1. NUCLEOTIDE METABOLISM Metabolism of purine nucleotides
Prof. Mária Sasvári
NUCLEOTIDE METABOLISM
Metabolism of purine nucleotides
Gergely Keszler
2009.

2. The biological role of nucleotides
Building blocks of nucleic acids (DNA and RNA)
Storage of biochemical energy (ATP and GTP)
Activation of biosynthetic precursors (UDP-glucose, CDP-choline)
Components of coenzymes (NAD, FAD, Coenzyme A etc.)
Regulation of metabolism (cAMP, cGMP)
6. Nucleotide analogues: anticancer and antiviral therapies

3. Terminology of nucleotides
phosphoanhydride bonds
phosphoester bond
N-glycosidic bond
Nucleotides are composed of: a nucleobase
a pentose
at least one phosphate group
a nucleoside

4. Ribo- and deoxyribonucleotides
(Ado)
(dAdo)
(Guo)
(Urd)
(Cyd)
(dGuo)
(Thd)
(dCyd)

5. Structures of nucleobases
N-containing, heterocyclic aromatic compounds;
substituted purine or pyrimidine rings
RNA
DNA

6. Nucleotide synthesis „de novo” salvage (recycling)
stepwise assembly
from small precursors
(C1 fragments, CO2, amino acids,
ribose-P)
salvage (recycling)
• base + ribose-P → nucleotide
(typical for purines) OR
• nucleoside + Pi → nucleotide
(typical for pyrimidines)

7. “de novo” and salvage reactions
The origin of ribose-P
glc-6-P
fru-6-P
PPP
O-P P 1’
P-O-H2C 5’ O
ATP
AMP
ri-5-P
PRPP synthetase
PRPP
Pyrimidine
“de novo” synthesis
Purine
“de novo” and salvage reactions

8.  “de novo” synthesis URINE
Intestine
Blood
brain, RBC, lymphocytes
Food 
RNA, DNA
polynucleotides
nucleotides
nucleosides
bases
“salvage reactions”
“de novo”
synthesis 
nucleotides
nucleosides
bases
 urate
nucleosides
bases
liver
DNA
RNA
urate 
URINE

9. Purine nucleotide synthesis
ATP 
ADP
AMP
GTP 
GDP
GMP
IMP
salvage
reactions
purine
bases
“de novo”
synthesis

10. The origin of the purine ring
IMP
CO2 
Asp 2 5 N
Glycine N 6 7 3 4 1 N N
N10formyl H4F
N10formyl H4F
Gln

11. “de novo” purine synthesis (5-phosphoribosyl-1-amine)
Gln N 10
formyl H 4 F
Glycine CO 2
Asp
“de novo” purine synthesis
PRPP 1.
Gln + H2O
Gln PRPP amidotransferase
Glu + PPi
NH3+ 9.
ri-5-P
PRA
(5-phosphoribosyl-1-amine)
Glycine 2.
ATP
ADP + Pi
GAR synthetase

12. “de novo” purine synthesisNH
NH3+ 9. O
ri-5-P
GAR
(5’PR-Glycinamide) 3.
N10formyl H4F
H4F
GAR formyltransferase

13. “de novo” purine synthesis (5’PR-formyl-glycinamide)NH O
ri-5-P O
FGAR
(5’PR-formyl-glycinamide) 4.
Gln
Glu
ATP
ADP + Pi
FGAM synthetase

14. “de novo” purine synthesis (5’PR-formylglycinamidine)NH O
ri-5-P
H2N HN
FGAM
(5’PR-formylglycinamidine) 5.
ATP
ADP + Pi
AIR synthetase

15. “de novo” purine synthesis (5’PR-5-amino-imidazole)
ri-5-P
H2N
AIR
(5’PR-5-amino-imidazole) 6.
CO2
AIR carboxylase

16. “de novo” purine synthesis (5’PR-4-Carboxy- 5-amino-imidazole)
-OOC N
H2N
ri-5-P
CAIR
(5’PR-4-Carboxy- 5-amino-imidazole) 7.
Asp
ATP
ADP + Pi
SAICAR synthetase

