2. 1. Nucleotide functions in cells
2. Origin of nucleotides
1. De novo synthesis of nucleotides
2. Salvage pathways of nucleotides
3. Catabolism of nucleotides
4. Nucleotide metabolism disorders
Learning Objectives
3. 1. NUCLEOTIDE FUNCTIONS IN CELLS
1.Structural components of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA)
are essential to the maintenance, protein synthesis and inheritance of the cell. Nucleic
acid precursors.
2. Activated intermediate transporters in the synthesis of UDP-glucose molecules or
CDP-
choline conjugated proteins.
3. Structural elements of essential coenzymes: FADH2, NADH, NADPH and CoA.
4. Second messengers in signal transduction: cAMP and cGMP.
5. Allosteric regulators of metabolic pathways.
6. ATP donor phosphoryl groups for protein kinases. ATP universal energy currency, GTP.
4. Bases can be methylated,
glycosylated,
acetylated and reduced
NITROGEN BASES NUCLEOSIDES NUCLEOTIDES:
Mono, di, triphosphate
Adenosin-triphosphate
ribonucleoside
mono
diphosphate
NUCLEOSIDES
NOMENCLATURE
1. NUCLEOTIDE FUNCTIONS IN CELLS
SUGARS
linkage occurs at C5
BASES
NUCLEOTIDES
The carbon numbers of the ribose
and deoxyribose are identified
with a prime (‘): phosphate ester
6. Natural polymers of nucleotides—nucleic acids.
• (a) Deoxyribonucleic acid (DNA) has 2-deoxyribose residues
and uses thymine but not uracil.
• Ribonucleic acid (RNA) has ribose residues and uses uracil
but not thymine.
• Both polymers are formed by C3 –C5
′ ′ phosphodiester bonds.
1. NUCLEOTIDE FUNCTIONS IN CELLS
Da Poian
7. ORIGIN OF NUCLEOTIDES
Purine and pyrimidine bases:
-De novo
-Nucleotides
salvage
pathway
2. ORIGIN OF NUCLEOTIDES
BASE NUCLEOSIDE
(BASE +
SUGAR)
NUCLEOTIDE
(BASE + SUGAR
+ Pi)
PURINES
Adenine (DNA, RNA) Adenosine AMP
Guanine (DNA, RNA) Guanosine GMP
Hypoxanthine Inosine IMP
PYRIMIDINES
Cytosine (DNA, RNA) Cytidine CMP
Uracil (RNA) Uridine UMP
Thymine (DNA) Thymidine dTMp
5%
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95%
8. 2.1 DE NOVO SYNTHESIS OF NUCLEOTIDES
• Several important precursors are shared by the de novo pathways for the synthesis of
pyrimidines and purines.
• Phosphoribosyl pyrophosphate (PRPP) is required.
• The structure of ribose is retained in the product nucleotide.
There is a specific amino acid precursor for each type of nucleotide synthesis pathway:
Glycine Purines
Aspartate Pyrimidines
Glutamine is the most important source of amino groups. It is involved in five different steps in
the de novo synthesis pathway.
Aspartate is also used as the source of an amino group in the purine pathway in two steps.
There is evidence, especially in the de novo purine pathway, that the enzymes are present as large,
multienzyme complexes in the cell.
The cellular pools of nucleotides (other than ATP) are quite small, roughly 1% or less of the amounts
required to synthesize the cell’s DNA. Cells must therefore continue to synthesize nucleotides during
nucleic acid synthesis and, in some cases, nucleotide synthesis may limit the rates of DNA replication
and transcription. Because of the importance of these processes in dividing cells, agents that inhibit
nucleotide synthesis have become particularly important in medicine.
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9. De novo synthesis of purine nucleotides: IMP, AMP and GMP
This process takes place mainly in the liver.
-Purine ring:
• 3 amino acids required (aspartate, glycine and
glutamine)
• Carbon dioxide (CO2)
• N10-formyl-THF (tetrahydropholate)
-Process:
• Addition of N and C to 5-phosphate ribose
preformed
• Highly complex process
-Requires:
• 5-phosphorybosil-1- pyrophosphate (PRPP)
• IMP
-High allosteric regulation
Origin of purine rings
With the exception of 3 atoms from glycine,
each C atom of the purine comes from a
different precursor in a different reaction.
