Nucleotides: Synthesis and Degradation

1. Nucleotides: Synthesis and Degradation

2. Nitrogenous Bases • Planar, aromatic, and heterocyclic • Derived from purine or pyrimidine • Numbering of bases is “unprimed”

3. Nucleic Acid Bases Pyrimidines Purines

4. Sugars • Pentoses (5-C sugars) • Numbering of sugars is “primed”

5. Sugars D-Ribose and 2’-Deoxyribose *Lacks a 2’-OH group

6. Nucleosides • Result from linking one of the sugars with a purine or pyrimidine base through an N-glycosidic linkage • Purines bond to the C1’ carbon of the sugar at their N9 atoms • Pyrimidines bond to the C1’ carbon of the sugar at their N1 atoms

7. Nucleosides

8. Phosphate Groups • Mono-, di- or triphosphates • Phosphates can be bonded to either C3 or C5 atoms of the sugar

9. Nucleotides • Result from linking one or more phosphates with a nucleoside onto the 5’ end of the molecule through esterification

10. Nucleotides • RNA (ribonucleic acid) is a polymer of ribonucleotides • DNA (deoxyribonucleic acid) is a polymer of deoxyribonucleotides • Both deoxy- and ribonucleotides contain Adenine, Guanine and Cytosine • Ribonucleotides contain Uracil • Deoxyribonucleotides contain Thymine

11. Nucleotides • Monomers for nucleic acid polymers • Nucleoside Triphosphates are important energy carriers (ATP, GTP) • Important components of coenzymes • FAD, NAD+ and Coenzyme A

12. Naming Conventions • Nucleosides: • Purine nucleosides end in “-sine” • Adenosine, Guanosine • Pyrimidine nucleosides end in “-dine” • Thymidine, Cytidine, Uridine • Nucleotides: • Start with the nucleoside name from above and add “mono-”, “di-”, or “triphosphate” • Adenosine Monophosphate, Cytidine Triphosphate, Deoxythymidine Diphosphate

13. In-Class Activities • Look at the Nucleotide Structures • Take the Nucleotide Identification Quiz • Be prepared to identify some of these structures on an exam. Learn some “tricks” that help you to distinguish among the different structures

14. Nucleotide Metabolism • PURINE RIBONUCLEOTIDES: formed de novo • i.e., purines are not initially synthesized as free bases • First purine derivative formed is Inosine Mono-phosphate (IMP) • The purine base is hypoxanthine • AMP and GMP are formed from IMP

15. Purine Nucleotides • Get broken down into Uric Acid (a purine) Buchanan (mid 1900s) showed where purine ring components came from: N1: Aspartate Amine C2, C8: Formate N3, N9: Glutamine C4, C5, N7: Glycine C6: Bicarbonate Ion

16. Purine Nucleotide Synthesis

17. Purine Nucleotide Synthesis at a Glance • ATP is involved in 6 steps • PRPP in the first step of Purine synthesis is also a precursor for Pyrimidine Synthesis, His and Trp synthesis • Role of ATP in first step is unique– group transfer rather than coupling • In second step, C1 notation changes from a to b (anomers specifying OH positioning on C1 with respect to C4 group) • In step 2, PPi is hydrolyzed to 2Pi (irreversible, “committing” step)

18. Coupling of Reactions • Hydrolyzing a phosphate from ATP is relatively easy G°’= -30.5 kJ/mol • If endergonic reaction released energy into cell as heat energy, wouldn’t be useful • Must be coupled to an exergonic reaction • When ATP is a reactant: • Part of the ATP can be transferred to an acceptor: Pi, PPi, adenyl, or adenosinyl group • ATP hydrolysis can drive an otherwise unfavorable reaction (synthetase; “energase”)

19. Purine Biosynthetic Pathway • Channeling of some reactions on pathway organizes and controls processing of substrates to products in each step • Increases overall rate of pathway and protects intermediates from degradation • In animals, IMP synthesis pathway shows channeling at: • Reactions 3, 4, 6 • Reactions 7, 8 • Reactions 10, 11

20. In Class Activity*** Calculate how many ATP equivalents are needed for the de novo synthesize IMP. Assume that all of the substrates (R5P, glutamine, etc) are available Note: You should be able to do this calculation for the synthesis of any of the nucleoside monophosphates

