BCHEM 354:

METABOLISM IV

LECTURER
F. O. MENSAH

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 COURSE OUTLINE
NITROGEN CYCLE
1.Nitrogen fixation
2.Utilization of ammonia
3.Amino acid biosynthesis
4.Biosynthesis of physiologically active amines

METABOLISM OF TETRAPYRROLES (The Porphyrins)

1.Synthesis of haem
2.Degradation of haem

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 NUCLEOTIDE METABOLISM
1.Synthesis of purine nucleotides
• IMP formation
• Formation of AMP and GMP from IMP
• Formation of nucleotide di- and triphosphates
• Control of purine nucleotide synthesis
2. Biosynthesis of Pyrimidine
• UMP formation
• Synthesis of UTP and CTP
• Control of pyrimidine nucleotide biosynthesis

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 3. Nucleotide Degradation
• Degradation of purines
• Degradation of pyrimidines
INBORN ERRORS ASSOCIATED WITH METABOLISM
1. Disorders associated with carbohydrate metabolism.
• Monosaccharides
• Glycogen
2. Diseases associated with lipid metabolism.
3. Diseases associated with amino acid metabolism.
4. Disorders of porphyrin metabolism.
5. Disorders of nucleotide metabolism.
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 CONTROL/REGULATION OF METABOLISM
1. Types of regulation
• Substrate availability
• Cofactor availability
• Enzyme concentration
• Compartmentalization
• Activation and inactivation of enzymes; e.g.
Allosterism
• Feedback inhibition
• Hormonal control

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 INORGANIC NITROGEN METABOLISM
• Both prokaryotic and eukaryotic cells need nitrogen for
growth and reproduction.

• Atmospheric nitrogen is the ultimate source of the

nitrogen present in proteins, nucleic acids and other
vital nitrogenous components of all living cells.

• All organisms can convert ammonia to organic

compounds. Fewer organisms synthesize ammonia
from inorganic nitrogen gas (N2) and nitrate ions (NO3-
), a soil component essential for growth of most plants.

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 • The reduction of nitrogen to ammonia called
biological nitrogen fixation, is carried out by only
certain microorganisms sometimes in symbiotic
relationship with plants.

• The ammonium compounds are further

transformed into nitrites (NO2-) and nitrates
(NO3-) in the soil and into nitrogenous
compounds in plants.

• Much of this nitrogen is returned to the soil or

the sea when plants and animals die and decay.

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 • A proportion of the nitrogen fixed by Nitrifying
bacteria is returned to the atmosphere as a result
of the activities of denitrifying bacteria.

• The interchange of nitrogen in the biosphere is

termed the nitrogen cycle.

• In nature, nitrogen may exist in a highly oxidized

form, nitrate, or a highly reduced form, ammonia.
(See scheme-relationship between inorganic and
organic nitrogen metabolism).

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 • The reduction of atmospheric nitrogen to ammonia
occurs in relatively few organisms.

• Fixation can be accomplished either by non-

symbiotic microbes that live independently or by
certain bacteria living in symbiosis with higher
plants.

• Examples of the former include aerobic organisms of

the soil (e.g. Azobacter), soil anaerobes (e.g.
Clostridium sp.), Photosynthetic bacteria (e.g.
Rhodospirillium rubrum) and Cyanobacteria (e.g.
Blue-green algae).
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 • The symbiotic organisms consist of bacteria called
Rhizobium living in symbiosis with leguminous
plants (e.g. beans, peas).

• After infection by a strain of Rhizobium specific for

a particular legume, a nodular tissue develops on
the root of the legume.

• The affecting bacteria assumes a modified form

called a bacteroid inside the cells of the infected
plants.

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 Components of nitrogen fixation
1. An electron donor or a powerful reductant.

2. An electron acceptor (nitrogen).

3. ATP together with a divalent cation, Mg2+.

4. Nitrogenase complex made up of two protein

components.
• A molybdenum non-haem iron protein (MoFe
protein).
• A non-haem iron protein (Fe protein).
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 • Nitrogen fixation occurs under anaerobic conditions.

• This is achieved in root nodules of plants infected

with Rhizobium. The plant synthesizes a
haemoglobin-like protein called leghaemoglobin.

• This protein binds any oxygen present thus

maintaining anaerobic environment.

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 The Nitrogenase complex
1. A reductase which provides electrons with high reducing
power and
2. a nitrogenase which uses these electrons to reduce
nitrogen to ammonia.

• Each component of the complex is an iron-sulphur

protein. The nitrogenase component is a molybdenum-
iron protein and the reductase is an iron protein. In most
nitrogen fixing organisms, reduced ferredoxin serves as
the source of high potential electrons.

• Reduced ferredoxin may be generated by photosynthetic

or oxidative processes depending on the species.

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 Stages in nitrogen fixation
1. Reduced ferredoxin transfers its electrons to the reductase
component of the complex.

2. ATP then binds to the reductase shifting its redox potential

and altering its conformation. The shift in redox potential (-0.3
to -0.4mV) allows the reductase to pass its electrons to the
nitrogenase component.

3. There is the transfer of electrons, ATP hydrolysis and

dissociation of the reductase from the nitrogenase
component.

4. Nitrogen binds to the nitrogenase component and is reduced

to ammonia.
NB: the reductase component dissociates from the nitrogenase
before nitrogen is converted to ammonia.

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 Stages in nitrogen fixation

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 16
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 Properties of Nitrogenase complex
1. The iron protein (reductase) is irreversibly
inactivated by oxygen. The Mo-Fe protein is more
stable.

2. The iron protein is extremely labile at 0◦C but not

20◦C.

3. The system requires a large input of energy in the

form of ATP.

4. The system requires electrons at very negative

potential.
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 Control of nitrogenase activity
• This is exerted at two levels; a course and fine
control
1.Course control: This involves the repression of the
nitrogenase system by ammonia. Ammonia has no
direct inhibitory effect in vitro but once it
accumulates in an organism, nitrogenase system is
repressed.

2.Fine control: ADP serves as competitive inhibitor of

nitrogenase. As ATP levels fall and ADP levels
increase in the cell, nitrogenase activity ceases. As a
result, the limited supply of energy is diverted to
other important functions.

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 Utilization of Ammonia
• High concentration of ammonia is quite toxic but at
lower levels it is a central metabolite serving as the
substrate for five reactions that convert it to organic
nitrogenous compounds.

• All organisms assimilate ammonia through reactions

that yield glutamate, glutamine and carbamoyl
phosphate.

• Enzymes that catalyze these reactions are glutamate

dehydrogenase, glutamine synthetase, and
carbamoyl phosphate synthetase respectively.
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 • Carbamoyl phosphate is used only in the
biosynthesis of arginine, urea and pyrimidine
nucleotides.

• Most of the nitrogen of amino acids and other

nitrogenous compounds are obtained from
glutamate and glutamine.

• Glutamate dehydrogenase catalyzes the synthesis

of glutamate from ammonia and α-ketoglutarate.

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 • Most bacteria and many plants contain an NADPH
specific form of the enzyme which catalyzes the
reaction.

• In animals glutamate dehydrogenase is located in

the mitochondrion suggesting its involvement in
energy generation.

• The enzyme is under allosteric control, inhibited

by ATP or GTP and stimulated by ADP or GDP. It is
activated under low energy conditions.

• In animal cells, the enzyme may function in the

catabolic direction supplying α-ketoglutarate for
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oxidation in the citric acid cycle. 22 22
 • Yeast and fungi contain both types of glutamate
dehydrogenase, one for nitrogen assimilation and
the other for catabolism.

• Another enzyme, glutamate synthase catalyzes

the synthesis of glutamate from glutamine and α-
ketoglutarate.

• Glutamate synthase is found in higher plants and

microorganisms. Glutamate dehydrogenase has a
high Km for ammonia. As a result glutamate
synthase is more active in glutamate biosynthesis
at a low ammonia concentration seen in most
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cells. 23 23
 • Glutamine synthetase catalyzes the generation of
biologically active amide nitrogen. Glutamate can
accept a second ammonia moiety to form
glutamine.

• Glutamine occupies a central role in nitrogen

metabolism. The amide nitrogen is used in the
biosynthesis of several amino acids e.g. Glutamate,
tryptophan and histidine, amino sugars, purines and
pyrimidine nucleotides.

• Glutamine promotes the regeneration of

mucoproteins in the intestinal epithelium as its
amide nitrogen is involved in the biosynthesis of
hexosamines.

