a powerful workout on metabolism of nucleic acid

1. Metabolism
of
Nucleic Acid
Dr Felix Okunlola

2. Objectives to know
1. Nitrogen bases i.e. purines & pyrimidines
2. Nucleosides
3. Nucleotides
4. Synthesis of purine nucleotides
5. Regulation of purine nucleotides synthesis
6. Inhibitors of purine nucleotide synthesis
7. Disorders of purine metabolism
8. Synthesis of pyrimidine nucleotides
9. Inhibitors of pyrimidine nucleotides synthesis
10. Disorders of pyrimidine nucleotide synthesis

3. Pyrimidines and Purines
Pyrimidine and purine are the names of the
parent compounds of two types of
nitrogen- containing heterocyclic aromatic
compounds.
N
N N
N
N
N
H
Pyrimidine Purine

4. Important Pyrimidines
• Pyrimidines that occur in DNA are
cytosine and thymine. Cytosine and
uracil are the pyrimidines in RNA.
HN
N
H
O
O
Uracil
HN
N
H
O
O
CH3
Thymine
HN
N
H
NH2
O
Cytosine

5. Important Purines
• Adenine and guanine are the
principal purines of both DNA and
RNA.
Adenine
N
N
NH2
N
N
H
Guanine
O
HN
N
H
N
N
H2N

6. Caffeine and Theobromine
• Caffeine (coffee) and theobromine
(coffee and tea) are naturally occurring
purines.
Caffeine
N
N
O
N
N
H3C
O
CH3
CH3
Theobromine
O
HN
N
N
N
CH3
CH3
O

7. Nucleosides
• Is a structure formed by the combination
of nitrogen base and sugar.
N2 base Sugars Nucleoside
Adenine Deoxyribose/Ribose Adenosine
Guanine Deoxiribose/Ribose Guanosine
Thymine Deoxyribose Thymidine
Cytosine Deoxyribose/Ribose Cytidine
Uracil Ribose Uridine

8. Nucleotides
• Nucleotides are phosphoric acid esters
of nucleosides.
Nucleoside Phosphoric acid Nucleotides
Adenosine Phosphoric acid Adenylate (AMP)
Guanosine Phosphoric acid Guanylate ( GMP)
Thymidine Phosphoric acid Thymidylate ( TMP)
Cytidine Phosphoric acid Cytidylate (CMP)
Uridine Phosphoric acid Uridylate (UMP)

9. Synthesis of purine
nucleotides
Synthesis
of purine
nucleotides
Denovo
synthesis
Synthesis of
purine base step
by step on the
ribose 5-
phosphate
Salvage
pathway
Addition of ribose
5-phosphate to
the preformed
purine bases or
addition of
phosphate to the
purine
nucleosides

10. Sources of different atoms of
purine ring

11. Denovo synthesis of
purine nucleotides
Tissue and site of synthesis
Tissues- major tissue is liver
Site- cytosol

23. Inhibitors
1. Sunfonamide
Are structural analogues of PABA
Act as competitive inhibitors of synthesis of folic
acid from PABA in bacteria.
They inhibit the reactions of purine nucleotide
synthesis requiring folic acid ( GAR transformylase
andAICAR transformylase)
Used as bacteriostatic drugs to control bacterial
infection.

24. 2. Methotrexate and Aminopterin
Are structural analogue of folic acid.
They act as a competitive inhibitors of
dihydrofolate reductase thus blocking the
biosynthesis of tetrahydrofolic acid.
They inhibit the reaction requiring folic
acid for purine nucleotide synthesis.
Used in t/t of cancers like leukemia
choriocarcinoma.

25. 3. Trimethoprim
structural analogue of folic acid.
Acts as a competitive inhibitors of
dihydrofolate reductase in bacteria thus
blocking the biosynthesis of tetrahydrofolic
acid.
Inhibit the reaction requiring folic acid to
purine nucleotide synthesis.
Used in the t/t of bacterial infections and
UTI.

