Metabolism of nucleotides

1. Metabolism of Nucleotides R. C. Gupta Department of Biochemistry National Institute of Medical Sciences Jaipur, India

2. Nucleotides containing purine and pyrimidine bases are essential for life They are required for the synthesis of DNA and RNA Many nucleotides, e.g. ATP, cAMP, cGMP etc, perform other important functions

3. Nucleotides are both taken in diet and are synthesized in the body Dietary nucleotides cannot be used in our body We are dependent exclusively on endogenously synthesized nucleotides

4. In food, nucleotides are present mainly in the form of nucleoproteins The protein part is degraded by proteolytic enzymes in the GIT The nucleic acids are hydrolysed by nucleases Digestion

5. DNA is hydrolysed by deoxyribonuclease and RNA by ribonuclease Polynucleotidases hydrolyse small poly- nucleotides into mononucleotides Nucleotidases hydrolyse nucleotides into nucleosides and phosphate

6. Nucleosides are absorbed by mucosal cells of intestine They are broken down to their final end products in intestinal mucosal cells The end products are released into circulation, and are excreted in urine

7. Synthesis of nucleotides Only endogenously synthesized purine and pyrimidine nucleotides are used for various purposes in our body There are two pathways for the synthesis of nucleotides: • De novo synthesis • Salvage pathway

8. De novo synthesis Nucleotides are synthesized afresh from amphibolic inter- mediates Bases or nucleosides released from catabolism of pre-existing nucleic acids and nucleotides are reutilized Salvage pathway

9. The salvage pathway is energy-efficient It prevents wastage of raw materials

10. Liver is the major site for de novo synthesis of purine nucleotides Synthesis can occur in several other tissues The synthesis occurs in the cytosol De novo synthesis of purine nucleotides

11. Different carbon and nitrogen atoms are provided by: Glycine Glutamine Aspartate Carbon dioxide Single-carbon moiety carried by tetrahydrofolate

12. The nitrogen at position 1 is provided by aspartate Carbon 2 comes from N10-formyltetra- hydrofolate Nitrogen 3 and nitrogen 9 come from the amide group of glutamine

13. Carbon atoms 4 and 5, and the nitrogen atom at position 7 are provided by glycine Carbon 6 comes from carbon dioxide Carbon 8 is provided by N5,N10-methenyl tetrahydrofolate

15. The first reaction is formation of 5- phosphoribosyl-1-pyrophosphate (PRPP) PRPP is required for the synthesis of pyrimidine nucleotides also By a series of reactions, PRPP is converted into inosine monophosphate

22. AMP and GMP are converted into ADP and GDP respectively by nucleoside monophosphate kinase ADP and GDP are converted into ATP and GTP respectively by nucleoside diphosphate kinase Formation of ATP and GTP

24. PRPP synthetase catalyses the first reaction of de novo synthesis It is an allosteric enzyme It is inhibited by AMP, ADP, GMP and GDP Regulation of de novo synthesis

25. PRPP synthetase is not unique to purine nucleotide synthesis PRPP is required for the synthesis of pyrimidine nucleotides as well Therefore, PRPP synthetase is not the main regulatory enzyme

26. PRPP glutamyl amidotransferase catalyses the first reaction unique to purine nucleotide synthesis This is the main regulatory enzyme It is allosterically inhibited by GMP

27. Synthesis of adenine and guanine nucleotides is cross-regulated Conversion of IMP into AMP requires GTP; conversion of IMP into GMP requires ATP This ensures a balanced production of AMP and GMP

28. Conversion of IMP into AMP and GMP is also regulated by allosteric mechanism AMP is an allosteric inhibitor of adenylo- succinate synthetase Similarly, GMP is an allosteric inhibitor of IMP dehydrogenase

29. Pre-existing purine bases and nucleosides may be salvaged to form new nucleotides Conversion of purine bases into nucleotides is catalysed by two enzymes: Synthesis of purine nucleotides by salvage pathway Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) Adenine phosphoribosyl transferase (APRT)

30. APRT acts on adenine HGPRT acts on hypoxanthine and guanine These enzymes transfer a phosphoribosyl group to their substrates PRPP provides the phosphoribosyl group

32. Purine nucleosides may be salvaged by adenosine kinase and deoxycytidine kinase Adenosine kinase acts on adenosine and deoxyadenosine Deoxycytidine kinase acts on deoxyadenosine, deoxyguanosine and deoxycytidine

34. The salvage pathway is regulated mainly by the availability of PRPP The available PRPP is used primarily for salvage reactions, and secondarily for de novo synthesis Regulation of salvage pathway

35. The sites of catabolism are intestinal mucosa, liver and kidneys The dietary purines are catabolized in intestinal mucosa Purines synthesized endogenously are catabolized in liver and kidneys Catabolism of purines

36. The pathway of purine catabolism is similar in all the tissues Adenosine and guanosine are the substrates for the catabolic enzymes The end product of purine catabolism is uric acid

37. N N N | Ribose N Adenosine deaminase H2O NH3 NH2 Adenosine 6Pi Ribose-1-P Inosine Purine nucleoside phosphorylase Hypoxanthine O2 + H2O Xanthine oxidase XanthineUric acid H2O2 N HN N H N O III Ribose N HN N N O II O O N H HN N H H N O II O N H HN N H N O II 6 6 O2 + H2O Xanthine oxidase H2O2 6 6

