1. Synthesis and Degradation of Nucleotides Part 1: September 1 st, 2009 Champion CS Deivanayagam Center for Biophysical Sciences and Engineering University of Alabama at Birmingham Birmingham, AL 35294-4400

2. Information Transfer in Cells The fundamental process of information transfer in cells.

3. Purines and Pyrimidines Note that the numbering are slightly different particularly where the glycosidic bonds are attached Gylocosidic bond

4. Nucleotide: purines and pyrimides linked to a ribose/de-oxy ribose sugar moiety

5. Two types of pathways leads to nucleotides Two types of pathways leads to nucleotides : 1. De novo pathway – Begins with their metabolic precursors: amino acids, ribose-5-phosphate, and CO2 – The free bases A, T, G, C, and U are not intermediates – Appears to be present in identical form in nearly all living organisms 2. Salvage pathway – Recycle the free bases and nucleosides released from nucleic acid breakdown – The free bases are intermediates

6. De Novo Purine Nucleotide Synthesis I. De Novo Purine Nucleotide Synthesis The initially synthesized purine derivative is inosine. Purines are initially formed as ribonucleotides rather than as free bases AMP (Adenosine monophosphate) GMP (Guanosine monophosphate)

7. John Buchanan (1948) "traced" the sources of all nine atoms of the purine ring Origin of the nine atoms: Bird feed containing selectively labeled atoms Examination of the isotope distribution in excreted uric acid (dove poop research!!!) N-1 from aspartic acid N-3, N-9 from glutamine C-4, C-5, N-7 from glycine C-6 from CO 2 C-2, C-8 from THF - one carbon units

9. 11 steps lead to the formation Of IMP (Ionosine mono-phospate)

10. The names of the enzymes can be a mouthful !!! In addition each of these enzymes have AKA’s (common name and EC names) It would be great if you could memorize them, however it is not necessary to memorize all the steps in this reaction What you need to learn from this lecture ? 1. What are the Committed steps that are unique in this synthesis cycle 2. What are the different feed back inhibition steps in this synthesis cycle 3. What steps can be utilized to develop inhibitors in this synthesis cycle 4. What are some of the diseases that are related to this synthesis cycle

12. A committed step is an effectively irreversible reaction in the biosynthesis pathway

13. Glutamine PRPP amidotransferase is subject to feedback inhibition GMP, GDP, GTP as well as AMP, ADP and ATP carry out this action. How? Through an allosteric site present on the enzyme. The G series of nucleotides at a Guanine-specific allosteric site on the enzyme and The A series of nucleotides at an Adenine-specific allosteric site on the enzyme Glutamine PRPP amidotransferase is also inhibited by ‘azaserine’ Azaserine acts as an irreversible inhibitor of glutamine-dependent enzymes by covalently attaching to nucleophilic groups in the glutamine-binding site. It is used as an anti-tumor agent.

14. Allosteric enzyme cartoon representation

16. (aka FGAM synthetase)

18. (aka Adenylosuccinate lyase) (aka IMP cyclohydrolase)

20. In vertebrates these reactions are coupled together One multifunctional polypeptide chain (110 kda) encodes for: GAR synthetase (step 3) GAR tranformylase (step 4) AIR synthetase (step 6) Another encodes for AIR carboxyalse (step 7) SACAIR synthetase (step 8) Another polypeptide chain 67 kD (organized as 135 kDa dimers) AICAR tranformylase (step 10) IMP synthase (step 11)

21. Folate Analogs as Antimicrobial and Anticancer Agents De novo purine biosynthesis depends on folic acid compounds at steps 4 and 10 For this reason, antagonists of folic acid metabolism indirectly inhibit purine formation and, in turn, nucleic acid synthesis, cell growth, and cell development Rapidly growing cells, such as infective bacteria and fast-growing tumors, are more susceptible to such agents Sulfonamides are effective anti- bacterial agents Methotrexate and aminopterin are folic acid analogs that have been used in cancer chemotherapy

