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

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)

Lecture 1 Metabolism of Nucleotides

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

Nucleoside and Nucleotide Nitrogenous base ribose Nitrogenous base ribose phosphate

Purines vs Pyrimidines

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

Section 1 Degradation of nucleic acid

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

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.

Section 2 Synthesis of Purine Nucleotides

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.

§ 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).

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

FH4 (or THF) N10—CHO—FH4

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

Step 1:Activation of ribose-5-phosphate OH 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

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

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

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

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

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

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

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

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

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

N10-CHOFH4

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.

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

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]

Purine nucleotide biosynthesis is regulated by feedback inhibition

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.

Section 3 Degradation of Purine Nucleotides

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

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.

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.

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

Advanced Gout Clinically Apparent Tophi 1 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

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

Section 4 Synthesis of Pyrimidine Nucleotides

§ 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)

1. Element source of pyrimidine base

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.

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

Step 3: ring closure to form dihydroorotate

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

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

The big picture

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

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

dTMP synthesis at the nucleoside monophosphate level.

§ 4. 2 Salvage pathway

Section 5 Degradation of Pyrimidine Nucleotides

Highly soluble products

Summary of purine biosynthesis IMP

Summary of pyrimidine biosynthesis UMP

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