Nucleotide Metabolism (and then, you know…)

1. Nucleotide Metabolism (and then, you know…)

2. 1865 – Gregor Mendel: proposes the existence of paired factors of heredity • 1869 – Friedrich Miescher: isolates nucleinand discovers that it was acidic and contained a lot of phosphate • 1928 –Fred Griffith: observes the transformation of non-pathogenic bacteria through exchange of material with pathogenic ones • 1944 – Oswald T. Avery, Colin McLeod and Maclyn McCarty: isolate the transforming agent -DNA • 1950 – Erwin Chargaff: the 1:1 ratio of adenine to thymine and guanine to cytosine • 1953 – James Watson and Francis Crick (with help from Maurice Wilkins and Rosalind Franklin): identify the double helical structure of DNA • 1977 – Frederick Sanger: DNA sequencing • 1996– Ian Wilmut: Dolly the Sheep • 2003 – The Human Genome Project: the sequencing of 3.2 billion base pairs along 23 DNA molecules measuring 2 meters • 2010 – Craig Venter: synthetic DNA

3. BIOSYNTHESIS AND DEGRADATION OF NUCLEOTIDES • Nucleotides are composed of a nitrogenous base, sugar and phosphate (s) • base and sugar only = nucleoside • Nucleotides are precursors of nucleic acids (DNA, RNA), energy rich compounds (ATP, GTP, UTP, CTP), coenzymes (NAD/P+ ), FAD, CoA, SAM, Vit B 12) and signaling molecules (cAMP, cGMP) • Small amounts of nucleic acids in the diet • Pancreatic ribonuclease and deoxyribonuclease change the nucleic acids into their nucleotide monomers • Intestinal phosphatase converts nucleotides into nucleosides • The greater portion of nucleosides is modified and excreted or recycled back to nucleotides

4. The bases of nucleotides are eitherpyrimidine or purine • Pyrimidines are characterized by a six-membered heterocyclic aromatic ring with two nitrogens • Purines have an additional five-membered imidazole ring fused with the aromatic ring of pyrimidines • The common naturally occurring pyrimidines are cytosine, uraciland thymine (5-methyuracil) • Purines: cytosine and guanine • Other naturally occurring purine derivatives include xanthine, hypoxanthine and uric acid

6. The sugar found in ribonucleotides is D-ribose and in deoxyribonucleotides is 2-deoxy D-ribose • The pentose sugars are attached to the nitrogenous bases through a β-N-glycosidicbond to give nucleosides

7. Inpentoses of nucleosides (and nucleotides), the carbon numbers are given a prime designation (,) to distinguish them from the numbered atoms of the bases • Nucleosides are named by adding –idineto the root name of pyrimidines and –osineto the root name of purines: cytidine, thymidine, uridine, adenosine, guanosine, inosine • The prefix deoxy- added in the case of deoxyribonucleosides • Deoxythymidine, deoxyuridine, …

8. Nucleosides are converted to nucleotides through the linking of a phosphate with the 5’ hydroxyl group of ribose or deoxyribose • Adenosine 5’-monophosphate (AMP), cytidine 5’ monophosphate (CMP),… • More phosphates could be added to give nucleoside di- and triphosphates ; or a single phosphate group can be esterified with two hydroxyls of the pentose to give a cyclic nucleotide

9. Two types of pathways lead to the synthesis of nucleotides: the de novo pathways and the salvage pathways • de novo synthesis begins with amino acids, ribose 5-phosphate and CO2 • Salvage pathways recycle free bases and nucleosides obtained from nucleic acid breakdown (from nucleic acid turnover or from the diet) • During de novo synthesis new bases are synthesized • purine nucleotides are constructed a few atoms at a time on a ribose-based structure • the framework for pyrimidine bases is first synthesized and is then attached to ribose and modified further • On the other hand, free bases can serve as intermediates in salvage pathways

10. Purine nucleotide synthesis • De novo: is initiated using an activated form of ribose • Ribose-5-phosphate from PPP reacts with ATP to produce 5-phospho-α-D-ribosyl-1-pyrophosphate (PRPP) • The pyrophosphate of PRPP is displaced by the amide nitrogen of glutamine in a reaction catalyzed by glutamine PRPP amidotransferase • The committed step of de novo purine synthesis