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 • In animals, glutamine synthetase is vital for
detoxifying ammonia formed from amino acid
catabolism especially in the brain.
• Glutamate and glutamine are two of the most
abundant free amino acids in brain cells as glutamine
can cross the blood brain barrier.

• The enzyme is regulated by two distinct mechanisms

1.Allosteric regulation by cumulative feedback
inhibition.
2.Covalent modification of the enzyme involving a
regulatory cascade.

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 Carbamoyl phosphate synthetase
• This catalyzes the synthesis of carbamoyl phosphate
using ammonia or glutamine as the nitrogen donor.

• The bacterial enzyme can catalyze both reactions but

glutamine is the preferred substrate. Eukaryotic cells
contain two forms of the enzyme.

• Form I is localized in the mitochondrion and has a

preference for ammonia as the substrate and is used
in the arginine and urea biosynthetic pathways.

• Form II found in the cytosol has a preference for

glutamine. It is involved in pyrimidine biosynthesis.
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 Nitrification
• Ammonia reaching the soil is rapidly oxidized to
nitrate which serves as the chief source of
nitrogen for non-fixing plants.

• The oxidation of ammonia is carried out by two

groups of bacteria called nitrifying bacteria.

• The first group, Nitrosomonas converts ammonia

to nitrite with oxygen as the oxidizing agent.

• The second group, Nitrobacter oxidizes nitrite to

nitrate.
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 NITRATE UTILIZATION
• As nitrate is abundant in the soil, plants and soil
organisms utilize it as the nitrogen source required for
their growth and development.

• The major route of incorporating inorganic nitrogen

into organic nitrogenous compounds involves the
reactions catalyzed by glutamine synthetase and
glutamate synthase.

• Higher plants and microorganisms must first reduce

nitrate to ammonia before it can be assimilated.

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 • The reduction is achieved in two steps;
1.The reduction of nitrate to nitrite. This is catalyzed
by a complex enzyme nitrate reductase. The
enzyme contains bound FAD, molybdenum and
cytochrome 557. Plants use NADH as the electron
donor while fungi and bacteria use NADPH.
• The electrons are first transferred to the enzyme
bound FAD, then to cytochrome 557, then to
molybdenum and finally to the substrate.
2. The reduction of nitrite to ammonia using nitrite
reductase.

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 • The enzyme is found in the chloroplast associated
with the thylakoid membrane so as to have easy
access to the NADPH produced by the light
reaction of photosynthesis.

• The enzyme contains an iron-haem protein called

sirohaem which is a partially reduced iron
porphyrin involved in the reduction.

• The electron donor in each step is ferredoxin.

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 Nitrate Assimilation
• The utilization of nitrogen in which aerobic organisms and
higher plants reduce nitrate to ammonia so as to
incorporate it into cell proteins is termed nitrate
assimilation.

• Ammonia is first converted to nitrate and then back to

ammonia before use possibly because ammonia is fairly
active metabolically and unstable and therefore cannot be
stored in plant tissue.

• Nitrates can however be absorbed and stored in the

vacuole and later reduced to ammonia and assimilated
when needed.
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 Nitrate Respiration
• Many microorganisms including E. coli use nitrate as
the terminal electron acceptor rather than oxygen.

• This process is known as nitrate respiration. Some

bacteria e.g. Pseudomonas denitrificans that utilize
nitrate respiration produce nitrogen instead of
ammonia.

• In this way, nitrogen is returned to the atmosphere as

nitrogen gas. The process is referred to as
denitrification.
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 • Recently (1980s), a group of bacteria have been
discovered that promote anaerobic ammonia oxidation,
or anammox, a process that converts ammonia (NH3)
and nitrite (NO2-) to Nitrogen (N2).

• It is believed that as much as 50% to 70% of the

ammonia-to-nitrogen conversion in the biosphere may
occur through this pathway.

• The obligate anaerobes that promote anammox are

providing some useful solutions to waste-treatment
problems.

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 • Bacteria promoting anammox were discovered in a
waste-treatment system in Delft, the Netherlands, in
the mid-1980s.

• A team of Dutch microbiologists led by Gijs Kuenen

and Mike Jetten began to study these bacteria which
were identified as belonging to an unusual bacterial
phylum, Planctomycetes.

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 BIOSYNTHESIS OF AMINO ACIDS
• Though essential amino acids occur in both animal and
vegetable proteins, many amino acids are synthesized by
pathways that are only present in plants and
microorganisms.

• Different proteins contain varying proportions of essential

amino acids. Examples, milk proteins contain all essential
amino acids in proportions required for proper human
nutrition.

• Bean protein contains an abundance of lysine but is

deficient in methionine.

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 • Wheat protein contains ample methionine but is
deficient in lysine.

• A balanced protein diet must therefore contain a

variety of different protein sources that complement
each other to supply the right amount of the essential
amino acids.

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 Biosynthesis of Non essential amino acids
• There are four metabolic intermediates or precursors
for the synthesis of non essential amino acids.

• These are pyruvate, oxaloacetate, α-ketoglutarate

and 3-phosphoglycerate.

• All the non essential amino acids except tyrosine are

synthesized by simple pathways leading from one of
these precursors.

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 Amino acid biosynthetic families, grouped by metabolic precursor

α – ketoglutarate Pyruvate
Glutamate Alanine
Glutamine Valine *
Proline Leucine *
Arginine Isoleucine *

3 – phosphoglycerate Oxaloacetate
Serine Aspartate
Glycine Asparagine
Cysteine Methionine *
Threonine *
Lysine *
Phosphoenolpyruvate and erythrose - 4 – Ribose – 5 – phosphate (PRPP)
phosphate Histidine *
Tryptophan *
Phenylalanine *
Tyrosine 

 Derived from phenylalanine in mammals

* Essential amino acids
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 Amino acids that utilize pyruvate, oxaloacetate and α-
ketoglutarate

• Pyruvate, oxaloacetate and α-ketoglutarate are the

keto acids that correspond to alanine, aspartate and
glutamate respectively.

• The synthesis of each of these amino acids is a one

step transamination reaction. Asparagine and
glutamine are synthesized from aspartate and
glutamate respectively by amidation.

• The enzyme for transamination is called

aminotransferase or transaminase.
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 • Glutamine synthetase catalyses the formation of
glutamine with ammonia as the amino group
donor.

• In the process, ATP is hydrolysed to ADP and Pi

with the formation of an unstable intermediate ɣ-
glutamyl phosphate.

• The synthesis of asparagine is catalyzed by

asparagine synthetase.

• It utilizes glutamine as its amino group donor and

hydrolyses ATP to AMP and PPi.
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 Synthesis of alanine from pyruvate

• Alanine participates in the glucose-alanine cycle as the

carrier of carbon for gluconeogenesis from muscle to
liver.
• Synthesis of Aspartate and Asparagine

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 • The nitrogen of aspartate is used in the biosynthesis of
urea and arginine.

• Aspartate is also used as a precursor in purine and

pyrimidine nucleotide biosynthesis.

• In plants and bacteria, aspartate is a precursor to

three amino acids by conversion to homoserine. This
finally forms methionine, threonine and isoleucine.

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 Synthesis of Glutamate and Glutamine
Glutamine synthetase
α – ketoglutarate aminotransferase glutamate glutamyl phosphate
inintermediate
Amino acid α - keto acid ATP ADP
NH3

Glutamine synthetase

Pi

Glutamine
Glutamine contributes its amide nitrogen for the synthesis of
other amino acids, nucleotides, amino sugars and
glycoproteins.

Reactions are catalyzed by a group of similar ATP dependent

ligases called amido transferases.
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 Amino acids that utilize Glutamate as precursor
• These are proline, ornithine and arginine.
Conversion of glutamate to proline involves the
following steps;

1. Reduction of ɣ-carboxyl group to an aldehyde.

2. Formation of an internal Schiff’s base.
3. Further reduction of the Schiff’s base to yield
proline.

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 • Synthesis of proline
Glutamate glutamyl kinaseglutamate -5- phosphate glutamate – 5 – semialdehyde
dehydrogenase

ATP ADP NAD(P)H NAD(P+)

Non – enzymatic

NAD(P)H NAD(P+)

Proline
∆ pyrroline – 5- carboxylate
Proline – 5 – carboxylate
reductase

• Proline is a precursor to hydroxyproline, an amino acid found in

structural proteins like collagen, the principal protein of connective
tissues.

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 48
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 • In E. coli, the conversion of glutamate to ornithine
and arginine involves ATP and a series of
intermediates however, in humans, the pathway
to ornithine is more direct.

