This document provides an overview of nucleotide metabolism. It discusses the structures of nucleic acids and nucleotides, as well as the degradation and synthesis pathways of purine and pyrimidine nucleotides. For purines, it describes the de novo synthesis pathway starting from ribose-5-phosphate, salvage pathways, regulation, and the formation of deoxyribonucleotides. For pyrimidines, it outlines the shorter de novo synthesis pathway and formation of UTP, CTP, and TMP. It also discusses nucleotide-related diseases and antimetabolite drugs used in cancer treatment.

1. Mr. G. Siluchali
Levy Mwanawasa Medical
University
Medical Biochemistry

2. N
N
N
N
H
N
N
Metabolism of Nucleotides

4. Contents
• Review: Structure of nucleic acid
• Degradation of nucleic acid
• Synthesis of Purine Nucleotides
• Degradation of Purine Nucleotides
• Synthesis of Pyrimidine Nucleotides
• Degradation of Pyrimidine Nucleotides

5. Nucleoside and Nucleotide
Nitrogenous base ribose
Nitrogenous base ribose phosphate
Nucleoside =
Nucleotide =

6. Purines vs Pyrimidines

7. pyrimidine purine
OR
Ribose
or
2-deoxyribose
N-b-glycosyl
bond
Structure of nucleotides

8. Section 1
Degradation of nucleic acid

9. Nucleoprotein
Nucleic acid Protein
Nucleotide
Nucleoside
Phosphate
Base Ribose
Nucleotidase
Nucleosidase
Degradation of nucleic acid
In stomach Gastric acid and pepsin
In small intestine Endonucleases: RNase and DNase

10. Significances of nucleotides
1. Precursors for DNA and RNA synthesis
2. Essential carriers of chemical energy, especially
ATP
3. Components of the cofactors NAD+, FAD, and
coenzyme A
4. Formation of activated intermediates such as
UDP-glucose and CDP-diacylglycerol.
5. cAMP and cGMP, are also cellular second
messengers.

11. Section 2
Synthesis of Purine Nucleotides

12. There are two pathways leading to
nucleotides
• De novo synthesis: The synthesis of nucleotides
begins with their metabolic precursors: amino
acids, ribose-5-phosphate, CO2, and one-carbon
units.
• Salvage pathways: The synthesis of nucleotide
by recycle the free bases or nucleosides released
from nucleic acid breakdown.

13. § 2.1 De novo synthesis
• Site:
– in cytosol of liver, small intestine and thymus
• Characteristics:
a. Purines are synthesized using 5-
phosphoribose(R-5-P) as the starting material
step by step.
b. PRPP(5-phosphoribosyl-1-pyrophosphate) is
active donor of R-5-P.
c. AMP and GMP are synthesized further at the
base of IMP(Inosine-5'-Monophosphate).

14. N10-
Formyltetrahydrofolate
N10-
Formyltetrahydrofolate
1. Element sources of purine bases
First, synthesis Inosine-5'-Monophosphate, IMP

15. FH4 (or THF)
N10—CHO—FH4

16. 2. Synthesis of Inosine Monophosphate (IMP)
• Basic pathway for biosynthesis of purine
ribonucleotides
• Starts from ribose-5-phosphate(R-5-P)
• Requires 11 steps overall
• occurs primarily in the liver

17. OH
1
ATP
AMP
2
Gln:PRPP amidotransferase
ribose phosphate pyrophosphokinase
Step 1:Activation of ribose-5-phosphate
Step 2: acquisition of purine atom N9
•Steps 1 and 2 are tightly
regulated by feedback inhibition
Committed step

18. 3
Step 3: acquisition of purine atoms C4, C5, and N7
glycinamide synthetase

19. 4
•Step 4: acquisition of purine atom C8
GAR transformylase

20. 5
Step 5: acquisition of purine atom N3

21. 6
•Step 6: closing of the imidazole ring

22. Carboxyaminoimidazole
ribonucleotide (CAIR)
7
Step 7: acquisition of C6
AIR carboxylase

23. Carboxyaminoimidazole
ribonucleotide (CAIR)
Step 8: acquisition of N1
SAICAR synthetase

24. Step 9: elimination of fumarate
adenylosuccinate lyase

25. Step 10: acquisition of C2
AICAR transformylase

26. Step 11: ring closure to form IMP
• Once formed, IMP is rapidly
converted to AMP and GMP (it does
not accumulate in cells).

