Zhihong LiDepartment of BiochemistryBiochemistry

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-assesmentFree 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

ContentsReview: Structure of nucleic acidDegradation of nucleic acidSynthesis of Purine NucleotidesDegradation of Purine NucleotidesSynthesis of Pyrimidine NucleotidesDegradation of Pyrimidine Nucleotides

Nucleoside and NucleotideNucleoside =

Nucleotide =

Purines vs Pyrimidines

pyrimidinepurineORStructure of nucleotides

Section 1 Degradation of nucleic acid

Nucleoprotein Nucleic acidProtein NucleotideNucleosidePhosphate Base Ribose NucleotidaseNucleosidaseDegradation of nucleic acid In stomach Gastric acid and pepsinIn small intestineEndonucleases: RNase and DNase

Significances of nucleotides1. Precursors for DNA and RNA synthesis 2. Essential carriers of chemical energy, especially ATP 3. Components of the cofactors NAD+, FAD, and coenzyme A4. 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 nucleotidesDe 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 synthesisSite: 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).

1. Element sources of purine basesFirst, synthesis Inosine-5'-Monophosphate, IMP

FH4 (or THF)N10CHOFH4

2. Synthesis of Inosine Monophosphate (IMP)Basic pathway for biosynthesis of purine ribonucleotidesStarts from ribose-5-phosphate(R-5-P)Requires 11 steps overalloccurs primarily in the liver

Gln:PRPP amidotransferaseribose phosphate pyrophosphokinaseStep 1:Activation of ribose-5-phosphateStep 2: acquisition of purine atom N95-,PRA Steps 1 and 2 are tightly regulated by feedback inhibitionCommitted step

Step 3: acquisition of purine atoms C4, C5, and N7glycinamide synthetase

Step 4: acquisition of purine atom C8GAR transformylase

Step 5: acquisition of purine atom N3

5-Step 6: closing of the imidazole ring

Step 7: acquisition of C6AIR carboxylase

Step 8: acquisition of N1SAICAR synthetase

5--4-Step 9: elimination of fumarateadenylosuccinate lyase

5--4-Step 10: acquisition of C2AICAR transformylase

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

The big picture

3. Conversion of IMP to AMP and GMPNote: GTP is used for AMP synthesis.Note: ATP is used for GMP synthesis.IMP is the precursor for both AMP and GMP.

ADPADPATPATPADPAMPATPGDPADPGTPATPADPGMPATP4. ADP, ATP, GDP and GTP biosynthesis

5. Regulation of de novo synthesisThe 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

2.2 Salvage pathwayPurine bases created by degradation of RNA or DNA and intermediate of purine synthesis can be directly converted to the corresponding nucleotides. The significance of salvage pathway :Save the fuel.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.

Purine Salvage PathwayAbsence of activity of HGPRT leads to Lesch-Nyhan syndrome.

Lesch-Nyhan syndromefirst described in 1964 by Michael Lesch and William L. Nyhan. there is a defect or lack in the HGPRT enzymeSex-linked metabolic disorder: only malesthe 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 goutin addition there are mental aberrationspatients will self-mutilate by biting lips and fingers off

Lesch-Nyhan syndrome

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.

Deoxyribonucleotide synthesis at the NDP level

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

1. Purine analogs6-Mercaptopurine (6-MP) is a analog of hypoxanthine.

hypoxanthine

6-MP6-MP nucleotidede novo synthesissalvage pathwayHGPRTamidotransferaseIMPAMP and GMP6-MP nucleotide is a analog of IMP

2. Amino acid analogsAzaserine (AS) is a analog of Gln.

3. Folic acid analogsAminopterin (AP) and Methotrexate (MTX)MTX

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

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

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.Uric acid

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

The uric acid and the goutUric acid Over 8mg/dl, in the plasmaGout, Urate crystallization in joints, soft tissue, cartilage and kidneyOut of bodyIn urineDiabetese nephrosis

Advanced GoutClinically Apparent Tophi11. 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, 1998.213

Allopurinol a suicide inhibitor used to treat GoutXanthine oxidaseXanthine oxidase

Section 4 Synthesis of Pyrimidine Nucleotides

shorter pathway than for purinesPyrimidine 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 ringrequires 6 steps (instead of 11 for purine)the product is UMP (uridine monophosphate)

4.1 De novo synthesis

1. Element source of pyrimidine base

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

Step 2: synthesis of carbamoyl aspartateATCase: aspartate transcarbamoylaseCarbamoyl 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 orotateQH2CoQ(a pyrimidine)

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

The big picture

3. UTP and CTP biosynthesis

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 synthesis at the nucleoside monophosphate level.

5. Regulation of de novo synthesis

4. 2 Salvage pathway

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

1. Pyrimidine analogs5-fluorouracil (5-FU) is a analog of thymine.

5-FU5-FdUMP5-FUTPdUMPdTMPSynthesis of RNADestroy structure of RNA

2. Amino acid analogsAzaserine (AS) inhibits the synthesis of CTP.

3. Folic acid analogsMethotrexate (MTX) inhibits the synthesis of dTMP.

4. Nucleoside analogsArabinosyl cytosine (ara-c) inhibits the synthesis of dCDP.

Section 5 Degradation of Pyrimidine Nucleotides

Highly soluble products

Summary of purine biosynthesisIMP

Summary of pyrimidine biosynthesisUMP

Summary of Nucleotide SynthesisPurines built up on ribosePRPP synthetase: key stepFirst, synthesis IMPPyrimidine rings built, then ribose addedCPS-II: key stepFirst, synthesis UMPSalvage is important

PointsSynthesis of Purine NucleotidesDe novo synthesis: Site, Characteristics, Element sources of purine basesSalvage pathway: definition, significance, enzyme, Lesch-Nyhan syndromeFormation of deoxyribonucleotide: NDP levelAntimetabolites of purine nucleotides: Purine, Amino acid, and Folic acid analogsDegradation of Purine NucleotidesUric acid, goutSynthesis of Pyrimidine NucleotidesDe novo synthesis: Characteristics, Element sources of pyrimidine basesSalvage pathwayAntimetabolites of pyrimidine nucleotidesCatabolism of Pyrimidine Nucleotides

****************************************************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.2The 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.

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