ASSOC. PROF. DR. CEMİLE KOCA ANKARA ATATÜRK TRAINING AND RESEARCH HOSPITAL

1. NUCLEOTIDE METABOLISM ASSOC. PROF. DR. CEMİLE KOCA ANKARA ATATÜRK TRAINING AND RESEARCH HOSPITAL

2. Biological functions of nucleotides 1. Building blocks of nucleic acids (DNA and RNA). 2. Involved in energy storage, muscle contraction, active transport, maintenance of ion gradients. 3.      Activated intermediates in biosynthesis (e.g. UDP-glucose, S-adenosylmethionine). 4.      Components of coenzymes (NAD+, NADP+, FAD, FMN, and CoA) 5.      Metabolic regulators: - Second messengers (cAMP, cGMP) - Phosphate donors in signal transduction (ATP)

3. NUCLEOSIDES- NUCLEOTIDES • Purines bond to the C1’ carbon of the sugar at their N9 atomsthrough an N-glycosidic linkage • Pyrimidines bond to the C1’ carbon of the sugar at their N1 atomsthrough an N-glycosidic linkage

4. Nucleotides b-glycosidic bond RNA- ribose (R) DNA – deoxyribose (dR)

5. Nucleoprotein Protein Nucleic acid Nucleases Nucleotide Nucleotidase Absorption Phosphate Nucleoside Nucleosidase Base Ribose Digestion and absorption of nucleotide Blood

6. BASES OF NUCLEOTIDES

7. Nucleobase structures

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

9. Nucleotides • Result from linking one or more phosphates with a nucleoside onto the 5’ end of the molecule through esterification • The phosphate groups are responsible for the negative charges associated with nucleotides, and cause DNA and RNA to be referred to as nucleic acids

10. Hypoxanthine Inosine Inosinate (IMP) Xanthine Xanthosine Xanthylate (XMP)

12. Two major routes for nucleotide biosynthesis Salvage Pathway – Synthesis from nucleosides or bases that become available through the diet or from degradation of nucleic acids De Novo Pathway – Synthesize purine and pyrimidine nucleotides from LMW precursors (“anew”) *good targets for anti-cancer/antibacterial/antiparasitic drugs dNTPs dNTPs

13. DE NOVO PURINE SYNTHESIS

14. DE NOVO PURINE SYNTHESIS • Purines are not made as free bases - but as nucleotides • First purine derivative formed isIMP : • Inosine Mono-phosphate (IMP) The purine base ishypoxanthine the “parent” purine nucleotide

15. Inosine Monofosfat (IMP) the “parent” purine nucleotide N-1from aspartic acid N-3, N-9 from glutamine C-4, C-5, N-7 from glycine C-6 from CO2 C-2, C-8 from THF - one carbon units

18. DE NOVO PURINE SYNTHESIS 1. Synthesis of 5-phosphoribosyl-1-pyrophosphate (PRPP) PRPP is a Central Metabolite in De NovoandSalvagePtw PRPP is an “activatedpentose” thatparticipates in the de novosynthesisandsalvage of purinesandpyrimidines Synthesis of PRPP from ATP andribose 5-phosphate is catalyzedbyPRPP synthetase (ribosephosphatepyrophosphokinase) 2 high energy phosphate equivalents are consumed

19. DE NOVO PURINE SYNTHESIS • Synthesis of 5′-phosphoribosylamine • commited step The amide group of glutamine replaces the pyrophosphate group attached to carbon 1 of PRPP (inversion of configuration – a to b) Enzyme: glutamine:phosphoribosyl pyrophosphate amidotransferase, is inhibited by the purine 5′-nucleotides AMP, GMP, and inosine monophosphate (IMP)—the end products of the pathway

20. Steps 1 and 2 are tightly regulated by feedback inhibition

21. Purine Nucleotide Synthesis

22. DE NOVO PURINE SYNTHESIS

23. Conversion of IMP to AMP and GMP • two-step, energy-requiring pathways • the synthesis of AMP requires GTP as an energy source, whereas the synthesis of GMP requires ATP • the first rxn in each pathway is inhibited by the end product of that pathway

24. Regulation of De NovoPurineSynthesis • İntracellularconcentration of PRPP is themostimportantregulator • Rate of AMP production increases with increasing concentrations of GTP; rate of GMP production increases with increasing concentrations of ATP

25. Conversion of nucleoside monophosphates to nucleoside diphosphates and triphosphates • Same enzyme act on all nucleotide di & triphophates Specific ATP-Dependent Kinase Non-Specific ATP-Dependent Kinase

26. SALVAGE PATHWAY FOR PURINES • Free purine bases, derived from the turnover of nucleotides or from the diet, can be attached to PRPP to form purine nucleosidemonophosphates Freeadenine, guanine, andhypoxanthine, can be reconvertedtotheircorrespondingnucleotidesbyphosphoribosylation Twokeytransferaseenzyme: adenosinephosphoribosyltransferase(APRT)& hypoxanthine-guaninephosphoribosyltransferase(HGPRT)