2.1 DE NOVO SYNTHESIS OF NUCLEOTIDES
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10. De novo synthesis of purine nucleotides: IMP, AMP, GMP
IMP (inosin-5’-monophosphate) synthesis from 5-phosphorybosilamine
IMP is the purine nucleotide precursor for AMP and GMP and contains hypoxanthine as base
A huge number of enzymatic reactions are involved
Glycine precursor
PRPP is required
Glutamine and Aspartate are required
ATP is consumed
2.1 DE NOVO SYNTHESIS OF NUCLEOTIDES
-AMP synthesis: GTP and Asp are required
Control mechanisms in purines supply Department of
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-GMP synthesis: ATP and Gln are required
11. De novo synthesis of purine nucleotides: IMP, AMP, GMP
Purine nucleotide synthesis
2.1 DE NOVO SYNTHESIS OF NUCLEOTIDES
Purine nucleotide synthesis regulation:
-Negative Feedback Regulation by:
1. PRPP synthetase AMP, ADP and GMP
2. PRPP amide transferase AMP, ADP, GMP, GDP
3. AMP synthesis requires GTP, and GMP requires ATP: Cell mechanism to keep both nucleotides balanced
ATP accumulation Increase in GMP synthesis Decrease in AMP
GTP Accumulation Increase in AMP synthesis Decrease in GMP
Harvey and Ferrier
*Purine synthesis inhibitors: antitumoural treatments Department of
Biochemistry and Molecular Biology
12. De novo synthesis of pyrimidine nucleotides: UMP, TMP, CTP
-Synthesis of the pyrimidine ring before binding to the PRPP that donates 5’ribose phosphate
- Pyrimidine ring: Ammonia from glutamine, carbon from CO2 and aspartate
-Step is regulated by carbamoyl phosphate synthetase (CPS) II:
Carbamoyl phosphate synthesis from CO2 and glutamine
Pyrimidine ring
1: Synthesis of carbamoyl
phosphate:
Carbamoyl Pi + aspartate
2: Synthesis of orotic acid:
Orotic acid + PRPP
3: Formation of a
pyrimidine nucleotide:
UTP synthesis
Pyrimidine nucleotides are
made from
aspartate, PRPP, and
carbamoyl phosphate
2.1 DE NOVO SYNTHESIS OF NUCLEOTIDES
Gln
Harvey and Ferrier
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13. PRPP
- +
UTP
2.1 DE NOVO SYNTHESIS OF NUCLEOTIDES
UMP
UTP
CTP
TTP Pyrimidine nucleotide biosynthesis is regulated by feedback inhibition
De novo synthesis of pyrimidine nucleotides: UMP, TMP, CTP
Summary of the differences between
carbamoyl phosphate synthetase
(CPS) I and II
C
P
S
I
I
i
s
i
n
h
i
b
i
t
e
d
b
y
Synthesis of CTP from UTP
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14. Deoxyribonucleotide synthesis
Reduction of NTPs to dNTPs:
Ribonucleotide reductase uses ATP as a donor of Pi groups as it is the most abundant energy donor.
ADP
dADP dGDP dCDP dUDP
GDP CDP
UDP
Ribonucleotide reductase
dATP dGTP dCTP dTTP
2.1 DE NOVO SYNTHESIS OF NUCLEOTIDES
Conversion of ribonucleotides to deoxyribonucleotides
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+
ATP
15. 2.2 SALVAGE PATHWAYS
Purine and pyrimidine bases are recycled by salvage pathways
Free purine and pyrimidine bases are constantly released in cells during the metabolic degradation of nucleotides.
PURINES
Free purines are in large part salvaged and reused to make nucleotides, in a pathway much simpler than the de novo
synthesis of purine nucleotides described earlier.
One of the primary salvage pathways consists of a single reaction catalyzed by hypoxanthine-guanine
phosphoribosyl transferase (HPGRT) to release free guanine and hypoxanthine (the deamination product of
adenine). The other enzyme participating in catalysis is adenine phosphoribosyl transferase (APRT), in which free
adenine reacts with PRPP to yield the corresponding adenine nucleotide.