• The Glutamate-5-semi-aldehyde is directly

transaminated to yield ornithine in a reaction
catalyzed by ornithine-δ-aminotransferase.

• Ornithine is converted to arginine by the urea

cycle.

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 Glutamate – 5 - semialdehyde ornithine δ aminotransferase L – ornithine urea cycle Arginine

Glutamate α – ketoglutarate

Amino acids that use 3-phosphoglycerate as precursor

• These are cysteine, serine and glycine.

• Serine is synthesized from 3-phosphoglycerate in a

pathway involving three reactions:

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 1. Conversion of 3-phosphoglycerate’s 2-OH to a ketone
yielding 3-phosphohydroxypyruvate, serine’s
phosphorylated keto acid analogue.

2. Transamination of 3-phosphohydroxypyruvate to
phosphoserine.

3. Hydrolysis of phosphoserine to serine.

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 3 – phosphoglycerate
dehydrogenase

• 3 – phosphoglycerate 3 – phosphohydroxy amino transferase 3 phospho serine

• pyruvate
• NAD+ NADH glutamate α – keto acid

Phosphoserine
phosphatase

Pi

Serine
In another pathway

3 – phosphoglycerate
phosphatase glycerate
dehydrogenase

Alanine

Hydroxypyruvate amino
3 – phosphoglycerate glycerate hydroxypyruvate serine transferase
pyruvate

Serine
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 In another pathway:

• Serine participates in glycine synthesis in two ways;

1. Direct conversion of serine to glycine by serine

hydroxymethyl transferase in a reaction that yields N5N10
methylene tetrahydrofolate.

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 2. Condensation of N5N10 methylene tetrahydrofolate
with carbon dioxide and ammonia catalyzed by
glycine synthase.

CO2 + NADH + NH3 Glycine synthase Glycine + NAD+

NH4 N5 N10 methylene THF

Cysteine biosynthesis

cystathione cystathionase
L – homocysteine Cystathionine Serine
L Serine H2O
H2O α – ketoglutarate + NH4+

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 Cysteine biosynthesis
• Plants and microorganisms utilize hydrogen sulphide
for cysteine synthesis using serine as the carbon
skeleton.

• Cysteine can also be produced during the degradation

of methionine.

• Thus cysteine synthesis is sustained by the availability

of dietary methionine.

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 BIOSYNTHESIS OF ESSENTIAL AMINO ACIDS
• Essential amino acids are also synthesized from
familiar metabolic precursors.

• Their synthesis only occurs in plants and

microorganisms and steps involved in the pathways
are usually more complex than those of the non
essential amino acids.

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 Essential amino acids and their precursors

Amino acid (s) Precursors

1. Lysine, Methionine, Threonine Aspartate

2. Valine, Leucine, Isoleucine, Pyruvate

3. Tryptophan, Phenylalanine, (Tyrosine) Phosphoenolpyruvate and Erythrose-4-

phosphate

4. Histidine Phosphoribosyl pyrophosphate (PRPP)

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 Methionine biosynthesis

• In plants and bacteria, cysteine provides the S group

for methionine synthesis. Carbon is provided by
homoserine.

• A metabolically activated form of methionine, S-

adenosylmethionine (adoMet), serves as an
activated methyl group donor and the donor of
propyl amino groups in biosynthesis of polyamines
e.g. spermine and spermidine in human semen.

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 Synthesis of Aromatic amino acids
• They are Phenylalanine, Tyrosine and Tryptophan

• A single branched pathway in microorganisms and

plants leads to the synthesis of phenylalanine,
tyrosine and tryptophan.

• All the carbons in the Phenylalanine and Tyrosine are

derived form erythrose-4-phosphate and PEP.

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 • Main intermediates of phenylalanine and tyrosine are
Shikimic acid and Chorismic acid, the branch point for
tryptophan biosynthesis.

• Chorismate is either converted to anthranilate and

then to tryptophan or to prephenate and onto either
tyrosine or phenylalanine.

• Mammals synthesize tyrosine by hydroxylating

Phenylalanine.

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 • E – 4 – P + PEP Shikimate Chorismate

prephenate Anthranilate

phenylalanine tryptophan
•tyrosine

Tyrosine utilization
Tyrosine plays important role in animal metabolism. It serves as
a precursor for
1. Thyroid hormones
2. Biological pigments called melanin
3. Catecholamines
• Tryptophan is a precursor to NAD+ and NADP+ synthesis.
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 Biosynthesis of Histidine
• Histidine is synthesized from an intermediate involved
in the biosynthesis of tryptophan, purine and
pyrimidine nucleotides called
phosphoribosylpyrophosphate (PRPP).

• The pathway is unbranched. Histidine metabolism

yields Histamine.

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 Use of aromatic amino acids in plants
• Lignin- rigid polymer in plants. Derived from phe and
tyr. Second to cellulose.

• Auxin(indole-3-acetate)- plant growth hormone

derived from trp.

• It also helps regulate a wide range of biological

processes in plants.

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 • Many commercially significant natural products are
obtained from phe and tyr.
These include:
• Tannins – inhibit oxidation in wines.

• Alkaloids – eg. morphine. Have potent physiological

effects.

• Flavouring of cinnamon oil, nutmeg, cloves, vanilla,

cayenne pepper and other products.

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BIOSYNTHESIS OF PHYSIOLOGICALLY ACTIVE AMINES
• Many basic compounds with amine functional groups
play important role in regulating mammalian metabolism.

• These amines produced in living systems and crucial for

life are called Biogenic amines, e.g. epinephrine,
norepinerphrine and dopamine (catecholamines),
serotonin, GABA and histamine.

• These are hormones and/or neurotransmitters derived

from amino acids. Most of them are formed by
decarboxylation reactions involving aromatic amino acids
or their derivatives.
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 Epinephrine, Norepinephrine and Dopamine
• These are called catecholamines because they are
amine derivatives of catechol.

• They are synthesized from tyrosine and are found in the

central nervous system, peripheral nervous system and
adrenal medulla.

• They function as excitatory neurotransmitters in the

brain.

• The conversion of tyrosine to catecholamine involves

the following steps;
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 1. Hydroxylation of tyrosine to L-DOPA (3,4-
dihydroxyphenylalanine) by Tyrosine hydroxylase. (DOPA is
also a product of Tyrosinase action however, tyrosinase is
found in the melanocytes but most catecholamine synthesis
occur in the adrenal medulla and CNS).

2. Decarboxylation of L-DOPA to Dopamine (3,4-

dihydroxyphenylethylamine). PALP-dependent.

3. Hydroxylation of dopamine to Norepinephrine by a copper

containing hydroxylase called Dopamine-β-hydroxylase
which requires ascorbic acid.

4. Methylation of amino group of norepinephrine by S-AM to

yield epinephrine.

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 69
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 Tyrosine
O2, NADPH+ H+

Tyrosine Hydroxylase

NADP+, H2O

L – DOPA ( 3, 4 dihydroxy Phenylalanine)

Epinephrine
AdoHcys
DOPA decarboxylase Phenylethanol amine N –
CO2 methyl transferase
O2
Dopamine (3 , 4 dihydroxyphenyl – Dopamine hydroxylase
AdoMet
ethylamine)
Ascorbic acid Nor epinephrine

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 • The synthesis of a particular catecholamine in a cell
depends on which enzymes of the pathway are
present.

• E.g. adrenal medulla functions to produce hormones

and therefore epinephrine is the dominant product.

• In some portions of the brain, norepinephrine is

more common.

• Parts of the nervous system that release dopamine as

neurotransmitter are called Dopaminergic neurons.

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 • Those that release adrenaline or noradrenaline are
called Adrenergic neurons.

• Control of catecholamine biosynthesis is mainly

governed by nervous activity.

• Increased nervous activity leads to increased

concentration of enzyme (monooxygenase) while
decrease in nervous activity decreases concentration
of enzyme.

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 ROLE OF CATECHOLAMINES
Epinephrine
1. It is the principal hormone governing the flight or
fight response to various stimuli. Norepinphrine and
epinephrine are released from storage vesicles in the
adrenal medulla in response to fright, exercise, cold
and low levels of blood glucose.

2. It stimulates glycogenolysis.

3. It triggers a variety of physiological events, e.g.

increasing depth and frequency of heart beat, (i.e. it
increases the output of the heart and blood
pressure).
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 4. Action of epinephrine causes fat breakdown and
release providing fuel for the muscle tissue. As a
result glucose uptake into muscle will decrease,
thereby, increasing blood glucose level.

5. It inhibits insulin secretion while stimulating

glucagon secretion.

