introduction of Purine and Pyrimidine metabolism, biosynthesis and degradation of nucleotides, biological functions and metabolic disorders, chemical analogues and therapeutic drugs, uric acid metabolism

1. NUCLEOTIDE METABOLISM
DIPESH TAMRAKAR
MSC. CLINICAL BIOCHEMISTRY

2. Overview
■ INTRODUCTION
■ BIOLOGICAL FUNCTION
■ BIOSYNTHESIS OF NUCLEOTIDES : PURINE & PYRIMIDINE
■ DEGRADATION OF NUCLEOTIDES : PURINE & PYRIMIDINE
■ METABOLIC ABNORMALITIES
■ CLINICAL MANIFESTATIONS
■ CHEMICAL ANALOGUES
■ SUMMARY

3. INTRODUCTION
■ Nucleoside: nucleic acid bases attached to pentose sugar
(D-ribose or 2-deoxy-D-ribose)
■ Nucleotides: nitrogenous base, a pentose sugar &
phosphate groups.
■ Purines: Adenine & Guanine
■ Pyrimidines: Cytosine in both DNA & RNA,
Thymine in DNA
Uracil in RNA

4. Metabolic functions of
nucleotides1. Role in Energy metabolism as ATP
2. Monomeric units of Nucleic Acids ( in RNA & DNA)
3. Physiological mediators of key metabolic processes:
– Adenosine important in control of coronary blood flow
– ADP in platelet aggregation
– cAMP & cGMP acts as 2nd messengers
– GTP in capping mRNA
4. GTP is precursor for formation of cofactor,
tetrahydobiopterin, NAD+, NADP+, FAD+ & their reduced
forms contains 5’-AMP as structural part
5. Serves as carrier of activated intermediates; required for
reactions
6. Many of the regulated steps of metabolic pathways are
controlled by intracellular concentrations of nucleotides.

6. Digestion of Nucleic Acids
■ The nucleic acids in the diet are
hydrolyzed to a mixture of nucleotides
by ribonuclease and deoxy
ribonuclease present in pancreatic and
intestinal secretions.
■ Then nucleotidases liberate the
phosphate from nucleotides.
■ The resulting nucleosides are
hydrolyzed by nucleosidases forming
free bases and pentose sugars.
■ The dietary purines and pyrimidines
are neither converted to nucleotides
nor incorporated into nucleic acids.
They are directly catabolized.

7. Biosynthesis of Nucleotides
Major site: LIVER
(Cytoplasm)
Two types of pathways
lead to nucleotides
1. The De novo
pathways: begins
with their metabolic
precursors (Amino
Acids, Ribose-5P,
CO2, NH3)
2. Salvage pathway:
Recycling of the free
bases and
nucleosides released
from nucleic acid
breakdown

8. DE NOVO SYNTHESIS
■ The purine ring structure is built up one or more atoms at a
time, attached to ribose throughout the process.
■ The pyrimidine ring structure is synthesized as orotate,
which gets attached to ribose-P and then converted to
common pyrimidine nucleotides .
■ The cellular pools of nucleotides are small (1% or less of
the amounts required to synthesize the cell's DNA) so cells
must continue to synthesize nucleotides during nucleic acid
synthesis

9. Purine De Novo Synthesis
■ De Novo Purine Nucleotide Synthesis begins with 5-
phosphoribosyl-1-pyrophosphate (PRPP)
■ The two parent purine nucleotides of nucleic acids are
adenosine 5’-monophosphate (AMP; adenylate) and
guanosine 5’-monophosphate (GMP; guanylate),
containing the purine bases adenine and guanine.
■ The detailed pathway of purine biosynthesis was worked
out primarily by Buchanan and G. Robert Greenberg in the
1950s.