27. N10-CHOFH4
N10-CHOFH4

28. 3. Conversion of IMP to AMP and GMP
Note: GTP is used for AMP synthesis.
Note: ATP is used for
GMP synthesis.
IMP is the precursor for both AMP and GMP.

29. kinase
ADP
kinase
ADP
ATP
ATP ADP
AMP
ATP
kinase
GDP
kinase
ADP
GTP
ATP ADP
GMP
ATP
4. ADP, ATP, GDP and GTP biosynthesis

30. 5. Regulation of de novo synthesis
The significance of regulation:
(1) Meet the need of the body, without
wasting.
(2) AMP and GMP control their respective
synthesis from IMP by a feedback
mechanism, [GTP]=[ATP]

31. • Purine nucleotide biosynthesis is regulated by feedback
inhibition

32. § 2.2 Salvage pathway
• Purine bases created by degradation of RNA or
DNA and intermediate of purine synthesis can be
directly converted to the corresponding nucleotides.
• The significance of salvage pathway :
– Save the fuel.
– Some tissues and organs such as brain and bone marrow
are only capable of synthesizing nucleotides by salvage
pathway.
• Two phosphoribosyl transferases are involved:
– APRT (adenine phosphoribosyl transferase) for adenine.
– HGPRT (hypoxanthine guanine phosphoribosyl
transferase) for guanine or hypoxanthine.

33. Purine Salvage Pathway
N
N
N
N
NH2
O
Guanine
N
N N
O
N
Hypoxanthine
O
OH
HO
2-O3POH2C
N
N N
O
N
IMP
O
OH
HO
2-O3POH2C
N
N
N
N
NH2
O
GMP
.
.
Adenine AMP
PRPP PPi
adenine
phosphoribosyl transferase
PRPP PPi
hypoxanthine-guanine
phosphoribosyl transferase
(HGPRT)
Absence of activity of HGPRT leads to Lesch-Nyhan syndrome.

34. Lesch-Nyhan syndrome
• first described in 1964 by Michael Lesch and William L.
Nyhan.
• there is a defect or lack in the HGPRT enzyme
• Sex-linked metabolic disorder: only males
• the rate of purine synthesis is increased about 200-fold
– Loss of HGPRT leads to elevated PRPP levels and stimulation
of de novo purine synthesis.
• uric acid level rises and there is gout
• in addition there are mental aberrations
• patients will self-mutilate by biting lips and fingers off

35. Lesch-Nyhan syndrome

36. § 2. 3 Formation of
deoxyribonucleotide
• Formation of deoxyribonucleotide involves
the reduction of the sugar moiety of
ribonucleoside diphosphates (ADP, GDP,
CDP or UDP).
• Deoxyribonucleotide synthesis at the
nucleotide diphosphate(NDP) level.

37. S
S
H2O
Mg2+
NADPH + H+
NADP+
SH
SH
thioredoxin
ribonucleotide
reductase
NDP
£¨N=A, G, C, U£©
dNDP
dNTP
ATP
ADP
kinase
O Base
CH2
H
OH
O
P P
O Base
CH2
OH
OH
O
P P
thioredoxin
thioredoxin
reductase
FAD
Deoxyribonucleotide synthesis at the NDP level

38. § 2. 4 Antimetabolites of purine
nucleotides
• Antimetabolites of purine nucleotides are
structural analogs of purine, amino acids and
folic acid.
• They can interfere, inhibit or block synthesis
pathway of purine nucleotides and further
block synthesis of DNA, RNA, and proteins.
• Widely used to control cancer.

39. 1. Purine analogs
• 6-Mercaptopurine (6-MP) is an analog of
hypoxanthine.
N
N N
H
N
OH
N
N N
H
N
SH
6-MP
hypoxanthine

40. 6-MP 6-MP nucleotide
de novo synthesis
salvage pathway
HGPRT
amidotransferase
IMP
AMP and GMP
-
-
-
-
-
• 6-MP nucleotide is a analog of IMP

41. 2. Amino acid analogs
• Azaserine (AS) is a analog of Gln.
H2N C CH2
O
CH2 CH
NH2
COOH Gln
C
O
CH2 CH
NH2
COOH AS
N
N CH2 O

