Understanding Nucleotide Metabolism in Biochemistry: Pathways and Significance

1. Biochemistry Ⅱ

2. Metabolism of Nucleotides

3. Contents • Review: Structure of nucleic acid • Degradation of nucleic acid • Synthesis of Purine Nucleotides • Degradation of Purine Nucleotides • Synthesis of Pyrimidine Nucleotides • Degradation of Pyrimidine Nucleotides

4. Nitrogenous base ribose Nitrogenous base ribose phosphate Nucleoside and Nucleotide Nucleoside = Nucleotide =

5. Purines vs Pyrimidines

6. N-b-glycosyl bond Ribose or 2-deoxyribose Structure of nucleotides pyrimidine OR purine

7. Section 1Degradation of nucleic acid

8. Degradation of nucleic acid Nucleoprotein In stomach Gastric acid and pepsin Nucleic acid Protein In small intestine Endonucleases: RNase and DNase Nucleotide Nucleotidase Phosphate Nucleoside Nucleosidase Base Ribose

9. Significances of nucleotides 1. Precursors for DNA and RNA synthesis 2. Essential carriers of chemical energy, especially ATP 3. Components of the cofactors NAD+, FAD, and coenzyme A 4. Formation of activated intermediates such as UDP-glucose and CDP-diacylglycerol. 5. cAMP and cGMP, are also cellular second messengers.

10. Section 2Synthesis of Purine Nucleotides

11. There are two pathways leading to nucleotides • De novo synthesis: The synthesis of nucleotides begins with their metabolic precursors: amino acids, ribose-5-phosphate, CO2, and one-carbon units. • Salvage pathways: The synthesis of nucleotide by recycle the free bases or nucleosides released from nucleic acid breakdown.

12. § 2.1 De novo synthesis • Site: • in cytosol of liver, small intestine and thymus • Characteristics: a. Purines are synthesized using 5-phosphoribose(R-5-P) as the starting material step by step. b.PRPP(5-phosphoribosyl-1-pyrophosphate) is active donor of R-5-P. c. AMP and GMP are synthesized further at the base of IMP(Inosine-5'-Monophosphate).

13. N10-Formyltetrahydrofolate N10-Formyltetrahydrofolate 1. Element sources of purine bases First, synthesis Inosine-5'-Monophosphate, IMP

14. FH4 (or THF) N10—CHO—FH4

15. 2. Synthesis of Inosine Monophosphate (IMP) • Basic pathway for biosynthesis of purine ribonucleotides • Starts from ribose-5-phosphate(R-5-P) • Requires 11 steps overall • occurs primarily in the liver

16. OH ATP 1 AMP 2 Step 1:Activation of ribose-5-phosphate Committed step ribose phosphate pyrophosphokinase Step 2: acquisition of purine atom N9 Gln:PRPP amidotransferase • Steps 1 and 2 are tightly regulated by feedback inhibition

17. 3 Step 3: acquisition of purine atoms C4, C5, and N7 glycinamide synthetase

18. 4 • Step 4: acquisition of purine atom C8 GAR transformylase

19. 5 Step 5: acquisition of purine atom N3

20. 6 • Step 6: closing of the imidazole ring

21. 7 Carboxyaminoimidazole ribonucleotide (CAIR) Step 7: acquisition of C6 AIR carboxylase

22. Carboxyaminoimidazole ribonucleotide (CAIR) Step 8: acquisition of N1 SAICAR synthetase

23. Step 9: elimination of fumarate adenylosuccinate lyase

24. Step 10: acquisition of C2 AICAR transformylase

25. Step 11:ring closure to form IMP • Once formed, IMP is rapidly converted to AMP and GMP (it does not accumulate in cells).

26. N10-CHOFH4 N10-CHOFH4

27. 3. Conversion of IMP to AMP and GMP Note: GTP is used for AMP synthesis. Note: ATP is used for GMP synthesis. IMP is the precursor for both AMP and GMP.