27. Hypoxanthine-guanine phosphoribosyltransferase IMP PRPP PPi Hypoxanthine-guanine phosphoribosyltransferase GMP PRPP PPi Adenine phosphoribosyltransferase Adenosine kinase AMP AMP PRPP ATP ADP PPi SALVAGE PATHWAY FOR PURINES Hypoxanthine Guanine Adenine Adenosine

28. Salvage of purine bases

29. Lesch-Nyhan Syndrome • Caused by a severe deficiency in HGPRT activity • X-linked recessivetrait occurring mostly in males

30. Lack of HGPRT activity in Lesch-Nyhan Syndrome causes a buildup of PRPP, which activates the synthesis of purine nucleotides h y p o x a n t h i n e - g u a n i n e p h o s p h o r i b o s y l t r a n s f e r a s e G u a n y l a t e + P P i G u a n i n e + P R P P H y p o x a n t h i n e + P R P P I nosinate + P P i • Excessive uric acid forms as a degradation product of purine nucleotides • increased PRPP, decreased IMP & GMP levels • glutamine:phosphoribosylpyrophosphateamidotransferase (the committed step in purine synthesis) has excess substrate and decreased inhibitors available, so de novo purine synthesis is increased • decreased purine reutilization+increased purine synthesis= increased degradation of purines and the production of large amounts of uric acid

31. Symptoms: • gouty arthritis due to uric acid accumulation (depositionof urate crystals in the joints) • severe neurological malfunctions including • mental retardation • aggressiveness • self-mutilation Basis of neurological aberrations is unknown

32. Conversion of ribonucleotides to deoxyribonucleotides • donors of the H atomsneeded for the reduction of the 2′-OH group are two sulfhydryl groups on the enzyme itself, which, during the reaction, form a disulfide bond • to reduce this disulfide bond thioredoxin is used • its sulfhydryl groups donate their H atoms to ribonucleotide reductase, then reduced back

33. Degradation of PurineNucleotides

34. Purine biodegradation in humans leads to uric acid

35. Purine catabolism leads to uric acid • Nucleotidasesandnucleosidasesrelease ribose and phosphates and leave free bases • Xanthine oxidase andguanine deaminaseroute everything to xanthine • Xanthine oxidase converts xanthine to uric acid • (xanthine oxidase can oxidize two different sites on the purine ring system)

36. Degradation of purines

37. DISEASES ASSOCIATED WITH DEFECTS IN PURINE METABOLISM • LESCH-NYHAN SYNDROME • HYPERURICEMIA • GOUT • KIDNEY STONES • SEVERE COMBINED IMMUNODEFECIENCY (SCID)

38. AdenosineDeaminase (ADA) Deficiency • AR inheritance • Excess adenosineis converted to its ribonucleotide& deoxyribonucleotide forms by cellular kinases • Increased dATP levels inhibit ribonucleotide reductase, thus preventing the production of all deoxyribose-containing nucleotides • So cells cannot make DNA and divide • Most severe form: severe combined immunodeficiencydisease (SCID) • involving a decrease in both T cells and B cells

39. Deoxynucleotide dGDP, dCDP, and dUDP synthesis are inhibited DNA synthesis is disturbed

41. HYPERURICEMIA plasma urate (uric acid) level greater than 7.0 mg/dL Normal plasma levels Females=2.4-6 mg/dL Males=3.4-7 mg/dL • Primary Hyperuricemia: an innate defect in purine metabolism and/or uric acid excretion • Secondary Hyperuricemia: increased availability of purines due to medications/ medical conditions or through diet

42. GOUT Gout is caused by precipitation of sodium urate crystals (tophi) in the joints resulting in inflammation and pain (gouty arthritis) tophaceous deposits in left ear

43. GOUT • Diagnosis of gout: aspiration and examination of synovial fluid from an affected joint • using polarized light microscopy to confirm the presence of needle-shaped monosodium urate crystals • Treatment: Allopurinol: an analog of hypoxanthine, is a potent inhibitor of xanthine oxidase - resulting in an accumulation of hypoxanthine and xanthine—compounds more soluble than uric acid

44. GOUT - Treatment • Colchicine –reduces inflammation • Uricosuric agents – increase renal excretion of uric acid (probenecid) • Allopurinol – inhibits uric acid synthesis • Low purine diet - Foods that are high in purine include: • Red meat and organ meats (eg. liver) • Yeasts and yeast extracts (eg. beer and alcoholic beverages) • Asparagus, spinach, beans, peas, lentils, oatmeal, cauliflower and mushrooms • Avoid caffeine and alcohol • Keep hydrated

45. 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

46. Pyrimidine Synthesis

48. Pyrimidine Synthesis • In contrast to purines, pyrimidines are not synthesized as nucleotides • the pyrimidine ring is completed first and then added to PRPP • With purines, the purine ring is built directly on the PRPP • Carbamoyl-phosphateandaspartateare the precursors of the six atoms of the pyrimidine ring • Mammals have two enzymes for carbamoyl phosphate synthesis – carbamoyl phosphate for pyrimidine synthesis is formed by carbamoyl phosphate synthetase II (CPS-II), a cytosolic enzyme

50. Carbamoyl Phosphate Synthetase II Carbamoyl Phosphate Synthetase II This is the committed step in pyrimidine synthesis in mammals