1. Nucleotide metabolism – Part 1 (purine biosynthesis)By
Henry Wormser, Ph.D
Professor of Medicinal Chemistry

2. Biological significance of nucleotide metabolism
Nucleotides make up nucleic acids (DNA and RNA)
Nucleotide triphosphates are the “energy carriers” in cells (primarily ATP)
Many metabolic pathways are regulated by the level of the individual nucleotides
Example: cAMP regulation of glucose release
Adenine nucleotides are components of many of the coenzymes
Examples: NAD+, NADP+, FAD, FMN, coenzyme A

3. Dietary nucleotides
do not contribute energy as do carbs, proteins and fats
are not incorporated into RNA or DNA unless given I.V.
normally metabolized to individual components (bases, sugar and phosphate)
purines are converted to uric acid which is then excreted

4. Medical significance of nucleotide metabolism
Anticancer agents:
Rapidly dividing cells biosynthesize lots of purines and pyrimidines, but other cells reuse them. Cancer cells are rapidly dividing, so inhibitor of nucleotide metabolism kill them
Antiviral agents
Zidovudine (Retrovir)
Lamivudine (Epivir)
Valacyclovir (Valtrex)

6. Structures of nucleotide building blocks and nucleotides

7. Structures of nucleotide building blocks and nucleotides
guanine: comes from guano; thymine –thymus gland

8. Ribonucleotide – phosphate = ribonucleoside

9. Biosynthesis of the purine nucleotide system

10. Synthesis of Inosine Monophosphate
Basic pathway for biosynthesis of purine ribonucleotides
Starts from ribose-5-phosphate which is derived from the pentose phosphate pathway
Requires 11 steps overall
occurs primarily in the liver

11. The big picture

12. Steps 1 thru 3 Step 1:Activation of ribose-5-phosphate
enzyme: ribose phosphate pyrophosphokinase
product: 5-phosphoribosyl-a-pyrophosphate (PRPP)
PRPP is also a precursor in the biosynthesis of pyrimidine nucleotides and the amino acids histidine and tryptophan

13. Step 1: purine synthesis

14. Steps 1 thru 3 Step 2: acquisition of purine atom 9
enzyme: amidophosphoribosyl transferase
displacement of pyrophosphate group by glutamine amide nitrogen (inversion of configuration – a to b
product: b-5-phosphoribosylamine
Steps 1 and 2 are tightly regulated by feedback inhibition

15. Step 2: purine synthesis: commited step

16. Steps 1 thru 3 Step 3: acquisition of purine atoms C4, C5, and N7
enzyme: glycinamide synthetase
b-phosphoribosylamine reacts with ATP and glycine
product: glycinamide ribotide (GAR)

17. Step 3 : purine synthesis

18. Steps 4 thru 6 Step 4: acquisition of purine atom C8
formylation of free a-amino group of GAR
enzyme: GAR transformylase
co-factor of enzyme is N10-formyl THF
Step 5: acquisition of purine atom N3
The amide amino group of a second glutamine is transferred to form formylglycinamidine ribotide (FGAM)
Step 6: closing of the imidazole ring or formation of 5-aminoimidazole ribotide

20. Step 6: purine synthesis

21. Step 7 Step 7: acquisition of C6 C6 is introduced as HCO3-
enzyme: AIR carboxylase (aminoimidazole ribotide carboxylase)
product: CAIR (carboxyaminoimidazole ribotide)
enzyme composed of 2 proteins: PurE and PurK (synergistic proteins)

22. Step 7: purine synthesis

23. Steps 8 thru 11 Step 8: acquisition of N1
N1 is acquired from aspartate in an amide condensation reaction
enzyme: SAICAR synthetase
product: 5-aminoimidazole-4-(N-succinylocarboxamide)ribotide (SAICAR)
reaction is driven by hydrolysis of ATP

24. Step 8: purine synthesis

25. Steps 8 thru 11 Step 9: elimination of fumarate
Enzyme: adenylosuccinate lyase
Product: 5-aminoimidazole-4-carboxamide ribotide (AICAR)
Step 10: acquisition of C2
Another formylation reaction catalyzed by AICAR transformylase
Product: 5-formaminoimidazole-4-carboxamide ribotide (FAICAR)

26. Step 9: purine synthesis

27. Step 10: purine synthesis

28. Step 11 cyclization or ring closure to form IMP water is eliminated
in contrast to step 6 (closure of the imidazole ring), this reaction does not require ATP hydrolysis
once formed, IMP is rapidly converted to AMP and GMP (it does not accumulate in cells

