This document discusses nucleotides, their synthesis and degradation. It covers the following key points: 1. Nucleotides are composed of a nucleoside (a nitrogenous base linked to a 5-carbon sugar) bound to one or more phosphate groups. They are the monomers that make up nucleic acids like RNA and DNA. 2. Purine nucleotides are synthesized de novo through a complex 10 step pathway beginning with phosphoribosyl pyrophosphate (PRPP) and ending with inosine monophosphate (IMP). Pyrimidine nucleotides can also be synthesized from PRPP. 3. Nucleotides can be broken down through both intracellular catabolism pathways that generate purine

1. Nucleotides: Synthesis and
Degradation
Tapeshwar Yadav
(Lecturer)
BMLT, DNHE,
M.Sc. Medical Biochemistry

2. Nitrogenous Bases
Planar, aromatic, and heterocyclic
Derived from purine or pyrimidine
Numbering of bases is “unprimed”

3. Nucleic Acid Bases
Purines Pyrimidines

4. Sugars
Pentoses (5-C sugars)
Numbering of sugars is “primed”

5. Sugars
D-Ribose and 2’-Deoxyribose
*Lacks a 2’-OH group

6. Nucleosides
Purine or pyrimidine base + Sugar through an N-
glycosidic linkage
Purines bind to the C1’ carbon of the sugar at their
N9 atoms
Pyrimidines bind to the C1’ carbon of the sugar at
their N1 atoms.

7. Nucleosides

8. Phosphate Groups
Mono-, di- or triphosphates
Phosphates bind at C3 or C5 atoms of the sugar

9. Nucleotides
Result from linking one or more phosphates with a
nucleoside onto the 5’ end of the molecule through
esterification

10. Nucleotides
RNA (ribonucleic acid) is a polymer of ribonucleotides
DNA (deoxyribonucleic acid) is a polymer of
deoxyribonucleotides
Both deoxy- and ribonucleotides contain Adenine,
Guanine and Cytosine
Ribonucleotides contain Uracil
Deoxyribonucleotides contain Thymine

11. Nucleotides
Monomers for nucleic acid polymers
Nucleoside Triphosphates are important energy carriers
(ATP, GTP)
cAMP
Important components of coenzymes
FAD, NAD+
and Coenzyme A

12. Naming Conventions
Nucleosides:
Purine nucleosides end in “-sine”
Adenosine, Guanosine
Pyrimidine nucleosides end in “-dine”
Thymidine, Cytidine, Uridine
Nucleotides:
Start with the nucleoside name from above and add
“mono-”, “di-”, or “triphosphate”
Adenosine Monophosphate, Cytidine Triphosphate,
Deoxythymidine Diphosphate

13. Digestion of Nucleic acids

15. Nucleotide Metabolism
PURINE RIBONUCLEOTIDES: formed de novo
i.e., purines are not initially synthesized as free bases
First purine derivative formed is Inosine Mono-phosphate
(IMP)
The purine base is hypoxanthine
AMP and GMP are formed from IMP

17. Most of the tissues
Liver
Cytosol
Multi enzyme complex

18. Purine Nucleotide Synthesis
OH
H
H
CH2
OH OH
H H
O
α
O2-
O3P
α-D-Ribose-5-Phosphate (R5P)
O
H
H
CH2
OH OH
H H
O α
O2-
O3P
5-Phosphoribosyl-α-pyrophosphate (PRPP)
P
O
O
O P
O
O
O
PRPP Synthase

19. O
H
H
CH2
OH OH
H H
O α
O2-
O3P
5-Phosphoribosyl-α-pyrophosphate (PRPP)
P
O
O
O P
O
O
O
H
NH2
H
CH2
OH OH
H H
O
β
O2-
O3P
β-5-Phosphoribosylamine (PRA)
PRPP Glutamyl amido transferase

20. H
NH2
H
CH2
OH OH
H H
O
β
O2-
O3P
β-5-Phosphoribosylamine (PRA)
H
NH
H
CH2
OH OH
H H
O
O2-
O3P
CO
H2C NH2
Glycinamide Ribotide (GAR)
GAR Synthetase

21. H
NH
H
CH2
OH OH
H H
O
O2-
O3P
CO
H2C NH2
Glycinamide Ribotide (GAR)
H2C
C
NH
O
CH
H
N
O
Ribose-5-Phosphate
Formylglycinamide ribotide (FGAR)
GAR formyl transferase
N
5
N
10
Methenyl (Formyl) THF

