1. Nucleotide Metabolism

2. Nucleotides Nucleotides are made from a nucleoside and phosphate Nucleosides from nitrogenous base and sugar molecule – The nitrogenous base can be Adenine and guanine – purines Cytosine, thymine or uracil – pyrimidine s

3. RNA contains Ribose while DNA contains 2'deoxy-D-Ribose Nucleoside: Nitrogenous base + ribose:

4. Nucleotides biological importance Forming nucleic acids, nucleotides energy metabolism and activation of intermediary metabolites Second messengers As sulfate and methyl donor Coenzymes Synthetic analogs used in the treatment of cancer, immunosuppression and viral infection

5. Polymerize to make DNA and RNA Energy currency of the cell e.g. ATP, GTP Act as carriers of active intermediates in various metabolic pathways e.g. GTP drives protein synthesis, CTP drives lipid synthesis, UTP drives carbohydrate metabolism (UDP- glucose in glycogen synthesis) Component of coenzymes e.g. FAD, NADH, NADPH Act as 2nd messengers e.g. cAMP and cGMP Allosteric regulation of various metabolic pathways e.g. ATP inhibits PFK-1 Functions of Nucleotides

6. Nitrogenous Bases Nitrogenous base: derivatives of Purines and pyrimidines DNA and RNA contain the same purine bases and the pyrimidine base Cytosine But Thymine found only in DNA and Uracil found only in RNA (A) (G) (C)(T) (U)

7. AB The pentose sugars

8. Nucleoside structure The nucleotide has three characteristic components  Nitrogenous base  Pentose sugar

9. Nucleotides structure The nucleotide has three characteristic components  Nitrogenous base  Pentose sugar  Phosphate

10. Sources of nucleotides The cellular pools (other than ATP) are quite small, – Amounts required to synthesize the cell’s DNA 1% or less available – Cells must continue to synthesize nucleotides – Nucleotide synthesis may limit the rates of DNA replication and transcription Nucleotide sources are – De novo synthesis and salvage pathways Important in dividing cells – Agents that inhibit nucleotide synthesis have become particularly important to modern medicine

11. Cont De novo synthesis takes place in liver and number of other tissues – The enzymes are found in the cytosol Has a very high expense – Makes salvage synthesis more preferable An easy alternative

12. Overview of Nucleotide Metabolism 12

13. DE NOVO biosynthesis of Purine Nucleotide Nitrogenous base molecule – Source of Nitrogen 1 from Aspartate 2 from Glutamine 1 from Glycine – Source of Carbon 2 from Glycine 2 from N 10 -Formyl tetrahydrofolate 1 from CO 2 The source of the pentose sugar From hexose monophosphate shunt

14. Sources of N and C for purine

15. Steps of purine de novo synthesis Not made as free bases but as nucleotides Inosine mono phosphate (IMP) – The first nucleotide synthesized IMP then converted to AMP and GMP In the first step Ribose 5 phosphate is converted into pyrophosphoribosyl-5- phosphate (PP ribose P) PP ribose P synthetase with ATP and Mg++ Concentration PRPP The major determinant of the overall rate The rate of PRPP synthesis depends both – on the availability of ribose 5-phophate and – on the activity of PRPP synthetase

16. Cont

18. 18

19. Important steps PRPP synthesis from ribose-5-phosphate and ATP is a regulatory step By PRPP synthetase Stimulated by ribose-5’ phosphate from the pentose phosphate pathway Inhibited by purine-5’-nucleotides Steps 4 and 10 – The dependence folic acid compounds – antagonists of folic acid metabolism indirectly inhibit purine formation. – This in turn affects, nucleic acid synthesis, cell growth, and cell division Clearly, rapidly dividing cells such as malignancies or infective bacteria are more susceptible to these antagonists than slower-growing normal cells.