29. Step 11: purine synthesis

31. Synthesis of adenine
and guanine nucleotides

32. Purine nucleoside diphosphates and triphosphates:
- to be incorporated into DNA and RNA, nucleoside
monophosphates (NMP’s) must be converted into
nucleoside triphosphates (NTP’s)
- nucleoside monophosphate kinases (adenylate & guanylate kinases)
- nucleoside diphosphate kinase

33. Regulation of purine nucleotide biosynthesis

35. The purine salvage pathway
Purine bases created by degradation of RNA or DNA and intermediate of purine synthesis were costly for the cell to make, so there are pathways to recover these bases in the form of nucleotides
Two phosphoribosyl transferases are involved:
APRT (adenine phosphoribosyl transferase) for adenine
HGPRT (hypoxanthine guanine phosphoribosyl transferase) for guanine or hypoxanthine

36. Salvage of purines
Adenine phosphoribosyltransferase (APRT)

37. Salvage of purines
Salvage is needed to maintain the purine pool (biosynthesis is not completely adequate, especially in neural tissue)
Hypoxanthine-guanine phosphoribosyltransferase (HGPRT)
Hypoxanthine + PRPP IMP + Ppi
Guanine + PRPP GMP + Ppi
Lack of HGPRT leads to Lesch-Nyhan syndrome. Lack of enzyme leads to overproduction of purines which are metabolized to uric acid, which damages cells

38. Lesch-Nyhan syndrome there is a defect or lack in the HGPRT enzyme
the rate of purine synthesis is increased about 200X
uric acid level rises and there is gout
in addition there are mental aberrations
patients will self-mutilate by biting lips and fingers off

39. Lesch-Nyhan syndrome

40. Salvage of purine bases

41. Next: Part 2 - biosynthesis of pyrimidine
nucleotides

42. Nucleotide metabolism – Part 2 (pyrimidine biosynthesis)By
Henry Wormser, Ph.D
Professor of Medicinal Chemistry

43. Synthesis of pyrimidine ribonucleotides
shorter pathway than for purines
base 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)

44. Origin of atoms in pyrimidine ring

45. The big picture

46. Step 1: synthesis of carbamoyl phosphate
Condensation of glutamine, bicarbonate in the presence of ATP
Carbamoyl phosphate synthetase exists in 2 types: CPS-I which is a mitochondrial enzyme and is dedicated to the urea cycle and arginine biosynthesis) and CPS-II, a cytosolic enzyme used here

47. Step 1: pyrimidine synthesis
CPS-II is the major site of regulation in animals: UDP and
UTP inhibit the enzyme and ATP and PRPP activate it
It is the committed step in animals

48. Step 2: synthesis of carbamoyl aspartate
enzyme is aspartate transcarbamoylase (ATCase)
catalyzes the condensation of carbamoyl phosphate with aspartate with the release of Pi
ATCase is the major site of regulation in bacteria; it is activated by ATP and inhibited by CTP
carbamoyl phosphate is an “activated” compound, so no energy input is needed at this step

49. Step 2: pyrimidine synthesis

50. Step 3: ring closure to form dihydroorotate
enzyme: dihydroorotase
forms a pyrimidine from carbamoyl aspartate
water is released in this process

51. Step 3: pyrimidine synthesis

52. the first 3 enzymatic reactions are catalyzed by 3 separate proteins/enzymes in E. coli
in animals, all 3 steps are found in a multifunctional enzyme (210 kD). This allows “channeling” of the substrates and products between active sites without releasing them to the medium where they could be degraded.
The acronym CAD is used as a name for the multienzyme: carbamoyl phosphate synthetase, aspartate transcarbamoylase and dihydroorotase
channeling also increases the overall rate of multistep processes

53. Step 4: oxidation of dihydroorotate to orotate
an irreversible reaction
enzyme: dihydroorotate dehydrogenase
oxidizing power is derived from quinones (thru coenzyme Q)

54. Step 4: pyrimidine synthesis

55. Step 5: acquisition of ribose phosphate moiety
enzyme: orotate phosphoribosyl transferase
ribose phosphate originates from PRPP
product is orotidine-5’-monophosphate (OMP)
orotate phosphoribosyl transferase is also used in salvage of uracil and cytosine to their corresponding nucleotide

