1. NUCLEOTIDE METABOLISM SITI ANNISA DEVI TRUSDA

2. Nucleotides are essential for all cells DNA/RNA synthesis  protein synthesis  cells proliferate Carriers of activated intermediates in the synthesis of carbohydrate, lipids and protein Structural component of several essential coenzymes (coA,FAD,NAD +,NADP + ) cAMP,cGMP  2 nd messenger in signal transduction pathway Important regulatory compound for many of the pathways of intermediary metabolism, inhibiting/activating key enzimes

3. Nucleotide structure Consist of: ◦ Nitrogenous base : purine & pyrimidine ◦ Pentose monosaccharide ◦ 1/2/3 phosphate groups DNA and RNA contain the same purine bases: A & G Pirimidine RNA : U & C DNA : T & C T& U differ by only one methyl group

4. Nucleosides Pentose sugar + Nitrogen Base = Nucleosides So, nucleotides = Nucleosides + Phosphate If the sugar is ribose : ribonucleosides If deoxyribose: deoxyribonucleosides Ribonucleosides of A,G,C,U: Adenosine,Guanosine,Cytidine,Uridine What are the deoxyribonucleosides for A,G,C,T?

5. Nucleotides  mono,di,tri esters of nucleosides 1 st phosphate group is attached by an ester linkage to the 5’OH of the pentose  nucleoside 5’phosphate/5’-nucleoside Type of pentose is added as prefix for nucleotide, can be ribose/deoxyribose e.g: 5’- ribonucleotide/5’-deoxyribonucleotide

6. 1 phosphate group + 5’-carbon of the pentose  nucleoside monophosphate(NMP) e.g AMP, CMP 2 or 3 phosphate group added to the nucleoside  nucleoside di/triphosphate e.g ADP/ATP The latter connected to the nucleotide by a high- energy bond Phosphate groups  (-) charge  DNA/RNA=nucleic acids

7. So, what is : ◦ Nucleoside? ◦ Nucleotide? ◦ Nucleic acid?

10. SYNTHESIS OF PURINE NUCLEOTIDES Source of purine ring: aspartic acid, glycine, glutamine, CO2,N 10 -formylTHF Synthesis of 5-phosphoribosyl-1- pyrophosphate (PRPP)  an activated pentose for synthesis of purine/pirimidine & salvage of purine bases  catalyzed by PRPP synthetase, from ATP & ribose 5-phosphate  this enzyme is activated by inorganic phosphat (Pi), inhibited by purine nucleotides  the sugar of PRPP is ribose  ribonucleotides as end product of purine synthetis

11. Purine synthesis is critical to fetal development, therefore defects in enzymes will result in a nonviable fetus. PRPP synthetase defects are known and have severe consequences (next slide) PRPP synthetase superactivity has been documented, resulting in increased PRPP, elevated levels of nucleotides, and increased excretion of uric acid.

12. Phosphoribosyl Pyrophosphate (PRPP) Synthetase Defects PRPP deficiency results in convulsions, autistic behavior, anemia, and severe mental retardation. Excessive PRPP activity causes gout (deposition of uric acid crystals), along with various neurological symptoms, such as deafness.

13. Synthesis of 5’-phosphoribosylamine  Amide group of glutamine replaces the pyrophosphate group at C1 of PRPP  the enzyme, glutamine:phosphoribosyl pyrophosphate amidotransferase is inhibited by the purine 5’-nucleotides AMP,GMP,IMP (end product)  Committed step  Rate of reaction also controlled by intracellular [] of glutamine and PRPP

14. Synthesis of inosine monophosphate,the “parent” of purine nucleotide  requires 4 ATP  2 steps require N 10 –formyltetrahydrofolate Conversion of IMP to AMP and GMP  2 step energy requiring pathway  synthesis of AMP requires GTP as energy source  synthesis of GMP requires ATP

15. Conversion of nucleoside monophosphates to nucleoside di and triphosphate  AMP + ATP ↔ 2 ADP GMP +ATP ↔ GDP + ADP  GDP + ATP ↔ GTP + ADP CDP + ATP ↔ CTP + ADP

16. Purine Synthesis

17. DAUR dr IMP  AMP & GMP IMP dehidrogenase XMP aminase Adenilosuksinat synthetase Adenilosuksinat lyase

18. Salvage Pathway of purines Purines that result from the normal turnover of cellular nucleic acids/diet can be reconverted into nucleoside triphosphates  salvage pathway 2 enzymes: Adenine phosphoribosyltransferase (APRT), and hypoxanthine-guanine phosphoribosyltransferase (HPRT) Both needs PRPP as the source of the ribose 5-phosphate

