De novo synthesis, salvage, and catabolism of purines and pyrimidines

1. Metabolism of Nucleotides
R. C. Gupta
Department of Biochemistry
National Institute of Medical Sciences
Jaipur, India

2. Nucleotides containing purine and
pyrimidine bases are essential for life
They are required for the synthesis of
DNA and RNA
Many nucleotides, e.g. ATP, cAMP, cGMP
etc, perform other important functions

3. Nucleotides are both taken in diet and
are synthesized in the body
Dietary nucleotides cannot be used in
our body
We are dependent exclusively on
endogenously synthesized nucleotides

4. In food, nucleotides are present mainly in
the form of nucleoproteins
The protein part is degraded by
proteolytic enzymes in the GIT
The nucleic acids are hydrolysed by
nucleases
Digestion

5. DNA is hydrolysed by deoxyribonuclease
and RNA by ribonuclease
Polynucleotidases hydrolyse small poly-
nucleotides into mononucleotides
Nucleotidases hydrolyse nucleotides into
nucleosides and phosphate

6. Nucleosides are absorbed by mucosal
cells of intestine
They are broken down to their final end
products in intestinal mucosal cells
The end products are released into
circulation, and are excreted in urine

7. Synthesis of nucleotides
Only endogenously synthesized purine
and pyrimidine nucleotides are used for
various purposes in our body
There are two pathways for the synthesis
of nucleotides:
• De novo synthesis
• Salvage pathway

8. De novo
synthesis
Nucleotides are synthesized
afresh from amphibolic inter-
mediates
Bases or nucleosides released
from catabolism of pre-existing
nucleic acids and nucleotides
are reutilized
Salvage
pathway

9. The salvage pathway is energy-efficient
It prevents wastage of raw materials

10. Liver is the major site for de novo
synthesis of purine nucleotides
Synthesis can occur in several other
tissues
The synthesis occurs in the cytosol
De novo synthesis of purine nucleotides

11. Different carbon and nitrogen
atoms are provided by:
Glycine
Glutamine
Aspartate
Carbon dioxide
Single-carbon moiety carried by
tetrahydrofolate

12. The nitrogen at position 1 is provided by
aspartate
Carbon 2 comes from N10-formyltetra-
hydrofolate
Nitrogen 3 and nitrogen 9 come from the
amide group of glutamine

13. Carbon atoms 4 and 5, and the nitrogen
atom at position 7 are provided by glycine
Carbon 6 comes from carbon dioxide
Carbon 8 is provided by N5,N10-methenyl
tetrahydrofolate

15. The first reaction is formation of 5-
phosphoribosyl-1-pyrophosphate (PRPP)
PRPP is required for the synthesis of
pyrimidine nucleotides also
By a series of reactions, PRPP is
converted into inosine monophosphate

22. AMP and GMP are converted into ADP
and GDP respectively by nucleoside
monophosphate kinase
ADP and GDP are converted into ATP
and GTP respectively by nucleoside
diphosphate kinase
Formation of ATP and GTP

24. PRPP synthetase catalyses the first
reaction of de novo synthesis
It is an allosteric enzyme
It is inhibited by AMP, ADP, GMP and
GDP
Regulation of de novo synthesis

25. PRPP synthetase is not unique to purine
nucleotide synthesis
PRPP is required for the synthesis of
pyrimidine nucleotides as well
Therefore, PRPP synthetase is not the
main regulatory enzyme

26. PRPP glutamyl amidotransferase
catalyses the first reaction unique to
purine nucleotide synthesis
This is the main regulatory enzyme
It is allosterically inhibited by GMP

27. Synthesis of adenine and guanine nucleo-
tides is cross-regulated
Conversion of IMP into AMP requires GTP;
conversion of IMP into GMP requires ATP
This ensures a balanced production of
AMP and GMP

28. Conversion of IMP into AMP and GMP is
also regulated by allosteric mechanism
AMP is an allosteric inhibitor of adenylo-
succinate synthetase
Similarly, GMP is an allosteric inhibitor of
IMP dehydrogenase

29. Pre-existing purine bases and nucleosides
may be salvaged to form new nucleotides
Conversion of purine bases into nucleotides
is catalysed by two enzymes:
Synthesis of purine nucleotides by
salvage pathway
Hypoxanthine-guanine
phosphoribosyl
transferase (HGPRT)
Adenine
phosphoribosyl
transferase (APRT)

30. APRT acts on adenine
HGPRT acts on hypoxanthine and guanine
These enzymes transfer a phosphoribosyl
group to their substrates
PRPP provides the phosphoribosyl group

32. Purine nucleosides may be salvaged by
adenosine kinase and deoxycytidine kinase
Adenosine kinase acts on adenosine and
deoxyadenosine
Deoxycytidine kinase acts on deoxyadeno-
sine, deoxyguanosine and deoxycytidine

34. The salvage pathway is regulated mainly
by the availability of PRPP
The available PRPP is used primarily for
salvage reactions, and secondarily for de
novo synthesis
Regulation of salvage pathway

35. The sites of catabolism are intestinal
mucosa, liver and kidneys
The dietary purines are catabolized in
intestinal mucosa
Purines synthesized endogenously are
catabolized in liver and kidneys
Catabolism of purines

36. The pathway of purine catabolism is
similar in all the tissues
Adenosine and guanosine are the
substrates for the catabolic enzymes
The end product of purine catabolism is
uric acid

