of
‘Refsum’s Disease’.
2. ω-oxidation (for long-chain and medium-
chain fatty acids)

Definition: Biological oxidation of fatty-acids at

the omega (ω) C-atom i.e. the C-atom
most distant from the –COOH group.

Discovery: First reported by Verkade and his group.

Occurence: In many organisms, including vertebrates.

Sub-cellular occurrence

In the endoplasmic reticulum (microsomes) of a liver

and kidney cell.

Salient features of ω-oxidation

• Only long-chain and medium-chain (10-12 C-atoms)

fatty acids serve as substrates
• This process involves the hydroxylation of a fatty acid’s
Cω atom by Cytochrome P450, a monooxygenase
that utilises NADPH and O2.

• The –OH group at the Cω atom so generated is

then oxidised to a –COOH to yield an α,
ω-dicarboxylic acid.

• This is then converted to a CoA derivative at its both

ends, and further oxidised via the β-oxidation pathway.
 Mechanism

α, ω
Industrial significance of ω-oxidation –
an example

Extremely important in the bacterial

biodegradation of both:

a. detergents derived from fatty acids and,

b. large amounts of oil spilled over the ocean surface.

(under aerobic conditions is estimated as high as 0.5 gm
day-1 m-2 of oil-surface).
‘Dicarboxylic aciduria’

• There is a genetic deficiency of the mitochondrial

medium-chain acyl-CoA dehydrogenase (MCAD).

• So, there is no β-oxidation of the medium-chain

acyl-CoAs.

• This results in a corresponding rise in their ω-oxidation

to C6-C10 dicarboxylic acids, which are eliminated in
the urine.
 Fatty acid biosynthesis

The pathway

• Site: Takes place in all organisms; prominent in

liver, adipose tissues, and mammary glands of
higher animals.

• Sub-cellular site: cytosol.

• Condition: Citrate must be present.

• Enzyme -complex (7 proteins)
needed : Fatty acid synthetase
complex
• This reaction proceeds by the sequential addition of
2-carbon units derived from acetyl-CoA.

• The single molecule of acetyl-CoA required in the

process serves as a primer or starter.

• One key intermediate is malonyl-CoA.

• During these reactions, the substrates are bound to a

low-molecular weight conjugated protein, known
as the acyl carrier protein (ACP).
biotin
 Bioenergetics
• The synthesis of fatty acids requires energy in the form
of ATP and reducing power in the form of NADPH.

• Example
synthesis of a C16 saturated fatty acid palmitic acid
requires 7 ATP and 14 NADPH.

8 acetyl-CoA + 7 ATP + 14 NADPH + 14 H+ palmitic acid + 14

NADP+ + 8 CoA +
6 H2O + 7 ADP +
7 Pi
 Regulation of fatty-acid
metabolism
1. Fatty acid synthesis is controlled in part, by
short-term regulation.

Example: acetyl-CoA carboxylase, that catalyses the first

rate-limitting, committed step in the pathway is:

a. inhibited by palmitoyl-CoA and by the glucagon-stimulated

cAMP-dependant increase in phosphorylation

b. activated by citrate and by insulin-stimulated

dephosphorylation
2. Lipid biosynthesis is also regulated by
long-term regulation operative for over hours
or days.

Example

a. insulin stimulates the synthesis of acetyl-CoA

carboxylase and fatty-acid synthase.

b. presence of polyunsaturated fatty acids in the diet

and starvation decrease the concentrations of these
enzymes.
3. Fatty acid metabolism is regulated largely by
hormones in the body, like epinephrine,
norepinephrine, glucagon and insulin.

4. Another control point that inhibits fatty acid

oxidation when fatty acid synthesis is stimulated
is the inhibition of Carnitine acyl transferase I (CAT I)
by malonyl CoA.
 Cholesterol biosynthesis

About Cholesterol
• The membranes of many eukaryotic cells contain sterols,
such as cholesterol.

• Cholesterol is made up of repeating units of the

unsaturated hydrocarbon isoprene (isoprenoid hydrocarbons).

• Cholesterol is also the precursor of steroid hormones and

bile acids.

• Its deposition in arteries has been associated with

cardiovascular disease and stroke.
O c c u r r e n c e : Cholesterol is synthesised in the liver.

The pathway

• Here there is a long chain carbon skeleton formation

from 5-carbon isoprenoid units that have the
carbon skeleton of isoprene (2-methyl-1, 3 –butadiene).

C-C-C-C
isoprene an isoprene unit
Cholesterol biosynthesis
 Abnormalities in cholesterol
metabolism
• High blood cholesterol levels is called ‘hypercholesterolemia’

• Results from the overproduction and / or underutilization

of LDL.

• This is known to be caused by two metabolic irregularities:

a. genetic disease familial hypercholesterolemia (FH) in which

there is a deficiency of functional LDL-receptors

b. the consumption of a high-cholesterol diet.