2 Enzymes: adenine phosphoribosyl transferase (APRT) and hypoxanthine-
guanine phosphoribosyl transferase (HGPRT).
These reactions use PRPP as the source of ribose 5-phosfate and are
irreversible.
If there is hypoxanthine, purine nucleotides are always synthesized by the
salvage pathway.
Hypoxanthine inhibits de novo synthesis.
*HGPRT Deficiency: Lesch-Nyhan Syndrome
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16. Pyrimidine nucleotides
Purine nucleotides
AMP IMP GMP
ADENOSINE INOSINE GUANOSINE
(BASES) HYPOXANTHINE GUANINE
XANTHINE
URIC ACID
HGPRT
SALVAGE
PATHWAY
95%
dTMP
CMP UMP
CYTIDINE URIDINE THYMIDINE
URACIL THYMINE
β-ALANINE β-AMINOBUTYRATE
+ CO2 + NH+
4
XANTHINE OXIDASE
5%
3. CATABOLISM OF NUCLEOTIDES
UREA CYCLE
Purines: These are degraded to uric acid. Uric acid is
the final product of purine degradation. It enters
the bloodstream and is excreted in urine.
Pyrimidines: These are completely degraded to β-
alanine and β-aminobutyric acid to enter the urea
cycle.
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17. Uric acid synthesis
PURINES XanthineUrate URINE excretion
Normal ratio of excretion: 0.6 g/24 h
The excreted product arises partly from ingested purines
and partly from turnover of the purine nucleotides of
nucleic acids.
3. CATABOLISM OF NUCLEOTIDES
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18. 4. NUCLEOTIDE METABOLISM ALTERATIONS
PURINES
Alterations in the salvage pathways of purine nucleotide synthesis
Lesch-Nyhan syndrome
HGPRT deficiency: Lesch-Nyhan syndrome: a rare recessive X-linked
disorder.
Consequences:
-Inability to rescue hypoxanthine or guanine.
-Increased degradation of purines Too much uric acid
(hyperuricaemia)
-Increase in PRPP levels and decrease in IMP and GMP levels.
-Increased de novo purine synthesis (no synthesis inhibitor).
-Formation of uric acid deposits in the kidneys, deposition of urate
crystals in the joints (gouty arthritis).
-Motor dysfunction, cognitive deficits and behavioural disorders (self-
mutilation).
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Children with this genetic disorder, which becomes manifest by the age of two,
are sometimes poorly coordinated and have intellectual deficits.
They are also extremely hostile and show compulsive self-destructive
tendencies: they mutilate themselves by biting off their fingers, toes, and lips.
This syndrome is a potential target for gene therapy.
Harvey and Ferrier
19. 4. NUCLEOTIDE METABOLISM ALTERATIONS
PURINES
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Harvey et al.,
Biosynthesis and
degradation of purine
nucleotides to uric acid
illustrating some of the
genetic diseases
associated with these
pathways.
20. Adenosine deaminase (ADA) deficiency leads to severe immunodeficiency disease in which T and B lymphocytes do not
develop properly.
A lack of ADA leads to a 100-fold increase in the cellular concentration of dATP, a strong inhibitor of ribonucleotide
reductase.
High levels of dATP produce a general deficiency of other dNTPs in lymphocytes.
Individuals with ADA deficiency lack an effective immune system and do not survive unless treated.
Current therapies include bone marrow transplant from a matched donor to replace the hematopoietic stem cells that
mature into B and T lymphocytes. However, transplant recipients often suffer a variety of cognitive and physiological
problems.
Enzyme replacement therapy, requiring once- or twice-weekly intramuscular injection of active ADA, is effective but
the therapeutic benefit often declines after 8 to 10 years and complications arise, including malignancies.
For many people, a permanent cure requires replacing the defective gene with a functional one in bone marrow
cells. ADA deficiency was one of the first targets of human gene therapy trials first assayed in 1990.
Immunodeficiciency syndrome (ADA)
PURINES
Alterations in the biosynthesis and degradation of purine nucleotides.
4. NUCLEOTIDE METABOLISM ALTERATIONS
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21. PURINES
Alterations in the degradation of purine nucleotides to uric acid.
Excess uric acid causes gout
Gout is a disease of the joints caused by an elevated concentration of
uric acid in the blood and tissues.
