For MBBS, MD, BDS students

1. NUCLEOTIDE CHEMISTRY AND
METABOLISM
(With ECE as per new CBME guidelines)
Prof (Dr) Viyatprajna Acharya MD, PhD

2. At the end of the class we’ll learn about…..
• Why nucleotides in Medicine??- An overview
• Nitrogenous base, nucleosides, nucleotides
and nucleic acids
• Structure of Nucleotides
• Nucleoside and nucleotide analogues
• Biologically important nucleotides
• Nucleotide metabolism
• Diseases associated with Purine and
Pyrimidine metabolism

3. Nucleotides are building blocks of nucleic
acids- DNA & RNA

4. As energy currency

5. Overview

6. Nucleotides & Medicine
• Nucleotide analogues as
anticancer agents- Azathioprine,
Cytarabine, 2F-dC etc
• Diseases associated with
nucleotide metabolism- Gout,
Lesch-Nyhan syndrome, ADA
deficiency, Orotic aciduria etc
• Pathogenesis of Cholera- altered G-
protein activity

7. Nucleotide structure
Nucleotides have three characteristic
components:
A phosphate group
A nitrogenous base
(pyrimidine or purine)
A pentose sugar

8. Nucleoside, Nucleotides….
• Nitrogenous base= Purine/ Pyrimidine
• Nucleoside= Nitrogenous base+ Ribose sugar
• Nucleotide= Nucleoside + Phosphoryl residue
• Purine- Adenine, Guanine
• Pyrimidine- Thymine, Cytosine, Uracil
• Nucleoside are N-glycosides- β-N-glycosidic
bond

9. Ribonucleotide & Deoxyribonucleotide

10. Ribose
Ribose (β-D-furanose)
is a pentose sugar (5-
membered ring).
Note numbering of
the carbons. In a
nucleotide, "prime" is
used in numbering
the sugar to
differentiate from
nitrogenous base
numbering.

11. An important
derivative of ribose
is 2'-deoxyribose,
or just
deoxyribose, in
which the 2' OH is
replaced with H.
Deoxyribose is in
DNA
(deoxyribonucleic
acid)
Ribose is in RNA
(ribonucleic acid).

12. Pyrimidine and purine Nucleus
Purines and pyrimidines are heterocycles with N atom

13. Adenine– 6-aminopurine Guanine– 2-amino-6-oxy purine

14. 4-Amino-1H pyrimidine-2-one; 5-methyl Uracil ; 2,4-dihydroxypyrimidine

15. Minor purine bases
• Hypoxanthine- 6-oxopurine
• Xanthine- 2,6-di-oxopurine
• Uric acid- 2,6,8-tri-oxopurine
• Methylated purine bases—
N-methyl adenine
N6N6-dimethyl adenine
7-methyl guanine
• Plant derivatives- Caffeine,
theophyllin of tea and
theobromine of Cocoa

16. Modified pyrimidine bases
Dihydrouracil
5-methyl cytosine

17. Tautomerism of bases
The oxo and amino groups show
Keto (Lactum) and enol (lactim)
Amine and imine tautomerism.
Physiological conditions favour keto and amine
form
A keto structure occurs when the hydrogen atom
bonds to a nitrogen atom within the ring.
An enol structure occurs when the hydrogen atom
bonds to a nearby oxygen atom that sticks out
from the ring.

18. Syn & Anti conformers
• There’s no freedom of rotation along β-N-
glycosidic bonds of nucleosides and
nucleotides
• Both exist in Syn & Anti conformation
• Anti-conformation predominates in nature

19. Nucleoside
Sugar + Base
H

20. Nucleotide structure
triphosphate
Ribose sugar
Base (adenine)

21. • Bases attach to the C-1' of ribose or deoxyribose
• The pyrimidines attach to the pentose via the N-1
position of the pyrimidine ring
• The purines attach through the N-9 position
• Some minor bases may have different attachments.