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 Dopamine
• It plays a role in CNS transmission possibly as an
inhibitory agent.

• Deficiency in dopamine production is believed to be

associated with Parkinson’s disease, a disorder
characterized by tremors, monotonous speech, loss
of memory and problem solving ability.

• Excess dopamine causes schizophrenia.

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 • Dopamine also appears to play a role in addictive
behaviour.
• When present in ‘proper amounts’, it causes
pleasant, satisfied feeling (‘doping’).
• The greater the amount, the more intense the
sensation (the ‘high’).
• Substances (drugs) that increase levels of dopamine
are cocaine, heroin, alcohol, nicotine, amphetamines,
marijuana.

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 Serotonin
• This is 5-hydroxytryptamine and is derived from
tryptophan.

• It is produced in the pineal gland where it serves as a

precursor to melatonin.

• It is found in highest concentration in blood platelets

and cells of the intestinal mucosa.

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 Tryptophan 5 monooxygenase
Tryptophan 5-hydroxytryptophan
(hydroxylase)

O2
CO2 decarboxylase

5-hydroxytryptamine,
(Serotonin)

A deficiency of serotonin has been associated with depression- involved

In bulimia and anorexia nervosa.

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 Functions
1. It plays possible regulatory roles in nervous system including
neurotransmission.

2. It is secreted by cells in the small intestines where it

regulates intestinal peristalsis. It also causes contraction of
smooth muscles of arterioles and bronchioles.

3. It is a potent vasoconstrictor and helps regulate blood

pressure. With thromboxane A2, it stimulates
vasoconstriction thereby reducing blood flow at injury site.

4. It is involved in the regulation of sleep, temperature and

blood pressure and affects mood and social behavior.

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 Histamine
• It is secreted in the mast cells which are present in
most tissues especially in connective tissues as a
result of allergic reaction or trauma.

• It is synthesized from histidine.

• Antihistamines compete with histamine for binding

to target cells.

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 Histidine Decarboxylase
Histamine

CO2

Functions

• It is involved in allergic responses. It is a potent

vasodilator released locally at site of trauma,
inflammation or allergic reaction.

• Release of histamine in trauma contributes to

extreme lowering of blood pressure that can lead
to shock.
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 • When secreted in the stomach, it promotes secretion
of gastric acid or hydrochloric acid and pepsin that aid
digestion.

• If response occurs frequently, it will result in chronic

heart burns and eventually erosion of tissue in
oesophagus and ulceration. It also contributes to
stomach ulcers.

• Histamine contributes to inflammatory symptoms and

colds and allergies including swollen mucous
membranes, congestion and excessive nasal
secretions.

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 • It also causes itchy skin rash responsible for symptoms
of hay fever.

• Antihistamines are used to treat allergies and other

inflammations or help relieve these symptoms. e.g.

• Ephedrine is a decongestant that helps to shrink

swollen mucous membranes and reduce nasal
secretions.

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 Gamma amino butyric acid (GABA) or 4-amino butyrate
• This is derived from PALP dependent decarboxylation of
glutamate.

Glutamate decarboxylase
Glutamate GABA

PALP, CO
CO2 CO2
• It functions as an inhibitory neurotransmitter and inhibits
transmission of impulse from one cell to another in the CNS.
• One class of tranquilizers, the benzodiazepines, relieve aggressive
behaviour and anxiety. These drugs are believed to enhance the
inhibitory activity of GABA.
• Its deficiency is linked with epileptic seizures.

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 TETRAPYRROLE BIOSYNTHESIS
• There are four classes of tetrapyrrole compounds in
biology;

1. Haem

2. Chlorophyll (of plants and photosynthetic bacteria).

3. Phycobilins (photosynthetic pigments of algae).

4. Cobalamines (vitamin B12 and its derivatives).

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 • All these compounds are synthesized from a common
precursor δ-aminolevulinic acid.

• The cyclic tetrapyrrole structure is called porphyrin.

The pathways are thought to be similar in animals
with the haem of animals being more established.

• The pathway involves seven reactions.

• The first occurs in the mitochondrion, followed by

three in the cytosol and finally three more in the
mitochondrion.

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 • Haem is produced in almost all mammalian tissues,
it’s synthesis being more pronounced in bone marrow
and liver as it is required for incorporation in to
haemoglobin and cytochromes respectively.

• Early studies indicated that all the nitrogen of haem

is obtained from glycine and all the carbons are
derived from succinate and glycine.

• The pathway is therefore referred to as succinate-

glycine pathway.

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 • Steps involved

1.Condensation of TCA cycle intermediate succinyl-CoA

with glycine followed by decarboxylation to yield δ-
aminolevulinic acid (ALA). Enzyme is δ-aminolevulinic
acid synthase.

Succinyl – CoA + Glycine α – amino –β- keto adipic acid

CoASH

δ – aminolevulinic acid synthase

CO2

δ – amino levulinic acid

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 • The enzyme ALA synthase is inhibited by excess
haem and is a rate limiting step in haem
biosynthesis.
• The enzyme is actually synthesized in the cytosol
and directed by mRNA from the nucleus.

2. Synthesis of substituted pyrrole compound-

Porphobilinogen (PBG).

In the presence of ALA dehydratase, two molecules of ALA

condense with the elimination of water to yield
Porphobilinogen. Reaction occurs in the cytosol.

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 2(δ – amino levulinic acid) ALA dehydratase Porphobilinogen

2H2O

• The ring structure bears one propionic acid and one

acetic acid residue. ALA dehydratase is a sulphydryl
enzyme and is very sensitive to inhibition by heavy
metals e.g. Lead.

• The elevation of ALA without the corresponding

elevation of porphobilinogen is an indication of lead
poisoning.

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 3. Four molecules of porphobilinogen condense to
form the first tetrapyrrole compound
Uroporphyrinogen III, the porphyrin nucleus.

• It involves a series of reactions catalyzed by

uroporphyrinogen synthase (alternatively called
porphobilinogen deaminase) and
uroporphyrinogen III cosynthase.

• The cosynthase actually directs the rapid synthesis

of the III isomer. Without it, uroporphyrinogen I
(symmetrical) is synthesized instead, though slowly.

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 4. Modification of side chains occur on the
Uroporphyrinogen III. This involves a series of
reactions;
• Uroporphyrinogen decarboxylase catalyses the
decarboxylation of all four acetate side chains (A) to
form methyl groups (M) yielding Coproporphyrinogen
III. The enzyme is inhibited by iron salts.
• Coproporphyrinogen III enters the mitochondrion and
is acted on by Coproporphyrinogen oxidase which
oxidatively decarboxylates two of the propionate side
chains (P) to vinyl groups (V). This yields
Protoporphyrinogen IX. The enzyme is specific for the
III isomer.

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 iii. Protoporphyrinogen oxidase oxidizes the methylene
groups linking the pyrrole rings to methenyl groups
producing protoporphyrin IX.
• All together, six carboxyl groups are lost as carbon
dioxide. Protoporphyrin IX, unlike the others is
highly water insoluble.

5. Ferrochelatase located in the inner mitochondrion

membrane catalyses the insertion of the ferrous
iron into protoporphyrin IX to form haem.

• The enzyme is inactivated by heavy metals like lead

and low concentration of iron.
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 Haem Biosynthesis

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 95
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 Co
Corriphyrin Cobalamine (Vit B12)

Fe Siroheme
Uroporphyrinogen III

Fe
Protoporphyrin IX Protohaem haem

Mg

Chlorophyll

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 Control of Haem biosynthesis
1. The main control point in haem biosynthesis is the ALA
synthase step i.e. the first step. Haem or its Fe3+ oxidation
product controls the enzyme’s activity through three
mechanisms;
a. Feedback inhibition
b. Inhibition of the transport of ALA synthase from its site of
synthesis in the cytosol to its site of action in the
mitochondrion.
c. Repression of ALA synthase synthesis.
2. Another mode of control of haem biosynthesis is
compartmentalization. While some reactions occur in the
mitochondrion, others occur in the cytosol.
3. Ferrochelatase is also inhibited by haem.

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 Role of Porphyrins
1. Chlorophyll in plants is a principal photoreceptor in
photosynthesis. It is involved in electron transfer.

2. In animals, haemoglobin is involved in the transport of

oxygen.

3. Transport of electrons to oxygen is done by the cytochrome

system.

4. Certain enzymes e.g. catalase and peroxidase contain the

porphyrin moiety. These are involved in catalytic reactions.

