Prof. Mária Sasvári
NUCLEOTIDE METABOLISM
Metabolism of purine nucleotides
Gergely Keszler
2009.
1
The biological role of nucleotides
1. Building blocks of nucleic acids (DNA and RNA)
2. Storage of biochemical energy (ATP and GTP)
3. Activation of biosynthetic precursors (UDP-glucose, CDP-choline)
4. Components of coenzymes (NAD, FAD, Coenzyme A etc.)
5. Regulation of metabolism (cAMP, cGMP)
6. Nucleotide analogues: anticancer and antiviral therapies
2
Terminology of nucleotides
phosphoanhydride bonds
phosphoester bond
N-glycosidic bond
Nucleotides are composed of: a nucleobase
a nucleoside
a pentose
at least one phosphate group
3
Ribo- and deoxyribonucleotides
(Ado)
(Guo)
(Urd)
(Cyd)
(dAdo)
(dGuo)
(Thd)
(dCyd)
4
Structures of nucleobases
N-containing, heterocyclic aromatic compounds;
substituted purine or pyrimidine rings
RNA
DNA
5
Nucleotide synthesis
„de novo”
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)
6
The origin of ribose-P
glc-6-P
PPP
fru-6-P
ATP
ri-5-P
PRPP synthetase
AMP
5’
P-O-H2C
O
O-P P
1’
PRPP
Purine
“de novo” and salvage reactions
Pyrimidine
“de novo” synthesis
7
Intestine
Food
Blood
brain, RBC, lymphocytes

RNA, DNA

“salvage reactions”
polynucleotides

nucleotides

nucleosides
bases
nucleosides
bases
“de novo”
synthesis

nucleotides

urate

URINE
liver
DNA
RNA
nucleosides
bases

urate
8
Purine nucleotide synthesis
ATP

ADP

AMP
GTP

GDP

GMP
IMP
purine
bases
salvage
reactions
“de novo”
synthesis
9
The origin of the purine ring
IMP
Asp
N6
7
N10formyl H4F
CO2

5
4
N
2
Glycine
N
1
N
3
N10formyl H4F
Gln
10
“de novo” purine synthesis
CO2

Asp
N Glycine
N
PRPP
Gln + H2O
N10formyl H4F
N
N
N10formyl H4F
1.
Gln PRPP amidotransferase
Gln
9.
Glu + PPi
NH3+
ri-5-P
PRA
(5-phosphoribosyl-1-amine)
Glycine
ATP
2. GAR synthetase
ADP + Pi
11
“de novo” purine synthesis
CO2

Asp
N10formyl
H4F
NH3+
N Glycine
N
9.
N
N
Gln
N10formyl H4F
9.
NH
O
ri-5-P
GAR
(5’PR-Glycinamide)
N10formyl H4F
3.
GAR formyltransferase
H4F
12
“de novo” purine synthesis
CO2

Asp
N10formyl
H4F
NH
N Glycine
N
9.
N
N
Gln
N10formyl H4F
O
NH
O
ri-5-P
FGAR
(5’PR-formyl-glycinamide)
Gln
4.
ATP
FGAM synthetase
Glu
ADP + Pi
13
“de novo” purine synthesis
CO2

Asp
N10formyl
H4F
NH
N Glycine
N
9.
N
N
Gln
N10formyl H4F
HHN
2N
NH
O
ri-5-P
FGAM
(5’PR-formylglycinamidine)
ATP
5.
AIR synthetase
ADP + Pi
14
“de novo” purine synthesis
CO2

Asp
N10formyl
H4F
N
N Glycine
N
9.
N
N
Gln
N10formyl H4F
H2N
N
ri-5-P
AIR
(5’PR-5-amino-imidazole)
CO2
6.
AIR carboxylase
15
“de novo” purine synthesis
CO2

Asp
N Glycine
N
N10formyl
H4F
9.
N
N
Gln
-OOC
N
N10formyl H4F
H2N
N
ri-5-P
CAIR
(5’PR-4-Carboxy- 5-amino-imidazole)
Asp
ATP
7.
SAICAR synthetase
ADP + Pi
16
“de novo” purine synthesis
CO2

Asp
N10formyl H4F
O
N Glycine
N
9.
N
N
succinyl- HN
N
N10formyl H4F
Gln
H2N
N
ri-5-P
SAICAR
(5’PR-succinyl-5-aminoimidazole-4-carboxamide)
8.
Adenylosuccinase (ASA)
fumarate
17
“de novo” purine synthesis
CO2

Asp
N10formyl H4F
O
N Glycine
N
9.
N
N
H2N
N10formyl H4F
N
Gln
H2N
N
ri-5-P
ACAIR
(5’PR-5-aminoimidazole-4-carboxamide
N10formyl H4F
9.
AICAR transformylase
H4F
18
“de novo” purine synthesis
CO2

Asp
N10formyl H4F
O
N Glycine
N
9.
N
N
H2N
N
N10formyl H4F
Gln
O
N
H
N
ri-5-P
FACAIR
5’PR-5-formamidoimidazole-4-carboxamide)
10
.
IMP cyclohydrolase
H2O
IMP
19
ATP

