Nucleotide metabolism

1. Nucleotide metabolism Chemistry 256

2. Pigeons excrete only uric acid (a purine), so see where its carbons and nitrogens come from • Sonne, Buchanan and Delluva (University of Pennsylvania), Biological Precursors of uric acid: I. The role of lactate, acetate and formate in the synthesis of the ureide groups of uric acid, J. Biol. Chem. (1948) Various precursor compounds were labeled with carbon-13 and nitrogen-15, and fed to the pigeons; the uric acid was then analyzed by mass spectrometry.

3. The key molecule to purine synthesis is a nucleotide analog Starting with α-D-ribose-5-phosphate, there is an 11-step synthesis of IMP. Six ATPs are used.

4. IMP is rapidly converted into AMP and GMP

5. Inhibitors of nucleotide synthesis are used as immunosupressants Mycophenolic acid has a structure similar to a purine, and is a competitive inhibitor of IMP dehydrogenase, which prevents rapidly proliferating cells the supply of GMP needed for DNA synthesis and energy use.

6. Kinases complete the phosphorylation • AMP + ATP « ADP + ADP • GMP + ATP « GDP + ADP In these reactions, ΔG ≈ 0, so the equilibrium position is governed by the needs of the organism. For instance, to produce more ATP through oxidative phosphorylation, some ATP might be “sacrificed” to make more ADP to ultimately produce more ATP.

7. Of course, there is plenty of feedback inhibition. There is also an example of feedforward activation.

8. Pyrimidine biosynthesis was not as easily elucidated Jones and Hager (Brandeis University), Source of carbamyl phosphate for pyrimidine biosynthesis in mouse Ehrlich ascites cells and rat liver, Science (1966) demonstrated that a version of carbamoyl phosphate synthetase (CPS II) exists in the cytosol and uses glutamine’s amide as the nitrogen source (not ammonia, as with CPS I).

9. So, between glutamine and aspartate, the pyrimidine ring is pretty much complete

10. The product is the nucleotide uridine monophosphate (UMP) 2 ATPs are used. In addition, a quinone is reduced as the molecule gets rid of two electrons.

11. Once UMP is produced, kinases phosphorylate it to UTP, then CTP synthetase converts it to CTP

12. An anti-parasite drug strategy is to prevent parasites from having a ready supply of CPS II Toxoplasma gondii (toxoplasmosis) is prevented from making UDP by blocking its CPS II by the drug but it won’t affect human CPS II. However, no such drug exists at present.

13. Similar to purine biosynthesis, pyrimidine biosynthesis has feedback inhibition and feedforward activation as well.

14. The next step: convert ribonucleotides to deoxyribonucleotides Another bit of evidence that RNA preceded DNA evolutionarily.

15. Ribonucleotide reductases (RNRs) are the enzymes that do the conversion In addition to the active site, the enzyme has several allosteric sites. Whether in bacteria or mammals, the enzyme normallly exists as inactive monomers, but, when the specificity site is occupied, the somewhat-active dimeric form is made. When the activity site is occupied, a more active tetramer is made. The hexamer is the most active version.

17. Why the extreme complexity of regulation in RNRs? • The lack of dNTPs is fatal to any organism. • The excess of dNTPs increases the mutation rate in any organism. • Between the two extremes, the organism must respond to periods of increased demands for dNTPs (replication) followed by periods of decreased demand. • Principal control: the concentration of nucleotides within the cell.

18. The state of oxidation of various cysteine side chains controls RNR activity – NADPH is the source of the transferred electrons

19. dTMP is made from dUMP by using THF for a methyl transfer

20. Nucleotide breakdown begins by removing the base from the sugar

21. Purine breakdown ultimately yields uric acid XMP is derived from IMP; both are intermediates in purine synthesis.

22. Purine breakdown in muscle tissue yields fumarate; important for energy production Kevin Tornheim and John Lowenstein (Brandeis Univ.), “The Purine Nucleotide Cycle”, Journal of Biological Chemistry 247 (1971).

23. The last step produces uric acid, which needs to be excreted. Not all animals can excrete uric acid, which is not very soluble in water (crystals in joints cause gout, for instance). Thus, depending on how much water the organism has to spare, various reactions increase solubility of the nitrogen to be excreted.

24. Pyrimidines can be salvaged for energy through malonyl-CoA Unlike purine degradation, pyrimidines are broken down through reduction, rather than oxidation. for either fatty acid biosynthesis or production of succinyl-CoA