Nucleotides, Purine Biosynthesis and Purine Catabolism

1. Nucleotides, Purine Biosynthesis and Purine Catabolism

2. LEARNING POINTS Understand nucleosides*, nucleotides, and their function in DNA and RNA Understand the structure and function of purines Understand the origin of atoms in the purine ring Understand the essential features of purine metabolism and catabolism Understand clinical aspects of purine metabolism and deficiencies *Keywords are highlighted in yellow

3. Nucleotides Chemical compound composed of three components: (1) heterocyclic base; (2) sugar (usually a pentose); and (3) one or more phosphate groups Glycosidic bond Base Phosphate Pentose sugar Adenosine monophosphate (AMP)

4. Building blocks for DNA and RNA

5. Energy Currency

6. Carriers for Activated Intermediates

7. Structural Components of: Coenzyme A Flavin adenine dinucleotide (FAD) NAD(P)+

8. Signaling Molecules

9. The Nitrogenous Bases In DNA: Adenine Guanine *Thymine* Cytosine In RNA: Adenine Guanine *Uracil* Cytosine

10. Hypoxanthine Xanthine Not typically found in DNA or RNA, but are important metabolic intermediates.

11. RNA is sensitive to alkaline degradation

12. Mechanism of RNA Hydrolysis Hydrolysis occurs by nucleophilic attack of the 2’-hydroxyl group on the polarized phosphate to yield a 2’-3’ cyclic phosphodiester intermediate (circled) that subsequently spontaneously hydrolyzes to a mix of 2’- and 3’-phosphomonoesters.

13. Two Important Points The phosphate groups are responsible for the net negative charge associated with DNA and RNA. The hydroxyl group at the 2’-position accounts for the greater ease with which RNA is degraded by alkali.

14. De novo purine synthesis

15. De novo purine synthesis • The purine ring is synthesized by a series of reactions that add the carbon and nitrogen atoms to a pre-formed ribose-5-phosphate. • The ribose-5-phosphate is synthesized as part of the Hexose MonoPhosphate pathway. • In humans, all necessary enzymes are found in the cytoplasm of the cell.

16. Source For Ribose-5-Phosphate

17. Conversion of Ribose-5-phosphate to PRPP • The pentose sugar is always a ribose, which may be reduced to deoxyribose after nucleotide synthesis is complete. • 5-Phosphoribosyl-1-pyrophosphate (PRPP) is also involved in synthesis of pyrimidine nucleotides, NAD+, and histidine biosynthesis.

18. First step of purine synthesis is committed step and ratelimiting step Intracellular concentrations of glutamine and PRPP control the reaction rate Inhibited by AMP, GMP, and IMP Requires 4 ATP molecules

19. Sulfonamide (PABA analogue)

20. Can synthesize folate Cannot synthesize folate

21. Methotrexate and Cancer • Affects rapidly growing cells • Adverse events include anemia, scaly skin, GI tract disturbances (diarrhea), and baldness • Resistance to MTX is caused by amplification of dihydrofolatereductase gene • Also used for treatment of rheumatoid arthritis and psoriasis at lower doses, though site of action is not through DHFR but inhibition of salvage pathways that lead to increased adenosine that inhibits T cell activation.

22. Need to Know That sulfonamides inhibit purine synthesis in bacteria by interfering with folate synthesis. That methotrexate inhibits purine synthesis by inhibiting dihydrofolate reductase. That IMP is the end product of de novo purine synthesis. AMP, GMP, and IMP inhibit the reaction. PRPP is an activator. Rate limiting step of the pathway and source of atoms for the purine ring

23. High levels shut down de novo purine synthesis Mycophenolic acid

24. Another Look at Regulation Fig 26.6

26. Purine Salvage Pathway • Purines from normal turnover of cellular nucleic acids • Purines obtained from the diet

27. Lesch-Nyhan Syndrome • Build up of hypoxanthine and guanine • Degradation of hypoxanthine and guanine results in increased uric acid • Excess uric acid in urine often results in orange crystals in the diaper of affected children • Severe mental retardation • Self-mutilation • Involuntary movements • Gout

28. Degradation of Purines

29. dATP builds up and inhibits ribonucleotide reductase. XMP Xanthosine Inhibition of ribonucleotide reductase results in no dNDP being produced. No synthesis of DNA in lymphocytes results in Severe Combined Immunodeficiency Syndrome (SCIDS).

30. Gout • Characterized by hyperuricemia and acute arthritic joint inflammation by deposition of uric acid crystals • Primary gout is genetic and mainly affects men over 30 • Secondary gout is associated with leukemia, polycythemia, HGPRT deficiency, renal insufficiency, lifestyle (rich foods)

31. Distribution of Serum Urate Values 40 Males Females 35 30 Urate crystallizes at a level of 6.8 mg/dL 25 Hyperuricemia defined at 7 mg/dL 20 Distribution, % Serum urate levels in 1515 men and 1670 women aged ≥30 in Taiwan 1991-1992 15 10 5 0 11 12 13 1 2 4 5 6 7 8 9 10 0 3 Serum urate, mg/dL Lin et al.J Rheumatol. 27, 1045-1050 (2000)

32. Higher Prevalence of Gout and Clinically Significant Hyperuricemia in Higher Age Groups 50 >18 45 18-44 40 45-64 35 65-74 30 75+ Prevalence rate per 1000 enrollees 25 20 15 10 5 0 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 Wallace et al. J Rheum.31, 1582-1587 (2004).

33. Common Foods With High Purine Content

34. Differences in Serum Urate Among Alcoholic Beverages Wine Total alcohol Liquor Beer 0.80 0.60 0.40 Difference in serum urate (mg/dL) 0.20 0.00 -0.20 Women BMI ≥25 kg/m2 BMI<25 kg/m2 Men Choi et al. Arthritis Rheum.50, S480 (2004)

35. Drugs That Promote Gout Caspi et al. Arth Rheum. 43, 103-108 (2000)

36. Treatments for gout: Acute attacks are treated with colchicine and indomethecin for 3 weeks. Long-term treatment with allopurinol reduces the amount of uric acid in circulation.