Nucleotides contain; Base + sugar + phosphoryl group

1. NUCLEOTIDES, NUCLEIC ACID STRUCTURE AND FUNCTION Prof. Dr. Yıldız DİNÇERCerrahpaşa Medical Faculty Department of Biochemistry

2. Nucleotides contain; • Base + sugar + phosphoryl group

3. Purines and pyrimidines are heterocyclic compounds including N atoms

4. Nucleosides contain; • Base + sugar (ribose or deoxyribose)

5. Sugar, D-ribose or 2’-deoxy D-ribose, is linked to base via a covalent β-N-glycosidic bond to N-9 of a purine or to N-1 of a pyrimidine • Nucleotides are termed ribonucleotides or deoxyribonucleotides based on whether the sugar is ribose or 2’-deoxyribose

6. DNA nucleotides

7. RNA nucleotides

8. Nucleotides are phosphorolated nucleosides • Mononucleotides are nucleotides singly phosphorolated on hydroxyl group of the sugar • AMP → Adenosine monophosphate • Adenine + ribose +phosphate • Additional phosphates linked by acid anhydride bonds to the existing phosphate of a mononucleotide form nucleoside di- and triphosphates

9. ADP → Adenosine diphosphate • Adenine + ribose +phosphate +phosphate • ATP → Adenosine triphosphate • Adenine + ribose +phosphate +phosphate + phosphate

10. Functions of nucleotides • *Nucleic acid biosynthesis • *Energy production and transduction • *Protein biosynthesis • *Regulatory cascades • *İntra- and intercellular signal transduction • *Biosynthesis some biomolecules

11. Some properties of nucleotides • 1.Mononucleotides have a negative charge at physiological pH • 2.Nucleotides absorb UV light • 3. Many coenzymes are nucleotide derivatives • 4. Synthetic nucleotide analogs are used in chemotherapy • 5. Nucleoside triphosphates have high group transfer potential • 6.Some nucleotides are involved in signal transduction

12. 1.Mononucleotides have a negative charge at physiological pH. The pKs of the primary and secondary phosphoryl groups are about 1.0 and 6.2, respectively • Nucleosides and or free purine or pyrimidine bases are uncharged at physiological pH

13. 2.Nucleotides absorb UV light. The conjugated double bonds of the heterocyclic bases of purines and pyrimidines ensure that nucleosides, nucleotides and polynucleotides absorb UV • Their spectra are pH- dependent but all common nucleotides absorb light at a wawelength close to 260 nm at pH 7,0 • Nucleotide and nucleic acid concentrations thus often are expressed in termes of ‘absorbance at 260 nm’

14. 3. Many coenzymes are nucleotide derivatives • AMP is present in many coenzymes

18. 4. Synthetic nucleotide analogs are used in chemotherapy • Chemically synthesized analogs of purines and pyrimidines, their nucleosides and their nucleotides find numerious applications in clinical medicine • Administration of an analog in which either the heterocyclic ring or the sugar moiety has been altered induces toxic effect when the analog is incorporated into specific cellular constituents

19. Their effects reflect one of two processes: • a)Inhibition by the drug of specific enzymes essential for nucleic acid synthesis • b)Incorporation of metabolites of the drug into nucleic acids, thus they effect the base pairing required for accurate replication

20. Examples • 5-fluoro uracil → thymine analog • 5-iodo deoxyuridine → thyminide analog • 6-mercaptopurine → purine analaog

21. 6-thioguanine → purine analaog • 8-azaguanine → purine analaog • 5 or 6-azauridine → pirimidine analaog • 6-azacytidine → pirimidine analaog

22. 4-hydroxypyrazolopyrimidine • (allopurinol) → purine analaog • Allopurinol inhibits de novo • purine biosynthesis and • xanthine oxidase activity. • It is used for treatment of • hyperuricemia and gout

23. The nucleoside, cytarabine (arabinosyl cytosine), in which arabinose replaces ribose, is used in chemotherapy and in treatment of viral infections • Azathioprine is catabolized to 6-mercaptopurine and is used during organ transplantation to suppress immunological rejection

24. 5. Nucleoside triphosphates have high group transfer potential because of acid anhydride bonds • High group transfer potential of nucleoside triphosphates allows them to participate as group transfer reagents in various reactions. • In these reactions, cleavage of an acid anhydride bond is coupled with a highly endergonic reaction such as protein synthesis or nucleic acid synthesis.

25. ADP and ATP are substrates and products,respectively, for oxidative phosphorylation • ATP serves as the major biologic transducer of free energy • It is the most abundant free nucleotide in mammalian cells (1 mM) • ATP donates some of its chemical energy by hydrolysis of the terminal phosphoanhydride bond

26. The hydrolytic cleavage of the terminal phosphoanhydride bond in ATP separates one of the three negatively charged phosphates and thus relieves some of the electrostatic repulsion in ATP • Released Pi is stabilized by the formation of several resonance forms not possible in ATP • ADP2- is the other product of hydrolysis and it immediately ionizes, releasing H+ into a medium of very low [H+]

28. For hydrolysis reactions with large, negative standard free energy changes, the products are more stable than the reactants

29. Transphosphorylations between nucleotides • Although we have focused on ATP as the cell’s energy currency and donor of phosphoryl groups, all other nucleoside triphosphates (GTP, UTP, CTP) and all the deoxynucleoside triphosphates (dATP, dGTP, dTTP and dCTP) are energetically equivalent to ATP • The free energy changes associated with hydrolysis of their phosphoanhydride linkages are very nearly identical with those for ATP

