Understanding Nucleotide Metabolism: Synthesis, Degradation, and Significance in Biochemistry Lectures

1. Biochemistry Ⅱ Zhihong Li（李志红） Department of Biochemistry

2. Main Topics

3. Lecture 1Metabolism of Nucleotides

5. 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

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

7. Purines vs Pyrimidines

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

9. Section 1Degradation of nucleic acid

10. 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

11. 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.

12. Section 2Synthesis of Purine Nucleotides

13. 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.

14. § 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).

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

16. FH4 (or THF) N10—CHO—FH4

17. 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

18. 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 5-磷酸核糖胺,PRA

19. 3 甘氨酰胺核苷酸 Step 3: acquisition of purine atoms C4, C5, and N7 glycinamide synthetase

20. 4 甲酰甘氨酰胺核苷酸 • Step 4: acquisition of purine atom C8 GAR transformylase

21. 5 甲酰甘氨咪核苷酸 Step 5: acquisition of purine atom N3

22. 6 • Step 6: closing of the imidazole ring 5-氨基咪唑核苷酸

23. 7 Carboxyaminoimidazole ribonucleotide (CAIR) 5-氨基-4-羧基咪唑核苷酸 Step 7: acquisition of C6 AIR carboxylase

24. Carboxyaminoimidazole ribonucleotide (CAIR) 5-氨基-4-(N-琥珀酸) -甲酰胺咪唑核苷酸 Step 8: acquisition of N1 SAICAR synthetase

25. Step 9: elimination of fumarate adenylosuccinate lyase 5-氨基-4-甲酰胺咪唑核苷酸

26. Step 10: acquisition of C2 AICAR transformylase 5-甲酰胺基-4-甲酰胺咪唑核苷酸

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

28. N10-CHOFH4 N10-CHOFH4

29. 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.

30. 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

31. 5. Regulation of de novo synthesis The significance of regulation: (1) Meet the need of the body, without wasting. (2) AMP and GMP control their respective synthesis from IMP by a feedback mechanism, [GTP]=[ATP]

32. Purine nucleotide biosynthesis is regulated by feedback inhibition

33. 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.

34. Section 3Degradation of Purine Nucleotides

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

36. 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.

37. 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.

38. 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

39. 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.

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

41. Section 4Synthesis of Pyrimidine Nucleotides

42. § 4.1 De novo synthesis • shorter pathway than for purines • Pyrimidine ring is made first, then attached to ribose-P (unlike purine biosynthesis) • only 2 precursors (aspartate and glutamine, plus HCO3-) contribute to the 6-membered ring • requires 6 steps (instead of 11 for purine) • the product is UMP (uridine monophosphate)

43. 1. Element source of pyrimidine base

44. Step 1: synthesis of carbamoyl phosphate • Carbamoyl phosphate synthetase(CPS) exists in 2 types: • CPS-I, a mitochondrial enzyme, is dedicated to the urea cycle and arginine biosynthesis. • CPS-II, a cytosolic enzyme, used here. It is the committed step in animals.

45. Step 2: synthesis of carbamoyl aspartate ATCase: aspartate transcarbamoylase • Carbamoyl phosphate is an “activated” compound, so no energy input is needed at this step.

46. Step 3: ring closure to form dihydroorotate

47. Step 4: oxidation of dihydroorotate to orotate CoQ QH2 (a pyrimidine)

48. Step 5: acquisition of ribose phosphate moiety Step 6: decarboxylation of OMP

49. The big picture

50. kinase kinase UMP UDP UTP ADP ATP ADP ATP 3.UTP and CTP biosynthesis