10. ■ John Buchanan "traced" the sources of all nine atoms of
purine ring

13. ■ This pathway is highly regulated by AMP & GMP; IMP is not
normally found to any extent in cells
■ PRPP is synthesized from ribose 5-phosphate generated by the
pentose phosphate pathway
■ Formation of 5-phosphoribosylamine is the committed and
regulated first step
■ The conversion of IMP to either AMP or GMP uses a two-step
energy-requiring pathway;
1. Conversion of IMP to AMP:
■ Conversion of inosinate to adenylate requires the insertion of an
amino group derived from aspartate
■ the synthesis of AMP requires guanosine triphosphate (GTP) as an
energy source
2. Conversion of IMP to GMP:
■ Guanylate is formed by the NAD-requiring oxidation of inosinate at
C-2, followed by addition of an amino group derived from
glutamine.
■ The synthesis of GMP requires ATP and ATP is cleaved to AMP and
PPi in the final step

16. REGULATION OF PURINE NUCLEOTIDE BIOSYNTHESIS
■ Regulated by feedback
inhibition by 3 major
mechanisms;
1. The first mechanism is
exerted on the reaction of
conversion of PRPP to 5-
phosphoribosylamine.
■ This reaction is catalyzed by
the allosteric enzyme
glutamine-PRPP
amidotransferase, which is
inhibited by the end products
IMP, AMP, and GMP.
2. PRPP synthetase regulated by
ADP

17. 3. An excess of GMP in the cell inhibits formation of
xanthylate from inosinate by IMP dehydrogenase, without
affecting the formation of AMP.
IMP dehydrogenase is the rate limiting enzyme and is
regulated by GMP acting as a competitive inhibitor of IMP
dehydrgense.
Adenylosuccinate synthase is rate limiting in conversion
of IMP to AMP with AMP acting as a competitive inhibitor.
■ Conversely, an accumulation of adenylate inhibits
formation of adenylosuccinate by adenylosuccinate
synthetase, without affecting the biosynthesis of GMP.
■ GTP is required in the conversion of IMP to AMP, whereas
ATP is required for conversion of IMP to GMP
■ So a reciprocal arrangement tends to balance the
synthesis of the two ribonucleotides.

18. Salvage Pathway for Purine
■ This pathway ensures the recycling of purines formed by
degradation of nucleotides
■ 2 pathways:1 pathway utilizes the bases- hypoxanthine,
guanine & adenine as substrates whereas other pathway
utilizes preformed nucleosides as substrate
■ PRPP is the starting material in this pathway; it is also a
substrate for de novo synthesis. Hence these two pathways
are closely inter-related.
■ The free purines are salvaged by two different enzymes;
– adenine phosphoribosyl transferase (APRTase) and
– hypoxanthine guanine phosphoribosyl transferase
(HGPRTase).
■ One of the primary salvage pathways consists of a single
reaction catalyzed by adenosine phosphoribosyl transferase
(APRTase), in which free adenine reacts with PRPP to yield
the corresponding adenine nucleotide:

19. ■ Free guanine and hypoxanthine (the deamination product
of adenine) are salvaged in the same way by
hypoxanthine-guanine phosphoribosyl transferase
(HGPRTase).
■ The pathway has special importance in tissues like RBCs
and brain where the de novo pathway is not operating.
■ The salvage pathway economizes intracellular energy
expenditure.
■ Absence of enzymes of salvage pathway produces
specific clinical syndromes
■ This pathway reactions are regulated by their end
products; IMP & GMP are competitive inhibitors of
HGPRTase and AMP is of APRTase

20. Degradation of purine nucleotide:
Degradation of AMP
■ Adenylate yields adenosine by loss of
phosphate through the action of 5’-
nucleotidase
■ Adenosine is deaminated to inosine by
adenosine deaminase
■ Inosine is hydrolyzed to hypoxanthine
(its purine base) and D-ribose.
■ Hypoxanthine is oxidized successively
to xanthine and then uric acid by
xanthine oxidase, a flavoenzyme with
an atom of molybdenum and four iron-
sulfur centers in its prosthetic group.
■ Molecular oxygen is the electron
acceptor in this complex reaction.

22. Degradation of GMP:
■ GMP catabolism also yields
uric acid as end product.
■ GMP is first hydrolyzed to
guanosine by enzyme 5’-
nucleotidase
■ Guanosine which is then
cleaved to free guanine by
nucleosidase
■ Guanine undergoes
hydrolytic removal of its
amino group to yield
xanthine by Guanine
deaminase
■ Xanthine is then converted
to uric acid by xanthine
oxidase

23. Uric acid:
■ Uric acid is the excreted end
product of purine catabolism
■ A healthy adult human
excretes uric acid at a rate of
about 0.6 g/24 hrs; the
excreted product arises in
part from ingested purines
and in part from turnover of
the purine nucleotides of
nucleic acids.
■ In most mammals, uric acid is
further degraded to allantoin
by the action of urate oxidase.