17. “de novo” purine synthesis
H2N
ri-5-P O HN
succinyl-
SAICAR
(5’PR-succinyl-5-aminoimidazole-4-carboxamide) 8.
Adenylosuccinase (ASA)
fumarate

18. “de novo” purine synthesis (5’PR-5-aminoimidazole-4-carboxamide
H2N
ri-5-P O
H2N
ACAIR
(5’PR-5-aminoimidazole-4-carboxamide 9.
N10formyl H4F
H4F
AICAR transformylase

19. “de novo” purine synthesis 5’PR-5-formamidoimidazole-4-carboxamide)
ri-5-P O
H2N
FACAIR
5’PR-5-formamidoimidazole-4-carboxamide)
10.
IMP cyclohydrolase
H2O
IMP

20. “de novo” purine synthesis and the purine nucleotide cycle
AMP
(6-amino)
GMP
(2-amino-6-oxo)
AMP DA
AMP+PPi
ASL GS
fumarate
ATP
Gln
Xanthylate
(2,6,-dioxo)
Adenylosuccinate
ASS
GTP
GDP+Pi
NAD+
H2O
NADH + H+
IMPDH
Asp
IMP(6-oxo)

21. The role of the purine nucleotide cycle Substrate level/oxidative
phosphorylation -
ADP + Pi  ATP
AMP
2 ADP
ATP
AMP
kinase
PNC
AMP DA
IMP
AMP
urate
Adenylosuccinate
severe Pi deficiency  [AMP]     hyperuricaemia
e.g. fructose intolerance

22. Muscle: high AMP DA level
Liver
ATP
AMP
IMP
NH3 +
glycolysis
NH3
inosine 
urate
inosine
urate
strenuous exercise: NH3 , urate 
Muscle AMP DA def.:
cramps, NH3, urate is NOT elevated

23. “de novo” purine synthesis
Summary
No free purine base during synthesis
Carbon donors: „C1 units” (N10-formyl-THF)
CO2
Glycine
N-donors: Asp
Gln
Gly
Energy: ATP for 1 IMP
Multifunctional proteins

24. Regulation of de novo purine synthesis+ +
“salvage” - -
Gln PRPP amidotransferase
+ PRPP
- IMP, GMP, AMP
PRPP synthetase
- ATP,GTP

25. Purine salvage reactions PRT (phosphorybosyl transferase)
base nucleotide
PRT (phosphorybosyl transferase)
+ ribose-P
APRT
adenine
AMP
PRPP
PPi
HGPRT
hypoxantine
guanine
PRPP
PPi
IMP
GMP

26. The Lesch-Nyhan syndrome linked to X-chromosome
HGPRT deficiency:
low GTP levels in the basal ganglia
Hyp/G + PRPP → IMP/GMP PPi
linked to X-chromosome
mental retardation
self-mutilation
aggression
hyperuricemia

27. Catabolism of purine nucleotides PNP (purine nucleoside
AMP
GMP Pi
5’nucleotidase B r
adenosine (6-amino)
guanosine
H2O
NH3
ADA
adenosine deaminase
ri-1-P Pi
inosine (6-oxo)
PNP (purine nucleoside
phosphorylase) B
hypoxantine
guanine

28. (2-oxo-6-amino-purine)
Purine salvage reactions
hypoxantine
(6-oxo-purine)
guanine
(2-oxo-6-amino-purine) E x c r e t i o n
H2O
NH3
guanase
H2O + O2
H2O2
xanthine
(2,6-dioxopurine)
xanthine oxidase
urate
(2,6,8-trioxopurine)
URINE
H2O + O2
H2O2

29. urate (dissociated anion)
Why is uric acid acidic?
uric acid (oxo)
uric acid (enol)
urate (dissociated anion)
well soluble
poor solubility
precipitates in joints,
initiates chemical arthritis
GOUT

30. Hyperuricemia (gout) Symptoms: acute gouty arthritis
urate crystals on the napkin (Lesch-Nyhan)
Na-urate crystals  kidney stones
urate in connective tissues and joints:
„tophus”, inflammation, pain
acute gouty arthritis
chronic gouty arthritis
Reason:
Urate has low solubility
(especially at acidic pH )