22. H2C
C
NH
O
CH
H
N
O
Ribose-5-Phosphate
Formylglycinamide ribotide (FGAR)
H2C
C
NH
O
CH
H
N
HN
Ribose-5-Phosphate
Formylglycinamidine ribotide (FGAM)
FAGM Synthetase

23. H2C
C
NH
O
CH
H
N
HN
Ribose-5-Phosphate
Formylglycinamidine ribotide (FGAM)
HC
C
N
CH
N
H2N
Ribose-5-Phosphate
4
5
5-Aminoimidazole Ribotide (AIR)
H2O +
AIR Synthetase

24. HC
C
N
CH
N
H2N
Ribose-5-Phosphate
4
5
5-Aminoimidazole Ribotide (AIR)
C
C
N
CH
N
H2N
OOC
Ribose-5-Phosphate
4
5
Carboxyamidoimidazole Ribotide (CAIR)
AIR Carboxylase

25. C
C
N
CH
N
H2N
OOC
Ribose-5-Phosphate
4
5
Carboxyamidoimidazole Ribotide (CAIR)
C
C
N
CH
N
H2N
C
N
H
O
HC
COO
CH2
COO
Ribose-5-Phosphate
4
5
5-Aminoimidazole-4-(N-succinylocarboxamide)
ribotide (SAICAR)
SAICAR Synthetase

26. C
C
N
CH
N
H2N
C
N
H
O
HC
COO
CH2
COO
Ribose-5-Phosphate
4
5
5-Aminoimidazole-4-(N-succinylocarboxamide)
ribotide (SAICAR)
Adeno succinate lyase
C
C
N
CH
N
H2N
Ribose-5-Phosphate
4
5
5-Aminoimidazole-4-carboxamide
ribotide (AICAR)
C
H2N
O

27. C
C
N
CH
N
NH
Ribose-5-Phosphate
4
5
5-Formaminoimidazole-4-carboxamide
ribotide (FAICAR)
C
H2N
O
C
H
O
C
C
N
CH
N
H2N
Ribose-5-Phosphate
4
5
5-Aminoimidazole-4-carboxamide
ribotide (AICAR)
C
H2N
O
AICAR Formyl transferase

28. C
C
N
CH
N
NH
Ribose-5-Phosphate
4
5
5-Formaminoimidazole-4-carboxamide
ribotide (FAICAR)
C
H2N
O
C
H
O
IMP cyclohydrolase
Inosine Monophosphate (IMP)
HN
HC
N
C
C
C
N
CH
N
O
4
5
HH
CH2
OH OH
H H
OO2-
O3P

29. Purine Nucleotide Synthesis
at a Glance
ATP is involved in 6 steps
PRPP in the first step of Purine synthesis is also a
precursor for Pyrimidine Synthesis, His and Trp
synthesis
Role of ATP in first step is unique– group transfer
rather than coupling
In second step, C1 notation changes from α to β
(anomers specifying OH positioning on C1 with respect
to C4 group)
In step 2, PPi is hydrolyzed to 2Pi (irreversible,
“committing” step)

30. Coupling of Reactions
Hydrolyzing a phosphate from ATP is relatively easy
∆G°’= -30.5 kJ/mol
If endergonic reaction released energy into cell as heat
energy, wouldn’t be useful
Must be coupled to an exergonic reaction
When ATP is a reactant:
Part of the ATP can be transferred to an acceptor: Pi,
PPi, adenyl or adenosinyl group
ATP hydrolysis can drive an otherwise unfavorable
reaction
(synthetase; “energase”)

31. IMP Conversion to AMP

32. IMP Conversion to GMP

33. Regulation of Purine Nucleotide
Biosynthesis
GTP is involved in AMP synthesis and ATP is involved in
GMP synthesis (reciprocal control of production)
PRPP is a biosynthetically “central” molecule (why?)
ADP/GDP levels – negative feedback on Ribose
Phosphate Pyrophospho synthetase
PRPP Glutamyl amido transferase is activated by
PRPP levels

34. Regulation of Purine Nucleotide
Biosynthesis
APRT activity has negative feedback at two sites
ATP, ADP, AMP bound at one site
GTP,GDP AND GMP bound at the other site
Rate of AMP production increases with increasing
concentrations of GTP; rate of GMP production increases
with increasing concentrations of ATP