11. Once 5-phospho-β-D-ribosylamine is formed, the building of the purine ring begins • First, the whole of a glycine molecule is added to 5-phospho-β-D-ribosylamine and phosphoribosylglycinamide is formed • After eight steps, the first purine nucleotide inosinemonophosphate (IMP) is produced • Additional nitrogen comes from glutamine and aspartate • N10-formyl THF and CO2 provide additional carbons • Four ATP molecules are used in the process The sources of the atoms in the purine ring

12. IMP is converted to AMP or GMP: • the keto group of IMP is replaced by an amino group (provided by aspartate) in AMP; requires GTP • the dehydrogenation of IMP using NAD+ yields xanthosinemonophosphate (XMP); XMP is then converted to GMP through the donation of the amino group of glutamine; requires ATP

14. Nucleoside triphosphates are the most common types of nucleotide in metabolism • The production of nucleoside diphosphates requires specific (for type of base) nucleoside monophosphatekinases whereas nucleoside triphosphate formation is catalyzed by nucleoside diphosphatekinases that are not specific for sugar or base • ATP+NMP→ADP+NDP • NTPD+NDPA →NDPD+NTPA • The regulation of de novo purine synthesis • The committed step is inhibited by excess levels of IMP, GMP and ATP • An excess of GMP inhibits the formation of XMP whereas an excess of AMP inhibits the synthesis of adenylosuccinate • ATP is needed for the synthesis of GMP and GTP is required for the synthesis of AMP • the pools of the two nucleotides are balanced

16. PRPP synthesis is allostericaly inhibited by ADP, GDP and other metabolites from other pathways which use PRPP as a precursor • Because the de novo pathway consumes much energy, cells synthesize a significant portion of their nucleotides by salvage pathways • Purine salvage pathways • Free purine bases or (deoxy) ribonucleosides can be changed back to mono nucleotides • The liver is the major site of purine nucleotide metabolism; it provides purines and purine nucleosides for other tissues • in some tissues, the de novo pathway produces little or no purine nucleotides – these tissues are dependent upon export from the liver

17. In most tissues AMP is changed by 5’-nucleotidase to adenosine • Adenosine can then be deaminated by adenosine deaminase to inosine and inosine can then be converted to IMP • In adenosine deaminase deficiency large concentrations of dATP (because adenosine deaminase acts also on deoxyadenosine) inhibit ribonucleotide reductase; consequently DNA synthesis is depressed • Severe combined immunodeficiency (SCID) - T and B lymphocytes are mainly affected • Adenosine can also be converted to AMP by adenosine kinase • Muscular work is accompanied by the production of ammonia, the immediate source of which is AMP • AMP is changed to IMP with the associated release of NH4+; the enzyme involved is known as AMP aminohydrolaseor AMP deaminase

18. IMP is changed back to AMP through the de novo pathway • The net effect is the generation of fumarate which can serve an anaplerotic role • This is known as the purine nucleotide cycle • It operates in the brain as well as in the muscles • Adenosine is thought to be a local hormone or autocoid • During strenuous activity, muscles release adenosine which has a vasodilatory effect leading to increased delivery of oxygen and nutrients • Adenosine slows the heart rate by blocking the flow of electrical current • Purine nucleoside phosphorylaseconverts inosine, guanosine and xanthosine to hypoxanthine, guanine and xanthine, respectively • Deficiency of purine nucleotide phosphorylase affects T cells

19. Purine bases can react with PRPP under the influence of phosphoribosyltransferases • Adenosine phosphoribosyltransferase catalyzes the reaction of adenine with PRPP to give AMP • Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) catalyses the reaction of guanine and hypoxanthine with PRPP • The deficiency of HGPRT causes the Lesch-Nyhan syndrome • guanine and hypoxanthine will be degraded instead of being salvaged • mental retardation, self-mutilation

