NUCLEOTIDE METABOLISM Metabolism of purin e nucleotides

1. Prof. Mária Sasvári NUCLEOTIDE METABOLISM Metabolism of purine nucleotides Gergely Keszler 2009.

2. The biological role of nucleotides • Building blocks of nucleic acids (DNA and RNA) • Storage of biochemical energy (ATP and GTP) • Activation of biosynthetic precursors (UDP-glucose, CDP-choline) • Components of coenzymes (NAD, FAD, Coenzyme A etc.) • Regulation of metabolism (cAMP, cGMP) • 6. Nucleotide analogues: anticancer and antiviral therapies

3. Terminology of nucleotides phosphoanhydride bonds phosphoester bond N-glycosidic bond Nucleotides are composed of: a nucleobase a pentose at least one phosphate group a nucleoside

4. (Ado) (Guo) (Urd) (Cyd) (dAdo) (dGuo) (Thd) (dCyd) Ribo- and deoxyribonucleotides

5. Structures of nucleobases N-containing, heterocyclic aromatic compounds; substituted purine or pyrimidine rings DNA RNA

6. Nucleotide synthesis salvage (recycling) • base + ribose-P → nucleotide (typical for purines) OR • nucleoside + Pi→ nucleotide (typical for pyrimidines) „de novo” stepwise assembly from small precursors (C1 fragments, CO2, amino acids, ribose-P)

7. glc-6-P fru-6-P 5’ ATP O P-O-H2C O-P P 1’ AMP PRPP Pyrimidine “de novo” synthesis The origin of ribose-P PPP ri-5-P PRPP synthetase Purine “de novo” and salvage reactions

8. Intestine Blood brain, RBC, lymphocytes liver nucleosides bases DNA RNA Food  RNA, DNA  polynucleotides  nucleotides  nucleosides bases “salvage reactions” “de novo” synthesis  nucleotides  nucleosides bases  urate urate  URINE

9. ATP  ADP  AMP GTP  GDP  GMP IMP purine bases salvage reactions Purine nucleotide synthesis “de novo” synthesis

10. Asp Glycine IMP Gln The origin of the purine ring CO2  2 5 N N 6 7 3 4 1 N N N10formyl H4F N10formyl H4F

11. “de novo” purine synthesis CO 2 ¯ Asp PRPP Glycine N N Gln + H2O 1. N N N formyl H F 10 N formyl H F 10 4 4 Gln 9. NH3+ ri-5-P Glycine ATP 2. ADP + Pi Gln PRPP amidotransferase Glu + PPi PRA (5-phosphoribosyl-1-amine) GAR synthetase

12. “de novo” purine synthesis NH3+ 9. O NH N10formyl H4F ri-5-P 3. H4F GAR (5’PR-Glycinamide) GAR formyltransferase

13. “de novo” purine synthesis NH O O NH ri-5-P Gln 4. ATP Glu ADP + Pi FGAR (5’PR-formyl-glycinamide) FGAM synthetase

14. “de novo” purine synthesis NH O H2N HN NH ri-5-P 5. ATP ADP + Pi FGAM (5’PR-formylglycinamidine) AIR synthetase

15. “de novo” purine synthesis N H2N N ri-5-P CO2 6. AIR (5’PR-5-amino-imidazole) AIR carboxylase

16. “de novo” purine synthesis -OOC N H2N N ri-5-P Asp 7. ATP ADP + Pi CAIR (5’PR-4-Carboxy- 5-amino-imidazole) SAICAR synthetase

17. “de novo” purine synthesis O HN succinyl- N H2N N ri-5-P SAICAR (5’PR-succinyl-5-aminoimidazole-4-carboxamide) 8. fumarate Adenylosuccinase (ASA)

18. “de novo” purine synthesis O N H2N N H2N ri-5-P N10formyl H4F 9. H4F ACAIR (5’PR-5-aminoimidazole-4-carboxamide AICAR transformylase

19. “de novo” purine synthesis O H2N N O N H N ri-5-P 10. FACAIR 5’PR-5-formamidoimidazole-4-carboxamide) IMP cyclohydrolase H2O IMP

20. “de novo” purine synthesis and the purine nucleotide cycle AMP DA AMP+PPi fumarate Gln Adenylosuccinate GDP+Pi NADH + H+ GTP NAD+ H2O Asp AMP (6-amino) GMP (2-amino-6-oxo) GS ASL ATP Xanthylate (2,6,-dioxo) ASS IMPDH IMP(6-oxo)

21. The role of the purine nucleotide cycle 2 ADP - AMP DA ATP + AMP IMP urate severe Pi deficiency  [AMP]     hyperuricaemia Substrate level/oxidative phosphorylation ADP + Pi ATP AMP kinase PNC AMP Adenylosuccinate e.g. fructose intolerance

22. Muscle: high AMP DA level Muscle Liver ATP AMP + NH3 NH3 IMP inosine urate glycolysis inosine   urate strenuous exercise: NH3, urate  Muscle AMP DA def.: cramps, NH3, urate is NOT elevated

23. “de novo” purine synthesis Summary No free purine base during synthesis Carbon donors: „C1 units” (N10-formyl-THF) CO2 Glycine N-donors: Asp Gln Gly Energy: 6 ATP for 1 IMP Multifunctional proteins

24. Regulation of de novo purine synthesis “salvage” “salvage” + + - - + PRPP - IMP, GMP, AMP Gln PRPP amidotransferase PRPP synthetase - ATP,GTP