20. Modification of IMP Ring Modification to produce GMP and AMP

21. Cont

22. ADPGDPCDPUDP dADPdGDPdCDPdUDP Ribonucleotide reductase RIBONUCLEOTIDES  DEOXYRIBONUCLEOTIDES

23. Hepatic purine nucleotide regulation Liver – major site of purine nucleotide biosynthesis. – provides purines and their nucleotides for salvage reaction Human brain – has a low level of PRPP amidotransferase – depends on exogenous purines Erythrocytes and polymorphonuclear leukocytes – cannot synthesize 5-phosphoribosylamine so utilize exogenous purines to form nucleotides

24. Cont Two mechanisms regulate conversion of IMP to GMP and AMP – AMP feedback regulates adenylo succinate synthetase and – GMP feedback inhibits IMP dehydrogenase For the conversion of IMP to adenylosuccinate in route to AMP requires GTP conversion of xanthinylate to GMP requires ATP

25. Cont Cross regulation between the pathways of IMP metabolism – to decrease synthesis of one purine nucleotide when there is a deficiency of the other nucleotide

27. Cont

28. The salvage pathway Far less energy requirement than de novo synthesis Recycles purine bases and nucleosides into nucleotide Two mechanisms – Single step direct conversion By phosphoribosylation Activated by – Adenosine phosphoribosyltransferase (APRT) giving rise to AMP – Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) that gives either IMP or GMP – Both enzymes use PRPP as the source of ribose-5-phosphate

29. – Direct phosphorylation of a purine ribonucloside (PuR) by ATP PuR + ATP → PuR-P + ADP – Adenosine kinase catalyzes phosphorylation of adenosine to AMP or of deoxyadenosine to dAMP Cont

31. Overview of purine metabolism

32. Catabolism of purine Uric acid – chief end-product of purine catabolism in man and in higher apes – Almost all tissue enzymes capable of breaking nucleoprotein down to nucleoside then oxidized to uric acid – Always excreted even on a purine free diet or in starvation – Other mammals degrade uric acid to allantoin by means of the enzyme, uricase, which is lacking in primates – endogenous and exogenous in origin

33. Cont Uricotelic – Organisms that form uric acid as the major nitrogenous waste product – Birds, amphibious and reptiles do not possess uricase activity. – These animals excrete uric acid and guanine as the end products of purine metabolism and nitrogen (protein) metabolism Ureotelic – Man and most of the mammals, – Urea is the main product of nitrogen metabolism

34. Cont Site – Occurs in liver, intestine, spleen, kidney, pancreas, skeletal and heart muscles – Starting metabolites are either adenosine or guanosine Steps – Adenosine is first converted into inosine by adsenosine deaminase – Ribose is then removed

35. Cont

36. Formation of uric acid Adenine deamination – by adenylate deaminase to form inosinic acid – adenylate deaminase is quite abundant in skeletal muscle – Adenosine can also be deaminated to form inosine Inosine give rise to free hypoxanthine, which may be reutilized for nucleic acid synthesis but is most frequently oxidized to xanthine by the enzyme xanthine oxidase present in greatest amount in liver, small intestine and kidney

37. Cont Xanthine oxidase – oxidizes xanthine to uric acid Free guanine is deaminated to form xanthine directly by the enzyme guanase, which is very active in most tissues – The liberated xanthine is then converted to uric acid by xanthine oxidase Some uric acid may be produced from nucleic acid by the bacterial flora of the intestinal tract, when it is absorbed directly excreted – minor contributor to the urinary uric acid on a normal diet

38. Excretion of uric acid Excreted in urine – About 80% – 0.5 - 0.6 gm/day The remaining in the bile. Gout (hyperuricemia): – A metabolic disease – characterized by increased levels of uric acid (as urates) in the blood (hyperuricemia) – above 8.0 mg/dL (normally it is 2.5 - 8 mg/dL) – With increased excretion of uric acid in the urine

39. Gout Sodium urate crystals – needle-shaped – referred to as tophi – deposited in soft tissues particularly in or about joints – Cause Acute inflammatory reactions called acute gouty arthritis – results in joint destruction Uric acid and urates precipitates in the kidney and urinary tract resulting in uric acid stone formation Gout is associated with morning stiffness that eases on walking opposite to osteoarthritis that is exaggerated by moving