22. Deoxyribonucleotides

23. Major deoxyribonucleotides

24. Ribonucleotide

25. The major ribonucleotides

26. Nucleotide nomenclature

27. Nucleoside triphosphates
• Esterification of further phosphate groups
• ATP- universal energy currency; formed during
oxidative processes by trapping the released
energy in the high energy phosphate bond
• cAMP – a phosphodiester linkage formed
between the 3’ and 5’ positions of ribose
group

28. Nucleotides are poly-functional acids
• Primary and secondary phosphoryl groups of
nucleosides have pKa values of about 1.0 and
6.2 respectively
• Can serve as both proton donors and
acceptors
• At physiological pH they bear negative charge

29. Nucleotides absorb UV rays
• Conjugated double bonds of purine and
pyrimidine absorb UV light
• At pH 7.0, all common nucleotides absorb
light at 260nm
• Mutagenic effect- chemical modification of
nucleotides occur

30. Purine , Pyrimidine and Nucleotide
analogues
• Allopurinol
• 5-Flurouracil
• Azathioprine
• Arabinosyladenine
• Arabinosylcytosine
• Azidothymidine

31. Biologically important free nucleotides
• High energy source– ATP, GTP
• Methyl donor– SAM
• Component of coenzymes– FAD, NAD

32. Non-hydrolysable NTP analogues are used
as research tools
Used in ATP utilising systems to study
different processes involving ATP.
Since ATP is not hydrolysed, it doesn’t disrupt
the system.

34. NUCLEOTIDE METABOLISM
DR.V.P.ACHARYA

35. Why should you care
about nucleotide metabolism?
1. It is catalyzed by amazingly intricate highly
regulated enzymatic machines.
2. Defects in these machines result in
disease. Some examples:
• Lesch-Nyhan Syndrome
• SCID
3. Many important therapeutic agents target
these machines. Some examples:
• Fluorouracil
• Sulfa drugs

36. Digestion of nucleic acid
Dietary nucleic acids and nucleotides
- non-essential; do not provide essential
components for the biosynthesis of
endogenous nucleic acids
- synthesised from amphibolic intermediates
- injected compounds can be incorporated

37. Overview of digestion of nucleic acids
DIETARY NUCLEIC ACIDS
↓
DNA & RNA
↓
MONONUCLEOTIDES
↓Nucleotidases, Phosphatases
NUCLEOSIDES
↓Phosphorylase
BASES + DEOXYRIBOSE/ RIBOSE
↓Oxidation
EXCRETED
Ribonuclease, Deoxyribonuclease,
Polynucleotidase

38. Overview of Nucleotide Metabolism
PRPP
Pyrimidine nucleotides Purine
nucleotides
β-alanine Uric acid
(useful metabolite) (toxic waste product)

39. Biosynthesis of purine nucleotides
Site: Most of the cells
Major organ- Liver
Intracellular location- cytoplasm
2 pathways:
1.De Novo pathway– New synthesis from amphibolic
intermediates
2. Salvage pathway
– by phosphorylation of free purine bases
-- by phosphorylation of purine nucleosides

40. Overview of de novo pathway
PRPP
11 steps Build purine ring onto the sugar
IMP (1st purine nucleotide)
AMP GMP
AMP kinase GMP Kinase
ADP GDP
Nucleoside diphosphate kinase
ATP GTP

41. Purine ring is assembled on ribose-5-
phosphate(Derived from PPP) from a variety of
precursors

42. de novo pathway

43. Formation of PRPP

47. IMP Conversion to AMP & GMP
Aspartate

48. Regulatory Control of Purine Biosynthesis
A. Concentration of PRPP
B. Feedback regulation at diff. sites
A. ↑PRPP → ↑Purine nucleotides
PRPP synthesis depends–
• availability of ribose-5-PO4
• on the activity of PRPP synthase

49. B. Feedback regulation
a. PRPP synthase– feedback inhibition by purine
nucleotides, AMP,GMP & IMP
b. Committed step—
PRPP →Phosphoribosylamine
PRPP glutamyl- amido transferase enz– inhibited by
AMP and GMP
c. AMP- feedback regulates adenylosuccinate synthase
GMP- feedback regulates IMP dehydrogenase
d. ATP and GTP cross-regulate

50. Control of de novo synthesis

51. ANTIFOLATE DRUGS AND GLUTAMINE
ANALOGUES
• C of step 4 & 10- added by Tetrahydrofolate
• Folate antagonists (Methotrexate)- ↓ 1C
transfers- anticancer drugs
• Azaserine (Gln antagonist)- ↓ step 2,5
• Diazonorleucine (Gln antagonist) - ↓ step 2
• 6- mercaptopurine - ↓ step 13 & 14
(Conversion of IMP to AMP and GMP)
• Mycophenolic acid - reversibly inhibits IMP
dehydrogenase- ↓ step 14