35. Regulation of Purine Biosynthesis
Above the level of IMP production:
Independent control
Synergistic control
Feed forward activation by PRPP
Below level of IMP production
Reciprocal control
Total amounts of purine nucleotides controlled
Relative amounts of ATP, GTP controlled

36. PRPP synthetase
Activated by Pi
Inhibited by Purine nucleo tides AMP, GMP and IMP.
Regulation of Purine Biosynthesis

37. Committed step :
PRPP Gluamy amido transferase
Activators
PRPP
Glutamine
Inhibitors (Allosteric)
AMP, GMP, (IMP)

38. Synthetic inhibitors :
Mercaptopurine
Metho trexate
Azaserine
Thioguanine
Azaguanine
Sulphonamides
Trimethoprim

39. Intracellular Purine Catabolism
Nucleotides broken into nucleosides by action of 5’-
nucleotidase (hydrolysis reactions)
Purine nucleoside phosphorylase (PNP)
Inosine  Hypoxanthine
Xanthosine  Xanthine
Guanosine  Guanine
Ribose-1-phosphate splits off
Can be isomerized to ribose-5-phosphate
Adenosine is deaminated to Inosine (ADA)

40. Intracellular Purine Catabolism
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.

41. Salvage pathway
Recycling of purines (nucleosides)
PRPP
Two enzymes
RBCs and Brain.
Saves energy expenditure

42. Purine Salvage
Adenine phosphoribosyl transferase (APRT)
Adenine + PRPP AMP + PPi
Hypoxanthine-Guanine phosphoribosyl transferase (HGPRT)
Hypoxanthine + PRPP IMP + PPi
Guanine + PRPP GMP + PPi
(NOTE: THESE ARE ALL REVERSIBLE REACTIONS)
AMP,IMP,GMP do not need to be re-synthesized de novo !

43. Adenosine Degradation

44. Guanosine Degradation
• Ribose sugar gets recycled (Ribose-1-Phosphate  R-5-P )
– can be incorporated into PRPP (efficiency)
• Hypoxanthine is converted to Xanthine by Xanthine Oxidase
• Guanine is converted to Xanthine by Guanine deaminase
• Xanthine gets converted to Uric Acid by Xanthine Oxidase
Guanosine
Guanine

46. Xanthine Oxidase
A homodimeric protein
Contains electron transfer proteins
 FAD
Mo-pterin complex in +4 or +6 state
 Two 2Fe-2S clusters
Transfers electrons to O2  H2O2
 H2O2 is toxic
 Disproportionated to H2O and O2 by catalase

47. 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.

48. A CASE STUDY : GOUT
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.

49. Gout
Impaired excretion or overproduction of uric acid
Uric acid crystals precipitate into joints (Gouty
Arthritis), kidneys, ureters (stones)
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

53. ALLOPURINOL is a XANTHINE OXIDASE inhibitor
A substrate ANALOG is converted to an inhibitor.
In this case a “SUICIDE-INHIBITOR”

54. ALCOHOL CONSUMPTION AND GOUT

55. Causes :
1. PRPP Amido transferase
2. PRPP Synthetase
3. Deficiency of enzymes of salvage pathway
4. Glucose-6-Phosphatase deficiency
Secondary hyper urecemia (Gout) :
1. Leukemia
2. Lymphoma
3. Polycythemia
4. Trauma
5. Starvation
6. Renal failure
7. Toxemias

56. Clinical features :
Arthritis
Tophi
Urolithiasis
Renal failure
Treatment :
Reduce Purine intake and alcohol
Probenecid
Allopurinol
Colchicine

57. Lesch-Nyhan Syndrome
A defect in production or activity of HGPRT.
It is an X-linked disorder.
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

59. Pyrimidine Ribonucleotide Synthesis
 Uridine Monophosphate (UMP) is synthesized first
CTP is synthesized from UMP
Pyrimidine ring synthesis completed first; then attached to
ribose-5-phosphate

60. 2 ATP + HCO3
-
+ Glutamine + H2O
CO
O PO3
-2
NH2
Carbamoyl Phosphate
2 ADP +
Glutamate +
Pi
Carbamoyl
Phosphate
Synthetase II
Pyrimidine Synthesis