24. Diseases associated with purine degradation; Gout:
■ Gout is a disorder characterized by high levels of uric acid in
blood (hyperuricemia)→ as a result of either the
overproduction or underexcretion of uric acid.
■ The hyperuricemia can lead to the deposition of
monosodium urate crystals in the joints, and an
inflammatory response to the crystals, causing first acute
and then progressing to chronic gouty arthritis.
■ Nodular masses of monosodium urate crystals (tophi) may
be deposited in the soft tissues, resulting in chronic
tophaceous gout
■ Formation of uric acid stones in the
kidney (urolithiasis) may also be seen.

26. Type of Gout:
2 types;
1. Primary gout
2. Secondary gout
1. Primary Gout:
■ In this, hyperuricaemia is not due to increased destruction
of nucleic acid.
■ The essential abnormality is increased formation of uric
acid from simple carbon and nitrogen compounds without
intermediary incorporation into nucleic acids.

27. ■ Primary Gout is further classified as;
a. Primary metabolic gout:
■ It is due to inherited metabolic defect in purine metabolism
leading to excessive rate of conversion of glycine to uric
acid.
■ X-linked recessive defects enhancing the de novo synthesis
of purines and their catabolism can also lead to
hyperuricaemia.
For example, defects of PRPP may make it feedback resistant.
b. Primary renal gout:
■ It is due to failure in uric acid excretion.

28. 2. Secondary Gout
a. Secondary metabolic gout:
■ It is due to secondary increase in purine catabolism in
conditions like leukemia, prolonged fasting and
polycythemia.
b. Secondary renal gout:
■ Due to defective glomerular filtration of urate due to
generalized renal failure.
c. In von-Gierke’s disease:
■ Deficiency of G-6-phosphatase to elevated rate of pentose
formation in HMP.
■ Pentose acts as a good substrate for PRPP synthetase and
enhances the synthesis of purines followed by their
catabolism to uric acid.

29. Diagnosis:
■ Aspiration and examination of synovial fluid from an
affected joint (or material from a tophus) using polarized
light microscopy to confirm the presence of needle-shaped
monosodium urate crystals

30. Management of Gout
■ By reducing dietary purine intake and restricting alcohol.
■ By increasing renal excretion of urate by uricosuric drugs,
which decrease the reabsorption of uric acid from kidney
tubules, e.g. probenecid
■ By reducing urate production by allopurinol, which is an
analogue of hypoxanthine.
■ Allopurinol is a competitive inhibitor of xanthine oxidase
thereby decreasing the formation of uric acid.
■ Xanthine oxidase converts allopurinol to alloxanthine. It is a
more effective inhibitor of xanthine oxidase. This is a good
example of ‘suicide inhibition'
■ Antiinflammatory agents like Colchicine, steroidal drugs
such as prednisone, and nonsteroidal drugs such as
indomethacin are used to treat Acute attacks of gout

31. Lesch-Nyhan syndrome:
■ This syndrome is a rare, X-linked, recessive disorder
associated with a virtually complete deficiency of hypo -
xanthine-guanine phosphoribosyltransferase (HGPRT).
■ This deficiency results in an inability to salvage
hypoxanthine or guanine, from which excessive amounts of
uric acid are produced
■ In patients with Lesch-Nyhan syndrome,
the hyperuricemia frequently results in
the formation of uric acid stones in the
kidneys (urolithiasis) and the deposition
of urate crystals in the joints (gouty
arthritis) and soft tissues.
■ In addition, the syndrome is
characterized by motor dysfunction,
cogenitive deficits, and behavioral
disturbances that include self-mutilation
(biting of lips and fingers)

32. ■ The gene for HGPRTase is on the Y chromosome; virtually
limited to males
■ In some study shows <2% of normal HGPRTase activity
causes mental retardation and <0.2% of normal causes
self –mutilation
■ Mutation in HGPRTase gene results in loss of HGPRTase
protein and HGPRTase activity
■ HGPRTase activity in brain has 10-20 times the level found
in liver, spleen or kidney and 4-8 times that found in RBC.
■ Treatment with allopurinol will decrease the amount of uric
acid formed, relieving some of the problems caused by
sodium urate deposits.
■ There in no treatment for the neurological problems; these
patients usually die from kidney failure, resulting from high
sodium urate deposits.