30. In preparation for their various biological roles, these other nucleotides are generated as the nucleoside triphosphate (NTP) forms by phosphoryl group transfer to the corresponding nucleoside diphosphates (NDPs) and monophosphates (NMPs) • ATP is the primary high-energy phosphate compound produced by catabolism in the processes of glycolysis, oxidative phosphorylation. Several enzymes carry phosphoryl groups from ATP to the other nucleotides

31. Nucleoside diphosphate kinases, found in all cells, catalyzes the reaction • Mg2+ • ATP+NDP(or dNDP) → ADP+NTP(or dNDP) • Although this reaction is fully reversible the relatively high ATP/ADP ratio in cells normally drives the reaction to the right, with the net formation of NTPs and dNTPs

32. Phosphoryl group transfers from ATP result in an accumulation of ADP. For example, when muscle is concracting vigorously ADP accumulates and interferes with ATP-dependent contraction. • During periods of intense demand for ATP, the cell lowers the ADP concentration, and at the same time acquires ATP, by the action of adenylate kinase: • Mg2+ • 2ADP → ATP + AMP • ←

33. This reaction is fully reversible, so after the intense demand for ATP ends, the enzyme can recycle AMP by converting it to ADP which can then be phosphorylated to ATP in mitochondria • A similar enzyme guanylate kinase, converts GMP to GDP at the expense of ATP. By-pathways such as these, energy conserved in the catabolic production of ATP is used to supply the cell with all required NTPs and dNTPs

34. 6.Some nucleotides are involved in signal transduction • cAMP and cGMP

35. cAMP (adenosine 3’,5’-monophosphate) • The cyclic phosphodiester cAMP is formed from ATP by the reaction catalyzed by adenylyl cyclase

36. cAMP is a second messenger in signal transduction • Adenylyl cyclase activity is regulated by complex interactions, many of which involve hormon receptors • As a second messenger, cAMP participates numerous regulatory functions by activating cAMP dependent protein kinases • cAMP is broken down by cAMP phosphodiesterase

37. Intracellular cAMP level is maintained by the interaction of adenylyl cyclase and phosphodiesterase

38. cGMP (guanosine 3’,5’-monophosphate) • cGMP is a second messenger in signal transduction that can act antagonistically to cAMP • cGMP is formed from GMP by guanylyl cyclase • Both adenylyl and guanylyl cyclases are regulated by effectors that include hormones • A phosphodiesterase hydrolyzes cGMP to GMP

39. An increase in the level of the cGMP as response to the nitric oxide serves as the main second messenger during events that characterize the relaxation of smooth muscle

40. Other functions of free nucleotides • 1. ADENOSINE DERIVATIVE NUCLEOTIDES • Adenosine 3’-phosphate 5’-phosphosulfate (active sulfate) • This is the sulfate donor for the formation of the sulfated proteoglycans or urinary metabolites of drugs excreted as sulfate conjugates

42. S-adenosylmethionine (SAM) • This is a form of active methionine • SAM serves as a methyl group donor for methylation reactions • SAM forms propylamine which is required in polyamine synthesis

44. 2. GUANOSINE DERIVATIVE NUCLEOTIDES • Guanosine nucleotides participate in the conversion of succinyl-CoA to succinate, a reaction that is coupled to the substrate level phosphorylation of GDP to GTP • GTP is required for • * activation of adenylyl cyclase (as a allosteric regulator) • **protein synthesis (as a energy source)

45. 3. HYPOXANTHINE DERIVATIVE NUCLEOTIDES • Hypoxanthine ribonucleotide is inosine monophosphate (IMP) • It is precursor for purine nucleotides • IMP is formed by deamination of AMP and amination of IMP re-forms AMP • Nucleotide inosine (hypoxanthine + ribose) is an intermediate in the purine solvage cycle

46. 4. URACYL DERIVATIVE NUCLEOTIDES • UDP-sugar derivatives participate in sugar epimerization • UDP-glucose is the glucosyl donor for biosynthesis of glycogen and glucosyldisaccharides • Other UDP-sugars act as sugar donors for biosynthesis of oligosaccharides of glycoproteins and proteoglycans • UDP-glucuronic acid is the glucuronyl group donor for conjugation reactions

47. 5. CYTOSINE DERIVATIVE NUCLEOTIDES • CTP is required for the biosynthesis of some phosphoglycerides • CTP is required for the biosynthesis of sphingomyelin and other substituted sphingosines

48. Polynucleotides • Mononucleotidesarecovalentlylinkedthroughphosphate-group ‘bridges’ • Specifically, the 5’-OH group of onenucleotideunit is joinedtothe 3’-OH group of thenextnucleotideby a phosphodiesterlinkageresultingtorelease of onemoleculewater • Thisresults in a dinucleotide • Thetermoligonucleotide is usedforpolymerscontaining 50 orfewernucleotides • Mononucleotides are covalently linked through phosphate-group ‘bridges’ • Specifically, the 5’-OH group of one nucleotide unit is joined to the 3’-OH group of the next nucleotide by a phosphodiester linkage resulting to release of one molecule water • This results in a dinucleotide • The term oligonucleotide is used for polymers containing 50 or fewer nucleotides

50. Polynucleotides are directional macromolecules • Since the phosphodiester bond links 3’- and 5’- carbons of adjacent monomers, each end of a polimers is distinct. They are reffered as ‘3’-end’ or ‘5’-end’ of polynucleotides • In the most representations displaying only the base sequences 5’ end is shown on the left and 3’ end is shown on the right: • 5’ GTATTGC 3’