Nucleotide Metabolism

(Purine and Pyrimidine Metabolism)

By : Darwish
 Introduction
• Nucleotides are important intracellular molecules.
• They are the building blocks of both DNA and RNA.
• Nucleotides also enter in the structure of many coenzymes.
• They are essential for life and health and are key elements in cell
physiology since they are :
Precursors of deoxyribonucleic acid (DNA) and ribonucleic acid
(RNA).
Components of coenzymes, e.g NAD(H), NADP(H), FMN(H2),
and CoA
 Energy currency, driving many metabolic processes, e.g. ATP and
GTP.
 Carriers in biosynthesis, e.g. UDP for carbohydrates and CDP for
lipids;
• De novo synthesis of nucleotides from basic
metabolites ,which is required in growing cells.
• Salvage pathways that recycle preformed bases
and nucleosides and provide an adequate supply
of nucleotides for cells at rest.
• Catabolic pathways for excretion of nucleotide
degradation products, a process that is essential to
limit.
• The accumulation of toxic levels of nucleotides
within cells: impaired elimination or increased
production of uric acid may produce gout.
 Nucleotide structure
• A nucleotide is composed of nitrogenous base, a
pentose sugar and one ,two, and three phosphate
groups.
• Both DNA and RNA contain the same Purine bases :
adenine and guanine.
• Both DNA and RNA contain the pyrimidine Cytosine
(C), but differ in their second pyrimidine base: DNA
contains thymine (T), whereas RNA contains Uracil
(U).
 PURINE METABOLISM

• De novo synthesis of the purine ring: synthesis of Inosine

monophosphate (IMP).
• Purines and pyrimidines are synthesized by both de novo and
salvage pathways.
• They can be synthesized by adding carbon and nitrogen atoms
to ribose-5-phosphate (De novo pathway) or can be obtained
through salvage pathway by using the preformed purines
(salvage pathway).
• All enzymes involved with purine nucleotide synthesis and
degradation are found in the cytosol of the cell.
 Pyrimidine and purine
Nucleotide bases in nucleic acids are pyrimidines or purines.

Know these!
The biosynthetic origins of Purine ring
atoms
 Sources of atoms in Purine ring
A. Amino acids:
1. Aspartate: gives N1
2. Glycine : gives C4,C5 and N7.
3. Glutamine : gives N3 and N9
B. CO2 : gives C6
C. FH4 (tetrahydrofolate): gives C2 and C8.
• The Purines are built upon a pre-existing ribose 5-phosphate.
• Liver is the major site for purine nucleotide synthesis.
• Erythrocytes, polymorphonuclear leukocytes and brain
cannot produce purines
 Biosynthesis of Purine nucleotides

At first produces Starts from ribose-

IMP 5-phosphate

Requires 11 steps
over all
 Steps of purine nucleotides

1. Synthesis of 5’-phosphoribosyl-1-
pyrophosphate (PRPP):
• The transfer of pyrophosphate (PP) from ATP to C-1 of
ribose 5-phosphate form 5-phosphoribosyl-1-
pyrophosphate (PRPP).
• PRPP also participates in the synthesis of pyrimidines and
in the salvage reactions of purine.
• Enzyme catalyzed this step is phosphoribosyl-
pyrophosphate kinase
Step 1:
• Step 2: acquisition of purine atom 9 or synthesis of 5’-
phosphoribosylamine:
• Enzyme: amidophosphoribosyl transferase
• Displacement of pyrophosphate group by glutamine amide
nitrogen (inversion of configuration – a to b).
• The enzyme PRPP glutamyl aminotransferase
• Product: β-5-phosphoribosylamine

Steps 1 and 2 are tightly regulated by feedback inhibition

Step 2:
• Step 3: acquisition of purine atoms C4, C5, and N7
• Enzyme: glycinamide synthetase (GAR synthetase)
• β-phosphoribosylamine reacts with ATP and glycine
• Product: glycinamide ribotide (GAR)
Step 3:
• Step 4: acquisition of purine atom C8
• Formylation of free α-amino group of GAR
• Enzyme: GAR transformylase
• co-factor of enzyme is N10-formyl THF
• Step 5: acquisition of purine atom N3
• The amide amino group of a second glutamine is transferred to
form formylglycinamidine ribotide (FGAM)

• Step 6: closing of the imidazole ring or

formation of 5-aminoimidazole ribotide
Step 6:
 Step 7

• Step 7: acquisition of C6
• C6 is introduced as HCO3-
• Enzyme: AIR carboxylase (aminoimidazole ribotide
carboxylase)
• Product: CAIR (carboxyaminoimidazole ribotide)
Step 7: purine synthesis
Steps 8

• Step 8: acquisition of N1
• N1 is acquired from aspartate in an amide condensation
reaction
• Enzyme: SAICAR synthetase
• Product: 5-aminoimidazole-4-(N-
succinylocarboxamide)ribotide (SAICAR)
• reaction is driven by hydrolysis of ATP
Step 8: Purine
synthesis
• Step 9: elimination of Fumarate
• Enzyme: adenylosuccinate lyase
• Product: 5-aminoimidazole-4-carboxamide ribotide (AICAR)
• Step 10: acquisition of C2
• Another formylation reaction catalyzed by AICAR
transformylase
• Product: 5-formaminoimidazole-4-carboxamide ribotide
(FAICAR).
Step 9:
Step 10: purine
synthesis
 Step 11