42. 3. Folic acid analogs
• Aminopterin (AP) and Methotrexate (MTX)
R＝H： AP
folic acid
N
N
N
N
NH2
H2N
CH2 N C
R O
NH C
H
COOH
R＝CH3： TXT
CH2 CH2 COOH
N
N
N
N
OH
H2N
CH2 N C
H O
NH C
H
COOH
CH2 CH2 COOH
MTX

43. folate FH2 FH4
NADPH + H+
NADP+
NADPH + H+
NADP+
FH2 reductase FH2 reductase
AP or MTX
- -
•The structural analogs of folic acid(e.g. MTX) are widely
used to control cancer (e.g. leukaemia).
•Notice: These inhibitors also affect the proliferation of
normally growing cells. This causes many side-effects
including anemia, baldness, scaly skin etc.

44. Section 3
Degradation of Purine Nucleotides

45. N
HC
N
C
C
C
N
CH
N
NH2
Ribose-P
AMP
HN
HC
N
C
C
C
N
CH
N
O
Ribose-P
IMP
HN
HC
N
C
C
C
N
H
CH
N
O
HN
C
N
H
C
C
C
N
H
CH
N
O
O
HN
C
N
H
C
C
C
N
H
C
N
O
O
O
GMP
Hypoxanthine
Uric Acid Xanthine
Xanthine Oxidase
(2,6,8-trioxypurine)
Adenosine
Deaminase
The end product of purine metabolism

46. • Uric acid is the excreted end product of
purine catabolism in primates, birds, and
some other animals.
• The rate of uric acid excretion by the normal
adult human is about 0.6 g/24 h, arising in
part from ingested purines and in part from
the turnover of the purine nucleotides of
nucleic acids.
• The normal concentration of uric acid in the
serum of adults is in the range of 3-7 mg/dl.
Uric acid

47. • The disease gout, is a disease of the joints,
usually in males, caused by an elevated
concentration of uric acid in the blood and tissues.
• The joints become inflamed, painful, and arthritic,
owing to the abnormal deposition of crystals of
sodium urate.
• The kidneys are also affected, because excess
uric acid is deposited in the kidney tubules.
GOUT

48. The uric acid and the gout
Uric acid 
Over 8mg/dl, in the plasma
Gout, Urate crystallization
in joints, soft tissue, cartilage and kidney
Hypoxanthine
Xanthine
Out of body
In urine
Diabetese nephrosis
……

49. Advanced Gout
Clinically Apparent Tophi
1
1. Photos courtesy of Brian Mandell, MD, PhD, Cleveland Clinic.
2. Photo courtesy of N. Lawrence Edwards, MD, University of Florida.
3. ACR Clinical Slide Collection on the Rheumatic Diseases, 1998.
2
1
3

50. HN
HC
N
C
C
C
N
H
CH
N
O
Hypoxanthine
HN
HC
N
C
C
C
N
H
N
H
C
O
Allopurinol
Allopurinol – a suicide inhibitor used to treat Gout
Xanthine oxidase
Xanthine oxidase

51. Section 4
Synthesis of Pyrimidine Nucleotides

52. • shorter pathway than for purines
• Pyrimidine ring is made first, then attached to
ribose-P (unlike purine biosynthesis)
• only 2 precursors (aspartate and glutamine, plus
HCO3
-) contribute to the 6-membered ring
• requires 6 steps (instead of 11 for purine)
• the product is UMP (uridine monophosphate)
§ 4.1 De novo synthesis

53. 1. Element source of pyrimidine
base
N
C
N
C
C
C
1
2
3
4
5
6
Asp
CO2
Gln

54. •Carbamoyl phosphate synthetase(CPS) exists in 2 types:
•CPS-I, a mitochondrial enzyme, is dedicated to the urea
cycle and arginine biosynthesis.
•CPS-II, a cytosolic enzyme, used here. It is the committed
step in animals.
Step 1: synthesis of carbamoyl
phosphate

55. Step 2: synthesis of carbamoyl aspartate
ATCase: aspartate transcarbamoylase
•Carbamoyl phosphate
is an “activated”
compound, so no
energy input is needed
at this step.