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 HAEM DEGRADATION
• Although cytochrome contains haem, the most
abundant porphyrin in vertebrates is the haem of
haemoglobin found in the erythrocytes.

• The erythrocytes lack nuclei and are therefore incapable

of renewal. In humans, erythrocytes have an average
life span of 120 days.

• Aged erythrocytes are removed from circulation, taken

to the spleen and degraded. Amino acids released from
the globin portion of the molecule are degraded or are
reutilized.
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 Steps involved
1. A mixed function oxidase (microsomal) catalyses the
oxidative cleavage of the ring and converts one of the
methenyl bridge carbon to carbon monoxide.

• This results in the formation of biliverdin IX a green linear

tetrapyrrole. Iron is also released in the process. The only
endogenous form of carbon monoxide in humans is the α-
methene carbon.

• A fraction of the CO is released via the respiratory tract.

Thus the measure of CO in an exhaled breath provides an
index to the quantity of haem that is degraded in an
individual.

2. Iron is transported to storage pools for utilization.

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 3. The biliverdin’s central methenyl bridge is reduced
to form the red-orange bilirubin. NB. The changing
colours of a healing bruise are visible manifestation
of haem degradation.

• The bilirubin is highly lipophilic and is insoluble in

aqueous solution. Its excretion therefore involves
the participation of several organs. It first forms
complex with serum albumin and is transported in
the blood to the liver.

• In the liver, its aqueous solubility is increased by

esterification of its two propionate side chains with
glucuronic acid yielding blilirubin-diglucuronide.

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 • This is then secreted into the bile and eventually into the
intestines.

• Bacterial enzymes (hydrolases) present in the large

intestines hydrolyze the glucuronic acid groups and in a
series of reactions convert bilirubin into several products
especially urobilinogen, a colourless linear tetrapyrrole.

• Some of the urobilinogen is reabsorbed and transported

through the blood stream to the kidney. There, it is
converted to the yellow urobilin and excreted. This gives
urine its yellow colour.

• Most of the urobilinogen however, is microbially

converted to a deeply red-brown stercobilin the major
pigment of faeces.

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 Haem Degradation

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 104
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 • As several organ systems are involved in the degradation of
haem, a few things can go wrong.

• When there is a defect in haem degradation, the highly

insoluble bilirubin accumulates in the blood and becomes
deposited under the skin and the white of the eye, giving a
characteristic yellow colour.

• This condition is known as Jaundice. It may arise as a result of

the following;

a. An abnormally high rate of red blood cell destruction i.e.

haemolytic jaundice. In this condition, liver has the capacity
to conjugate and excrete over 3000mg/day whereas the
normal production is only 300mg/day. Bilirubin is produced
faster than liver can conjugate it so more bilirubin is excreted.

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 b. Liver dysfunction in acute or chronic liver disease i.e.
hepatocellular jaundice. In this condition, there is damage of
liver cells; e.g. in cirrhosis or hepatitis.

• This results in decrease in bilirubin uptake and production of

conjugated bilirubin. Urine is dark in colour and stools are
pale clay colour.

• Plasma levels of aspartate transferase (AST) and alanine

transferase (ALT) are elevated and the patient experiences
nausea and anorexia.

c. Bile duct obstruction i.e. obstructive jaundice e.g. gall stone

or hepatic tumor. This causes gastrointestinal pain, nausea
and stools are pale clay colour.
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 • New born infants especially when premature often
become jaundiced. This is because their livers do not
yet make sufficient bilirubin glucuronyl transferase so
bilirubin diglucuronide formation is impaired.

• Such infants can be treated by exposing them to light

from fluorescent lamp or to brief periods of sunlight.

• This photochemically converts bilirubin to more

soluble isomers that can be degraded and excreted by
the infants.

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 Causes of jaundice

Haemolysis

Production of bilirubin

JAUNDICE

Excretion of bilirubin

Bile duct
Liver damage
obstruction
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 NUCLEOTIDE METABOLISM
• Nucleotides are phosphate esters of pentose in which a
nitrogenous base is linked to C1 of the sugar residue.

• There are two main types; ribonucleotides found in RNA

containing D-ribose residues and deoxyribonucleotides found in
DNA contain 2’ deoxyribose.

• The phosphate group may be linked to the C3 or C5 of the

pentose to yield 3' nucleotide or 5' nucleotide respectively.
Nucleotides without the phosphate group are called nucleosides.

• The N-glycosidic linkage involving the C1 of the pentose is in the β

configuration. The nitrogenous bases are planar, aromatic
heterocyclic molecules which are mostly derivatives of either
purine or pyrimidine.
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 purine pyrimidine

Deoxyribonucleic acid
Ribonucleic acid
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 • The major purine components of nucleic acids are
adenine (A) and guanine (G) and those of pyrimidine
are cytosine (C) and uracil (U) (in RNA) and thymine
(T) (in DNA).
• In purines, the glycosidic bond is linked to N9 while in
pyrimidines, it is linked to N1.

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 112
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 PYRIMIDINES

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 Synthesis of purine nucleotides
• Early studies by John Buchanan and Robert Greenberg
(1948) revealed the reactions involved in the de novo
synthesis of purine nucleotides.

• The studies began with observation that birds

excreted most of their excess nitrogenous compounds
in the form of uric acid, an oxidized purine which is
insoluble in water and therefore can easily be
isolated.

• Researchers administered isotopically labeled

compounds to pigeons. They then crystallized uric
acid from their droppings and by selective chemical
degradation, determined which portions were labeled
by the precursor.
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 • In doing so, they were able to identify low molecular
weight precursors to purine.

• Next, two related antibiotics, azaserine and 6-diazo-5-

oxonorleucine were found to be potent inhibitors in
purine nucleotide synthesis.

• These compounds are structural analogues of glutamine

and were found to be irreversible inhibitors of a class of
enzymes, the glutamine amido transferase which
catalyzes the ATP dependent transfer of the amido
nitrogen of glutamine to an acceptor.

• Three of such reactions occur in purine nucleotide

synthesis.
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 Uric acid
azaserine

AICAR
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 • In later experiments in bacteria, a red compound, an
oxidized product of 5-aminoimidazole-4-carboxamide
ribonucleotide (AICAR) was excreted in large quantities
when the bacteria were treated with sulphonamide drug
like sulfanilamides.

• Since sulphonamides block the synthesis of folate

coenzymes, the accumulation of AICAR under these
conditions suggested the participation of a folate
coenzyme in the reaction.

• AICAR resembles an incomplete purine nucleotide

suggesting that purine ring is assembled while already
attached to the ribonucleotide moiety. These studies
revealed the pattern of purine nucleotide synthesis.

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 Steps involved in Purine nucleotide synthesis

• The synthesis of purines involves the stepwise addition

of the individual atoms to carbon 1 of ribose-5-P to
form the key intermediate, inosine-5’-monophosphate
(IMP) also called inosinic acid.

• This is the 5’ ribonucleotide of the purine base

hypoxanthine.

• During purine synthesis, the smaller of the two rings (5

membered) is completed before the larger one.

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 119
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 Synthesis of IMP
This involves eleven reactions

1. Activation of ribose-5-P to yield PRPP

• Purine synthesis begins with the activation of α-D-ribose-5-P
(from PPP) by ATP to yield 5-phosphoribosylpyrophosphate
(PRPP).

• Enzyme is ribose phosphate pyrophosphokinase (or PRPP

synthetase). PRPP is also a precursor for the biosynthesis of
pyrimidines, histidine and tryptophan.

• The activity of the enzyme will therefore depend on the

concentration of metabolites including PPi, 2,3-
bisphosphoglycerate (activators) and ADP and GDP (mixed
inhibitors). It is a regulatory but not a committed step.

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 2. Introduction of purine atom N9 (displacement of phosphoribosyl group
of PRPP)
• The initial committed step in purine biosynthesis is the
reaction of PRPP with glutamine to form 5-
phosphoribosylamine (PRA).

• Enzyme is amidophosphoribosyltransferase.

• The pyrophosphate group of PRPP is displaced by the amide

nitrogen of glutamine. In the course of the reaction, the α-
configuration of the ribose is inverted to β hence
establishing the anomeric form of purine nucleotide that will
eventually be formed.

• Enzyme is subject to feed back inhibition by purine

nucleotides. The reaction is brought to completion by the
eventual hydrolysis of the released PPi.
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 3. Introduction of Purine atom C4, C5, N7

•The carboxyl group of glycine forms an amide

linkage with the amino group of PRA yielding
glycinamide ribonucleotide (GAR) also called 5-
phosphoribosyl glycinamide.

