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Metabolism of nucleotides

  1. Metabolism of Nucleotides Guided by Dr. D V Bhale ( prof. & HOD) By Roshan Kumar Mahat (PG Student) Department Of Biochemistry
  2. Objectives to know 1. 2. 3. 4. 5. 6. 7. Nitrogen bases i.e. purines & pyrimidines Nucleosides Nucleotides Synthesis of purine nucleotides Regulation of purine nucleotides synthesis Inhibitors of purine nucleotide synthesis Disorders of purine metabolism
  3. 8. Synthesis of pyrimidine nucleotides 9. Inhibitors of pyrimidine nucleotides synthesis 10. Disorders of pyrimidine nucleotide synthesis
  4. Pyrimidines and Purines Pyrimidine and purine are the names of the parent compounds of two types of nitrogencontaining heterocyclic aromatic compounds. N N N N Pyrimidine N Purine N H
  5. Important Pyrimidines • Pyrimidines that occur in DNA are cytosine and thymine. Cytosine and uracil are the pyrimidines in RNA. NH O 2 O CH3 HN O HN HN N H Uracil O O N H Thymine N H Cytosine
  6. Important Purines • Adenine and guanine are the principal purines of both DNA and RNA. NH2 O N N N Adenine N H N HN H2N N Guanine N H
  7. Caffeine and Theobromine • Caffeine (coffee) and theobromine (coffee and tea) are naturally occurring purines. O H3C N N O O CH3 N CH3 Caffeine N N HN O CH3 N CH3 Theobromine N
  8. Nucleosides • Is a structure formed by the combination of nitrogen base and sugar. N2 base Sugars Nucleoside Adenine Deoxyribose/Ribose Adenosine Guanine Deoxiribose/Ribose Guanosine Thymine Deoxyribose Thymidine Cytosine Deoxyribose/Ribose Cytidine Uracil Ribose Uridine
  9. Nucleotides • Nucleotides are phosphoric acid esters of nucleosides. Nucleoside Phosphoric acid Nucleotides Adenosine Phosphoric acid Adenylate (AMP) Guanosine Phosphoric acid Guanylate ( GMP) Thymidine Phosphoric acid Thymidylate ( TMP) Cytidine Phosphoric acid Cytidylate (CMP) Uridine Phosphoric acid Uridylate (UMP)
  10. Synthesis of purine nucleotides Denovo synthesis Synthesis of purine base step by step on the ribose 5phosphate Salvage pathway Addition of ribose 5-phosphate to the preformed purine bases or addition of phosphate to the purine nucleosides Synthesis of purine nucleotides
  11. Sources of different atoms of purine ring
  12. Denovo synthesis of purine nucleotides Tissue and site of synthesis Tissues- major tissue is liver Site- cytosol
  13. Inhibitors 1. Sunfonamide  Are structural analogues of PABA  Act as competitive inhibitors of synthesis of folic acid from PABA in bacteria.  They inhibit the reactions of purine nucleotide synthesis requiring folic acid ( GAR transformylase and AICAR transformylase)  Used as bacteriostatic drugs to control bacterial infection.
  14. 2. Methotrexate and Aminopterin Are structural analogue of folic acid. They act as a competitive inhibitors of dihydrofolate reductase thus blocking the biosynthesis of tetrahydrofolic acid. They inhibit the reaction requiring folic acid for purine nucleotide synthesis. Used in t/t of cancers like leukemia choriocarcinoma.
  15. 3. Trimethoprim structural analogue of folic acid. Acts as a competitive inhibitors of dihydrofolate reductase in bacteria thus blocking the biosynthesis of tetrahydrofolic acid. Inhibit the reaction requiring folic acid to purine nucleotide synthesis. Used in the t/t of bacterial infections and UTI.
  16. 4. 6-mercaptopurine  is a structural analogue of purine bases.  is converted to 6- thioionosine monophosphate by the enzyme HGPRT, called lethal synthesis.  6-thio IMP inhibits the conversion of IMP to AMP and GMP.  6-thio IMP also feed back inhibits glutamine PRPP amidotransferase. Used as an anticancer drug.
  17. 5. Thioguanine Is a guanine analogue.  It is converted to 6-thio GMP by the enzyme HGPRT.  6-thio GMP inhibits the conversion of IMP to GMP.  Also inhibits glutamine PRPP amidotransferase.  Used as an anticancer drug.
  18. 6. Azaserine  is a structural analogue of glutamine.  is a glutamine antagonist.  inhibits the enzyme reactions in purine and pyrimidine nucleotide synthesis that utilize glutamine as a substrste.  it is highly toxic to the cells so it is not used clinically as a drug.
  19. Regulation 1. Intracellular conc. Of PRPPdepends upon 2 factors i.e. its synthesis & utilization. Synthesis depends on Availability of R-5-P.  Action of enzyme PRPP synthetase. Utilization depends on Denovo synthesis.  Salvage pathway.