56. Step 5: pyrimidine synthesis

57. Step 6: decarboxylation of OMP
enzyme: OMP decarboxylase
product: uridine monophosphate (UMP)
in animals, steps 5 and 6 are catalyzed by a single polypeptide with 2 active sites

58. Step 6: pyrimidine synthesis

60. The big picture again

61. Orotic aciduria
an inherited human disease caused by a deficiency in the multifunctional enzyme that catalyzes the last 2 steps in the pyrimidine synthesis
large amounts of orotic acid in urine
retarded growth and severe anemia
treat by administration (injection) of uridine and/or cytidine

62. Leflunomide (Arava)
Leflunomide is an isoxazole immunomodulatory agent which inhibits dihydroorotate dehydrogenase) and has antiproliferative activity. Several in vivo and in vitro experimental models have demonstrated an anti-inflammatory effect.
It is currently used as a DMARD in patients with serious rheumatoid arthritis

63. Leflunomide (Arava)

64. Activation of leflunomide
Opening of the isoxazole yields a reactive compound which
can then inhibit the enzyme dihydroorotate dehydrogenase

65. Synthesis of uridine and cytidine triphosphate
(in bacteria, ammonia donates the amino group)

66. Regulation of pyrimidine nucleotide biosynthesis
UTP and CTP are feeback inhibitors of CPS II

67. Formation of deoxyribonucleotides
All pathways shown previously led to synthesis of ribonucleotides
dADP, dGDP, dUDP and dCDP are all synthesized by the same enzyme
Synthesized from nucleoside diphosphate (not mono or triphosphate) by
ribonucleotide reductase

68. Synthesis of dTMP Methylation of d-UMP via N5,N10-methylene THF
Reaction inhibited by 5-fluorouracil (Efudex)

72. Activation of 5-fluorouracil

75. Dihydrofolate reductase

76. Next - Part 3: catabolism

77. Nucleotide metabolism – Part 3 (nucleotide degradation)By
Henry Wormser, Ph.D
Professor of Medicinal Chemistry

78. Nucleotide degradation

79. Degradation
of AMP

81. PENTOSTATIN previously called deoxycoformycin (DCF)
a purine analog with a 7-membered-ring
potent inhibitor of adenosine deaminase
ADA is a key enzyme which regulates
adenosine levels in cells
indicated for refractory hairy cell leukemia
other uses: chronic lymphocytic leukemia
and lymphomas

82. Adenosine deaminase

83. ADA deficiency In the absence of ADA lymphocytes are destroyed
deoxyadenosine is not destroyed, is converted to dAMP and then into dATP
dATP is a potent feedback inhibitor of deoxynucleotide biosynthesis
this leads to SCID (severe combined immunodeficiency disease)
Infants with this deficiency have a high fatality rate due to infections

84. ADA deficiency
treatment consists of administering pegylated ADA which can remain in the blood for 1 – 2 weeks
more efficient is gene therapy: replacing the gene that is missing or defective
gene therapy has been performed on selected patients

85. Degradation of
GMP and XMP

87. CATABOLISM OF PURINES

89. GOUT a disorder associated with abnormal amounts of urates in the body
early stage: recurring acute non-articular arthritis
late stage: chronic deforming polyarthritis and eventual renal complication
disease with rich history dating back to ancient Greece

90. GOUT once fashionable to associate gout with intelligence
people with gout:
Isaac Newton
Benjamin Frankin
Martin Luther
Charles Darwin
Samuel Johnson

91. Gout prevails mainly in adult males
rarely encountered in premenopausal women
symptoms are cause by deposition of crystals of monosodium urate monohydrate (can be seen under polarized light)
usually affect joints in the lower extremities (the big toe is the classic site)

92. Gout

93. Four Stages of Gout 1. asymptomatic hyperuricemia
2. acute gouty arthritic attacks
3. asymptomatic intercritical period
4. tophaceous gout (characterized by the formation of tophi in joints)
podagra (big toe)
cheiagra (wrist) according to Hippocrates
gonadra (knee)

94. Diagnostic features
usually affect joints in the lower extremities ( 95%)
onset is fast and sudden
pain is usually severe; joint may be swollen, red and hot
attack may be accompanied by fever, leukocytosis and an elevated ESR

95. Drugs which may induce hyperuricemia
niacin
thiazides and other diuretics
low dose aspirin
pyrazinamide
ethambutol
cyclosporine
cytotoxic drugs

96. Non-pharmacological approaches
Avoid purine rich foods:
red meat and organ meat (liver, kidneys)
shellfish, anchovies, mackerel, herring
meat extracts and gravies
peas and beans, aspargus, lentils
beer, lager, other alcoholic beverages
Weight loss
Control alcohol (binge drinking)