•The reaction is ATP dependent and is also the

step in purine biosynthesis in which more than one
purine ring atom is acquired.

•Enzyme is GAR synthetase.

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4.Introduction of purine atom C8

• A formyl group is transferred from N10 formyl THF to GAR’s free

amino group to form formyl glycinamide ribonucleotide (FGAR).

• Enzyme is GAR transformylase.

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 5. Introduction of purine atom N3

• The amido group of a second glutamine is transferred

to the growing purine ring to form formyl
glycinamidine ribonucleotide (FGAM) or 5-
phosphoribosyl-N-formylglycinamide.

• Enzyme is FGAR amidotransferase or FGAM

synthetase.

• Reaction is ATP dependent.

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 6. Formation of purine imidazole ring
• FGAM undergoes ring closure by a dehydration
reaction that requires ATP to form the imidazole ring
called aminoimidazole ribonucleotide (AIR).

• Enzyme is AIR synthetase or FGAM cyclase.

• The aromatization of the imidazole ring is facilitated

by the tautomeric shift of the reactant from it’s imine
to its enamine form.

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 7. Introduction of C6

• The C6 of purine is introduced as CO2 in a

Carboxylation reaction that yields
carboxylaminoimidazole ribonucleotide (CAIR).
The reaction is neither biotin nor ATP dependent.

• The reaction has an unfavourable equilibrium and

is driven by its coupling to the exergonic reaction
further along the pathway.

• Enzyme is AIR carboxylase.

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 8. Introduction of NI

• Purine N1 is provided by aspartate in an amide

forming condensation reaction yielding N – succinylo-
5 – aminoimidazole – 4- carboxamide ribonucleotide
(SAICAR) or 5-aminoimidazole-4-(N-
succinylocarboxamide) ribonucleotide.

• Enzyme is SAICAR synthetase. (Reaction is ATP

dependent and resembles step 3).

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 9.Elimination of fumarate

• SAICAR is cleaved with the release of fumarate to

yield 5 – aminoimidazole – 4 – carboxamide
ribonucleotide ( AICAR).
• Enzyme is SAICAR lyase or adenylosuccinate lyase.
(Reactions 8 and 9 chemically resemble reactions in
urea cycle in which citrulline is aminated to form
arginine.
• In both pathways, aspartate’s amino group is
transferred to an acceptor through an ATP driven
coupling reaction followed by the elimination of
aspartate’s carbon skeleton as fumarate).

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 10.Introduction of C2
• N10 formyl THF transfers a single carbon unit to the
growing structure to yield 5-formylaminoimidazole-4-
carboxamide ribonucleotide (FAICAR). Enzyme is
AICAR transformylase.
• In bacteria, this reaction and 4 are indirectly inhibited
by sulphonamides which prevent the synthesis of
folate by competing with its para-amino benzoate
component.
• Animals including humans obtain them from diet.
They are therefore not affected by sulphonamides.

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 11. Cyclization to IMP
• In the final step, there is ring closure to form IMP with
the elimination of water. Reaction does not require
ATP hydrolysis.
• Enzyme is IMP synthase or IMP cyclohydrolase.

Synthesis of Adenine and Guanine ribonucleotides

from IMP.
• IMP serves as a branch point for purine nucleotide
biosynthesis. It does not accumulate in the cell, but is
rapidly converted to AMP and GMP.

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 Synthesis of IMP

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 132

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 Synthesis of AMP
AMP differs from IMP by the replacement of the
keto group with an amino group. It involves two
reactions
• Amino group of aspartate is linked to the C6 of IMP
to yield adenylosuccinate. The reaction is driven by
the hydrolysis of GTP to GDP and Pi. Enzyme is
adenylosuccinate synthetase.(Similar to reaction 8).

• Fumarate is split off adenylosuccinate to yield AMP.

Enzyme is adenylosuccinate lyase. (Resembles step
9 of IMP pathway).

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 Synthesis of GMP
1. IMP is dehydrogenated to form xanthosine
monophosphate (XMP). NAD+ is required for the
oxidation. Enzyme is IMP dehydrogenase.
2. XMP is converted to GMP by the transfer of
glutamine amide nitrogen to replace the keto
group of C2.
• The reaction is driven by ATP hydrolysis to AMP and
PPi and subsequently to 2Pi.

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 Asp + GTP
IMP NAD+ + H2O
IMP dehydrogenase
Adenylosuccinate synthetase
NADH + H+
GDP + Pi

Adenylosuccinate Xanthosine mono phosphate ( XMP)

Glutamine, ATP, H2O
Adenylosuccinate lyase
GMP synthetase or XMP
aminase Glutamate, AMP+ PPi or ADP + pi
Fumarate

AMP (adenylate) GMP (guanylate)

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 Synthesis of nucleotide di- and tri- phosphates from the
monophosphates
• The nucleotides are active in metabolism primarily as
the nucleoside triphosphates.

• The conversion of AMP and GMP to their corresponding

di- and tri- phosphates occurs by two successful
phosphorylation reactions.

1. Conversion to diphosphates. This involves base

specific ATP dependent nucleoside monophosphate
kinases.

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 Nucleoside monophosphate

• NMP + ATP NDP + ADP

kinase

• GMP+ ATP GDP + ADP

Guanylate kinase

• AMP + ATP 2 ADP

Adenylate kinase

• The nucleoside monophosphate kinases do not

discriminate between ribose and deoxyribose in the
substrate.

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 2. Conversion of diphosphates to triphosphates. This
is catalysed by nucleoside diphosphate kinase.

The enzyme is highly active but non specific with regard to

the base on either of its substrates and to the sugar residues,
whether ribose or deoxyribose. ATP is the phosphate donor
for most NDP.

NDP kinase
GDP + ATP GTP + ADP
kinase

N1DP + N2TP N1TP + N2DP

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 • Phosphorylation of ADP to ATP occurs through energy
releasing metabolic reactions, i.e. substrate level
phosphorylation and oxidative phosphorylation.

• ATP can also be formed from ADP through the action

of adenylate kinase in the reverse direction.

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 Control of purine nucleotide metabolism

• The metabolism of nucleic acids is well regulated. This

is indicated by the increased rate of nucleotide
synthesis during cell proliferation.

• Pathways leading to IMP, ATP and GTP are individually

regulated in most cells so as not only to control the
total amount of purine nucleotide produced, but also
to coordinate the relative amounts of ATP and GTP.

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 • IMP pathway is regulated at its first two reactions;
synthesis of PRPP and phosphorybosylamine.
• The first enzyme in the pathway, ribose phosphate
pyrophosphokinase is inhibited by both GDP and ADP.
• The enzyme catalyzing the second reaction,
amidophosphoribosyl transferase is also subject to
feedback inhibition
• The enzyme requires glutamine as substrate as well.

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 • It displays a hyperbolic kinetics with respect to
glutamine as substrate and sigmoidal with respect to
PRPP as substrate.
• Glutamine concentration has little effect in regulating
IMP synthesis because its intracellular concentration
is close to its Km and its concentrations vary relatively
little.
• However, intracellular concentration of PRPP varies
widely and can be between 10 to 100 times less than
the Km for PRPP.
• As a result, PRPP plays important role in regulating
purine nucleotide synthesis.

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 • The enzyme binds ATP, ADP and AMP at one inhibitory
site and GTP, GDP and GMP at another.

• The rate of IMP production is therefore independently

but synergistically controlled by the levels of adenine and
guanine nucleotides.

• Amidophosphoribosyl transferase is allosterically

stimulated by PRPP.

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Glutamine PRPP amido tranferase activity as a function of a) glutamine b) PRPP

• A) B)

PRPP amido transferase activity

PRPP amido transferase activity

Glutamine concentration PRPP concentration

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 • In the presence of IMP, AMP or GMP, the enzyme
forms a dimer, that is much less active.

• PRPP favours active monomer formation.

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 • A second level of control is immediately below the branch
point from IMP to GMP and AMP.

• AMP and GMP are each competitive inhibitors of IMP

during their own synthesis, thus slowing excessive build
up of these products.

• In addition, the rate of synthesis of adenine and guanine

nucleotides is coordinated.

• The hydrolysis of GTP for AMP synthesis from IMP and the
hydrolysis of ATP for GMP synthesis from IMP is thus a
good control mechanism.
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 • This reciprocal activity serves to balance the
production of AMP and GMP which are required in
roughly equal mounts in nucleic acid biosynthesis.

• The rate of synthesis of GMP increases with

increasing ATP concentrations whereas that of AMP
increases with increasing concentrations of GTP.

