Nucleotide metabolism, purine metabolism, pyrimidine metabolismSalvage pathway, Denovo synthesis of Purine, Gout( Primary and secondary), lesch-nyhan syndrome, Orotic aciduria,ADA deaminase deficiency,

1. Nucleotide Metabolism
•Digestion and Absorption of
Nucleotides
•Synthesis of Nucleotide:
1. Denovo Synthesis
2. Salvage Pathway
•Disorders related to Purine and
pyrimidine Metabolism
Anup Shamsher Budhathoki
Lecturer
Department of Biochemistry
National Medical college, Birgunj,Nepal

2. Overview of digestion And Absorption
of nucleotides
• Ribonucleases and
Deoxyribonucleases from
pancreas degrades Nucleic acid to
oligonucleotides
• pancreatic Phosphodiesterase
further hydrolyze oligonucleotides
into 3’ and 5’mononucleotides
• Nucleotidase from intestinal
mucosal cells removes phosphate
and produce nucleosides
• Nucleotidase breaks nucleosides
into free bases and sugars
• Only negligilble amount of
nitrogen bases are reutilized for
nucleotide synthesis.

3. Purine synthesis
Two types of pathways lead to purine nucleotides synthesis:
1.Denovo Synthesis of Purine Nucleotides
Amino acids( Glycine, Glutamine and Aspartate) , CO2 and
one carbon donors are required for synthesis of new
molecule of purine nucleotide
Synthesis of purine Nucleotide occurs in most of the tissue
but predominantly in liver
 Purine monophosphates(IMP, GMP,AMP) are synthesized
instead of free purine
2.Salvage Pathway
Salvage pathways recycle the free purines and purine
nucleosides released from nucleic acid breakdown.

4. Formation of Purine ring
C and N atom of Purine ring

5. Steps of Denovo Synthesis of Purine
nucleotides
• Step Zero ( Preparatory Step)
Formation of PRPP

6. Steps of Denovo synthesis of Purine

7. Steps of Denovo synthesis of purine nucleotide up to IMP

8. Enzyme for synthesis of purine nucleotide
1. PRPP glutamyl amindotransferase
2. GAR synthase
3. GAR transformylase
4. FGAR amido tranferase
5. AIR synthetase
6. AIR carboxylase
7. Succinyl carboxamide synthetase ( SAICAR synthetase)
8. Adenylosuccinate lyase( SAICAR lyase)
9. Formyl transferase
10.IMP synthase

9. Steps of Synthesis of AMP and GMP from IMP

10. Regulation of Denovo synthesis of Purine Nucleotides

12. Overview of purine metabolism
A
5

13. Salvage pathway and its significance
Salvage pathways recycle the free purines and purine
nucleosides released from nucleic acid breakdown.
Significance of salvage pathway:
1. This pathway ensures the recycling of purines formed by
degradation of nucleotides. Nucleosides and deoxy-
nucleosides can also be salvaged.
2.The pathway utilizes less energy compared to denovo
synthesis of purine nucleotide
3. All tissues are not capable of de novo synthesis of purine
nucleotides, e.g. erythrocytes, Neutrophils and the brain
cells. These cells lack the enzyme PRPP- glutamyl
amidotransferase. Therefore salvage pathway provides
purine nucleotides.

14. Salvage Pathway for synthesis of purine nucleotide
•

15. Salvage Pathway for synthesis of purine nucleotide
Ribonucleoside kinase

16. Catabolism of Purine/ Uric acid synthesis
Uric acid is the end product of
purine metabolism in
humans.

17. Case
• A 40-year-old male presented with severe pain,
redness and swelling of the base of the first
metatarsophalangeal joint in the night after a bout of
alcohol consumption. The patient was in usual state
of health until early in the morning when he woke up
with severe pain in his right big toe. The patient
denies any trauma to the toe and no previous history
of such pain in other joints. On examination, he had
mild fever 38.2°C. The right big toe was swollen,
warm, red, and exquisitely tender. Serum uric acid
was 9.7 mg/dL. What is the likely diagnosis? What is
the pathogenesis of the condition? How would you
make a definite diagnosis?

18. DISORDERS OF PURINE METABOLISM
1. HYPERURICEMIA AND GOUT
• The normal concentration of uric acid in the serum
Men : 3-7 mg/dl
Women: 2.5-6 mg/dl
The daily excreation of uric acid is about 500-700 mg.
Hyperuricemia is uric acid concentration > 7mg/dl in
men and 6mg/dl in women
Gout is a metabolic disorder associated with the
overproduction of uric acid and deposition of
monosodium urate crystals in joints and other tissues.

