NUCLEOTIDE METABOLISM

1. NUCLEOTIDE METABOLISM SITI ANNISA DEVI TRUSDA

2. Nucleotides are essential for all cells • DNA/RNA synthesisproteinsynthesis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2nd 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 • 1st 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/deoxyribosee.g: 5’-ribonucleotide/5’-deoxyribonucleotide

6. 1 phosphate group + 5’-carbon of the pentosenucleosidemonophosphate(NMP) e.g AMP, CMP • 2 or 3 phosphate group added to the nucleosidenucleosidedi/triphosphatee.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,N10-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 purinesynthetis

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 synthetasesuperactivity 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 inosinemonophosphate,the “parent” of purine nucleotide requires 4 ATP 2 steps require N10 –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 Adenilosuksinat synthetase IMP dehidrogenase XMP aminase 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(exonucleasenon spesific enzyme)  mononucleotide • By phosphomonoesterase (nucleotidase)result:nucleosideand orthophosphate. • Nucleosidaphosphorylase result: baseand 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 ureidopropionic (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, ribonucleotidereductase 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 xanthineoxidase.

29. SYNTHESIS OF DEOXYRIBONUCLEOTIDES • Deoxyribonucleotides required for DNA synthesis (2’-deoxyribonucleotides) • Enzyme: ribonucleotidereductase • Inhibitor : dATP • Needed a coenzyme : thioredoxin • Thioredoxin is regenerated by thioredoxinreductase • 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Uridinemonophosphate (UMP) • Synthesis of uridinetriphosphate and cytidinetriphosphate CTP is produced by amination of UTP • Synthesis of thymidinemonophosphate from dUMP

33. Orotatfosforibosiltransferase Orotidilate dekarboksilase CTP synthetase UMP kinase 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 oroticaciduria)

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 succinylcoA

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

39. Nucleic Acid Metabolism Overview

40. HaturNuhun See you next time