26. 4. 6-mercaptopurine
is a structural analogue of purine bases.
is converted to 6- thioionosine
monophosphate by the enzyme HGPRT,
called lethal synthesis.
6-thio IMP inhibits the conversion of IMP
to AMP and GMP.
6-thio IMP also feed back inhibits
glutamine PRPP amidotransferase.
Used as an anticancer drug.

27. 5. Thioguanine
Is a guanine analogue.
It is converted to 6-thio GMP by the
enzyme HGPRT.
6-thio GMP inhibits the conversion of IMP
to GMP.
Also inhibits glutamine PRPP
amidotransferase.
Used as an anticancer drug.

28. 6. Azaserine
is a structural analogue of glutamine.
is a glutamine antagonist.
inhibits the enzyme reactions in purine and
pyrimidine nucleotide synthesis that utilize
glutamine as a substrste.
it is highly toxic to the cells so it is not
used clinically as a drug.

29. Regulation
1. Intracellular conc. Of PRPP-
depends upon 2 factors i.e. its synthesis & utilization.
Synthesis depends on-
Availability of R-5-P.
Action of enzyme PRPP synthetase. Utilization
depends on-
Denovo synthesis.
Salvage pathway.

30. 2. Activity of enzyme PRPP amidotransferase.
Increased activity of PRPP amidotransferase
leads to Increased synthesis of AMP amd GMP,
which feedbackly inhibit the enzyme PRPP
amidotransferase.
3. Both AMP and GMP inhibit their own
formation by feedback inhibition of
adenylosuccinate synthetase and IMP
dehydrogenase.

31. Salvage pathway
It refers to the formation of purine nucleotides
by the
1.Addition of ribose phosphate ( from PRPP)
to the preformed purine bases.
2.Addition of phosphate to the preformed
purine nucleosides.

32. Significance
Salvage pathway provide a pathway for the
utilization of purine bases derived from
diet (exogenous) and normal turnover of
the nucleic acids.
In erythrocytes, denovo syntheis of purine
nucleotides does not occur because of
absence of PRPP amidotransferase.
The requirement of purine nucleotides is
met by the salvage pathway.

33. Synthesis of purine nucleotides from purine
bases
Catalyzed by HGPRT and
APRT.
Adenine + PRPP AMP + PPi
Hypoxanthine + PRPP IMP + PPi
Guanine + PRPP GMP+ PPi
APR
T
HGPR
T
HGPR
T

34. Synthesis of purine nucleotides from
purine nucleosides
Adenosine + ATP AMP+ ADP
Adenosine kinase

35. Degradation of purine nucleotides

36. Disorders of purine metabolism
1. Gout
2. Lesch nyhan syndrome
3. Immunodeficiency associated with
purine metabolism
4. Infantile autism

37. Gout
Metabolic disorders associated with
overproduction of uric acid.
At physiological form, uric acid is found
in more soluble form as sodium urate.
In severe hyperuricemia, crystal of sodium
urate get deposited in the soft tissues,
particularly in joints. Such deposits are
commonly known as tophi.

38. This causes inflammation of joints
resulting in gouty arthritis.
The prevalence of gout is about 3 per 1000
persons, mostly affecting males.
Post menopausal women, however are as
susceptable as men for this disease.
Historically, gout was found to be
associated with high living, over eating and
alcohol consumption.
Lead poisoning also causes gout by
decreasing uric acid excretion.

39. Types of gout
Gout
Primary
gout
Metabolic
gout
Renal gout
Secondary
gout
Metabolic
gout
Renal gout

40. Primary metabolic gout
It is an inborn error of purine
metabolism due to overproduction of
uric acid.
Causes:
1.Increased activity of PRPP synthetase
2.Overactivity of PRPP amidotransferase
3.HGPRT deficiency
4.Glucose 6-phosphatase deficiency
5.Elevation of glutathione reductase

41. Primary renal gout
It is due to failure of uric acid excretion
from the body so that uric acid level in
the body gets increased.