  20. 2. Activity of enzyme PRPP amidotransferase. Increased activity of PRPP amidotransferase leads to Increased synthesis of AMP amd GMP, which feedbackly inhibit the enzyme PRPP amidotransferase. 3. Both AMP and GMP inhibit their own formation by feedback inhibition of adenylosuccinate synthetase and IMP dehydrogenase.
  21. Salvage pathway It refers to the formation of purine nucleotides by the 1. Addition of ribose phosphate ( from PRPP) to the preformed purine bases. 2. Addition of phosphate to the preformed purine nucleosides.
  22. Significance Salvage pathway provide a pathway for the utilization of purine bases derived from diet (exogenous) and normal turnover of the nucleic acids. In erythrocytes, denovo syntheis of purine nucleotides does not occur because of absence of PRPP amidotransferase. The requirement of purine nucleotides is met by the salvage pathway.
  23. Synthesis of purine nucleotides from purine bases Catalyzed by HGPRT and APRT. Adenine + PRPP AMP + PPi APRT Hypoxanthine + PRPP Guanine + PRPP HGPRT HGPRT IMP + PPi GMP+ PPi
  24. Synthesis of purine nucleotides from purine nucleosides Adenosine + ATP AMP+ ADP Adenosine kinase
  25. Degradation of purine nucleotides
  26. Disorders of purine metabolism 1. Gout 2. Lesch nyhan syndrome 3. Immunodeficiency associated with purine metabolism 4. Infantile autism
  27. Gout Metabolic disorders associated with overproduction of uric acid. At physiological form, uric acid is found in more soluble form as sodium urate. In severe hyperuricemia, crystal of sodium urate get deposited in the soft tissues, particularly in joints. Such deposits are commonly known as tophi.
  28. This causes inflammation of joints resulting in gouty arthritis. The prevalence of gout is about 3 per 1000 persons, mostly affecting males. Post menopausal women, however are as susceptable as men for this disease. Historically, gout was found to be associated with high living, over eating and alcohol consumption. Lead poisoning also causes gout by decreasing uric acid excretion.
  29. Types of gout Gout Primary gout Metabolic gout Renal gout Secondary gout Metabolic gout Renal gout
  30. Primary metabolic gout It is an inborn error of purine metabolism due to overproduction of uric acid. Causes: 1. Increased activity of PRPP synthetase 2. Overactivity of PRPP amidotransferase 3. HGPRT deficiency 4. Glucose 6-phosphatase deficiency 5. Elevation of glutathione reductase
  31. Primary renal gout It is due to failure of uric acid excretion from the body so that uric acid level in the body gets increased.
  32. Secondary metabolic gout Secondary gout is due to secondary to certain diseases like leukemia, polycythemia, lymphoma, psoriasis and increased tissue breakdown like in trauma, starvation etc.
  33. Secondary renal gout It is due to secondary to defective glomerular filtration of urate due to generalized renal failure.
  34. Tratment of gout Is by 1. Use of colchicine & uricosuric drugs.  To remove urates from the joint, colchine is the drug of choice.  To remove the urates from the body, urocosuric drugs such as probenecid, sulfinpyrazole, salicylates etc are used. 2. Use of allopurinol-inhibits the activity of enzyme xanthine oxidase as a result of which uric acid is not produced.
  35. Lesch Nyhan syndrome  Fist described tn 1964 by Michael Lesch( a medical student) and William L. Nyhan (his teacher).  It is X linked metabolic disorder since the structural genes for HGPRT is located on the X chromosome.  It affects only males and is characterized by excessive uric acid production and neurological abnormalities such as mental retardation, aggressive behaviour, learning disability etc.  The patients of this disorder have an irresistible urge to bite their fingers and lips,ofen causing self-mutilation.
  36. Biochemical basis HGPRT deficiency spares the utilization of PRPP through salvage and the accumulated PRPP takes part in the purine biosynthesis by the denovo pathway finally leading to hyperuricemia. The biochemical basis for neurological abnormalities are big enegma till date. Indeed, it is surprising that the deficiency of a single enzyme can cause such an abnormal behavioural changes.
  37.  few explanations are putforth in this regard. Neurological symptoms may be due to decreased availability of purines to the developing brain which has a low capacity for denovo purine synthesis and hence depends on purine salvage pathway for the supply of purine nucleotides it requires.
  38. Treatment  allopurinol is used to treat hyperuricemia but it has no effect on the neurological menefestation in theses patients. Treatment for the neuro-behavioural features are limited to behavioural therapy and providing protective physical device to prevet self-mutilation.
  39. Immunodeficiency diseases associated with purine metabolism Two different immunodeficiency disorders associated with degredation of purine nucleotides are known. The enzyme defects are adenosine deaminase and purine nucleoside phosphorylase, involved in uric acid synthesis.
  40. The deficiency of ADA causes SCID involving T- cell and usually B- cell dysfunction. It is explained that ADA deficiency results in the accumulation of Datp which is an inhibitor of ribonucleotide reductase and thus DNA synthesis, replication are adversely affected. Different modes of t/t such as blood transfusion, bone marrow transplantation are tried to cure the diseases but with limited effects.