21. Purine catabolism • Hypoxanthine is oxidized to xanthine by xanthineoxidase • Guanine is deaminated by guanine aminohydrolaseto xanthine • Xanthine is converted to uric acid by xanthineoxidase • Xanthineoxidase is a flavoenzyme with an atom of molybdenum and four iron-sulfur centers; it produces H2O2 • in some plants xanthine is trimethylated to caffeine • During periods of extended wakefulness, metabolic activities in the brain lead to the accumulation of adenosine • Adenosine induces sleepiness; caffeine promoteswakefulness by blocking the interaction of extracellular adenosine with neuronal receptors • In primates uric acid is excreted in the urine as it is • In other mammals urateoxidasechanges uric acid to allantoin; in other organisms allantoin to allantoate to urea to NH4+

23. Urate is present in the serum of humans at levels near precipitation • This may be because of the use of urate as an anti-oxidant • Gout (from gutta, “drop”) is a common disorder resulting from hyperuricemia: > 7 mg/dL in males, > 6 mg/dL in females • It causes inflammation of the joints– gouty arthritis • Crystals of sodium urate are deposited in the joints • The deposition attracts macrophages that engulf the crystals • Lysosomal membranes of the macrophages are ruptured by the crystals; this results in the release of lysosomal enzymes and leukotriene B4 into the tissues • Histamine is released from mast cells • Visible structures known as tophi may form near joints causing deformities • The deposition of urate crystals within the renal tubules causes impaired renal function

24. Primary gout results from genetic defects in purine metabolism • An overactive PRPP synthase leading to an increase in purine synthesis • Deficiency of HGPRT means less salvage reactions • Glucose-6-phosphatase deficiency leads to the accumulation of glucose-6-phosphate which will be diverted to ribose-5-phosphate and then to PRPP • Secondary or acquired gout is caused by various factors that can cause an overproduction of urate or its underexcretion by the kidneys • Gout can be treated with dietary control and drugs • Allopurinolis a suicide inhibitor of xanthineoxidase • Allopurinol is changed to alloxanthine (oxypurinol) which is a competitive inhibitor of xanthineoxidase • Hypoxanthine and xanthine can be excreted in the urine

25. Allopurinol can also react with PRPP producing allopurinolribonucleotide thereby reducing purine synthesis • Colchicineinhibits the polymerization of tubulin into microtubules • The division and movement of and phagocytosis by white blood cells is inhibited • Lekotriene B4 and histamine release is curtailed • In earlier times gout was thought to be associated with rich diets and excessive consumption of alcoholic drinks • Lead salts used to be added as preservatives in alcoholic drinks; pots lined with lead-containing materials • Lead inhibits guanine aminohydrolase leading to the accumulation of guanine in the joints – Saturnine gout • The bone stores Pb+2 because it is divalent just like Ca+2; through time Pb+2 is transported to other tissues including the kidney • treatment by chelating agents like ethylenediaminetetraaceticacid (EDTA)

27. The de novo biosynthesis of pyrimidine nucleotides • The synthesis of pyrimidines begins with the formation of carbamoyl phosphate by CPS II – a cytosolicisozyme • Uses glutamine as nitrogen donor instead of free ammonia • Carbamoyl phosphate reacts with aspartate to give carbamoyl aspartate; the enzyme is aspartate transcarbamoylase (ATCase) • The closure of the pyrimidine ring is then catalyzed by dehydration through the action of dihydroorotase • Dihyrdroorotate is then oxidized to orotate by dihydroorotate dehydrogenase ( a flavoprotein) • the NADH produced can enter the electron transport chain • CPS II, aspartate transcarbamoylase and dihydroorotate dehydrogenase are part of a single trifunctional protein known as CAD • At this point, the basic framework of pyrimidine bases has been constructed

29. Orotate and PRPP react under the catalysis of orotatephosphoribosyltransferaseto yield orotidine-5-phosphate (OMP) • OMP is then decarboxylated to uridylate (UMP) by OMP decarboxylase • UMP is phosphorylated to UTP • UTP receives an amide group from glutamine to become cytidinetriphosphate (CTP) • Both orotatephosphoribosyltransferase and OMP decarboxylase activities are located on a protein called UMP synthase • The deficiency of UMP synthase leads to a condition as oroticaciduria - orotate in the urine, retarded growth, anemia • Can be treated with uridine which inhibits the production of orotate and provides building blocks for nucleic acid synthesis