33. Hypouricemia: Adenosine deaminase (ADA) deficiency:
■ A deficiency of ADA results in an accumulation of
adenosine, which is converted to its ribonucleotide or
deoxyribonucleotide forms by cellular kinases.
■ As dATP levels rise, ribonucleotide reductase is inhibited,
thus preventing the production of all deoxyribose-containing
nucleotides
■ Consequently, cells cannot make DNA and divide.
■ The dATP and adenosine that accumulate in ADA deficiency
lead to developmental arrest and apoptosis of lymphocytes.

34. ■ This deficiency causes severe combined immunodeficiency
(SCID) involving T-cell and usually B-cell dysfunction
■ It is estimated that in the United States, ADA deficiency
accounts for approximately 14% of all cases of SCID.
■ Treatment requires either bone marrow transplantation
(BMT) or enzyme replacement therapy (ERT).
■ Without appropriate treatment, ADA deficient children
usually die before 2 years of age

35. De novo synthesis of pyrimidine
■ The common pyrimidine ribonucleotides are cytidine 5’-
monophosphate (CMP; cytidylate) and uridine 5’-
monophosphate (UMP; uridylate), which contain the
pyrimidines cytosine and uracil.
■ De novo pyrimidine nucleotide biosynthesis proceeds in a
somewhat different manner from purine nucleotide
synthesis; i.e the six-membered pyrimidine ring is made
first (orotate) and then attached to ribose 5-phosphate.
■ This process require carbamoyl phosphate, which is also
an intermediate in the urea cycle.
■ Carbamoyl phosphate required in urea synthesis :is made
in mitochondria by carbamoyl phosphate synthetase I
■ Carbamoyl phosphate required in pyrimidine biosynthesis :
is made in cytosol by carbamoyl phosphate synthetase II

36. ■ The sources of the atoms in the pyrimidine ring are
glutamine, CO2, and aspartic acid

40. Regulation of pyrimidine synthesis
■ Pyrimidine nucleotide biosynthesis is
regulated by feedback inhibition:
1. Regulation at the level of CPS II
(enzyme 1) : inhibited by UTP & are
activated by PRPP.
2. Aspartate transcarbamoylase
(enzyme 2): inhibited by CTP, and
activated by ATP.
3. Orotidylate (OMP) decarboxylase:
inhibited by UMP.
■ The requirement of ATP for CTP
formation and the stimulatory effect
of GTP on CTP synthetase ensure a
balanced synthesis of purine and
pyrimidine nucleotides.
.

41. Salvage of pyrimidines
■ Few pyrimidine bases are salvaged in human cells.
■ There are 2 enzymes that catalyze the reactions of salvage
pathway. They are uracil phosphoribosyl transferase (UPRT)
and thymidine kinase.
■ The salvage of pyrimidine nucleosides is the basis for using
uridine in the treatment of hereditary orotic aciduria.
41

42. Degradation of pyrimidine
nucleotides■ The first step of the catabolism of pyrimidines is
dephosphorylation to the nucleosides by 5’-
nucleotidases.
■ Pyrimidine nucleosides are then phosphorolysed into
free pyrimidines and pentose 1 phosphate with the help
of Pi and nucleoside phosphorylases.
Degradation of Cytosine and Uracil:
■ Cytosine will form uracil by deaminase
■ Uracil, is then reduced to 5,6-dihydrouracil by
dihydrouracil dehydrogenase using NADPH.
■ 5,6-dihydrouracil is converted by hydropyrimidine
hydrase to produce β-ureidopropionic acid.
42

43. ■ The next step is further hydrolysis by β-ureidopropionase
into CO2, NH3 and β-alanine.
■ The β-alanine can either be used in the synthesis of An
serine, or CoA or can be oxidised to acetate, NH3 and CO2
43

44. Degradation of thymine
■ Thymine released from thymidine or produced by the
deamination of 5-methylcytosine is reduced to
dihydrothymine by an NADH dependent dehydrogenase
■ Dihydrothymine undergoes hydrolysis by hydrase to give β-
ureidoisobutyric acid.
■ The β-ureidoisobutyric acid is hydrolysed by β-
ureidoisobutyrase into CO2, NH3 and β-amino-isobutyrate.
44