21. IMP Conversion to AMP

22. IMP Conversion to GMP

23. Regulatory Control of Purine Nucleotide Biosynthesis • GTP is involved in AMP synthesis and ATP is involved in GMP synthesis (reciprocal control of production) • PRPP is a biosynthetically “central” molecule (why?) • ADP/GDP levels – negative feedback on Ribose Phosphate Pyrophosphokinase • Amidophosphoribosyl transferase is activated by PRPP levels • APRT activity has negative feedback at two sites • ATP, ADP, AMP bound at one site • GTP,GDP AND GMP bound at the other site • Rate of AMP production increases with increasing concentrations of GTP; rate of GMP production increases with increasing concentrations of ATP

24. Regulatory Control of Purine Biosynthesis • Above the level of IMP production: • Independent control • Synergistic control • Feedforward activation by PRPP • Below level of IMP production • Reciprocal control • Total amounts of purine nucleotides controlled • Relative amounts of ATP, GTP controlled

25. Purine Catabolism and Salvage • All purine degradation leads to uric acid (but it might not stop there) • Ingested nucleic acids are degraded to nucleotides by pancreatic nucleases, and intestinal phosphodiesterases in the intestine • Group-specific nucleotidases and non-specific phosphatases degrade nucleotides into nucleosides • Direct absorption of nucleosides • Further degradation Nucleoside + H2O  base + ribose (nucleosidase) Nucleoside + Pi  base + r-1-phosphate (n. phosphorylase) NOTE: MOST INGESTED NUCLEIC ACIDS ARE DEGRADED AND EXCRETED.

26. Intracellular Purine Catabolism • Nucleotides broken into nucleosides by action of 5’-nucleotidase (hydrolysis reactions) • Purine nucleoside phosphorylase (PNP) • Inosine  Hypoxanthine • Xanthosine  Xanthine • Guanosine  Guanine • Ribose-1-phosphate splits off • Can be isomerized to ribose-5-phosphate • Adenosine is deaminated to Inosine (ADA)

27. Intracellular Purine Catabolism • Xanthine is the point of convergence for the metabolism of the purine bases • Xanthine  Uric acid • Xanthine oxidase catalyzes two reactions • Purine ribonucleotide degradation pathway is same for purine deoxyribonucleotides

28. Adenosine Degradation

29. Xanthosine Degradation • Ribose sugar gets recycled (Ribose-1-Phosphate  R-5-P ) • – can be incorporated into PRPP (efficiency) • Hypoxanthine is converted to Xanthine by Xanthine Oxidase • Guanine is converted to Xanthine by Guanine Deaminase • Xanthine gets converted to Uric Acid by Xanthine Oxidase

30. Xanthine Oxidase • A homodimeric protein • Contains electron transfer proteins • FAD • Mo-pterin complex in +4 or +6 state • Two 2Fe-2S clusters • Transfers electrons to O2 H2O2 • H2O2 is toxic • Disproportionated to H2O and O2 by catalase

31. THE PURINE NUCLEOTIDE CYCLE AMP + H2O  IMP + NH4+(AMP Deaminase) IMP + Aspartate + GTP  AMP + Fumarate + GDP + Pi(Adenylosuccinate Synthetase) COMBINE THE TWO REACTIONS: Aspartate + H2O + GTP  Fumarate + GDP + Pi+ NH4+ The overall result of combining reactions is deamination of Aspartate to Fumarate at the expense of a GTP

32. Purine Nucleotide Cycle*** In-Class Question: Why is the purine nucleotide cycle important in muscle metabolism during a burst of activity?

33. Uric Acid Excretion • Humans – excreted into urine as insoluble crystals • Birds, terrestrial reptiles, some insects – excrete insoluble crystals in paste form • Excess amino N converted to uric acid • (conserves water) • Others – further modification : Uric Acid  Allantoin  Allantoic Acid  Urea  Ammonia

34. Purine Salvage • Adenine phosphoribosyl transferase (APRT) Adenine + PRPP  AMP + PPi • Hypoxanthine-Guanine phosphoribosyl transferase (HGPRT) Hypoxanthine + PRPP  IMP + PPi Guanine + PRPP  GMP + PPi (NOTE: THESE ARE ALL REVERSIBLE REACTIONS) AMP,IMP,GMP do not need to be resynthesized de novo !