52. de novo pathway

53. Salvage pathway
Significance: Provides purines to tissues that are
incapable to produce them by de novo
pathway.
Human brain--↓PRPP amidotransferase
RBC & WBC– can’t synthesise 5-
phosphoribosylamine
Economizes cell energy expenditure

54. Salvage– property recovered from loss
Purines, purine ribonucleosides, purine
deoxyribonucleosides– mononucleotides
Metabolic degradation of nucleic acids and
nucleotides– free purines and pyrimidines
formed → large part salvaged and remake
purine nucleotides
2 mechanisms—
a. Phosphoribosylation of purine bases
b. Phosphoribosylation of purine nucleosides

55. a. Phosphoribosylation of purine bases
Pu +PR−PP →PRP +PPi

57. b. Phosphoribosylation of purine nucleosides
PuR +ATP → PuR−P +ADP
Kinase enzyme involved.

58. Synthesis of deoxyribonucleotides
• By reduction of ribonucleotide diphosphates
• Enzyme complex active during cell division
preparation when DNA is being synthesised
• Reduction requires thioredoxin, thioredoxin
reductase and NADPH

61. DE NOVO SYNTHESIS OF PYRIMIDINE NUCLEOTIDES
CMP,UMP, TMP
N1, C4, C5, C6 : Aspartate
C2 : HCO3
-
N3 : Glutamine amide
Nitrogen

62. Overview of de novo Pyrimidine synthesis

63. • Pyrimidine ring synthesis completed
first; then attached to ribose-5-
phosphate
• Uridine Monophosphate (UMP) is
synthesized first
–CTP is synthesized from UMP

68. Difference between CPS-I and CPS-II
Characteristics CPS-I CPS-II
Cellular location Mitochondria Cytosol
Pathway involved Urea cycle Pyrimidine
synthesis
Source of nitrogen Ammonia Glutamine
Allosteric activator N-acetylglutamate
(NAG)
Nil

69. 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

70. Regulatory Control of Pyrimidine Synthesis
• Animals – regulation at Carbamoyl phosphate
synthetase II
UDP and UTP inhibit enzyme; ATP and PRPP
activate it
UMP and CMP competitively inhibit OMP
Decarboxylase
Aspartate transcarbamoylase (step 2)– feedback
inhibited by CTP & activated by ATP
the first three and the last two enzymes of the
pathway are regulated by coordinate repression
and derepression.
Purine & Pyrimidine nucleotide biosynthesis are
coordinately regulated

71. UMP and CMP competitively inhibit OMP
decarboxylase

72. Salvage pathway for Pyrimidine nucleotide synthesis
Uridine and cytidine, and deoxythymidine and
deoxycytidine– respective nucleotides

73. A smile at your enemy disarms him

74. Catabolism of Purine nucleotides
Xanthine is the point of convergence for the
metabolism of the purine bases
Xanthine  Uric acid
Xanthine oxidase catalyzes two reactions
Purine ribonucleotide degradation
pathway is same for purine
deoxyribonucleotides

76. Uric Acid Excretion
• Humans – excreted into urine as insoluble
crystals
• Serum uric acid level— 4-7 mg/dl
• Birds, terrestrial reptiles, some insects –
excrete insoluble crystals in paste form
– Excess amino N converted to uric acid
(conserves water)
• Others – further modification :
Uric Acid  Allantoin  Allantoic Acid  Urea  Ammonia

77. A CASE STUDY : GOUT
• A 45 year old man awoke from sleep with a painful
and swollen right great toe. On the previous night
he had eaten a meal of fried liver and onions, after
which he met with his poker group and drank a
number of beers.
• He saw his doctor that morning, “gouty arthritis”
was diagnosed, and some tests were ordered. His
serum uric acid level was elevated at 8.0 mg/dl (NL
< 7.0 mg/dl).
• The man recalled that his father and his
grandfather, both of whom were alcoholics, often
complained of joint pain and swelling in their feet.

78. • The doctor recommended that the man use NSAIDs
for pain and swelling, increase his fluid intake (but
not with alcohol) and rest and elevate his foot. He
also prescribed Allopurinol.
• A few days later the condition had resolved and
Allopurinol had been stopped. A repeat uric acid
level was obtained (7.1 mg/dl). The doctor gave the
man some advice regarding life style changes.