19. • Monosodium urate deposits in joints are commonly known
as tophi.
• This causes inflammation in the joints resulting in a
painful gouty arthritis. Typical gouty arthritis affects first
metatarsophalangeal joint.(GREAT TOE).
• Sodium urate &/or uric acid may also precipitate in
kidneys & ureters that result in renal damage & stone
formation.
• Historically, gout was found to be often associated with
high living, over-eating & alcoho consumption.
• The prevalence of gout is about 3 / 1,000 persons, mostly
affecting males.
Sign and symptoms of Gout

20. Clinical features:
•Attacks are precipitated by alcohol intake.
Often patient have few drinks , go to sleep
symptomless , but are awakened during early
hours by severe joint pains
•Diagnosis
Synovial fluid shows birefringent crystals under
polar microscope is diagnostic.

21. Diagnosis

22. • GOUT IS OF TWO TYPES
1. PRIMARY GOUT.
It is an inborn error of metabolism due to
overproduction of uric acid.
This is mostly related to over production of purine
nucleotides.
• PRPP synthetase : in normal circumstances , PRPP
synthetase is under feedback control by purine
nucleotides ( ADP & GDP ).
However, varient forms of PRPP synthetase-which are
not subjected to feedback regulation-have been
detected. This leades to increased production of purines.

23. • PRPP glutamylamidotransferse :
The lack of feedback control of this enzyme by purine
nucleotides also leads to their elevated synthesis.
• HGPRT deficiency : This is an enzyme of purine salvage
pathway, & its defect causes Lesch-Nyhan syndrome. This
disorder is associated with increased synthesis of purine
nucleotides by a two fold mechanism.
Firstly, decreased utilization of purines ( Hypoxanthine &
guanine ) by salvage pathway, resulting in the
accumulation & divertion of PRPP for purine nucleotides.
Secondly, the defect in salvage pathway leads to
decreased levels of IMP & GMP causing impairment in
the tightly controlled feedback regulation of their
production.

24. Glucose 6-phosphatase dificiency:
Intype I glycogen storage disease ( von-gierke’s ), glucose-6-
phosphate cannot be converted to glucose due to the
deficiency of glucose-6-phosphatase.
This leads to the increased utilazation of glucose-6-phosphate
by HMP shunt resulting in elevated levels of ribose-5-
phosphate & PRPP &, ultimately, purine overproduction.
von gierke’s disease is also associated with increased activity
of glycolysis.
Due to this, lactic acid accumulates in the body which
interferes with the uric acid excretion through renal tubules.

25. ELEVATION OF GLUTATHIONE REDUCTASE :
varient of glutathione reductase generates more
NADP+ which is utilized by HMP shunt .
This leads to increased ribose 5-phosphate and PRPP
synthesis.

26. Secondary gout:
Secondary hyperuricemia is due to various diseases causing
increased synthesis or decreased excretion of uric acid.
Increased degradation of nucleic acids (hence more Uric acid
formation) is observed in various cancers (leukemias,
polycythemias, lymphomas, etc).
Psoriasis and increased tissue breakdown (trauma,
starvation etc).
Decreased excretion of uric acid in Chronic Kidney
Disease.

27. Areas for deposition of Mono sodium urate Crystals

28. Treatment Strategies in Gout

29. Mechanism of action Allopurinol In Treatment of Gout
Allopurinol, an analog of hypoxanthine (Fig. 43.16B). Allopurinol is a
competitive inhibitor of xanthine oxidase thereby decreasing the
formation of uric acid. Xanthine and
hypoxanthine are more soluble and so are excreted more easily.
Xanthine oxidase converts allopurinol to
alloxanthine. It is a more effective inhibitor of xanthine oxidase. This is a
good example of ‘suicide inhibition'

30. (Alloxanthine)

31. Treatment of Gout:
The drug of choice for the treatment of primary gout is
allopurinol.
This is a structural analog of hypoxanthine that
competitively inhibits the enzyme xanthine oxidase.
Further allopurinol is oxidized to alloxanthine by xanthine
oxidase.
Alloxanthine, in turn is a more potent inhibitor of xanthine
oxidase. This type of inhibition is referred to as suicide
inhibition.

32. Inhibition of xanthine oxidase by allopurinol leads to the
accumulation of hypoxanthine and xanthine.
These two compounds are more soluble than uric acid,
hence easily excreted.
Besides the drug therapy, restriction in dietary intake of
purines and alcohol is advised.
Consumption of plenty of water will also be useful.

33. Pseudogout:
The clinical manifestations of pseudo gout are similar to
gout.
This disorder is caused by the deposition of calcium
pyrophosphate crystals in joints.
Further serum uric acid concentration is normal in pseudo
gout.

34. Lesch-Nyhan syndrome
This disorder is due to the deficiency of hypoxanthine-
guanine phosphoribosyltransferase (HGPRT) , an enzyme
of purine salvage pathway .
Lesch-nyhan syndrome is a sex-linked metabolic disorder
since the structural gene for HGPRT is located ontlhe X-
chromosome.
Mutations that decrease or abolish hypoxanthine-guanine phosphoribosyl
transferase activity include deletions, frame-shift mutations, base substitutions,
and aberrant mRNA splicing.
It affects only the males and is characterized by excessive
uric acid production (often gouty arthritis).

