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

3. Main Topics Metabolism of Nucleotides (4h) DNA replication; RNA transcription; Protein synthesis (12h) Gene expression and regulation (4h); Recombinant DNA technology (4h) Signal transduction; Oncogene (6h) Diabetes mellitus (2h); Lipoproteins Metabolism (4h) Cholesterol Metabolism (2h); Bile acids Metabolism (2h) Examination Heme Synthesis (2h); Bile Pigments Metabolism (2h) Liver function tests (2h); Metabolism of xenobiotics (2h) Plasma Proteins and Immuno Proteins (2h) Free Radicals and Antioxidants (2h) Mineral Metabolism:(micro-elements) (2h) Water and Electrolyte Balance; Acid Base Balance (4h) Hormones (6h) Biochemical changes during Pregnancy (2h)

4. Lecture 1 Metabolism of Nucleotides

6. 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

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

8. pyrimidinepurineOR Ribose or 2-deoxyribose N-  -glycosyl bond Structure of nucleotides

9. Purines vs Pyrimidines

11. Section 1 Degradation of nucleic acid

12. Nucleoprotein Nucleic acidProtein Nucleotide NucleosidePhosphate BaseRibose Nucleotidase Nucleosidase Degradation of nucleic acid In stomach Gastric acid and pepsin In small intestine Endonucleases: RNase and DNase

13. 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.

14. Section 2 Synthesis of Purine Nucleotides

15. There are two pathways leading to nucleotides De novo synthesis: The synthesis of nucleotides begins with their metabolic precursors: amino acids, ribose-5-phosphate, CO 2, and one-carbon units. Salvage pathways: The synthesis of nucleotide by recycle the free bases or nucleosides released from nucleic acid breakdown.

16. § 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).

17. N 10 -Formyltetrahydrofolate 1. Element sources of purine bases First, synthesis Inosine-5'-Monophosphate, IMP

18. 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

19. OH 1 ATP AMP 2 Gln:PRPP amidotransferase ribose phosphate pyrophosphokinase Step 1:Activation of ribose-5-phosphate Step 2: acquisition of purine atom N9 5- 磷酸核糖胺,PRA Steps 1 and 2 are tightly regulated by feedback inhibition Committed step

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

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

22. FH 4 (or THF) N 10 —CHO—FH 4

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

24. 6 5- 氨基咪唑核苷酸 Step 6: closing of the imidazole ring

25. 5- 氨基 -4- 羧基咪唑核苷酸 Carboxyaminoimidazole ribonucleotide (CAIR) 7 Step 7: acquisition of C6 AIR carboxylase

26. 5- 氨基 -4-(N- 琥珀酸 ) - 甲酰胺咪唑核苷酸 Carboxyaminoimidazole ribonucleotide (CAIR) Step 8: acquisition of N1 SAICAR synthetase

27. 5- 氨基 -4- 甲酰胺咪唑核苷酸 Step 9: elimination of fumarate adenylosuccinate lyase

28. 5- 甲酰胺基 -4- 甲酰胺咪唑核苷酸 Step 10: acquisition of C2 AICAR transformylase

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

30. The big picture

31. N 10 -CHOFH 4

32. 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.

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

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

35. Purine nucleotide biosynthesis is regulated by feedback inhibition

36. Regulation of de novo synthesis of purine nucleotides

37. § 2.2 Salvage pathway Purine bases created 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.

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

39. 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

40. Lesch-Nyhan syndrome

41. § 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.

42. Deoxyribonucleotide synthesis at the NDP level

43. § 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.

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

45. 6-MP6-MP nucleotide de novo synthesis salvage pathway HGPRT amidotransferase IMP AMP and GMP - - - - - 6-MP nucleotide is a analog of IMP

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

47. 3. Folic acid analogs Aminopterin (AP) and Methotrexate (MTX) MTX

48. 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.

49. Sulfanilamides Sulfanilamides (also known as sulfa drugs) is a competitive inhibitor of p-aminobenzoic acid (PABA). p-aminobenzoic acid (PABA) is required by many bacteria to produce THF. These bacteria require THF for their growth and division.