40. Cont Types of gout: A) Primary: In this case, gout is the primary disease and is either metabolic or renal. – Primary metabolic gout constitutes most of the cases and is due to inherited autosomal or X- linked recessive metabolic defects – Primary renal gout is a rare condition and is due to an inherited defect in the kidneys leading to decreased secretion of urates

41. Cont Primary metabolic gout is an arthrites characterized excessive production of purines occurring mostly in males Excessive purine synthesis has been found to be due to deficiency (partial) of hypoxanthine-guanine phosphoribosyl transferase HGPRT Decreased/partial HGPRT activity – increased HX and G – decreased IMP and GMP- inhibitors for purine synthesis – accumulation of PRPP – Increased purine synthesis – Increased purine degradation – Increased uric acid level

42. Cont Secondary: In this case, gout is a complication of other diseases and is either metabolic or renal: – Secondary metabolic gout is due to increased turnover of nucleic acid and uric acid production secondary to increased cell destruction – Secondary renal gout is due to decreased excretion of uric acid as in severe renal failure

43. Biochemical bases for the treatment of gout Antiinflammatory agents: Anti-inflammatory uricosuric drugs, e.g., salicylates, cortisone, probenecid and ACTH, act also to increase uric acid excretion by inhibiting its renal tubular reabsorption. Inhibition of xanthine oxidase by the structural analogs of hypoxanthine, e.g., allopurinol, Dietary control by avoidance of nucleic acid-rich diets, e.g., liver and meat Dairy products, eggs and meat of young animals are more suitable.

44. KIDNEY STONES When uric acid is present in high concentrations in the blood, it may precipitate as a salt in the kidneys. The salt can form stones, which can in turn cause pain, infection, and kidney damage.

45. X-Linked recessive disorder Complete HGPRT deficiency Characterized by mental deficiency, aggression, self- destructive behavior, characterized by lip and finger biting. Since high urate levels are present in the blood, individuals with this condition are also prone to gout and kidney stones LESCH-NYHAN SYNDROME

46. Nucleotide antimetabolites as anticancer and antiviral drugs © Michael Palmer 2014

47. Biosynthesis of pyrimidines The synthesis of pyrimidine ring – starts with the formation of carbamoyl phosphate – by carbamoyl phsphate synthetase – from glutamine, ATP and CO 2 – in the cytosol of the cell Carbamoyl phosphate – reacts with aspartate to form carbamoyl aspartate – by aspartate transcarbamoylase – with the lose of water, is converted into dihydroorotic acid (DHOA) – by the enzyme dihyroorotase

48. Synthesis of Carbamoyl Phosphate

49. Sources of atoms

50. De Novo synthesis of pyrimidines

51. Cont Dihydroorotic acid on dehydrogenation by dihydroorotate dehydrogenase utilizing NAD as coenzyme is converted into orotic acid (OA) which is by the action of orotate phosphoribosyl transferase, converted into orotidine monophospate (OMP). This on subsequent decarboxylation by orotidylic acid decarboxylase forms uridine monophsphate (UMP) By further phosphorylation, UMP is converted into UDP and then to UTP UTP is converted into CTP in the reaction catalyzed by CTP synthetase utilizing ATP and glutamine

52. Cont The enzyme ribonucleotide reductase converts UDP into deoxyuridine diphophate (dUDP) which is converted into dUMP. This is by the action of thymidylate synthetase with N5,N10-methylene H4 folate, converted into thymidine monophosphate (TMP)

53. Salvage pyrimidine synthesis

54. Catabolism of pyrimidines Liver is the main site for the catabolism of pyrimidines CO 2 is released from the pyrimidine nucleus representing a major pathway for the catabolism of uracil, cytosine and thymine The major end products of cytosine, uracil and thymine are alanine and aminoisobutric acid respectively

56. Orotic acidurea  Because the products of pyrimidine catabolism are soluble, few disorders result from excess levels of their synthesis or catabolism.  Two inherited disorders affecting pyrimidine biosynthesis are the result of deficiencies in the bi-functional enzyme catalyzing the last two steps of UMP synthesis, orotate phosphoribosyl transferase and OMP decarboxylase.  These deficiencies result in orotic aciduria that causes retarded growth, and severe anemia.