Zhihong Li （李志红） Department of Biochemistry

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

8. Purines vs Pyrimidines

10. Section 1Degradation of nucleic acid

11. 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

12. 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.

13. Section 2Synthesis of Purine Nucleotides

14. 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.

15. § 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).

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

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. FH4 (or THF) N10—CHO—FH4

22. 5 甲酰甘氨咪核苷酸 Step 5: acquisition of purine atom N3

23. 6 • Step 6: closing of the imidazole ring 5-氨基咪唑核苷酸

24. 7 Carboxyaminoimidazole ribonucleotide (CAIR) 5-氨基-4-羧基咪唑核苷酸 Step 7: acquisition of C6 AIR carboxylase

25. 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

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

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

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

29. N10-CHOFH4 N10-CHOFH4

30. 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.

31. 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

32. 5. Regulation of de novo synthesis The significance of regulation: (1) Fulfill the need of the body, without wasting. (2) [GTP]=[ATP]

33. Purine nucleotide biosynthesis is regulated by feedback inhibition

34. § 2.2 Salvage pathway • Purine basescreated by degradation of RNA or DNA and intermediate of purine synthesis were costly for the cell to make, so there are pathways to recover these bases in the form of nucleotides. • The significance of salvage pathway : • a. Save the fuel. • b. 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.

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

36. 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

37. Lesch-Nyhan syndrome

38. § 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.

39. Deoxyribonucleotide synthesis at the NDP level

40. § 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 RNA, DNA, and proteins. Widely used to control cancer.

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

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

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

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

45. 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.

46. Section 3Degradation of Purine Nucleotides

48. Adenosine Deaminase (2,6,8-trioxypurine)

49. 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.

50. 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.