Chapter 8

1. Chapter 8 Nucleotide Metabolism

2. Nucleotides are bilding blocks of nucleic acids. They are non-essential nutrients , because they can be synthesized in the body. Nucleic acids occur in the nucleoprotein.

3. Dietary nucleoprotein is digested in the stomach to yield protein and nucleic acids. Nucleic acids are further digested in the small intestine to generate nucleotides. Nucleotides are absorbed into intestinal mucosa cells , where they are degraded to three components : base , pentose and phosphate.

4. Pentose is absorbed but base is degraded and excreted. • Fnuctions of Nucleotides • They serve as building blocks of nucleic acids. • ATP plays an important pole in energy transformation. • ATP , ADP, and AMP may function as allosteric regula- • tors and participate in regulation of many metabolic path- • ways. ATP involves in covalent modification of enzymes.

5. 4. CAMP and cGMP are second messengers. 5. They are components of some coenzymes , such as ADP in FAD , ADP in HSCoA etc. 6 . They are carriers of active metabolic intermediates such as UDPG , SAM , CDP-DG etc.

6. Purine Nucleotide Metabolism Anabolism There are two pathways of synthesis of purine nucleotides : the de novo synthesis pathway and the salvage pathway. The former is the main synthesis pathway of nucleotides , the latter is important I brain and bone marrow.

7. The de novo synthesis of purine nucleotide means using phosphoribose , amino acids , one carbon units and CO2 as raw materials to synthesize purine nucleotide from the beginning. fig 8-2

8. The pathway can be divided into two stages. Stage one : formation of inosine monophosphate ( IMP ) Stage two : conversion of IMP to either AMP or GMP Stage One PRPP synthetase R5P + ATP---------------------------PRPP + AMP amidotransferase PRPP + Gln---------------------------PRA + Glu

9. Once PRA is formed , the building of the purine ring structure begins. In nine successive reactions the first purine nucleotide IMP is formed. fig. 8-3

10. Stage Two The conversion of IMP either to AMP or GMP requires two reactions. GTP,Mg++,adenylosuccinate synthase IMP + Asp-------------------------------adenylosuccinate adenylosuccinate lyase Adenylosuccinate-----------------------AMP + fumarate

11. IMP dehydrogenase IMP + H2O + NAD+---------------XMP + NADH + H+ ATP, Mg++, GMP synthase XMP + Gln------------------------------------GMP + Glu Nucleoside triphosphates are the most common nucleotide used in metabolism. ATP is synthesized from ADP and Pi via oxidative phos- phorylation or substrate level phosphorylation.

12. ADP is synthesized from AMP in a reaction catalyzed by adenylate kinase. AMP + ATP------------------------- 2ADP Other NTPs are also synthesized in ATP-requiring reactions catalyzed by corresponding NMP kinases. NMP + ATP-------------------------NDP + ADP NDP kinase catalyzes the formation of NTP. NDP + ATP-------------------------NTP + ADP

13. Regulation of de novo Pathway PRPP activates amidotransferase. IMP , AMP and GMP inhibit PRPP synthetase. AMP inhibits conversion of IMP to GMP and GMP inhibits conversion of IMP to GMP. ATP stimulates conversion of IMP to GMP and GTP stimulates conversion of IMP to AMP. That ensures a balanced synthesis of both families of purine nuc- leotides.

14. Salvage Pathway of Purine Nucleotides Many cells have mechanisms to retrieve purine bases and purine nucleosides. They are used to synthesize purine nu- cleotides. This is the salvage pathway.

15. From Base to Nucleotides APRT A + PRPP--------------------------------AMP + ppi HGPRT H + PRPP-------------------------------- IMP + ppi HGPRT G + PRPP--------------------------------GMP + ppi

16. From Nucleoside to Nucleotide AR kinase AR + ATP--------------------------------AMP + ADP In comparison to de novo pathway, salvage pathway is energy-saving. In brain and bone marrow tissues salvage pathway is the only pathway of nucleotide synthesis. Deficiency of HGPRT causes Lesch Nyhan syndrome.

17. Formation of Deoxynucleotides Deoxynucleotides are formed by reducing ribonucleotide diphosphates. ribonucleotide reductase NDP + NADPH + H+-----------------dNDP + H2O + NADP+ In the reaction hydrogen atoms are not directly donated by NADPH. Thioredoxin, a protein with two sulfhydryl groups mediates the trans- fer of hydrogen atoms from NADPH to ribonucleotide reductase.