 Biosynthesis of Triacylglycerols
(TAGs)
Site

• It takes place in vertebrates:

a. mostly in the cytosol of the cells of adipose tissue and
liver
b. to a lesser extent in other tissues, like kidneys.

• It also takes place in higher plants.

• Bacteria contain smaller amounts of TAGs.

Pathway:

Storage:
After their biosynthesis, TAGs are stored in:
a. adipose tissues in animals, or,
b. in nuts, fruits and seeds in plants.
Biosynthesis of Phospholipids and
Glycolipids

There are:

• 2 categories of phospholipids, glycerophospholipids

and sphingophospholipids

• 2 categories of glycolipids, glyceroglycolipids

and sphingoglycolipids.
 Biosynthesis of
Glycerophospholipids

These are found in eukaryotic membranes and in transport

lipoproteins.

1. In Eukaryotes

2. In mammals

Site: Biosynthesis of phospholipids (PLs) takes place on the

surface of the smooth endoplasmic reticulum (SER) of the
cells of mostly all tissues, including liver.
 Pathways:
Biosynthesis of Phosphatidyl Choline
(PC) – 2 pathways
1. The mechanism of activated phosphate ester formation
here is the same as phosphatidyl ethanolamine biosynthesis.
2. By direct methylation of the amino group
of phosphatidylethanolamine by the methyl group
donor S-adenosyl-homocysteine (SAM) in a 3-step process.
Biosynthesis of Phosphatidyl Ethanolamine (PE)
The mechanism of activated phosphate ester formation here is the
same as phosphatidyl choline biosynthesis.
Biosynthesis of Phosphatidyl Serine (PS)
This is synthesised from phosphatidyl ethanolamine by a
head-group exchange reaction catalysed by phosphatidyl
ethanolamine:serine transferase.

Head-group
exchange
reaction
S torage

After their biosynthesis, some of the newly formed

PLs remain within the SER membrane, but most move

from that site to other locations to perform their activities.

In most eukaryotes:
Biosynthesis of Phosphatidyl Glycerol
Here, the hydrophobic tail is activated rather than the polar
head group.
 Biosynthesis of Cardiolipin (Diphosphatidyl
Glycerol) and Phosphatidyl Inositol (PI)
While Cardiolipin makes up 20% of lipids in the mitochondrial inner
membrane, found in bacterial membrane, and is an important
phospholipid first isolated from heart tissue, PI acts as a precursor of
second messengers.

a. In animal tissues
b. In bacteria
 Biosynthesis of Plasmalogens and
Alkylacylglycerophospholipids (Ether lipids)

Present in eukaryotic membranes.

 Biosynthesis of
glycerophospholipids
In E. coli
Biosynthesis of S p hing o p ho sp ho lip id s
(like S phingom yelin )

• Only one major phospholipid contains ceramide (N-acyl-

sphingosine) as its hydrophobic tail – sphingomyelin (N-acyl-
sphingosine phosphocholine).

• It is an important structural lipid of nerve cell membranes.

• This biosynthesis takes place only in higher eukaryotes,

like animals.
Biosynthesis of Sphingophospholipids (like
Sphingomyelin)
Biosynthesis of Sphingoglycolipids

Biosynthesis of Ceramide (N-acyl sphingosine)

This biosynthesis occurs from the precursors palmitoyl-CoA
and serine.
 Biosynthesis of Cerebrosides

• Galactocerebroside (1-β-galactoceramide)
and glucocerebroside (1-β-glucoceramide) are the
two most common cerebrosides.

• Galactocerebroside is a common component of brain

lipids, whereas glucocerebroside is the precursor
of globosides and gangliosides.

• Both are synthesised from ceramide by the addition

of a globosyl unit from the corresponding UDP-hexose.
Biosynthesis of Cerebrosides
 Biosynthesis of Globosides and
Gangliosides

• Gangliosides are important lipids in the neuronal

membranes, particularly at the synapses.

• Biosynthesis of both globosides (neutral ceramide

oligosaccharides) and gangliosides (acidic, sialic
acid-containing ceramide oligosaccharides) are catalysed
by a series of glycosyl transferases.
Biosynthesis of Globosides and
Gangliosides
Sphingoglycolipid Degradation and
Lipid Storage Diseases ( Lipidoses )

• Sphingoglycolipids are lysosomally degraded by a series

of enzymatically mediated hydrolytic reactions.

• The hereditary absence of one of these enzymes results

in a sphingolipid storage disease (lipidose).

• One most common such condition is Tay-Sach’s disease.

• Others include Fabry’s disease, Gaucher’s disease,

and Niemann-Pick disease.
 Tay-Sach’s disease
• Autosomal recessive deficiency in hexosaminidase A,
which hydrolyses N-acetyl-galactosamine from
ganglioside GM2.

• Results in the neuronal accumulation of GM2 as

shell-like inclusions, which causes mental retardation.

• Although infants born with Tay-Sach’s disease at

first appear normal, by about one year of age,
they become progressively weaker, retarded, and
blinded until they die, usually by the age of three years.