20. Degradation of Purine Nucleotides Purine Nucleotides from ingested nucleic acids or turnover of cellular nucleic acids is excreted by humans as uric acid. Humans excrete about 0.6 g uric acid every 24 hours. Degradation of dietary nucleic acids occurs in the small intestine by pancreatic enzymes

22. Digestion of dietary nucleic acids In the stomach: low pH denatures DNA&RNA In small intestine: break down phosphodiester bond by endonuclease (pancreas)  oligonucleotide By phosphodiesterase(exonuclease non spesific enzyme)  mononucleotide By phosphomonoesterase (nucleotidase)  result: nucleoside and orthophosphate. Nucleosida phosphorylase  result: base and ribose-1-phosphate. ribose-1-phosphate

23. The nucleoside then absorbed by intestinal mucosal cells If the base or nucleoside is unused, it will be reused in salvage pathways, the base will be degraded: uric acid ureidopropionicureidopropionic (purin) (pyrimidine).

24. Diseases associated with purine degradation

25. Gout Elevated uric acid levels in the blood Uric acid crystals will form in the extremities with a surrounding area of inflammation. This is called a tophus and is often described as an arthritic “great toe”. Can be caused by a defect in an enzyme of purine metabolism or by reduced secretion of uric acid into the urinary tract. tophus

26. Adenosine Deaminase (ADA) and Purine Nucleoside Phosphorylase (PNP) Deficiency.  accumulation of adenosine wich is converted to its ribonucleotide or deoxyribonucleotide form by cellular kinases As dATP level rise, ribonucleotide reductase is inhibited  ↓ production of all deoxyribose containing nucleotides  cells cannot make DNA and divide. Most severe form: severe combined immunodeficiency disease (SCID)  lack of T and B cells

27. A deficiency of either ADA or PNP causes a moderate to complete lack of immune function. Affected children cannot survive outside a sterile environment. They may also have moderate neurological problems, including partial paralysis of the limbs. When a compatible donor can be found, bone marrow transplant is an effective treatment.

28. Lesch-Nyhan Syndrome Hypoxanthine Guanine Phosphoribosyltransferase (HGPRT) deficiency X-linked genetic condition Severe neurologic disease, characterized by self- mutilating behaviors such as lip and finger biting and/or head banging Up to 20 times the uric acid in the urine than in normal individuals. Uric acid crystals form in the urine. Untreated condition results in death within the first year due to kidney failure. Treated with allopurinol, a competitive inhibitor of xanthine oxidase.

29. SYNTHESIS OF DEOXYRIBONUCLEOTIDES Deoxyribonucleotides required for DNA synthesis (2’-deoxyribonucleotides) Enzyme: ribonucleotide reductase Inhibitor : dATP Needed a coenzyme : thioredoxin Thioredoxin is regenerated by thioredoxin reductasethioredoxin reductase Regulation of ribonucleotide reduction is controlled by allosteric feedback mechanisms.

30. PYRIMIDINE SYNTHESIS AND DEGRADATION Source of pyrimidine ring: glutamine, CO2, aspartic acid Synthesis of carbamoyl phosphate  from glutamine & CO2, enzyme: carbamoyl phosphate synthetase II (CPS II), inhibited by UTP  activated by ATP and PRPP

31. Synthesis of orotic acid  formation of carbamoylaspartate  dihydroorotate  orotic acid (mind the enzymes!!) Formation of a pyrimidine nucleotide : orotidine 5’-monophosphate (OMP)  the parent of pyrimidine mononucleotide  OMP  Uridine monophosphate (UMP) Synthesis of uridine triphosphate and cytidine triphosphate  CTP is produced by amination of UTP Synthesis of thymidine monophosphate from dUMP

33. Orotat fosforibosiltransferase Orotidilate dekarboksilase UMP kinase CTP synthetase Nukleosida diphosphat kinase

34. Pyrimidine Synthesis Production of Uridine 5’-monophosphate (UMP) from orotate is catalyzed by the enzyme UMP synthase

35. Orotic Aciduria Deficiency in UMP synthetase activity Due to the demand for nucleotides in the process of red blood cell synthesis, patients develop the condition of megaloblastic anemia, a deficiency of red blood cells. Pyrimidine synthesis is decreased and excess orotic acid is excreted in the urine (hence the name orotic aciduria)

36. Degradation of pyrimidine nucleotides Unlike the purine rings, which are not cleaved in human cells, the pyrimidine ring can be opened and degraded to highly soluble structures, such as β -alanine, and β -aminoisobutyrate, which can serve as precursors of acetyl coA and succinyl coA

38. SALVAGE OF PYRIMIDINES Pyrimidine salvage defects have not been clinically documented

39. Nucleic Acid Metabolism Overview