22. Step 12: Synthesis of Adenine and Guanine Ribonucleotides: a). AMP is made from IMP in two steps. The first step converts IMP to adenylosuccinate. The second step is catalyzed by adenylosuccinate lyase that produces AMP. b). The formation of GMP from IMP requires oxidation at C-2 of the purine ring, followed by a glutamine-dependent amidotransferase reaction that replaces the oxygen on C-2 with an amino group to yield 2-amino, 6-oxy purine nucleoside monophosphate – i.e., GMP. The second reaction is catalyzed by GMP synthetase, shown here.

23. The Purine Biosynthetic Pathway is Regulated at Several Steps Allosteric regulation occurs in the first two steps, and AMP and GMP are competitive inhibitors in the two branches at right.

24. Can Cells Salvage Purines? Nucleic acid turnover (synthesis and degradation) is an ongoing process in most cells Purines that are obtained from diet and turn over and not degraded can be reconverted to nucleosided tri-phosphates and be reused. Nucleotides are then converted to nucleosides by specific nucleotidases and non-specific phosphotases NMP + H 2 O  nucleoside + P i Nucleosides are hydrolyzed by nucleosidases or nucleoside phophorylases to release the purine base Nucleoside + H2O  Nucleosideases  base + ribose Nucleoside + Pi  Nucleoside phosphorylase  base + ribose-1-P Salvage pathways collect hypoxanthine and guanine and recombine them with PRPP to form nucleotides in the HGPRT reaction

25. Major pathways of purine catabolism in animals.

27. Animals Oxidize Uric Acid to Different Excretory Products

28. HGPRT Converts Bases Back to Nucleotides Notice: PRPP is also involved in the salvage pathway. HGPRT - Hypoxanthine-guanine phosphoribosyltransferase

29. Lesch-Nyhan Syndrome – HGPRT Deficiency Leads to a Severe Disorder Victims of Lesch-Nyhan syndrome experience severe arthritis due to accumulation of uric acid, as well as retardation, and other neurological symptoms. Lesch-Nyhan syndrome results from a complete deficiency in HGPRT. Absence of HGPRT is the cause of Lesch-Nyhan syndrome In L-N, purine synthesis is increased 200-fold and uric acid is elevated in blood

30. Major pathways of purine catabolism in animals.

31. Lack of Adenosine Deaminase is One Cause of Severe Combined Immunodeficiency Syndrome SCID is a group of related disorders involving diminished immune responses. 30% of SCID patients lack the enzyme adenosine deaminase. In the absence of ADA, deoxyadenosine is not deaminated to deoxyinosine as normal (above).

32. Gout is a Disease Caused by an Excess of Uric Acid Xanthine oxidase (XO) in liver, intestines (and milk) can oxidize hypoxanthine (twice) to uric acid Humans and other primates excrete uric acid in the urine, but most N goes out as urea Birds, reptiles and insects excrete uric acid and for them it is the major nitrogen excretory compound Gout occurs from accumulation of uric acid crystals in the extremities Precipitation and deposition of uric acid causes arthritic pain and kidney stones Causes: impaired excretion of uric acid and deficiencies in HGPRT Allopurinol, an analog of hypoxanthine, is a potent inhibitor of xanthine oxidase. Allopurinol binds tightly to xanthine oxidase, preventing uric acid formation. Hypoxanthine and xanthine do not accumulate to harmful concentrations because they are more soluble and thus more easily excreted.

33. Summary of disorders of Purine Metabolism: DisorderDefect Comments Gout PRPP synthase/ Hyperuricemia HGPRT Lesch Nyhanlack of HGPRT Hyperuricemia syndrome SCID ADA High levels of dAMP von Gierke’s diseaseglucose 6-phosphatase Hyperuricemia Reading assignment:

34. Tomorrow: 1. Pyrimindine synthesis/degradation 2. Deoxyribonucleotide synthesis