The joints become inflamed, painful and arthritic, owing to the
abnormal deposition of sodium urate crystals.
The kidneys are also affected, as excess uric acid is deposited in the
kidney tubules. Gout occurs predominantly in males. Its precise cause is
not known but it often involves an underexcretion of urate.
A genetic deficiency of one or another enzyme of purine metabolism
may also be a factor in some cases.
Gout is effectively treated by a combination of nutritional and drug
therapies. Patients exclude foods especially rich in nucleotides and
nucleic acids from the diet.
Major alleviation of the symptoms is provided by the drug allopurinol,
which inhibits xanthine oxidase, the enzyme that catalyzes the conversion
of purines to uric acid. When xanthine oxidase is inhibited, the excreted
products of purine metabolism are xanthine and hypoxanthine, which are
more water-soluble than uric acid and less likely to form crystalline
deposits.
Allopurinol, an inhibitor of xanthine oxidase.
Hypoxanthine is the normal substrate of xanthine
oxidase.
Allopurinol is slightly different to hypoxanthine
and acts as an enzyme inhibitor.
At the active site, allopurinol is converted to
oxypurinol, a strong competitive inhibitor that
remains tightly bound to the reduced form of the
enzyme.
4. NUCLEOTIDE METABOLISM ALTERATIONS
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22. PURINES
Other related pathologies
Roughly 10% of the human population (and up to 50% of people in impoverished communities) suffer from folic
acid deficiency.
When the deficiency is severe, the symptoms can include heart disease, cancer, and some types of brain
dysfunction.
At least some of these symptoms arise from a reduction of thymidylate synthesis, leading to an abnormal
incorporation of uracil into DNA. Uracil is recognized by DNA repair pathways and is cleaved from the DNA.
The presence of high levels of uracil in DNA leads to strand breaks that can greatly affect the function and
regulation of nuclear DNA, ultimately causing the observed effects on the heart and brain, as well as increased
mutagenesis that leads to cancer.
Many chemotherapeutic agents target enzymes in nucleotide biosynthetic pathways
The growth of cancer cells is not controlled in the same way as cell growth in most normal tissues. Cancer
cells have greater requirements for nucleotides as precursors of DNA and RNA, and consequently are generally
more sensitive than normal cells to inhibitors of nucleotide biosynthesis.
A growing array of important chemotherapeutic agents – for cancer and other diseases – act by inhibiting one
or more enzymes in these pathways.
PURINE SYNTHESIS INHIBITORS
- are used to treat several types of cancer
-are extremely toxic for tissues with a high rate of replication, including bone marrow, the skin,
the gastrointestinal tract, the immune system and follicular hair.
Folic acid analogues (i.e. methotrexate):
individuals taking such anticancer drugs may suffer from:
4. NUCLEOTIDE METABOLISM ALTERATIONS
anaemia, scaly skin, gastrointestinal tract disorders, immunodeficiency, and hair loss. Department of
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23. ■The purine ring system is built up step by step, beginning with 5-phosphoribosylamine. The
amino acids glutamine, glycine and aspartate furnish all the nitrogen atoms of purines. Two ring-
closure steps form the purine nucleus.
■Pyrimidines are synthesized from carbamoyl phosphate and aspartate, and ribose 5-phosphate
is then attached to yield the pyrimidine ribonucleotides.
■Nucleoside monophosphates are converted into their triphosphates by enzymatic
phosphorylation reactions. Ribonucleotides are converted into deoxyribonucleotides by
ribonucleotide reductase, an enzyme with novel mechanistic and regulatory characteristics. The
thymine nucleotides are derived from dCDP and dUMP.
■ Uric acid and urea are the end products of purine and pyrimidine degradation.
■Free purines can be salvaged and rebuilt into nucleotides. Genetic deficiencies in certain
salvage enzymes cause serious disorders, such as Lesch-Nyhan syndrome and ADA deficiency.
■ The accumulation of uric acid crystals in the joints, possibly caused by another genetic
deficiency,
results in gout.
■Enzymes of the nucleotide biosynthetic pathways are targets for an array of
chemotherapeutic agents used to treat cancer and other diseases.
BIOSYNTHESIS AND DEGRADATION OF NUCLEOTIDES. SUMMARY.
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