18. Then the enzyme catalyzes the reduction of NDP, to form dNDP. NDP reductase NDP + thioredoxin ( SH )2 --------dNDP + thioredoxin (-S-S-) The regeneration of reduced thioredoxin is catalyzed by thioredoxin reductase. thioredoxin (-S-S-) +NADPH +H+ thioredoxin ( SH )2+NADPH NDP reductase is an allosteric enzyme, Its activity is controled by various NTPs and dNTPs.

19. Antimetabolites of Purine Nucleotides Antimetabolites of purine nucleotides are analogues of purine, amino acids or folic acid. They either act as competitive inhibitors of enzymes in purine nucleotides synthesis or can be incorporated into purine nucleo- tides. Thus they block purine nucleotides synthesis or interfere in nuc- leic acids synthesis.

20. 6-MP and 6-MG are purine analogues 6-MP can be converted to 6-MP nucleotide in the body. 6-MP nucleotide is structurally similar to IMP and inhibits conversion of IMP to AMP and GMP. It also blocks synthesis of PRA from PRPP , synthesis of AMP fromA , synthesis of GMP and IMP from G and H respectively.

21. Azaserine and diazonorleucine are amino acid analogues. They are analogues of Gln and interfere with Gln in purine nucleotide de novo synthesis. Aminopterine and MTX are folic acid analogues. They inhibit DHF reductase and block transfer of one carbon unit. Thus purine nucleotide systhesis is blocked.

22. Catabolism of Purine Nucleotide AMP undergoes hydrolysis and deamination, the A residue is converted to H. H is oxidized , yielding X and X is oxidizd , yielding uric acid. GMP is hydrolyzed and G is released. G is converted to X and X is oxidized yielding uric acid.

23. In the human body the purine ring can not be degraded. Uric acid contains the purine ring and is less soluble in water. Certain genetic defects in purine metabolism can cause high blood levels of uric acid and results in a disease known as gout.

24. In the disease sodium urate crystals are deposited in and around joints and in the kidney. Allopurinol is used to treat gout. It is an analogue of H and inhibits xanthine oxidase which catalyzes the oxidation of H and X. Thus it inhibits uric acid formation.

25. Pyrimidine Nucleotide Metabolism There are also two synthesis pathways of pyrimidine nucleotides: denovo and salvage pathway. De Novo Synthesis Pathway In de novo pathway the pyrimidine ring is assembled first and then linked to ribose phosphate. The carbon and nitrogen atoms in the pyrimidine ring are derived from bicarbonate, aspartate, and glutamine. fig 8-9

26. It begins with the formation of carbamoyl phosphate. Carbamoyl phosphate synthetase II in cytoplasm catalyzes the reaction. Gln + HCO3¯+ 2ATP----------------CP + Glu + 2ADP + Pi Carbamoyl phosphate reacts with aspartate to form carbamoyl aspartate . The closure of the pyrimidine ring is then calalyzed dihydroorotase.

27. The product undergoes oxidation, addition of ribose phosphate and decarboxylation, and is converted to UMP. fig 8-10 UMP is a precursor for the other pyrimidine nucleotides. Two sequential phosphorylation reactions form UTP which then accepts an amide nitrogen from Gln to form CTP. UMP-------UDP--------UTP UTP + Gln---CTP + Glu

28. d dUMP is produced by dephosphorylation of dUDP. The methylation of dUMP yields dTMP ( TMP ). Regulation of de novo Pathway In mammalian UMP inhibits CPS II. Puring and pyrimidine nucleotide synthesis are coodinately regulated.

29. Salvage Pathway Pyrimidine phosphoribosyl transferase catalyzes the following reaction. Py + PRPP-------------- PyMP + ppi Uridine kinase catalyzes the formation of UMP from uridine and ATP. UR + ATP------------- UMP + ADP

30. Antimetabolites of Pyrimidine Nucleotides 5FU is structurally similar to T. It is converted to FdUMP or FUTP in the body. FdUMP blocks the synthesis of dTMP. FUTP can be incorporated in an RNA molecule in the form of FUMP and thus inactivates it.

31. The machanism of amino acid and folic acid analogues interfering with pyrimidine nucleotide synthesis is similar to that with purine nucleotide synthesis. Some nucleoside analogues such as arabinocytidine and cyclocytidine are also important antimetabolites. Arabinocytidine inhibits reauction of CDP to dCDP.

32. Catabolism of Pyrimidine Nucleotide Pyrimidine nucleotides are hydrolyzed, yielding the building blocks of pyrimidine, ribose and phosphate. C and U are degraded to CO2, H2O and β alanine. T is degraded to CO2, H2O and β aminoisobutyric acid. fig 8-11