45. Disorders of Pyrimidine Metabolism; Orotic Aciduria
■ It is an autosomal recessive disease.
Cause:
■ Results from absence of either or both of the enzymes, both
orotate phosphoribosyltransferase (OPRTase) and OMP
decarboxylase.
It is of 2 types:
1. Type I orotic aciduria:
■ Due to genetic disorder of a protein acting as both orotate
phosphoribosyltransferase and OMP decarboxylase.
■ Orotate fails to be converted to uridylate.
2. Type II orotic aciduria: due to defect of OMP decarboxylase
 Orotic aciduria may also occur in ornithine
transcarbamoylase deficiency (urea cycle enzyme) as
carbamoyl phosphate accumulates due to defective
45

46. Orotic Aciduria
Features:
■ Poor growth
■ Megaloblastic anaemia
■ Does not improve with vitamin B12 of folic acid
■ Excretion of large amount of orotate in urine
Treatment:
■ can be successfully treated by feeding cytidine or uridine.
46

47. Many Chemotherapeutic Agents Target Enzymes In
The Nucleotide Biosynthetic Pathways

48. ■ 5-Fluorouracil (5-FU)
 Acts on thymidylate synthase.
 Fluorouracil itself is not the enzyme inhibitor.
 Salvage pathways metabolically converts it to 5-
Flurodeoxyuridine Monophosphate, which becomes
permanently bound to the inactivated Thymidylate
Synthase; for this reason it is called ‘sucide inhibitor’.
■ Methotrexate
 is an inhibitor of DHFRase.
 folate analog ,acts as a competitive inhibitor
 the enzyme binds methotrexate with about 100 times
higher affinity than dihydrofolate

49. ■ Aminopterine:
 Another folate analog
identical to methotrexate,
except it lacks methyl group
■ Trimethoprim:
 Folate analog
 Potent antibacterial activity
because of selective inhibition
of bacterial dihydrofolate
reductase
Azaserine:
 Are glutamine analogs, Inhibit
glutamine amidotransferases.
 Glutamine is a nitrogen donor
in at least half a dozen
separate reactions in
nucleotide biosynthesis
Inhibited by 5-
Fluorouracil

50. Compounds that interfere with Cellular Nucleotide
metabolism■ De novo synthesis of purine and pyrimidine nucleotides is
critical to normal cell replication, maintenance, and function.
■ Regulation of these pathways is important since disease
states arise from defects in the regulatory enzymes
■ They act as competitive inhibitors of the naturally occurring
nucleotides that are used to synthesize DNA.
■ When wrong bases are incorporated, the DNA becomes
functionally inactive, thereby cell division is arrested.
■ So they are useful as anticancer drugs.
■ Few examples are:
1. Glutamine amidotransferase inhibitor
■ Glutamine analogs like Azaserine and Acivine competitively
inhibit pathways in which glutamine is metabolized
(potential antineoplastic agent)

51. 2. PRPP Amidotransferase inhibitor
■ Eg: Mercaptopurine (immunosupressive drug) inhibits purine
synthesis by inhibiting PRPP amidotransferase &
HGPRTase(leukemia, lymphoma, Psoriatic arthritis)
3. Para Aminobenzoic acid (PABA)analogues:
■ Eg. Sulphonamides
■ They are structural analog of PABA which competitively
inhibits bacterial synthesis of folic acid in bacteria
■ Since tetrahydro folate is requires for purine synthesis, sulfa
drugs slow this pathway in bacteria
■ Human can’t synthesize folic acid, so human cells are not
affected.

52. 4. IMP dehydrogenase inhibitor:
■ Eg. Mycophenolate- it Prevents synthesis of GMP from IMP
by inhibiting IMP dehydrogense
■ Rapidly Deprives proliferating B- and T-lymphocytes
■ In immunosuppresent used to prevent graft rejection
5. Folic acid analogs:
■ Eg. Methotrexate, Pyrimethamine
■ Inhibit reduction of DHF to THF (DHFRase)
■ So, Limit the amount of THF available for purine synthesis
■ Used in treatment of certain cancers

54. Adverse effects of anti-cancer drugs
■ Inhibitors of human purine synthesis are extremely toxic to
tissues, especially to developing structures such as in a
fetus, or to cell types that normally replicate rapidly eg. bone
marrow, skin, GI tract, immune system, or hair follicles.
■ As a result, individuals taking such anticancer drugs can
experience adverse effects including;
– GI disturbance
– anaemia
– scaly skin
– Immunodificiencies
– hair loss(baldness)

55. Summary:

57. Thank-you