50. Sulfanilamide acts as a competitive inhibitor to enzymes that convert PABA into folic acid, resulting in a depletion of this cofactor. The depletion of this cofactor results in retarded growth and eventual death of the bacteria. Mammals absorb their folic acid from their diets, so sulfanilamide exerts no effects on them. Mammal: Folic acid (diet) DHF THF Bacteria: PABA DHF THF

51. Section 3 Degradation of Purine Nucleotides

53. ( 2,6,8-trioxypurine ) Adenosine Deaminase

54. Uric acid is the excreted end product of 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. Uric acid

55. 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. GOUT

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

57. Advanced Gout Clinically Apparent Tophi 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. 2 1 3

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

59. Adenosine Deaminase(ADA) and Severe Combined Immunodeficiency Disease (SCID) SCID is caused by an Adenosine Deaminase Deficiency (ADA) –Gene is located on chromosome #22 –ADA is involved in purine degradation –Accumulation of nucleotide metabolites = TOXIC to developing T lymphocytes –Deficiency results in failure to develop functional T and B lymphocytes –Patients cannot withstand infection  die if untreated

60. Boy in the Bubble

61. Gene therapy September 14, 1990 @ NIH, French Anderson and R. Michael Blaese perform the first GT Trial –Ashanti (4 year old girl) Her lymphocytes were gene-altered (~10 9 )  used as a vehicle for gene introduction using a retrovirus vector to carry ADA gene –Cynthia (9 year old girl) treated in same year

62. Section 4 Synthesis of Pyrimidine Nucleotides

63. 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 HCO 3 - ) contribute to the 6-membered ring requires 6 steps (instead of 11 for purine) the product is UMP (uridine monophosphate) § 4.1 De novo synthesis

64. 1. Element source of pyrimidine base

65. 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. Step 1: synthesis of carbamoyl phosphate

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

67. Step 3: ring closure to form dihydroorotate

68. Step 4: oxidation of dihydroorotate to orotate

69. Step 5: acquisition of ribose phosphate moiety ribose phosphate originates from PRPP

70. Step 6: decarboxylation of OMP

71. The big picture

72. 3. UTP and CTP biosynthesis UDP ADP UTP ATPADP UMP ATP kinase

73. 4. Formation of dTMP The immediate precursor of thymidylate (dTMP) is dUMP. The formation of dUMP either by deamination of dCMP or by hydrolyzation of dUDP. The former is the main route. dTMP dTDP dTTP dUMP dUDP dCMPdCDP N5,N10-methylene- tetrahydrofolic Acid ATP ADP dTMP synthetase UDP

74. dTMP synthesis at the nucleoside monophosphate level.

75. 5. Regulation of de novo synthesis

76. § 4. 2 Salvage pathway

77. § 4. 3 Antimetabolites of pyrimidine nucleotides Antimetabolites of pyrimidine nucleotides are similar with them of purine nucleotides.

78. 1. Pyrimidine analogs 5-fluorouracil (5-FU) is a analog of thymine.

79. 5-FU 5-FdUMP 5-FUTP dUMP dTMP Synthesis of RNA Destroy structure of RNA

80. 2. Amino acid analogs Azaserine (AS) inhibits the synthesis of CTP. 3. Folic acid analogs Methotrexate (MTX) inhibits the synthesis of dTMP.

81. 4. Nucleoside analogs Arabinosyl cytosine (ara-c) inhibits the synthesis of dCDP.

82. Section 5 Degradation of Pyrimidine Nucleotides

83. Highly soluble products

84. Summary of purine biosynthesis IMP

85. Summary of pyrimidine biosynthesis UMP

86. Summary of Nucleotide Synthesis Purines built up on ribose –PRPP synthetase: key step –First, synthesis IMP Pyrimidine rings built, then ribose added –CPS-II: key step –First, synthesis UMP Salvage is important

87. Points Synthesis of Purine Nucleotides –De novo synthesis: Site, Characteristics, Element sources of purine bases –Salvage pathway: definition, significance, enzyme, Lesch- Nyhan syndrome –Formation of deoxyribonucleotide: NDP level –Antimetabolites of purine nucleotides: Purine, Amino acid, and Folic acid analogs Degradation of Purine Nucleotides –Uric acid, gout Synthesis of Pyrimidine Nucleotides –De novo synthesis: Characteristics, Element sources of pyrimidine bases –Salvage pathway –Antimetabolites of pyrimidine nucleotides Catabolism of Pyrimidine Nucleotides