42. Secondary metabolic
gout
Secondary gout is due to secondary to
certain diseases like leukemia,
polycythemia, lymphoma, psoriasis and
increased tissue breakdown like in
trauma, starvation etc.

43. Secondary renal gout
It is due to secondary to defective
glomerular filtration of urate due
to generalized renal failure.

44. Tratment of gout
Is by
1. Use of colchicine & uricosuric drugs.
To remove urates from the joint, colchine
is the drug of choice.
To remove the urates from the body,
urocosuric drugs such as probenecid,
sulfinpyrazole, salicylates etc are used.
2. Use of allopurinol-inhibits the activity of
enzyme xanthine oxidase as a result of
which uric acid is not produced.

45. Lesch Nyhan syndrome
Fist described tn 1964 by Michael Lesch( a
medical student) and William L. Nyhan (his
teacher).
It is X linked metabolic disorder since the
structural genes for HGPRT is located on the X
chromosome.
It affects only males and is characterized by
excessive uric acid production and neurological
abnormalities such as mental retardation,
aggressive behaviour, learning disability etc.
The patients of this disorder have an irresistible
urge to bite their fingers and lips,ofen causing
self-mutilation.

46. Biochemical basis
HGPRT deficiency spares the utilization of
PRPP through salvage and the accumulated
PRPP takes part in the purine biosynthesis
by the denovo pathway finally leading to
hyperuricemia.
The biochemical basis for neurological
abnormalities are big enegma till date.
Indeed, it is surprising that the deficiency
of a single enzyme can cause such an
abnormal behavioural changes.

47.  few explanations are putforth in this
regard.
 Neurological symptoms may be due to
decreased availability of purines to the
developing brain which has a low capacity
for denovo purine synthesis and hence
depends on purine salvage pathway for the
supply of purine nucleotides it requires.

48. Treatment
allopurinol is used to treat hyperuricemia
but it has no effect on the neurological
menefestation in theses patients.
Treatment for the neuro-behavioural
features are limited to behavioural therapy
and providing protective physical device
to prevet self-mutilation.

49. Immunodeficiency diseases associated
with purine metabolism
Two different immunodeficiency
disorders associated with degredation of
purine nucleotides are known.
The enzyme defects are adenosine
deaminase and purine nucleoside
phosphorylase, involved in uric acid
synthesis.

50. The deficiency of ADA causes SCID
involving T- cell and usually B- cell
dysfunction.
It is explained that ADA deficiency
results in the accumulation of Datp which
is an inhibitor of ribonucleotide reductase
and thus DNA synthesis, replication are
adversely affected.
Different modes of t/t such as blood
transfusion, bone marrow transplantation
are tried to cure the diseases but with
limited effects.

51. But, like in any other inborn error, the real hope
for the future is only gene therapy.
In 1990, a 5 year old girl suffering from SCID
was successfully cured by transfecting the
ADA gene into stem cells of the patients.
This is considered as landmark in the history of
trating inborn errors of metabolism.
The deficiency of purine nucleoside
phosphorylase is associated with impairement
of T cell function but has no effect on B cell
function.
It is believed that d GTP inhibits the
development of normal T-cells.

52. Infantile Autism
Recently it was observed that children
suffering from infantile autism exihibited
increased excretion of uric acid but
surprisingly the serum concentrations are
within normal limits.
The biochemical basis for this is
unknown.
An oral dose of uridine is tried in the t/t.

53. Synthesis of pyrimidine
nucleotides
Synthesis
of
pyrimidine
nucleotides
Denovo
synthesis
Synthesis of
pyrimidine
nucleotide refers
to the formation
of pyrimidine ring
structure
followed by the
addition of ribose
phosphate.
Salvage
pathway
Formation of
pyrimidine
nucleotides from
pyrimidine
bases

54. Sources of different atoms of prrimidine
rings

55. Denovo synthesis of pyrimidine
nucleotides
Tissue and site of synthesis
Mainly occurs in the liver.
The reaction occurs in cytosol and
mitochondria. The formation of orotate from
dihydroorotate occurs ie mitochondria and
all other reactions occur in the cytosol.