37. N
N
N
|
Ribose
N Adenosine
deaminase
H2O NH3
NH2
Adenosine
6Pi Ribose-1-P
Inosine
Purine nucleoside
phosphorylase
Hypoxanthine
O2
+
H2O
Xanthine
oxidase
XanthineUric acid
H2O2
N
HN
N
H
N
O
III
Ribose
N
HN
N
N
O
II
O
O
N
H
HN
N
H
H
N
O
II
O N
H
HN
N
H
N
O
II
6
6
O2
+
H2O
Xanthine
oxidase
H2O2
6
6

38. Guanosine
Guanase
Guanine
O2
+
H2O
Xanthine
oxidase
XanthineUric acid
H2O2
Pi Ribose-1-P
Purine nucleoside
phosphorylase
O
O
N
H
HN
N
H
H
N
O
II
O N
H
HN
N
H
N
O
II
6
6
H2N
I
Ribose
N
HN
N
N
O
II
H2N
N
HN
N
H
N
O
II
H2O
NH3

39. Uric acid is released from tissues into
circulation
Circulating uric acid is excreted in urine

40. In some species, uric acid is the end
product of protein catabolism as well
These species include amphibians, birds
and reptiles
Uric acid is the main vehicle for excretion
of nitrogenous waste in these organisms
Such organisms are said to be uricotelic

41. In many organisms, including man, urea
is the major form of nitrogenous waste
Such organisms are said to be ureotelic

42. De novo synthesis of pyrimidines
occurs in several tissues
The pathway is located in cytosol with
the exception of one reaction
The exception is synthesis of orotate,
which occurs in mitochondria
De novo synthesis of pyrimidine
nucleotides

43. The first reaction in de novo synthesis
is formation of carbamoyl phosphate
It is formed from glutamine, CO2 and
ATP

44. Formation of carbamoyl phosphate is also
the first reaction in synthesis of urea
But this reaction occurs in mitochondria in
urea cycle
The amino group comes from ammonia
instead of glutamine

46. Carbamoyl
aspartate
H2N
O
||
HO−C
CH2
CHC
O COOHN
H
Carbamoyl
phosphate
Carbamoyl group transferred to aspartate
Second reaction
Aspartate
transcarbamoylase
PiO~
NH2
I
C
O
COOH
CH2
CH−NH2
COOH
Aspartate

48. Dihydro-orotate Orotate
N
H
HN
O
||
C
CH
CC
O
HN
O
||
C
CH2
CHC
O COOHN
H
COOH
Dihydro-orotate
dehydrogenase
NADH+H+NAD+
Dihydro-orotate oxidized to orotate
Fourth reaction

51. UMP is the first pyrimidine nucleotide to
be formed
Cytidine and thymidine nucleotides are
formed from UMP
For the formation of cytidine nucleotides,
the amino group is provided by glutamine

52. First, UMP is converted into UDP, and
UDP into UTP
The 4-oxy group is replaced by amino
group to form CTP

54. Thymidine nucleotides occur only in DNA
The sugar in thymidine nucleotides is
deoxyribose
The synthesis occurs from UDP
Thymidylate

55. The ribose residue of UDP is reduced to
deoxyribose by ribonucleotide reductase
Deoxyuridine diphosphate (dUDP) is
dephosphorylated to dUMP
dUMP is methylated to form dTMP (also
termed TMP) by thymidylate synthetase

56. The methyl group is provided by N5, N10-
methylene-H4-folate
During this reaction:
Methylene group is reduced
to methyl group
Tetrahydrofolate is oxidised
to dihydrofolate

58. For continued synthesis of TMP, dihydro-
folate has to be reduced to tetrahydrofolate
This reaction is catalysed by dihydrofolate
reductase
Amethopterin and aminopterin are
competitive inhibitors of this enzyme
They inhibit TMP synthesis and decrease
cell division

59. Regulation occurs by allosteric mechanism
The regulatory enzymes are:
Carbamoyl phosphate
synthetase
Aspartate
transcarbamoylase
Regulation of de novo synthesis

60. Carbamoyl phosphate synthetase is
activated by PRPP, and inhibited by UTP
Aspartate transcarbamoylase is inhibited
by CTP

61. Human beings lack enzymes that can
salvage free pyrimidine bases
However, pyrimidine nucleosides can be
salvaged and used for nucleotide synthesis
Synthesis of pyrimidine nucleotides by
salvage pathway

62. Uridine and cytidine are phosphorylated by
a common enzyme, uridine-cytidine kinase
Thymidine (deoxythymidine) is phospho-
rylated by thymidine kinase
Deoxycytidine is phosphorylated by deoxy-
cytidine kinase

65. Deoxyribonucleotides are required for DNA
synthesis
They are synthesized from ribonucleotides
Formation of deoxyribonucleotides

66. Formation of deoxyribo-
nucleotides requires:
Ribonucleotide reductase
Thioredoxin (a protein having
two sulphydryl groups)
Thioredoxin reductase
(a flavoprotein)

67. Ribose residue reacts with the sulphydryl
groups of thioredoxin
Ribonucleotide reductase removes an
oxygen atom from ribose and two
hydrogen atoms from thioredoxin as water
Thioredoxin is oxidized in this reaction

68. Oxidised thioredoxin is reduced by
thioredoxin reductase
The hydrogens atom are provided by
NADPH

70. This enzyme system can act on ADP,
GDP, CDP and UDP
The products are dADP, dGDP, dCDP
and dUDP
dADP is the inhibitor of this reaction while
ATP is its activator

71. Catabolism of pyrimidines
Cytosine and uracil are catabolised to b-
alanine, carbon dioxide and ammonia
b-Alanine may be utilized in the body or is
excreted in urine

73. Thymine is catabolised to b-aminoiso-
butyrate, carbon dioxide and ammonia
b-Aminoisobutyrate is excreted in urine
Ammonia released from pyrimidines and
purines is disposed off as urea