30. The sources of the atoms in the pyrimidine ring

31. The regulation of de novo pyrimidine synthesis • CPS II is activated by PRPP and inhibited by UTP • As cells approach the S-phase of the cell cycle CPS II becomes more sensitive to activation by PRPP and less sensitive? to inhibition by UTP • phosphorylation of CPS II by MAP kinase at a specific site leads to an easily activated enzyme while phosphorylation at another site would lead to an easily inhibited enzyme • The ATCase reaction is the committed step of pyrimidine synthesis; its regulation in bacteria has been discussed under allosteric regulation of enzymes • inhibited by CTP and activated by ATP • The synthesis of thymidylate • DNA contains thymine instead or uracil • The route to thymidine is through deoxyribonucleotides

32. Deoxyribonucelotides are synthesized from the ribonucleotides by reduction of the 2’-carbon of D-ribose • The enzyme involved is ribonucleotide reductase • The substrates are diphosphonucleotides • The reducing equivalents are ultimately donated by NADPH via thioredoxinorglutaredoxin • UDP is changed to dUDP; dUDP is phosphorylated to dUTP and then pyrophosphate is removed from dUTP to produce dUMP • Or dCDP can be changed to dCMP which is deaminated to dUMP • dUMP receives a methyl group from N5,N10- methylene THF to give dTMP which will be phosphorylated to dTTP • the enzyme is thymidylatesynthase

36. The thymidylatesynthase reaction converts THF to dihydrofolate (DHF) • THF is regenerated through the action of dihydrofolate reductase with NADPH providing the reducing equivalents • Rapidly dividing cells require ample supplies of thymidylate for DNA synthesis; nucleotide biosynthesis can be targeted to design important drugs • azaserineis an antibiotic that inhibits glutamine amidotransferases • fluorouracil is changed in the body to fluorodeoxyridylate (FdUMP) which in turn binds to thymidylatesynthase inhibiting it • Examples of mechanism-based inactivation • methotrexate (amethopterin) andaminopterinare competitive inhibitors of dihydrofolate reductase • Fluorouracil, methotrexate and aminopterin are important anticancer agents; trimethoprim- is an antibiotic with a higher affinity for bacterial DHFR

38. Pyrimidine salvage pathways • Mammalian cells reutilize a small amount of free pyrimidine bases • Pyrimidine phosphorylase can synthesize uridine and cytidine (through the reaction of uracil and cytosine with ribose-1-P) but it cannot use thymine • A ribonucleoside containing thymine is not normally synthesized in the body • Instead, thymine will react with deoxyribose-1-Pto form thymidine • The nucleosides uridine, cytidine, thymidine and deoxycytidine are phosphorylated to their respective nucleotides • Thymidine kinase (TK) levels increase significantly during the S phase of the cell cycle

39. Salvage of pyrimidine bases • Pyrimidine catabolism • The pyrimidine ring can be degraded unlike that of purines • Cytosine is degraded through uracil • Cytidine and deoxycytidine are first deaminated to uridine and deoxyuridine or: • dCMP (deoxycytidylate) can be deaminated to dUMP (deoxyuridylate) and 5’-nucleotidase converts dUMP to uridine

40. Uridine and cytidine are changed to uracil and cytosine by pyrimidine phosphorylase • dTMP changed to thymine by the successive actions of 5’-nucleotidase and nucleoside phosphorylase • In addition to CO2 and NH4+ (which enters the urea cycle), the degradation of uracil gives β - alanineand thymine gives β- aminoisobutyrate • β - alanine and β-aminoisobutyrate can be excreted • Or β - alanine can further be processed to malonyl-CoA and β-aminoisobutyrate to methylmalonyl-CoA • In some cases β-aminoisobutyrate is produced in excessive amounts – genetic predisposition (Chinese or Japanese ancestry) and leukemia (massive cell destruction)