• Cyclization or ring closure to form IMP

• water is eliminated in contrast to step 6
(closure of the imidazole ring), this reaction
does not require ATP hydrolysis.
• once formed, IMP is rapidly converted to
AMP and GMP (it does not accumulate in
cells
Step 11: purine
synthesis
IMP
 Summary of the purine biosynthesis pathway

A. Synthesis of 5’-phosphorisyl-1-pyrophosphate (PRPP)

• The transfer of pyrophosphate (PP) from ATP to C1 of ribose 5-phosphate
forms 5phosphoribosyl-1-pyrophosphate.
B. Synthesis of 5-phosphoribosylamide: the synthesis of 5-
phosphoribosylamide from PRPP and glutamine is done by replacement of
pyrophosphate from PRPP by the amide nitrogen of glutamine.
C. Synthesis of inosine-monophosphate (IMP): condensation and
acquisition reaction of glycine, aspartate, glutamine,Co2 and folate with 5-
phosphoribosylamide form inosine monophosphate.
D. Conversion of IMP to AMP and GMP:
• The synthesis of AMP requires GTP as an energy source, first reaction in
each pathway is inhibited by the end product of the pathway.
 Regulation of purine de novo
synthesis
• Purine synthesis utilize 6 ATP molecules, and requires
glycine,glutamine,aspartate, and tetrahydofolate
(THF) derivatives.
• The regulation of this pathway depends on :
a. Availability of ribose 5-phosphate
b. Activity of PRPP synthase, which feedback, regulated by
AMP,ADP,GMP,GDP.
c. Activity of PRPP glutamyl amidotransferase, which feed
back, regulated by IMP and AMP
 salvage pathway for purine
biosynthesis
Requires
Purine
bases and Two enzymes
PRPP
HGPRT (hypoxanthine
APRT (adenine
guanine phosphoribosyl
phosphoribosyl
transferase) for guanine
transferase) for adenine
or hypoxanthine
 Salvage pathway for purine nucleotide
formation

• Enzymes of de novo synthesis of purine are deficiency in

some tissues like brain cells, red cells and white blood cells.
• In addition to de novo synthesis, cells can use preformed
nucleotides obtained from the diet or from the breakdown of
endogenous nucleic acids through salvage pathways.
• In mammals, there are two enzymes in the purine salvage
pathway.
• Adenine phosphoribosyl transferase (APRT) converts free
adenine into AMP.
• Hypoxanthineguanine phosphoribosyl transferase
(HGPRT) catalyzes a similar reaction for both
hypoxanthine (the purine base in IMP) and guanine.
• The sources of purines for this pathway are either bases
synthesized in liver or those derived from cellular
breakdown
• The reaction proceed as shown in the following figures
• Lesch Nyhan syndrome: it is one type of hyperuricemia
plasma uric acid) resulting form deficiency of hypoxanthine
guanine phosphoribosy transferase (HGPRTase) enzyme.
Catabolism of Purine nucleotide

• Sources and disposal of uric acid is the end-product of purine

catabolism in humans.
• Uric acid, the end product -of purine catabolism in humans, is not
metabolized and must be excreted.
• Net excretion of total uric acid in normal human is about 500
mg/day
• Normal human plasma uric acid average 2-7 mg/dl.
• In mammals other than higher primates,uric acid is further oxidized
into a highly water soluble end product called allantion. This
reaction is catalyzed by uricase enzyme, which is lacked in human.
Nucleotide degradation
Steps
CATABOLISM OF PURINES
 Disorders of purine catabolism