31. Reasons for hyperuricemia
1. PRPP overproduction
as a consequence of mutation
at the allosteric site of PRPP synthase,
the enzyme cannot be inhibited
overproduction of ribose-5-P
PPP ri
-5-P
PRPP gl
-6-P fr
e.g. gl-6-phosphatase deficiency
(von Gierke’s disease)
Gl- 6-P  
fr- 6-P  
ri- 5-P 

32. Reasons for hyperuricemia Decreased adenine, guanine reutilization
2. Absence of purine salvage reactions
e.g. HPRT deficiency
Decreased adenine, guanine reutilization 
increased excretion

33. Reasons for hyperuricemia
3. Low ATP level, disturbed ATP metabolism
strenuous exercise
fructose intolerance (phosphate trap)
see before

34. Reasons for hyperuricemia overproduction of purines
4. Secondary reasons:
tissue damage
cancer, cell damage
Overproduction of organic anions
(lactate, ketone bodies, drug derivatives) 
DNA breakdown
overproduction of purines

35. Medication of gout: allopurinol
hypoxanthine
Xanthine oxidase
xanthine
allopurinol
alloxanthine
oxopurinol
Competitive inhibitors
Hypoxanthine and xanthine in urine (better solubility)

36. Allopurinol, a special purine analogue
N-7 and C-8 have been scrambled up
Blocks xanthine oxidase, the enzyme
catalyzing the oxidation of
xanthine to uric acid – cures gout

37. Enzyme deficiency: ADA / PNP / (ADA + PNP) Symptoms: immunodeficiency,
“NON-HIV AIDS”
Reason:
B/T lymphocyte deficiency
Mechanism:
adenosine   dATP (ATP) 
dATP  inhibits ribonucleotide reductase
 inhibits DNA synthesis
 promotes apoptosis
Treatment: ADA enzyme therapy,
gene therapy

38. ADA ADA Adenosine deaminase functions on the outer surface
of red and white blood cell membranes (ectoenzyme)
ADA
ADA
binding glycoprotein
(„complexing factor”)

39. A1 A2 The pathogenesis of SCID - selective lymphotoxicity Gi Gs
1. Extracellular accumulation of (deoxy)adenosine
(d)Ado A1 A2 Gi Gs
dAdo
cAMP 
cAMP 
Inhibition of the SAM/SAH cycle
Impaired DNA synthesis

40. The pathogenesis of SCID - selective lymphotoxicity apoptosis
2. Intracellular accumulation of (deoxy)adenosine triphosphate
Inhibition of
ribonucleotide
reductase
Inhibition of
cell proliferation
dCK
dATP 
dAdo
dAdo
dAMP AK
DNA strand breaks
Inhibition of
DNA polymerases
Lymphocyte-
selectivity!!
apoptosis

41. Clinical manifestation of SCID
 Recurring, opportunistic infections: candidiasis, Pneumocystis-pneumonia
 Absence of lymph nodes, no thymic shadow upon chest X-ray examination
 Severe impairment of both humoral and cellular immunity:
 lower than 500/μl total lymphocyte count
 very low plasma immunoglobulin levels
 Untreated patients die before their age of 2 years

42. Treatment of ADA deficiency
1. Treatment of symptoms
 Infections
antibiotics, antiviral and antifungal drugs
 Immunoglobulin supplementation
Maternal immunoglobulins are effective in the first
few weeks of life

43. Treatment of ADA deficiency
2. Enzyme substitution
 Red blood cell transfusion
 Polyethylene-glycol-conjugated recombinanat ADA
(PEG-ADA): intramuscular injection
costs: 250,000 USD a year

44. Treatment of ADA deficiency
3. Gene therapy
Principle: Introduction of the normal allele into the
patients’ own stem cells
Ex vivo: Stem cells are transfected and trans-
planted into the patient

45. Ex vivo gene therapy

46. Ashanti da Silva: the first patient in the
world treated by retrovirus-mediated
ADA gene therapy
The introduced ADA gene functioned fine
for a few months. Later on, PEG-ADA
substitution therapy must have been
restarted due to inactivation of the gene.