• The conversion of IMP to GMP is regulated by the

enzyme IMP dehydrogenase which is the rate limiting
enzyme. GMP acts as a competitive inhibitor of the
enzyme.

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 Regulation of purine nucleotide synthesis

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 Use of adenine nucleotides in coenzyme biosynthesis

• Purine nucleotides play metabolic roles in the

synthesis of coenzymes especially those containing
the adenylate moiety.

• These include flavin nucleotides and nicotinamide

nucleotides and coenzyme A.

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 Metabolic functions of nucleotides
1. Role in energy metabolism: Nucleotides play
important role as energy currency of the cell.
Example ATP is principal form of chemical energy in
the cell.
• It drives phosphorylation reactions and is also
involved in muscle contraction, active transport
and maintenance of ion gradient.

• It also serves as phosphate donor for the

synthesis of other nucleoside-5’- triphosphate.

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 2. Monomeric units of nucleic acids; RNA and DNA
are made up of sequences of nucleotide units. DNA
and RNA are involved in protein synthesis and cell
proliferation.
3. Physiological mediators; Nucleosides and
nucleotides serve as physiological mediators of key
metabolic processes. Example: cAMP and cGMP act
as second messengers and signal transduction
involves GTP binding protein ( G – proteins).
4. Precursor function: GTP is precursor for the
formation of cofactor tetrabiopterin required for
some hydroxylation reactions (nitric oxide
generation).

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 5. Structural component of coenzymes; NAD+ and NADP+
and FAD and their reduced forms and CoA all contain 5’
AMP moiety in their structure.

6. Activated intermediates; Nucleotides serve as carriers of

activated intermediates in the synthesis of some
carbohydrates, lipids and proteins. Example UDP – glucose
is involved in glycogen synthesis, GDP – mannose, GDP –
fucose , UDP – galactose and CMP – sialic acid are all
involved in glycoprotein synthesis, eg. blood group
oligosaccharides.

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 7. Allosteric effectors ( important regulatory
compounds) : Many of the regulatory steps in
intermediary metabolism are controlled by
intracellular concentration of nucleotides inhibiting or
activating key enzymes.

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 Salvage of Purines

• Most cells have an active turn over of many of their

nucleic acids especially RNA. These through
degradative processes release adenine, guanine and
hypoxanthine.
• Cells have developed means of converting free
purines to their corresponding nucleotides through
salvage pathways.
• Though purine synthesis is almost identical in all cells,
the salvage pathways are diverse in character and
distribution.
• In mammals purines are mostly salvaged by two
different enzymes.

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 1. Adenine phosphoribosyl transferase (APRT): This
mediates AMP formation through the transfer of
adenine to PRPP with the release of PPi.
Adenine + PRPP AMP +PPi
2. Hypoxanthine guanine phosphoribosyl transferase (
HGPRT): This catalyses the analogous reaction for
both hypoxanthine and guanine.
Hypoxanthine + PRPP IMP + PPi
Guanine + PRPP GMP + Ppi

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 1. Erythrocytes do not have glutamine PRPP amido
transferase and hence cannot synthesize PRA, the
first unique metabolite in the pathway of purine
nucleotide synthesis.

2. They therefore depend on purine phosphoribosyl

transferase and other salvage pathways to replenish
their nucleotide pools.

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 BIOSYNTHESIS OF PYRIMIDINES
• Pyrimidine biosynthesis involves a simpler process
than that of purines.
• Isotopic labeling experiments have shown that N1,
C4, C5 and C6 of the pyrimidine ring are all derived
from aspartate.
• C2 is derived from HCO3 ( CO2) and N3 is obtained
from glutamine. C2 and N3 are derived by the
formation of carbamoyl phosphate.
• The pathway for Pyrimidine biosynthesis is almost
identical in all organisms studied.

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 There are two major distinctions from purine
biosynthesis
• The pyrimidine ring is assembled as a free base with
conversion to a nucleotide occurring after the base
formation when the base, orotic acid is converted to
orotidine monophosphate (OMP).
• The pathway is unbranched. Uridine triphosphate,
(UTP) one of the common ribonucleotide triphosphate
is an end product of the pathway.
• It is also the substrate for the formation of cytidine
triphosphate (CTP), the other end product .

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 Background History
• The pathway for the de novo biosynthesis of
Pyrimidine ribonucleotide was established from
studies on mutants of bread mould Neurospora
crassa.

• These organisms are unable to synthesize

Pyrimidines and therefore require both cytosine and
uracil in their growth medium.

• They were found to grow normally when supplied

with the pyrimidine orotic acid ( Uracil – 6- carboxylic
acid).

• These observations led to the elucidation of a six

reaction pathway for UMP biosynthesis.
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 Synthesis of UMP

1 Synthesis of Carbamoyl Phosphate

• Carbamoyl phosphate is synthesized from HCO3- and
the amide nitrogen of glutamine.

• Enzyme is cytosolic carbamoyl phosphate

synthetase II. Reaction does not require biotin.

• Two molecules of ATP are required; one provides

the phosphate group and the other energizes the
reaction.

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 • In the synthesis of arginine in the urea
cycle, the carbamoyl phosphate used in the
process is synthesized by carbamoyl
phosphate synthetase I which uses
ammonia as the nitrogen source and is
located in the mitochondria.

• Prokaryotes have one carbamoyl

phosphate synthetase which supplies both
pyrimidine and arginine biosynthesis and
utilizes glutamine.
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 2. Synthesis of Carbamoyl Aspartate
• Carbamoyl phosphate condenses with aspartate to
form carbamoyl aspartate.

• Enzyme is aspartate transcarbamoylase (ATCase) or

aspartate carbamoyl transferase.

• Reaction does not require ATP as the carbamoyl

phosphate is intrinsically activated. This is a
committed step in pyrimidine biosynthesis.

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 • In enteric bacteria, the enzyme is inhibited by the
end product CTP and activated by ATP.

• This is a mechanism to keep purine and pyrimidine

biosynthesis in a balance.

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 3. Ring closure to form Dihydro orotate
• An intramolecular condensation occurs with the
elimination of water to yield the ring structure of the
pyrimidine ring, Dihydro orotate.
Enzyme is Dihydro orotase.

4. Oxidation of Dihydro orotate

Dihydro orotate is irreversibly oxidized to orotate.
Enzyme is Dihydro orotate dehydrogenase. In
eukaryotes, the enzyme contains FMN and non haem
iron and is associated with the inner mitochondrial
membrane where quinones supply oxidizing power.

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 5. Acquisition of the Ribose Phosphate moiety
• Orotidine 5’ monophosphate ( OMP) is produced
from a reaction between orotate and PRPP.

• Enzyme is orotate phosphoribosyl transferase.

The reaction is driven by the hydrolysis of the released
PPi. This reaction fixes the anomeric form of
pyrimidine nucleotides in the beta configuration.

• The enzyme also acts to salvage other pyrimidine

bases. e.g. Uracil and cytosine by converting them to
their corresponding nucleotides.

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 6. Decarboxylation to form UMP
• In the final step, OMP is decarboxylated to form
UMP.

• Enzyme is OMP decarboxylase or orotidylate

decarboxylase and requires no cofactors.

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 Synthesis of UTP and CTP
• The synthesis of UTP from UMP is analogous to
purine nucleotide triphosphates. The process involves
a sequential action of a nucleoside monophosphate
kinase and NDP kinase.

UMP kinase UDP + ADP

• CTP is formed byUDP
UMP + ATP
amination
kinase of UTP catalysed by CTP
UDP + ATP UTP + ADP
synthetase. In animals, the amino group is donated
by glutamine and in bacteria by ammonia.

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 2ATP + HCO3-+ GLUTAMINE + H2O
Carbamoyl phosphate
synthetase II
2 ADP + Glu + Pi
Carbamoyl phosphate CTP
Pi, ADP, Glu
Aspartate CTP synthetase
ATCase Gln, ATP

Pi
Uridine triphosphate (UTP)
Carbamoyl aspartate ADP
Nucleoside
diphosphate kinase
ATP
Dihydro orotase Uridine diphosphate (UDP)
H2O
ADP
UMP kinase
Dihydro orotate ATP

Uridine monophosphate (UMP)

NAD+ CO2
Dihydro orotate
dehydrogenase OMP
PRPP PPi decarboxylase
NADH + H+

Orotate Orotidine – 5’ –
Orotate phosphoribosyl
transferase monophosphate
(OMP)

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 Control of Pyrimidine nucleotide biosynthesis.
• In bacteria, Pyrimidine biosynthesis is primarily regulated at
reaction 2, the ATCase reaction.