97. Pharmacological management of gout
based on the premise that the hyperuricemia is due to both overproduction and underexcretion of uric acid
symptomatic relief of pain is also achieved with analgesics (i.e. indomethacin)
drugs used:
analgesics (NSAIDs)
uricosuric agents
xanthine oxidase inhibitors

98. Therapy of acute gout treat with colchicine or NSAIDs avoid aspirin
do not treat with allopurinol or uricosuric drugs
uric acid lowering agents should never be started or stopped during acute attack
pain resolution occurs within hrs

99. Colchicine
a non-basic alkaloid from the seeds and corms of Colchicum autumnale
(Meadow Safron)

100. COLCHICINE used in the symptomatic treatment of acute attacks of gout
decreases leukocyte motility, decreases phagocytosis and lactic acid production
not used in other forms of arthritis
a very potent drug
can cause severe GI distress and abdominal pain

101. Probenecid (Benemid)
A uricosuric agent

102. Probenecid (Benemid) inhibits the tubular reabsorption of uric acid
it can also inhibit the tubular excretion of certain organic acid via the transporter
used in gout to promote the elimination of uric acid (not effective in acute attack)
also used to enhance plasma concentration of certain antiinfectives (beta lactams)

103. ALLOPURINOL (Zyloprim)
prevention of attacks of gouty arthitis and nephropathy
also used during chemotherapy of cancer and to prevent recurrent calcium oxalate calculi
metabolized to oxypurinol (also an inhibitor of xanthine oxidase)
inhibits the metabolism of certain anticancer drugs (6-MP, azathioprine)

104. Allopurinol (Zyloprim)
An inhibitor of xanthine oxidase; prevents the formation of uric acid from
precursorial purines

106. Fate of uric acid
in human and other primates uric acid is the final product of purine degradation and is excreted in the urine
the same is true in bird, reptiles and many insects
in other mammals uric acid is oxidized to allantoin (urate oxidase)
teleost (bony) fish convert allantoin to allantoic acid
cartilaginous fish and amphibian further degrade allantoic acid to urea
and finally marine invertebrates decompose urea to ammonia

108. Rasburicase (Elitek) A recombinant form of uric
acid oxidase. Used for initial management of plasma uric acid levels in pediatric patients with leukemia, lymphoma, and solid tumor malignancies who are receiving anticancer therapy expected to result in tumor lysis and subsequent elevation of plasma uric acid.

109. Catabolism of a pyrimidine

112. Formation of deoxyribonucleotides
ribonucleotide reductase studied by JoAnne Stubbe (Wisconsin, then MIT)
very complex enzyme; contains:
Tyrosine radical
2 non-heme irons
Two catalytically active cysteine residues
Cys are reduced by other proteins – thioredoxin
Ribo. Reductase is the therapeutic target of the anticancer drug hydroxyurea

113. Mechanism of ribonucleotide reductase
Free radical mechanism involving tyrosyl residues and cysteine residues on the enzyme
The enzyme is a dimer of dimers:
R1 – a dimer of identical a subunits (85 kD each)
R2 – a dimer of identical b subunits (45 kD each)

117. Reduction of the disulfide bond in ribonucleotide reductase
2 proteins can perform this reductive reaction:
Thioredoxin (ubiquitous 12 kD monomer)
Glutaredoxin which functions similarly to thioredoxin. Oxidized glutredoxin is reduced by glutathione (g-glutamylcysteinylglycine)

118. Regeneration of thioredoxin and ribonucleotide reductase

119. HYDROXYUREA (Hydrea) inhibits the enzyme ribonucleotide reductase
this enzyme causes ribonucleotides to be converted to deoxyribonucleotides
DNA synthesis cannot occur
cell are killed in the S phase
drug holds other cells in the G1 phase
primarily used to treat chronic myelogenous leukemia
cancer cell develop resistance by:
increasing quantity of inhibited enzyme
decreasing sensitivity of enzyme for inhibitor
used orally
major side effect is leukopenia

120. GEMCITABINE (Gemzar)
Another inhibitor of ribonucleotide reductase:indicated for non-small cell lung cancer (usually with cisplatin) also first line treatment for non-resectable pancreatic cancer

123. The purine nucleotide cycle for anaplerotic replenishment of citric acid cycle intermediates in skeletal muscle

124. For quiz review: check out this website

125. The end