  41. But, like in any other inborn error, the real hope for the future is only gene therapy. In 1990, a 5 year old girl suffering from SCID was successfully cured by transfecting the ADA gene into stem cells of the patients. This is considered as landmark in the history of trating inborn errors of metabolism. The deficiency of purine nucleoside phosphorylase is associated with impairement of T cell function but has no effect on B cell function. It is believed that d GTP inhibits the development of normal T-cells.
  42. Infantile Autism Recently it was observed that children suffering from infantile autism exihibited increased excretion of uric acid but surprisingly the serum concentrations are within normal limits. The biochemical basis for this is unknown. An oral dose of uridine is tried in the t/t.
  43. Synthesis of pyrimidine nucleotides Synthesis of pyrimidine nucleotides Denovo synthesis Synthesis of pyrimidine nucleotide refers to the formation of pyrimidine ring structure followed by the addition of ribose phosphate. Salvage pathway Formation of pyrimidine nucleotides from pyrimidine bases
  44. Sources of different atoms of prrimidine rings
  45. Denovo synthesis of pyrimidine nucleotides Tissue and site of synthesis Mainly occurs in the liver. The reaction occurs in cytosol and mitochondria. The formation of orotate from dihydroorotate occurs ie mitochondria and all other reactions occur in the cytosol.
  46. Pyrimidine Synthesis O - 2 A T P + H C O 3 + G lutam in e + H 2 O C 2 ADP + O C arbam oyl Phosphate Synthetase II G lutam ate + Pi C C CH PRPP PPi C O C N H O PO 3 N COO 2- O 3P O CH2 NH2 C C O HN O CH HN O H H OH O ro tate P h o sp h o rib o sy l T ran sferase OH H H COO -2 O ro tid ine-5'-m onophosphate (O M P ) O rotate C arbam oyl P hosphate R educed Q uinone A spartate A spartate T ranscarbam oylase (A T C ase) O C CH2 CH N H C CH2 HN D ihydroorotase O 2- CH N H CH N O H 2O C O CH HN C C C O O NH2 CO2 Q uinone Pi HO OM P D ecarboxylase D ihydroorotate D ehydrogenase O 3P O CH2 O COO H OH COO H OH H H D ih y d ro o ro tate C arb am o y l A sp artate U ridine M o nophosphate (U M P )
  47. 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 C4 in animals
  48. UDP Ribonucleotide reductase dUDP dUMP dUDP dUMP Thymidylate synthetase N5,10 formyl THF dTMP formyl THF
  49. Regulation of pyrimidine synthesis  In bacteria, aspartate transcarbamoylase catalyses a committed step in pyrimidine biosynthesis.  Aspartate transcarbamoylase is a good example of an enzyme controlled by feedback mechanism by the end product CTP.  In certain bacteria, UTP also inhibits aspartate transcarbamoylase. ATP, however stimulates aspartate transcarbamoylase activity.
  50. Carbamoyl phosphate synthase II is the regulatory enzyme of pyrimidine synthesis in animals. It is activated by PRPP and ATP and inhibited by UDP and UTP. OMP decarboxylase inhibited by UMP and CMP, also controls pyrimidine formation.
  51. Inhibitors of pyrimidine synthesis Sulfonamides Methotrexate Trimethoprim 5-fluorouracil Fluorocytosine
  52. Salvage pathway Salvage pathway of pyrimidine nucleotide synthesis refers to the formation of pyrimidine nucleotides from pyrimidine bases. Significance Salvage pathway provide a pathway for the utilization of pyrimidine bases derived from diet(exogenous) and normal turnover of nucleic acids.
  53. Enzymes and reactions There are 2 enzymes that catalyze the reactions of salvage pathway. They are uracil phosphoribosyl transferase (UPRT) and thymidine kinase. Uracil + PRPP UMP + PPi UPRT Thymidine + ATP Thymidine kinase TMP+ ADP
  54. Disorders of pyrimidine metabolism Disorders of pyrimidine metabolism includes: Orotic aciduria Reye’s syndrome
  55. Orotic aciduria Is a rare metabolic disorder characterized by the excretion of orotic acid in urine, severe anemia and retarded growth. It is due to the deficiency of the enzymes orotate phosphoribosyl transferase and OMP decarboxylase of pyrimidine synthesis. Both these enzymes activities are present on a single protein as domains (bifunctional enzyme).
  56. Treatment Feeding diet rich in uridine or cytidine is an effective t/t of orotic aciduria. These compounds provide pyrimidine nucleotides required for DNA and RNA synthesis.
  57. Reye’s syndrome Is considered as a secondary orotic aciduria. It is believed that a defect in ornithine transcarbamoylase (of urea cycle) causes the accumulation of carbamoyl phosphate. This is then diverted for the increased synthesis and excretion of orotic acid.