79. Gout
Impaired excretion or overproduction of uric acid
Uric acid crystals (monosodium urate) precipitate
into joints (Gouty Arthritis), kidneys, ureters
(stones)
Lead impairs uric acid excretion – lead poisoning
from pewter drinking goblets
Fall of Roman Empire?
Xanthine oxidase inhibitors inhibit production of
uric acid, and treat gout
Allopurinol treatment – hypoxanthine analog that
binds to Xanthine Oxidase to decrease uric acid
production

83. Causes of hyperuricemia
Increased uric acid formation Decreased uric acid excretion
Primary (genetic)
Enzyme defects in
PRPP synthase
PRPP aminotransferase
HGPRTase
Secondary
Due to increased:
Dietary intake
Nucleic acid turnover
ATP breakdown
Deficiency of
glucose-6-
phosphatase
Primary
idiopathic
Secondary
Renal
insufficiency
Metabolic acidosis
Lactic acidosis
Ketoacidosis
Starvation
DM
Increased tubular
reabsorption

84. Lesch -Nyhan Syndrome
A defect in production or activity of
HGPRT
↓
↓ salvage pathway
↓
Causes increased level of Hypoxanthine and Guanine ( in degradation
to uric acid)
Also, PRPP accumulates
↓
Stimulates production of purine nucleotides (and thereby increases their
degradation)
• Causes gout-like symptoms, but also neurological symptoms 
spasticity, aggressiveness, self-mutilation
• First neuropsychiatric abnormality that was attributed to a
single enzyme

85. Lesch Nyhan syndrome
Self Confident Men
Get Heavy Success
•Self-mutilation
•Choreoathetosis
•Mental retardation
•Gout
•Hyperuricemia
•Spasticity

86. HYPOURICEMIA
• Ser. Uric acid < 2.0 mg/dl
• Decreased production or increased excretion
• No symptoms or pathology- hence no tt
required
• Causes: Uricosuric drugs, X-ray contrast
materials, TPN hyperalimentation, neoplastic
disease, hepatic cirrhosis, DM, SIADH, Fanconi
syndrome and Lesch Nyhan syndrome

87. Adenosine deaminase deficiency
• SCID- severe combined immunodeficiency
disease
• RAG-1 0r2 mutation
• B- &T- cells are dysfunctional
• Deoxyadenosine and adenosine abundant→
inhibits further production of precursors for DNA
synthesis, especially dCTP
• Hypouricemia- defective breakdown of purines
• ‘Bubble baby syndrome’
• 1st disease to be cured by gene therapy

89. Catabolism of pyrimidines
• CMP and UMP degraded to bases similarly to
purines
Dephosphorylation
Deamination
Glycosidic bond cleavage
• Uracil reduced in liver, forming b-alanine
– Converted to malonyl-CoA  fatty acid synthesis
for energy metabolism

91. • Overproduction of pyrimidine catabolites is
only rarely associated with clinically
significant abnormalities
• Catabolites highly water soluble
• Since N5,N10-methylene-tetrahydrofolate is
required for thymidylate synthesis, disorders
of folate and vitamin B12 metabolism result
in deficiencies of TMP.

92. β- hydroxybutyric aciduria
• Total or partial deficiency of
dihydropyrimidine dehydrogenase
Uraciluria - thyminuria
Disorder of pyrimidine catabolism
Formation of β- alanine and β- aminoisobutyrate
Serious neurological disorder

93. Orotic aciduria
• Type-I--↓OPRTase and OMP decarboxylase
• Type-II-- ↓ OMP decarboxylase
• Autosomal recessive disorder
• Accompanies Reye syndrome also
• Allopurinol- competitively inhibits orotic acid
metabolism
• 6-Azauridine also accumulates orotic acid by inhibiting
orotidylate decarboxylase
• Retarded growth and megaloblastic anemia
• ↓ feedback inhibition of the enzyme
• Tt. – feeding cytidine or Uridine

95. Pseudouridine metabolism
• From RNA
• Not metabolised
• Excreted unchanged in urine
• It was first isolated from urine

97. More ppt on Medical Biochemistry on
www.vpacharya.com