35. Symptoms in Lesch-nyhan syndrome
 Neurological abnormalities such as mental
retardation, aggressive behavior, learning disability etc.
 The patients of this disorder have an irretible urge to bite their fingers and
lips, often causing self-mutilation.
 HGPRT deficiency results in the accumulation of PRPP and decrease in GMP
and IMP, ultimately leading to increased synthesis and degradation of
purines.This may result in gout.
 Uric acid stones in kidney (Urolithiasis)

36. Biochemical bases for the neurological symptoms
 The biochemical bases for the neurological
symptoms observed in Lesch-Nyhan syndrome is
not clearly understood.
 This may be related to the dependence fo brian on
the salvage pathway for de novo synthesis of purine
nucleotides.

37. Severe Combined
Immunodeficiency
Disorder(SCID)
It is associated with severe
immunodeficiency where both T and B cells
are deficient.
It is an inherited autosomal recessive
disease.
ADA deficiency leads to accumulation of
adenosine and dATP; this would inhibit
further production of precursors for DNA
synthesis especially dCTP.
Lymphocytes usually contain high levels of
ADA. Therefore, ADA deficiency is mainly
manifested as reduced lymphocytes. This
leads to impaired cellular and humoral
immunity.

38. Biosynthesis of Pyrimidine

39. •
Carbamoyl Phosphate
2ATP
2ADP
Carbamoyl Aspartate
Orotidine monophosphate
Orotic acid
Dihydro orotic acid
Uridine monophosphate
Uridine diphosphate
Uridine triphosphate
Glutamine + HCO3-
Aspartate
Pi
H2O
NAD+
NADH
PRPP
PPi
CO2
ATP
ADP
ATP
ADP
Glutamine
Glutamate
Carbamoyl phosphate synthase II
Aspartyl transcarbamoylase
Dihydro orotase
Dihydro orotate dehydrogenase
Orotate phosphoribosyl transferase
OMP decarboxylase
Cytidine triphosphate
ATP
ADP
ADP
UMP kinase
Nucleoside diphosphate kinase
CTP synthetase
Steps for Denovo synthesis of pyrimidine

40. Formation of TMP from UDP: dUMP is
substrate for TMP synthesis. dUDP is
dephosphorylated to d UMP.

41. Methylation of dUMP:
This occurs at C5 by N5,N10methyleneTHF, forming
TMP.
This reaction is catalysed by Thymidylate synthase.

42. Regulation of Pyrimidine synthesis

43. Salvage pathway
The pyrimidines (like purines) can also serve as precursors in
the salvage pathway to be converted to the respective
nucleotides.
This reaction is catalysed by pyrimidine phospshoribosyl
transferase which utilizes PRPP as the source of ribose 5-
phosphate.

44. SALVAGE PATHWAYOF PYRIMIDINE SYNTHESIS
Pyrimidine base + PRPP
pyrimidine phosphoribosyl
transferase
Pyrimdine nucleotide + PPi

45. Degradation of Pyrimidine Nucleotides
Thymidine
CMP TMP
H2O
pi
H2O
pi pi
Ribose 1-p
Nucleotidase
Phosphorylase
Deaminase
Uridine
Uracil
pi
Ribose 1-p
Phosphorylase
Hydratase

46. CASE
• A 3-year-old girl presented with megaloblastic anemia
, which did not improve despite blood transfusions.
There was no response to B12, folate and pyridoxine
therapy. Urinalysis revealed presence of a crystalline
sediment, which was identified to be orotic acid. Very
high levels of orotate (above 1.0 g/day, normal being
< 1.4 mg/day) were excreted. Enzyme assays were
done and showed deficiency of orotate
phosphoribosyl transferase (OPRTase). What is the
likely condition? What is the pathogenesis of the
findings?

47. Orotic aciduria
• absence of either or both of the enzymes, OPRTase and OMP
decarboxylase. It is an autosomal recessive disease.
• There is retarded growth and megaloblastic anemia. The
rapidly growing cells are more affected and hence Synthesis of
pyrimidine nucleotides the anemia
• Urinary tract obstruction due to accumulation of crystals
• decreased production of UMP,UDP therefore loss of regulation
and overproduction of orotic acid resulting in orotic acid uria
• The condition can be successfully treated by feedingcytidine or
uridine. They may be converted to UTP which can act as
feedback inhibitor.

48. Other causes for Orotic aciduria
• Orotic aciduria may also occur in ornithine
transcarbamoylase deficiency (urea cycle enzyme) as
carbamoyl phosphate accumulates due to defective
conversion to citrulline. Carbamoyl phosphate shifts from
mitochondria to cytosol and results in overproduction of
orotic acid.
• Allopurinol competes with orotic acid for the enzyme
orotate phosphoribosyl transferase leading to orotic
aciduria and orotidinuria.