28. kinase kinase kinase kinase 4. ADP, ATP, GDP and GTP biosynthesis ATP AMP ADP ADP ATP ADP ATP GTP GDP GMP ADP ATP ADP ATP

29. 5. Regulation of de novo synthesis The significance of regulation: • Meet the need of the body, without wasting. (2) AMP and GMP control their respective synthesis from IMP by a feedback mechanism, [GTP]=[ATP]

30. Purine nucleotide biosynthesis is regulated by feedback inhibition

31. § 2.2 Salvage pathway • Purine basescreated by degradation of RNA or DNA and intermediate of purine synthesis can be directly converted to the corresponding nucleotides. • The significance of salvage pathway : • Save the fuel. • Some tissues and organs such as brain and bone marrow are only capable of synthesizing nucleotides by salvage pathway. • Two phosphoribosyl transferases are involved: • APRT (adenine phosphoribosyl transferase) for adenine. • HGPRT (hypoxanthine guanine phosphoribosyl transferase) for guanine or hypoxanthine.

32. Purine Salvage Pathway Absence of activity of HGPRT leads to Lesch-Nyhan syndrome.

33. Lesch-Nyhan syndrome • first described in 1964 by Michael Lesch and William L. Nyhan. • there is a defect or lack in the HGPRT enzyme • Sex-linked metabolic disorder: only males • the rate of purine synthesis is increased about 200-fold • Loss of HGPRT leads to elevated PRPP levels and stimulation of de novo purine synthesis. • uric acid level rises and there is gout • in addition there are mental aberrations • patients will self-mutilate by biting lips and fingers off

34. Lesch-Nyhan syndrome

35. § 2. 3 Formation of deoxyribonucleotide • Formation of deoxyribonucleotide involves the reduction of the sugar moiety of ribonucleoside diphosphates (ADP, GDP, CDP or UDP). • Deoxyribonucleotide synthesis at the nucleoside diphosphate(NDP) level.

36. Deoxyribonucleotide synthesis at the NDP level

37. § 2. 4 Antimetabolites of purine nucleotides • Antimetabolites of purine nucleotides are structural analogs of purine, amino acids and folic acid. • They can interfere, inhibit or block synthesis pathway of purine nucleotides and further block synthesis of DNA, RNA, and proteins. • Widely used to control cancer.

38. 1. Purine analogs • 6-Mercaptopurine (6-MP) is a analog of hypoxanthine.

39. - - - - - de novo synthesis • 6-MP nucleotide is a analog of IMP amidotransferase IMP 6-MP 6-MP nucleotide AMP and GMP HGPRT salvage pathway

40. 2. Amino acid analogs • Azaserine (AS) is a analog of Gln.

41. 3. Folic acid analogs • Aminopterin(AP)andMethotrexate (MTX) MTX

42. The structural analogs of folic acid(e.g. MTX) are widely used to control cancer (e.g. leukaemia). • Notice: These inhibitors also affect the proliferation of normally growing cells. This causes many side-effects including anemia, baldness, scaly skin etc.

43. Section 3Degradation of Purine Nucleotides

44. Adenosine Deaminase The end product of purine metabolism (2,6,8-trioxypurine)

45. Uric acid • Uric acid is the excreted end productof purine catabolism in primates, birds, and some other animals. • The rate of uric acid excretion by the normal adult human is about 0.6 g/24 h, arising in part from ingested purines and in part from the turnover of the purine nucleotides of nucleic acids. • The normal concentration of uric acid in the serum of adults is in the range of 3-7 mg/dl.

46. GOUT • The disease gout, is a disease of the joints, usually in males, caused by an elevated concentration of uric acid in the blood and tissues. • The joints become inflamed, painful, and arthritic, owing to the abnormal deposition of crystals of sodium urate. • The kidneys are also affected, because excess uric acid is deposited in the kidney tubules.

47. Hypoxanthine Xanthine The uric acid and the gout Out of body In urine Uric acid  Over 8mg/dl, in the plasma Diabetese nephrosis …… Gout, Urate crystallization in joints, soft tissue, cartilage and kidney

48. Advanced GoutClinically Apparent Tophi 2 1 3 1 1. Photos courtesy of Brian Mandell, MD, PhD, Cleveland Clinic. 2. Photo courtesy of N. Lawrence Edwards, MD, University of Florida. 3. ACR Clinical Slide Collection on the Rheumatic Diseases, 1998.

49. Allopurinol – a suicide inhibitor used to treat Gout Xanthine oxidase Xanthine oxidase

50. Section 4Synthesis of Pyrimidine Nucleotides