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

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

3. Lecture 1 Metabolism 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. Nucleoside and Nucleotide
Nitrogenous base ribose
Nitrogenous base ribose phosphate

7. Purines vs Pyrimidines

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

9. Section 1 Degradation 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 2 Synthesis 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: Characteristics:
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. First, synthesis Inosine-5'-Monophosphate, IMP
1. Element sources of purine bases
N10-Formyltetrahydrofolate
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. Step 1:Activation of ribose-5-phosphateOH 1
ATP
AMP
Step 1:Activation of ribose-5-phosphate
Committed step
ribose phosphate pyrophosphokinase 2
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. Step 3: acquisition of purine atoms C4, C5, and N7
甘氨酰胺核苷酸 3
glycinamide synthetase

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

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

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

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

24. Step 8: acquisition of N1 Carboxyaminoimidazole ribonucleotide (CAIR)
-甲酰胺咪唑核苷酸
Carboxyaminoimidazole
ribonucleotide (CAIR)
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

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

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
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 3 Degradation of Purine Nucleotides

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

36. Uric acid
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.
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. The uric acid and the gout
Hypoxanthine
Xanthine
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 Gout Clinically Apparent Tophi1 2 1 3
The advanced gout stage is often referred to as chronic tophaceous gout to indicate the presence of this clinical manifestation, which will remain unresolved in the absence of urate-lowering therapy.
Tophi are characterized by solid urate deposits in connective tissues that produce irregular nodularities and joint destruction. In addition, the skin overlying the tophi may become ulcerated and exude a white, chalky material.
Shown here are some common sites of tophi, including dermal tophi on the finger, periarticular tophi on the hands, and tophi on the helix of the ear.
The patient who was experiencing the intradermal tophi on the knees was diagnosed and treated by multiple generalists and specialists for osteoarthritis. This photo was taken during his self-referred first visit to a rheumatologist and reinforces the point that gout is a disease that is under-recognized.1 The patient with polyarticular involvement of his hands had been misdiagnosed and treated for rheumatoid arthritis for 8 years.2
The tophi exhibited on this slide are clinically apparent, but this may not always be the case, as was seen in the previous case study examples of the tophi that formed in the bone of the knee and the palm of the hand.
1. Patient case study courtesy of Brian Mandell, MD, PhD, Cleveland Clinic.
2. Patient case study courtesy of N. Lawrence Edwards, MD, University of Florida.
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

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

41. Section 4 Synthesis 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. 3. UTP and CTP biosynthesis
UDP
ADP
UTP
ATP
UMP
kinase

51. 4. Formation of dTMP dTMP dTTP
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
dCMP
dCDP
N5,N10-methylene-tetrahydrofolic Acid
ATP
ADP
dTMP synthetase
UDP

52. dTMP synthesis at the nucleoside monophosphate level.

53. § Salvage pathway

54. Section 5 Degradation of Pyrimidine Nucleotides

55. Highly soluble products

56. Summary of purine biosynthesis
IMP

57. Summary of pyrimidine biosynthesis
UMP

58. 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