38. Guanosine Guanase Guanine O2 + H2O Xanthine oxidase XanthineUric acid H2O2 Pi Ribose-1-P Purine nucleoside phosphorylase O O N H HN N H H N O II O N H HN N H N O II 6 6 H2N I Ribose N HN N N O II H2N N HN N H N O II H2O NH3

39. Uric acid is released from tissues into circulation Circulating uric acid is excreted in urine

40. In some species, uric acid is the end product of protein catabolism as well These species include amphibians, birds and reptiles Uric acid is the main vehicle for excretion of nitrogenous waste in these organisms Such organisms are said to be uricotelic

41. In many organisms, including man, urea is the major form of nitrogenous waste Such organisms are said to be ureotelic

42. De novo synthesis of pyrimidines occurs in several tissues The pathway is located in cytosol with the exception of one reaction The exception is synthesis of orotate, which occurs in mitochondria De novo synthesis of pyrimidine nucleotides

43. The first reaction in de novo synthesis is formation of carbamoyl phosphate It is formed from glutamine, CO2 and ATP

44. Formation of carbamoyl phosphate is also the first reaction in synthesis of urea But this reaction occurs in mitochondria in urea cycle The amino group comes from ammonia instead of glutamine

46. Carbamoyl aspartate H2N O || HO−C CH2 CHC O COOHN H Carbamoyl phosphate Carbamoyl group transferred to aspartate Second reaction Aspartate transcarbamoylase PiO~ NH2 I C O COOH CH2 CH−NH2 COOH Aspartate

48. Dihydro-orotate Orotate N H HN O || C CH CC O HN O || C CH2 CHC O COOHN H COOH Dihydro-orotate dehydrogenase NADH+H+NAD+ Dihydro-orotate oxidized to orotate Fourth reaction

51. UMP is the first pyrimidine nucleotide to be formed Cytidine and thymidine nucleotides are formed from UMP For the formation of cytidine nucleotides, the amino group is provided by glutamine

52. First, UMP is converted into UDP, and UDP into UTP The 4-oxy group is replaced by amino group to form CTP

54. Thymidine nucleotides occur only in DNA The sugar in thymidine nucleotides is deoxyribose The synthesis occurs from UDP Thymidylate

55. The ribose residue of UDP is reduced to deoxyribose by ribonucleotide reductase Deoxyuridine diphosphate (dUDP) is dephosphorylated to dUMP dUMP is methylated to form dTMP (also termed TMP) by thymidylate synthetase

56. The methyl group is provided by N5, N10- methylene-H4-folate During this reaction: Methylene group is reduced to methyl group Tetrahydrofolate is oxidised to dihydrofolate

58. For continued synthesis of TMP, dihydrofolate has to be reduced to tetrahydrofolate This reaction is catalysed by dihydrofolate reductase Amethopterin and aminopterin are competitive inhibitors of this enzyme They inhibit TMP synthesis and decrease cell division

59. Regulation occurs by allosteric mechanism The regulatory enzymes are: Carbamoyl phosphate synthetase Aspartate transcarbamoylase Regulation of de novo synthesis

60. Carbamoyl phosphate synthetase is activated by PRPP, and inhibited by UTP Aspartate transcarbamoylase is inhibited by CTP

61. Human beings lack enzymes that can salvage free pyrimidine bases However, pyrimidine nucleosides can be salvaged and used for nucleotide synthesis Synthesis of pyrimidine nucleotides by salvage pathway

62. Uridine and cytidine are phosphorylated by a common enzyme, uridine-cytidine kinase Thymidine (deoxythymidine) is phosphorylated by thymidine kinase Deoxycytidine is phosphorylated by deoxycytidine kinase

65. Deoxyribonucleotides are required for DNA synthesis They are synthesized from ribonucleotides Formation of deoxyribonucleotides

66. Formation of deoxyribonucleotides requires: Ribonucleotide reductase Thioredoxin (a protein having two sulphydryl groups) Thioredoxin reductase (a flavoprotein)

67. Ribose residue reacts with the sulphydryl groups of thioredoxin Ribonucleotide reductase removes an oxygen atom from ribose and two hydrogen atoms from thioredoxin as water Thioredoxin is oxidized in this reaction

68. Oxidised thioredoxin is reduced by thioredoxin reductase The hydrogens atom are provided by NADPH

70. This enzyme system can act on ADP, GDP, CDP and UDP The products are dADP, dGDP, dCDP and dUDP dADP is the inhibitor of this reaction while ATP is its activator

71. Catabolism of pyrimidines Cytosine and uracil are catabolised to b- alanine, carbon dioxide and ammonia b-Alanine may be utilized in the body or is excreted in urine

73. Thymine is catabolised to b-aminoisobutyrate, carbon dioxide and ammonia b-Aminoisobutyrate is excreted in urine Ammonia released from pyrimidines and purines is disposed off as urea

Metabolism of nucleotides

Metabolism of nucleotides

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Metabolism of nucleotides