56. -
2 A T P + H C O 3 + G lutam ine + H 2 O
C
O
O 3
PO
-2
N H 2
C arbam oyl Phosphate
N
H
2
C
N
H
C
H
C
H
2
H O C
C O
O
O
O
C arbam oyl A spartate
H
N
C
N
H
C
H
C H 2
C
C O
O
O
O
D ihydroorotate
H
N
C
N
H
C
C
H
C
C O
O
O
O
O rotate
H N
C
N
C
C H
C
C O
O
O
O
H
H
O H O H
H H
O
2-
O 3 P O C H
2
O rotidine-5 '-m onophosphate
(O M P)
H N
C
N
C H
C H
C
O
O
H
H
O H O H
H H
O
O C H
2
2-
O 3 P
U ridine M onophosphate (U
M P)
2 A D P +
G lutam ate +
Pi
C arbam oyl
Phosphate
Synthetase II
A spartate
Transcarbam oylase (A
TCase)
A spartate
Pi
H 2 O
D ihydroorotase
Q uinone
R educed
Q uinone
D ihydroorotate D
ehydrogenase
PR P P PPi
O rotate Phosphoribosyl T
ransferase
C O 2
O M P
D ecarboxylase
Pyrimidine Synthesis

59. UMP  UTP and CTP
• Nucleoside monophosphate kinase catalyzes
transfer of Pi to UMP to form UDP; nucleoside
diphosphate kinase catalyzes transfer of Pi from
ATP to UDP to form UTP
• CTP formed from UTP via CTP Synthetase
driven by ATP hydrolysis
– Glutamine provides amide nitrogen for C4 in
animals

60. UDP dUDP
dUDP dUMP
dUMP dTMP
N5,10 formyl THF formyl THF
Ribonucleotide reductase
Thymidylate synthetase

61. Regulation of pyrimidine synthesis
In bacteria, aspartate transcarbamoylase catalyses
a committed step in pyrimidine biosynthesis.
 Aspartate transcarbamoylase is a good example
of an enzyme controlled by feedback mechanism
by the end product CTP.
In certain bacteria, UTP also inhibits aspartate
transcarbamoylase. ATP, however stimulates
aspartate transcarbamoylase activity.

62. Carbamoyl phosphate synthase 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.

63. Inhibitors of pyrimidine
synthesis
Sulfonamides
Methotrexate
Trimethoprim
5-fluorouracil
Fluorocytosine

64. Salvage pathway
Salvage pathway of pyrimidine nucleotide
synthesis refers to the formation of
pyrimidine nucleotides from pyrimidine
bases.
Significance
Salvage pathway provide a pathway for the
utilization of pyrimidine bases derived
from diet(exogenous) and normal turnover
of nucleic acids.

65. Enzymes and reactions
There are 2 enzymes that catalyze the
reactions of salvage pathway. They are
uracil phosphoribosyl transferase
(UPRT) and thymidine kinase.
Uracil + PRPP UMP +
PPi
Thymidine + ATP TMP+ ADP
UPR
T
Thymidine
kinase

66. Disorders of pyrimidine metabolism
Disorders of pyrimidine metabolism
includes:
Orotic aciduria
Reye’s syndrome

67. Orotic aciduria
Is a rare metabolic disorder characterized by the
excretion of orotic acid in urine, severe anemia
and retarded growth.
It is due to the deficiency of the enzymes orotate
phosphoribosyl transferase and OMP
decarboxylase of pyrimidine synthesis.
Both these enzymes activities are present on a
single protein as domains (bifunctional
enzyme).

68. Treatment
Feeding diet rich in uridine or cytidine is
an effective t/t of orotic aciduria.
These compounds provide pyrimidine
nucleotides required for DNA and RNA
synthesis.

69. Reye’s syndrome
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.

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