61. CO
O PO3
-2
NH2
Carbamoyl Phosphate
NH2
C
N
H
CH
CH2
C
COO
O
HO
O
Carbamoyl Aspartate
Aspartate
Transcarbamoylase
(ATCase)
Aspartate
Pi

62. NH2
C
N
H
CH
CH2
C
COO
O
HO
O
Carbamoyl Aspartate
Pyrimidine Synthesis
HN
C
N
H
CH
CH2
C
COO
O
O
Dihydroorotate

63. HN
C
N
H
CH
CH2
C
COO
O
O
Dihydroorotate
HN
C
N
H
C
CH
C
COO
O
O
Orotate

64. HN
C
N
H
C
CH
C
COO
O
O
Orotate
HN
C
N
C
CH
C
COO
O
O
HH
CH2
OH OH
H H
O
O2-
O3P
β
Orotidine-5'-monophosphate
(OMP)

65. HN
C
N
C
CH
C
COO
O
O
HH
CH2
OH OH
H H
O
O2-
O3P
β
Orotidine-5'-monophosphate
(OMP)
HN
C
N
CH
CH
C
O
O
HH
CH2
OH OH
H H
O
O2-
O3P
β
Uridine Monophosphate
(UMP)
CO2
OMP
Decarboxylase

66. 2 ATP + HCO3
-
+ Glutamine + H2O
CO
O PO3
-2
NH2
Carbamoyl Phosphate
NH2
C
N
H
CH
CH2
C
COO
O
HO
O
Carbamoyl Aspartate
HN
C
N
H
CH
CH2
C
COO
O
O
Dihydroorotate
HN
C
N
H
C
CH
C
COO
O
O
Orotate
HN
C
N
C
CH
C
COO
O
O
HH
CH2
OH OH
H H
O
O2-
O3P
β
Orotidine-5'-monophosphate
(OMP)
HN
C
N
CH
CH
C
O
O
HH
CH2
OH OH
H H
O
O2-
O3P
β
Uridine Monophosphate
(UMP)
2 ADP +
Glutamate +
Pi
Carbamoyl
Phosphate
Synthetase II
Aspartate
Transcarbamoylase
(ATCase)
Aspartate
Pi
H2O
Dihydroorotase
Quinone
Reduced
Quinone
Dihydroorotate
Dehydrogenase
PRPP PPi
Orotate Phosphoribosyl
Transferase
CO2
OMP
Decarboxylase
Pyrimidine Synthesis

67. UMP Synthesis Overview
2 ATPs needed: both used in first step
One transfers phosphate, the other is hydrolyzed to ADP
and Pi
2 condensation rxns: form carbamoyl aspartate and
dihydroorotate (intramolecular)
Dihydroorotate dehydrogenase is an intra-mitochondrial
enzyme; oxidizing power comes from quinone reduction
Attachment of base to ribose ring is catalyzed by OPRT;
PRPP provides ribose-5-P
PPi splits off PRPP – irreversible
Channeling: enzymes 1, 2, and 3 on same chain; 5 and 6 on
same chain

68. OMP DECARBOXYLASE : THE MOST
CATALYTICALLY PROFICIENT ENZYME
FINAL REACTION OF PYRIMIDINE PATHWAY
ANOTHER MECHANISM FOR
DECARBOXYLATION
A HIGH ENERGY CARBANION INTERMEDIATE
NOT NEEDED
NO COFACTORS NEEDED !
SOME OF THE BINDING ENERGY BETWEEN OMP
AND THE ACTIVE SITE IS USED TO STABILIZE THE
TRANSITION STATE
“PREFERENTIAL TRANSITION STATE BINDING”

70. 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 C4in animals

72. Regulation of Pyrimidine Synthesis
Differs between bacteria and animals
Bacteria – regulation at ATCase rxn
Animals – regulation at carbamoyl phosphate synthetase II
UDP and UTP inhibit enzyme; ATP and PRPP activate it
UMP and CMP competitively inhibit OMP Decarboxylase
*Purine synthesis inhibited by ADP and GDP at ribose phosphate
pyrophosphokinase step, controlling level of PRPP  also
regulates pyrimidines