• In E. Coli, ATP is an allosteric stimulator of the enzyme while

the end product, CTP is an inhibitor.

• In many bacteria, UTP is a major ATCase inhibitor.

• In bacteria, another mode of regulating pyrimidine

biosynthesis is through the control of the synthesis of ATCase
and the other enzymes which are mainly regulated by UTP
concentration.

• The higher the UTP concentration, the lower the rate of

transcription of the genes.
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 • Another site for control is the amido transferase, CTP
synthetase step which converts UTP to CTP. The
enzyme is allosterically inhibited by the end product
CTP and activated by GTP.

• In animals, ATCase is not a regulatory enzyme,

Pyrimidine biosynthesis is controlled by the activity of
carbamoyl phosphate synthetase II which is inhibited
by UDP and UTP and activated by ATP and PRPP.
• At a second control level in the mammalian pathway,
UMP and to a lesser extent, CMP are competitive
inhibitors of OMP decarboxylase.

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 • In all organisms, the rate of production of OMP varies
with the availability of the precursor PRPP.
• The level of PRPP in turn depends on the activity of
ribose phosphate pyrophosphokinase which is
inhibited by ADP and GDP.
• Thus, ADP and GDP levels will eventually regulate
pyrimidine and general nucleotide biosynthesis.

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 Control of Pyrimidine Nucleotide Biosynthesis

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 Salvage of pyrimidines.

• Pyrimidines can be synthesized from degradative

intermediates of the pathway by reactions involving
pyrimidine phosphoribosyl transferases.

Pyrimidine + PRPP Pyrimidine nucleoside – 5’- MP + PPi

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 Nucleotide degradation
• Most food stuffs are of cellular origin and therefore
contain nucleic acids.

• Dietary nucleic acids survive the acid medium of the

stomach. However, in the duodenum, under the action
of pancreatic nucleases and intestinal
phosphodiesterases, they are degraded to their
component nucleotides.

• The nucleotides are ionic and cannot transverse the

cell membranes. As a result, they are hydrolysed to
nucleosides by a variety of group specific
nucleotidases and non specific phosphatases.
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 • The nucleosides may either be directly absorbed by
the intestinal mucosa or first undergo further
degradation to free bases and ribose or ribose – 1- P
through the action of nucleosidases and nucleoside
phosphorylases.

Nucleosidase
Nucleoside + H2O Base + Ribose

Nucleoside phosphorylase
Nucleoside + Pi Base + Ribose -1-P

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 • Radioactive labeling experiments have demonstrated
that only a small fraction of the bases of ingested
nucleic acids are incorporated into tissue nucleic
acids.

• This suggests that the de novo nucleotide

biosynthesis mainly satisfies an organism’s need for
nucleotides.

• Ingested bases are therefore mainly degraded and

excreted. Cellular nucleic acids are also subject to
degradation as part of the continual turn over of
nearly all cellular components.
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 Catabolism of purines
• Degradative pathways of purines vary between
organisms and tissues of the same organism.
• The intermediates of these pathways may be different
but all eventually lead to the production of uric acid.
• The intermediates in the pathway may be re-used for
nucleotide synthesis through salvage reactions.
• The ribose -1- P ( product of Purine Nucleotide
Phosphorylase (PNP) reaction) is isomerised by
phosphoribomutase to ribose – 5 – P, the PRPP
precursor.

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 • In the pathway, adenosine is deaminated to inosine,
catalysed by adenosine deaminase.
• Hydrolysis of the Nucleoside monophosphates, IMP
and GMP yield inosine and guanosine respectively.
• These are then acted on by PNP to yield hypoxanthine
and guanine respectively.
• Guanine deaminase catalyses the deamination of
guanine to xanthine.
• The enzyme is abundant in mammalian brain and
liver.

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 • Hypoxanthine is oxidized to xanthine and xanthine to
uric acid by xanthine oxidase.
• Xanthine oxidase which oxidizes several other
heterocyclic nitrogenous compounds contains bound
FAD, Mo and non – heme iron.
• Electrons from the substrates are passed from each of
the carriers and eventually reduce oxygen to
hydrogen peroxide.
• Xanthine oxidase is abundant in milk and liver

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 Fate of Uric acid
• Purine catabolism in primates ends with uric acid
which is excreted.

• However, most animals further oxidize the purine ring

to allantoin and then to allantoic acid.

• This is either excreted (in some bony fishes) or

further catabolised to urea (most fishes, amphibians,
mollusks) or ammonia ( some marine invertebrates).

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 Fate of uric acid

H2O
Urate oxidase allantoinase
Allantoicase
Uric acid Allantoin Allantoic acid Urea
H2O
2H2O + O2 CO2 + H2O2 H2O
Glyoxylic acid
(Uricase) Urease

CO2 + NH3

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 • Primates (including humans) as well as birds, some
reptiles, Dalmatian dogs and insects, lack urate
oxidase and hence excrete uric acid as the final
product.
• In other terrestrial animals including all other
mammals, uric acid is further oxidized to allantoin and
excreted.
• In many animals other than mammals, allantoin is
metabolized further to urea or ammonia and carbon
dioxide.
• In fish, allantoicate (allantoate) is the end product of
purine degradation.
• Allantoicate is further degraded to glyoxylate and urea
by microorganisms and some amphibians.
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 • Tubular secretion of urate is inhibited by organic acids
such as lactic and oxo acids and by ketones and
thiazide diuretics.

• 75% of urate leaving the body is in urine.

• 25% passes into intestinal lumen where it is broken

down by intestinal bacteria (process called uricolysis).

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 GOUT
• Uric acid and its urate salts are quite insoluble in
water.

• Excessive accumulation of uric acid in the blood

(hyperuricemia) and body fluids leads to a clinical
condition called gout.

• Increased urate levels in the blood causes its

precipitation as crystals of sodium urate in the
synovial fluid of joints.

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 • These precipitates cause inflammation resulting in
excruciatingly painful arthritis which if untreated
eventually leads to severe degeneration of the joints.
• Gout is mistakenly usually associated with excess of
high living, i.e. eating and drinking purine rich foods
(liver, wine, high meat diet, alcohol, etc.)
• Sodium urate may also precipitate in the kidneys and
ureters as stones resulting in renal damage and
urinary tract obstruction.

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 • Other causes of gout include:
❖ Impaired uric acid excretion.

❖Excessive biosynthesis of purine nucleotides far above the

bodies’ needs or capacity for disposition. This has been
attributed to several specific enzyme defects like HGPRT
deficiency, cancer, etc.
Urinary excretion of urate is slightly lower in males than in
females, which may contribute to the higher incidence of
hyperuricemia in men.

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 Causes of Hyperuricemia

Purine synthesis

1
Body purine nucleotides Tissue nucleic acids
3
2
Dietary purines

Purines
• Factors that may contribute
to hyperuricemia 4

1. Inc. synthesis of purine Uric acid

2. Inc. intake of purines
3. Inc. turnover of nucleic acids
4. Inc. rate of urate formation 5 Intestinal uricolysis
5. Reduced rate of excretion by bacteria 25%
Renal excretion
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 Treatment of gout
• It may be successfully treated by the administration of
the antimetabolite allopurinol, a structural analogue
of hypoxanthine which acts by strongly inhibiting
xanthine oxidase.

• Since hypoxanthine and xanthine are more soluble

than uric acid, and can thus be more readily excreted,
their accumulation, following allopurinol treatment is
not deleterious/ harmful.

• It may be necessary to reduce dietary purine intake

e.g.. Red meats, rarely effective by itself.

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 hypoxanthine

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 • An acute attack of gout can be treated with non-
steroidal anti-inflammatory drugs (NSAIDs). Eg.
Aspirin, ibuprofen, indomethacin.
• Renal excretion of urate can be increased with
uricosuric drugs such as probenecid, sulfinpyrazone or
benzbromarone.

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 Catabolism of pyrimidines
• In animal cells pyrimidine nucleotides are degraded to
their component bases.

• Like purines, the reactions occur through

dephosphorylation, deamination and glycosidic bond
cleavages.

• The resulting uracil and thymine are then broken

down in the liver through reduction.

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 • End products of pyrimidine catabolism, β- alanine
and β-amino isobutyrate are amino acids and are
metabolized as such.

• They are converted through transamination and

activation reactions to malonyl CoA and methyl
malonyl CoA for further utilization.

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 Catabolism of pyrimidines

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