A. Hyperuricemia:
• Definition : hyperuricemia is a condition in which serum urate level is
increased above normal level (2-7 mg/dl) and exceeds its solubility limit
• Causes of hyperuricemia:
I. Over activity of PRPP synthase: this leads to purine overproduction and
excretion.
II. Decreased HGPRTase (hypoxanthine guanine phosphoribosyl transferase)
enzyme (lesch Nyhan syndrome): this enzymatic defect leads to
hyperuricemia
III. Increase-Glucose 6-phosphate (von Gierke,s disease): it is one type of
glycogen storage diseases due to deficiency of glucose-6-phosphatase. This
enzymatic defect leads to hyperuricemia
• Treatment of hyperuricemia:
a. Treatment of the cause
b. Allopurinol : it is a structural analogue of the
hypoxanthine that competitively inhibits xanthine
oxidase enzyme then decreasing formation of uric acid.
B. Hypouricemia:
• It is rare condition associated with xanthine oxidase
deficiency. It is due to either genetic defect or severe
liver damage.
• It resulting in excess excretion of xanthine and
hypoxanthine and patient may show xanthine renal stone
 Effect of hyperuricemia/Gout
a. Tophi formation: increased insoluble urate leads to
crystallization of sodium urate in soft tissues and joints,
which results in formation of deposits called : tophi .
b. The tophi causes an inflammatory reaction called gouty
arthritis.
c. The joints that firstly affected are small joints especially
those of big toes
d. Renal stone: deposition of urate crystals in renal tubules
may lead to stone formation e.g kidney stone.
e. Lesch Nyhan syndrome: is characterized by
hyperuricemia, uric acid renal stone, neurological
disorders and mental retardation
Gout
 Biosynthesis of Pyrimidine
• Sources of atoms in pyrimidine ring:
a. Aspartate: gives N1, C4, C5 and C6
b. Carbamoyl phosphate: gives C2 and N3
Synthesis of pyrimidine:
• It begins with the formation of carbamoyl phosphate from
glutamine, ATP and CO2.
• This reaction is catalyzed by cytosolic carbamoyl phosphate
synthase II.
• Pyrimidine rings is first synthesis where in purine ring is built upon
a pre-existing ribose 5-phosphate.
Nucleotide bases in nucleic acids are pyrimidines or purines.
 Steps
• Glutamine transfers its amido nitrogen to CO2 to produce
carbamoyl phosphate.
• This reaction is ATP-dependent and is catalysed by cytosomal
enzyme carbamoyl phosphate synthetase ll (CPSII).
• CPS ll is activated by ATP and PRPP and inhibited by UTP.
• Carbamoyl phosphate synthetase| (CPSl ) is a mitochondrial
enzyme which synthesizes carbamoyl phosphate from
ammonia and CO2 and, in turn urea protein metabolism.
• Carbamoyl phosphate condenses with aspartate to
form carbamoyl aspartate.
• This reaction is catalysed by aspartate
transcarbamoylase.
• Dihydroorotase catalyses the pyrimidine ring
closure with a loss of H2O.
• The three enzymes-CPS ll, aspartate
transcarbamoylase and dihydroorotase are the
domains (functional units) of the same protein.
• This is a good example of a multifunctional
enzyme.
• The next step in pyrimidine synthesis is an NAD+ dependent
dehydrogenation, leading to the formation of orotate.
• Ribose5 –phosphate is now added to orotate to produce
orotidine monophosphate (OMP).
• This reaction is catalysed by orotate
phosphoribosyltransferase, and enzyme comparable with
HGPRT in its function.
• OMP undergoes decarboxylation to uridine mono-phosphate
(UMP).
• Orotate phosphoribosyltransferase and OMP decarboxylase
are domains of a single protein.
• A defect in this bifunctional enzyme causes orotic Aciduria .
• By an ATP-dependent kinase reaction, UMP is converted to
UDP which serves as a precursor for the synthesis of dUMP,
dTMP, UTP and CTP.
• Ribonucleotide reductase converts UDP to dUDP by a
thioredoxin-dependent reaction.
• Thymidylate synthetase catalyses the transfer of a methyl
group from N5, N10-methylene tetrahydrofolate to produce
deoxythymidine monophosphate( dTMP).
• UDP undergoes an ATP-dependent kinase reaction to produce
UTP.
• Cytidine triphosphatase (CTP) is synthesized from UTP by
amination.
• CTP synthetase is the enzyme and glutamine provides the
nitrogen
Pyrimidine synthesis
 Regulation of pyrimidine
synthesis
• Carbamoyl phosphate synthetase II (CPS II)
is the regulatory enzyme of pyrimidine
synthesis in animals.
• It is activated by PRPP and ATP and
inhibited by UDP and UTP.
• OMP decarboxylase, inhibited by UMP and
CMP, also controls pyrimidine formation.
 Salvage pathways

• Mammalian cells cannot use free pyrimidine bases

to form pyrimidine nucleotides
• They can convert (salvage) pyrimidine
nucleosides ; uridine ,cytidine and thymidine to
their respective mononucleotides.
 Catabolism of Pyrimidine

• The catabolism of pyrimidines occurs mainly in the liver.

• β-alanine is the major end product of both cytosine and
uracil.
• β-aminoisobutyric acid is the major end product of
thymine.
• The release of respiratory CO2 from the pyrimidine
nucleus (C2) occurs in the catabolism of all three
pyrimidine bases.
 Steps of catabolism of
Pyrimidine
• Steps : the overall reactions are summarized on the
board
 Metabolism disorder of
pyrimidine
A. Orotic aciduria : This is a rare metabolic
disorder characterized by the excretion of orotic
acid in urine, severe anemia and retarded growth.
• lt is due to the deficiency of the enzymes orotate
phosphoribosyl transferase and OMP
decarhoxylase of pyrimidine synthes.
• Both these enzyme activities are present on a single
protein as domains (bifunctional enzyme).
• Feeding diet rich in uridine and/or cytidine is an effective
treatment for orotic aciduria.
• These compounds provide (through phosphorylation)
pyrimidine nucleotides required for DNA and RNA
synthesis
B. Reye's syndrome : This is considered as a secondary
orotic aciduria.
• It is believed that a defect in ornithine
transcarbamoylase(of urea cycle) causes the
accumulation of carbamoyl Phosphate
• This is then diverted for the increased synthesis and
excretion of orotic acid.