25. Purine salvage reactions base nucleotide AMP adenine PPi PRPP hypoxantine guanine PPi PRPP PRT (phosphorybosyl transferase) + ribose-P APRT HGPRT IMP GMP

26. The Lesch-Nyhan syndrome HGPRT deficiency: low GTP levelsin the basal ganglia Hyp/G + PRPP → IMP/GMP + PPi linked to X-chromosome mental retardation self-mutilation aggression hyperuricemia

27. B AMP GMP -p r 5’nucleotidase Pi Pi B r H2O NH3 Pi Pi ri-1-P ri-1-P Catabolism of purine nucleotides guanosine adenosine (6-amino) ADA adenosine deaminase inosine (6-oxo) PNP (purine nucleoside phosphorylase) B hypoxantine guanine

28. Purine salvage reactions H2O guanase NH3 H2O + O2 H2O + O2 H2O2 H2O2 urate (2,6,8-trioxopurine) URINE hypoxantine (6-oxo-purine) guanine (2-oxo-6-amino-purine) E x c r e t i o n xanthine (2,6-dioxopurine) xanthine oxidase

29. poor solubility precipitates in joints, initiates chemical arthritis GOUT Why is uric acid acidic? uric acid (oxo) uric acid (enol) urate (dissociated anion) well soluble

30. Hyperuricemia (gout) Symptoms: • urate crystals on the napkin (Lesch-Nyhan) • Na-urate crystals  kidney stones • urate in connective tissues and joints: „tophus”, inflammation, pain acute gouty arthritis chronic gouty arthritis Reason: Urate has low solubility (especially at acidic pH )

31. gl -6-P PPP fr -6-P ri -5-P PRPP Reasons forhyperuricemia 1. PRPP overproduction • as a consequence of mutation • at the allosteric site of PRPP synthase, • the enzyme cannot be inhibited • overproduction of ribose-5-P e.g. gl-6-phosphatase deficiency (von Gierke’s disease) Gl- 6-P   fr- 6-P   ri- 5-P 

32. 2. Absence of purine salvage reactions Reasons forhyperuricemia e.g. HPRT deficiency Decreased adenine, guanine reutilization  increased excretion

33. Reasonsforhyperuricemia • 3. Low ATP level, disturbed ATP metabolism • strenuous exercise fructose intolerance (phosphate trap) see before

34. 4. Secondary reasons: • tissue damage • cancer, cell damage • Overproduction of organic anions • (lactate, ketone bodies, drug derivatives) Reasonsforhyperuricemia  DNA breakdown overproduction of purines

35. Competitive inhibitors Medication of gout: allopurinol Xanthine oxidase xanthine hypoxanthine allopurinol alloxanthine oxopurinol Hypoxanthine and xanthine in urine (better solubility)

36. Allopurinol, a special purine analogue N-7 and C-8 have been scrambled up Blocks xanthine oxidase, the enzyme catalyzing the oxidation of xanthine to uric acid – cures gout

37. ADA / PNP / (ADA + PNP) Symptoms: Reason: Mechanism: Treatment: ADA enzyme therapy, Enzyme deficiency: immunodeficiency, “NON-HIV AIDS” B/T lymphocyte deficiency adenosine   dATP (ATP)  dATP  inhibits ribonucleotide reductase  inhibits DNA synthesis  promotes apoptosis gene therapy

38. Adenosine deaminase functions on the outer surface of red and white blood cell membranes (ectoenzyme) ADA ADA binding glycoprotein („complexing factor”)

39. The pathogenesis of SCID - selective lymphotoxicity 1. Extracellular accumulation of (deoxy)adenosine (d)Ado A2 A1 Gi Gs dAdo cAMP  cAMP  Inhibition of the SAM/SAH cycle Impaired DNA synthesis

40. The pathogenesis of SCID - selective lymphotoxicity 2. Intracellular accumulation of (deoxy)adenosine triphosphate Inhibition of ribonucleotide reductase Inhibition of cell proliferation dCK dATP  dAdo dAdo dAMP AK DNA strand breaks Inhibition of DNA polymerases Lymphocyte- selectivity!! apoptosis

41. Clinical manifestation of SCID  Recurring, opportunistic infections: candidiasis, Pneumocystis-pneumonia  Absence of lymph nodes, no thymic shadow upon chest X-ray examination  Severe impairment of both humoral and cellular immunity:  lower than 500/μl total lymphocyte count  very low plasma immunoglobulin levels  Untreated patients die before their age of 2 years

42. Treatment of ADA deficiency 1. Treatment of symptoms  Infections antibiotics, antiviral and antifungal drugs  Immunoglobulin supplementation Maternal immunoglobulins are effective in the first few weeks of life

43. Treatment of ADA deficiency 2. Enzyme substitution  Red blood cell transfusion  Polyethylene-glycol-conjugated recombinanat ADA (PEG-ADA): intramuscular injection costs: 250,000 USD a year

44. Treatment of ADA deficiency 3. Gene therapy Principle: Introduction of the normal allele into the patients’ own stem cells Ex vivo: Stem cells are transfected and trans- planted into the patient

45. Ex vivo gene therapy

46. Ashanti da Silva: the first patient in the world treated by retrovirus-mediated ADA gene therapy The introduced ADA gene functioned fine for a few months. Later on, PEG-ADA substitution therapy must have been restarted due to inactivation of the gene.