73. CPS, ATC & DHOase multi enzyme complex
OPRTase & OMP decarboxylase single functional complex.

74. Salvage : PRPP and phospho ribosyl transferase
Nucleoside phosphorylase.

75. Degradation of Pyrimidines
CMP and UMP degraded to bases similarly to purines
Dephosphorylation
Deamination
Glycosidic bond cleavage
Uracil reduced in liver, forming β-alanine
Converted to malonyl-CoA  fatty acid synthesis for energy
metabolism

77. Deoxyribonucleotide Formation
Purine/Pyrimidine degradation are the same for
ribonucleotides and deoxyribonucleotides
Biosynthetic pathways are only for ribonucleotide
production
Deoxyribonucleotides are synthesized from
corresponding ribonucleotides

78. Formation of Deoxyribonucleotides
Reduction of 2’ carbon done via a free radical
mechanism catalyzed by “Ribonucleotide Reductases”
E. coli RNR reduces ribonucleoside diphosphates (NDPs) to
deoxyribonucleoside diphosphates (dNDPs)
Two subunits: R1 and R2
 A Heterotetramer: (R1)2 and (R2)2 in vitro

81. Thioredoxin
Physiologic reducing agent of RNR
Cys pair can swap H atoms with disulfide formed regenerate
original enzyme
Thioredoxin gets oxidized to disulfide
Oxidized Thioredoxin gets reduced by NADPH ( final electron acceptor)
mediated by thioredoxin reductase

82. Thymine Formation
Formed by methylating deoxyuridine monophosphate
(dUMP)
UTP is needed for RNA production, but dUTP not
needed for DNA
If dUTP produced excessively, would cause substitution errors
(dUTP for dTTP)
dUTP hydrolyzed by dUTPase
(dUTP diphosphohydrolase) to dUMP  methylated at
C5 to form dTMP rephosphorylate to form dTTP

83. dUMP dTMP
NADPH + H+
NADP+
SERINE
GLYCINE
REGENERATION OF N5
,N10
METHYLENETETRAHYDROFOLATE
DHFN5
,N10
– METHYLENE-THF
THF
dihydrofolate reductase
serine hydroxymethyl
transferase
thymidylate synthase

84. dUMP dTMP
NADPH + H+
NADP+
SERINE
GLYCINE
INHIBITORS OF N5
,N10
METHYLENETETRAHYDROFOLATE
REGENERATION
DHFN5
,N10
– METHYLENE-THF
THF
dihydrofolate reductase
serine hydroxymethyl
transferase
thymidylate synthase
METHOTREXATE
AMINOPTERIN
TRIMETHOPRIM
FdUMP
X
X

85. Anti-Folate Drugs
Cancer cells consume dTMP quickly for DNA
replication
Interfere with thymidylate synthase rxn to decrease dTMP
production
(fluorodeoxyuridylate – irreversible inhibitor) – also affects rapidly
growing normal cells (hair follicles, bone marrow, immune system,
intestinal mucosa)
Dihydrofolate reductase step can be stopped
competitively (DHF analogs)
Anti-Folates: Aminopterin, methotrexate, trimethoprim

86. ADENOSINE DEAMINASE DEFICIENCY
IN PURINE DEGRADATION, ADENOSINE  INOSINE
ENZYME IS ADA
ADA DEFICIENCY RESULTS IN SCID
“SEVERE COMBINED IMMUNODEFICIENCY”
SELECTIVELY KILLS LYMPHOCYTES
BOTH B- AND T-CELLS
MEDIATE MUCH OF IMMUNE RESPONSE
ALL KNOWN ADA MUTANTS STRUCTURALLY PERTURB
ACTIVE SITE

87. THE PURINE NUCLEOTIDE CYCLE
AMP + H2O  IMP + NH4
+
(AMP Deaminase)
IMP + Aspartate + GTP  AMP + Fumarate + GDP + Pi
(Adenylosuccinate Synthetase)
COMBINE THE TWO REACTIONS:
Aspartate + H2O + GTP  Fumarate + GDP + Pi+ NH4
+
The overall result of combining reactions is deamination of Aspartate to
Fumarate at the expense of a GTP

88. Orotic Aciduria
Caused by defect in protein chain with enzyme activities
of last two steps of pyrimidine synthesis
Increased excretion of orotic acid in urine
Symptoms: retarded growth; severe anemia
Only known inherited defect in this pathway (all
others would be lethal to fetus)
Treat with uridine/cytidine