56. Step 3: ring
closure to form
dihydroorotate

57. Step 4: oxidation of
dihydroorotate to orotate
QH2
CoQ
(a pyrimidine)

58. Step 5: acquisition of ribose phosphate
moiety
Step 6: decarboxylation of OMP

59. The big picture

60. 3. UTP and CTP biosynthesis
UDP
ADP
UTP
ATP ADP
UMP
ATP
kinase kinase

61. 4. Formation of dTMP
The immediate precursor of thymidylate (dTMP) is dUMP.
The formation of dUMP either by deamination of dCMP or
by hydrolyzation of dUDP. The former is the main route.
dTMP dTDP dTTP
dUMP
dUDP dCMP dCDP
N5,N10-methylene-
tetrahydrofolic Acid
ATP ATP
ADP ADP
dTMP synthetase
UDP

62. dTMP synthesis at the nucleoside
monophosphate level.
dUMP
dUDP
dCMP
dTMP
H2O
Pi
H2O
NH3
NADPH
NADP+
thymidylate synthase HN
N
O
O
R 5' P
d
CH3
reductase
HN
N
O
O
R 5' P
d
+ H+
FH2
FH4
N5
, N10
-CH2-FH4 FH2

63. § 4. 2 Salvage pathway
+ ATP
+ ATP
+ ATP
UMP
CMP
dTMP + ADP
dCMP + ADP
uridine
cytidine
deoxythymidine
deoxycytidine
thymidine kinase
deoxycytidine kinase
uridine-cytidine kinase
+ ADP
+ PRPP + PPi
uracil
thymine
orotic acid
pyrimidine phosphate
ribosyltransferase UMP
dTMP
OMP

64. § 4. 3 Antimetabolites of
pyrimidine nucleotides
• Antimetabolites of pyrimidine
nucleotides are similar with those of
purine nucleotides.

65. 1. Pyrimidine analogs
• 5-fluorouracil (5-FU) is a analog of
thymine.
HN
N
H
O
O
F
HN
N
H
O
O
CH3
thymine
5-FU

66. 2. Amino acid analogs
• Azaserine (AS) inhibits the synthesis of
CTP.
3. Folic acid analogs
• Methotrexate (MTX) inhibits the
synthesis of dTMP.

67. 4. Nucleoside analogs
• Arabinosyl cytosine (ara-c) inhibits
the synthesis of dCDP.
N
N
NH2
O
ara-c
O
H
OH H
H
CH2OH
H OH
N
N
NH2
O
cytosine
O
H
OH OH
H
CH2OH
H H

68. Section 5
Degradation of Pyrimidine Nucleotides

69. H2O
H2O
H2N CH2 CH2 COOH H2N CH2 CH COOH
CH3
N
N
H
O
NH2
H2O NH3
HN
N
H
O
O
CH2
CH2
NH2
N
H
O
HOOC
HN
N
H
O
O
CH3
CH2
CH
NH2
N
H
O
HOOC
CH3
cytosine uracil
thymine
¦Â
-ureidopropionate
¦Â
-ureido-
isobutyrate
CO2 + NH3
¦Â
-alanine ¦Â
-aminoisobutyrate
Highly soluble products

70. Summary of purine biosynthesis
dATP
dGTP
AMP
GMP
ADP
GDP
dADP
dGDP
IMP
ATP
GTP

71. CTP
Summary of pyrimidine biosynthesis
UDP UTP
CDP
dUDP
dCDP
dUMP
dCMP
dTMP
UMP
dTDP dTTP
dCTP

72. Summary of Nucleotide Synthesis
• Purines built up on ribose
– PRPP synthetase: key step
– First, synthesis IMP
• Pyrimidine rings built, then ribose added
– CPS-II: key step
– First, synthesis UMP
• Salvage is important

73. Points
• Synthesis of Purine Nucleotides
– De novo synthesis: Site, Characteristics, Element sources of
purine bases
– Salvage pathway: definition, significance, enzyme, Lesch-
Nyhan syndrome
– Formation of deoxyribonucleotide: NDP level
– Antimetabolites of purine nucleotides:
• Purine, Amino acid, and Folic acid analogs
• Degradation of Purine Nucleotides
– Uric acid, gout
• Synthesis of Pyrimidine Nucleotides
– De novo synthesis: Characteristics, Element sources of
pyrimidine bases
– Salvage pathway
– Antimetabolites of pyrimidine nucleotides
• Catabolism of Pyrimidine Nucleotides