ADP

AMP
GTP

GDP

GMP
IMP
“de novo” purine synthesis
and the purine nucleotide cycle
AMP
AMP DA
(6-amino)
GMP
(2-amino-6-oxo)
AMP+PPi
GS
ASL
ATP
fumarate
Gln
Xanthylate
Adenylosuccinate
ASS
(2,6,-dioxo)
GDP+Pi
NADH + H+
IMPDH
GTP
Asp
NAD+
H2 O
IMP(6-oxo)
20
The role of the purine nucleotide cycle
ADP + Pi  ATP
2 ADP
AMP
kinase
ATP
+
AMP
Substrate level/oxidative
phosphorylation
AMP DA
IMP
urate
PNC
Adenylosuccinate
severe Pi deficiency  [AMP]     hyperuricaemia
21
e.g. fructose intolerance
Muscle: high AMP DA level
Muscle
Liver
ATP
AMP + glycolysis
NH3
NH3
IMP
inosine
urate
inosine


urate
strenuous exercise: NH3 , urate 
Muscle AMP DA def.: cramps, NH3, urate is NOT elevated
22
“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:
6 ATP for 1 IMP
Multifunctional proteins
23
AMP of de novo purine
GMP
Regulation
synthesis
“salvage”
ATP

ADP

AMP
IMP
+
+
GTP

GDP

GMP
“salvage”
- IMP
Gln PRPP amidotransferase + PRPP
- IMP, GMP, AMP
PRPP synthetase
- ATP,GTP
24
Purine salvage reactions
ATP

ADP

AMP
PRT (phosphorybosyl transferase)
base
+ ribose-P
nucleotide
APRT
AMP
adenine
PRPP
PPi
HGPRT
hypoxantine
guanine
PRPP
GTP

GDP

GMP
IMP
purine
bases
salvage
reactions
“de novo”
synthesis
IMP
GMP
PPi
25
The Lesch-Nyhan syndrome
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
26
Catabolism of purine nucleotides
AMP
B
r -p
5’nucleotidase
Pi
adenosine deaminase
Pi
B
adenosine (6-amino)
H2O
ADA
GMP
r
guanosine
NH3
inosine (6-oxo)
Pi
PNP (purine nucleoside
Pi
phosphorylase)
ri-1-P
ri-1-P
hypoxantine
guanine
B
27
Purine salvage reactions
hypoxantine
(6-oxo-purine)
guanine
(2-oxo-6-amino-purine)
E H 2 O + O2
x
xanthine
c
(2,6-dioxopurine)
H2O2
r
xanthine oxidase H2O + O2
e
t
H2 O2
i
o
urate
n
(2,6,8-trioxopurine)
H2 O
guanase
NH3
URINE
28
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
29
Hyperuricemia (gout)
Symptoms:
 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 )
30
Reasons for hyperuricemia
1. PRPP overproduction
• as a consequence of mutation
at the allosteric site of PRPP synthase,
the enzyme cannot be inhibited
gl-6-P
•overproduction of ribose-5-P
PPP
fr-6-P
e.g. gl-6-phosphatase deficiency
(von Gierke’s disease)
Gl- 6-P   fr- 6-P   ri- 5-P 
ri-5-P
PRPP
31
Reasons for hyperuricemia
2. Absence of purine salvage reactions
ATP

ADP

AMP
e.g. HPRT deficiency
Decreased adenine, guanine reutilization

purine
increased excretion
bases
GTP

GDP

GMP
IMP
salvage
reactions
“de novo”
synthesis
32
Reasons for hyperuricemia
3. Low ATP level, disturbed ATP metabolism
• strenuous exercise
• fructose intolerance (phosphate trap)
2 ADP
see before
AMP
kinase
ATP +
AMP
AMP DA
AMPS
IMP
urate
33
Reasons for hyperuricemia
4. Secondary reasons:
• tissue damage
• cancer, cell damage

DNA breakdown
overproduction of purines
• Overproduction of organic anions
(lactate, ketone bodies, drug derivatives)
34
Medication of gout: allopurinol
hypoxanthine
Xanthine oxidase
allopurinol
alloxanthine
xanthine
oxopurinol
Competitive inhibitors
Hypoxanthine and xanthine in urine (better solubility)
35
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
36
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
37
Adenosine deaminase functions on the outer surface
of red and white blood cell membranes (ectoenzyme)
binding glycoprotein
(„complexing factor”)
38
The pathogenesis of SCID - selective lymphotoxicity
1. Extracellular accumulation of (deoxy)adenosine
(d)Ado
A2
A1
Gi
Gs
dAdo
cAMP 
cAMP 
Inhibition of the SAM/SAH cycle
Impaired DNA synthesis
39
The pathogenesis of SCID - selective lymphotoxicity
2. Intracellular accumulation of (deoxy)adenosine triphosphate
Inhibition of
ribonucleotide
reductase
Inhibition of
cell proliferation
dCK
dAdo
dAdo
dATP 
dAMP
AK
DNA strand breaks
Lymphocyteselectivity!!
Inhibition of
DNA polymerases
apoptosis
40
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
41
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
42
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
43
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 transplanted into the patient
44
Ex vivo gene therapy
45
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.
46