35. A CASE STUDY : GOUT • A 45 YEAR OLD MAN AWOKE FROM SLEEP WITH A PAINFUL AND SWOLLEN RIGHT GREAT TOE. ON THE PREVIOUS NIGHT HE HAD EATEN A MEAL OF FRIED LIVER AND ONIONS, AFTER WHICH HE MET WITH HIS POKER GROUP AND DRANK A NUMBER OF BEERS. • HE SAW HIS DOCTOR THAT MORNING, “GOUTY ARTHRITIS” WAS DIAGNOSED, AND SOME TESTS WERE ORDERED. HIS SERUM URIC ACID LEVEL WAS ELEVATED AT 8.0 mg/dL (NL < 7.0 mg/dL). • THE MAN RECALLED THAT HIS FATHER AND HIS GRANDFATHER, BOTH OF WHOM WERE ALCOHOLICS, OFTEN COMPLAINED OF JOINT PAIN AND SWELLING IN THEIR FEET.

36. A CASE STUDY : GOUT • THE DOCTOR RECOMMENDED THAT THE MAN USE NSAIDS FOR PAIN AND SWELLING, INCREASE HIS FLUID INTAKE (BUT NOT WITH ALCOHOL) AND REST AND ELEVATE HIS FOOT. HE ALSO PRESCRIBED ALLOPURINOL. • A FEW DAYS LATER THE CONDITION HAD RESOLVED AND ALLOPURINOL HAD BEEN STOPPED. A REPEAT URIC ACID LEVEL WAS OBTAINED (7.1 mg/dL). THE DOCTOR GAVE THE MAN SOME ADVICE REGARDING LIFE STYLE CHANGES.

37. Impaired excretion or overproduction of uric acid Uric acid crystals precipitate into joints (Gouty Arthritis), kidneys, ureters (stones) Lead impairs uric acid excretion – lead poisoning from pewter drinking goblets Fall of Roman Empire? Xanthine oxidase inhibitors inhibit production of uric acid, and treat gout Allopurinol treatment – hypoxanthine analog that binds to Xanthine Oxidase to decrease uric acid production Gout

41. ALLOPURINOL IS A XANTHINE OXIDASE INHIBITORA SUBSTRATE ANALOG IS CONVERTED TO AN INHIBITOR, IN THIS CASE A “SUICIDE-INHIBITOR”

42. ALCOHOL CONSUMPTION AND GOUT Choi HK, Atkinson K, Karlson EW et al. . 2004. “Alcohol intake and risk of incident gout in men: a prospective study”. Lancet 363: 1277-1281

43. A defect in production or activity of HGPRT Causes increased level of Hypoxanthine and Guanine ( in degradation to uric acid) Also,PRPP accumulates stimulates production of purine nucleotides (and thereby increases their degradation) Causes gout-like symptoms, but also neurological symptoms  spasticity, aggressiveness, self-mutilation First neuropsychiatric abnormality that was attributed to a single enzyme Lesch-Nyhan Syndrome

44. 25% of autistic patients may overproduce purines To diagnose, must test urine over 24 hours Biochemical findings from this test disappear in adolescence Must obtain urine specimen in infancy, but it’s difficult to do! Pink urine due to uric acid crystals may be seen in diapers Purine Autism

45. IN-CLASS QUESTION*** • IN von GIERKE’S DISEASE, OVERPRO- DUCTION OF URIC ACID OCCURS. THIS DISEASE IS CAUSED BY A DEFICIENCY OF GLUCOSE-6-PHOSPHATASE. • EXPLAIN THE BIOCHEMICAL EVENTS THAT LEAD TO INCREASED URIC ACID PRODUCTION? • WHY DOES HYPOGLYCEMIA OCCUR IN THIS DISEASE? • WHY IS THE LIVER ENLARGED?

46. Pyrimidine Ribonucleotide Synthesis • Uridine Monophosphate (UMP) is synthesized first • CTP is synthesized from UMP • Pyrimidine ring synthesis completed first; then attached to ribose-5-phosphate N1, C4, C5, C6 : Aspartate C2 : HCO3- N3 : Glutamine amide Nitrogen

47. Pyrimidine Synthesis

48. UMP Synthesis Overview • 2 ATPs needed: both used in first step • One transfers phosphate, the other is hydrolyzed to ADP and Pi • 2 condensation rxns: form carbamoyl aspartate and dihydroorotate (intramolecular) • Dihydroorotate dehydrogenase is an intra-mitochondrial enzyme; oxidizing power comes from quinone reduction • Attachment of base to ribose ring is catalyzed by OPRT; PRPP provides ribose-5-P • PPi splits off PRPP – irreversible • Channeling: enzymes 1, 2, and 3 on same chain; 5 and 6 on same chain

49. OMP DECARBOXYLASE : THE MOST CATALYTICALLY PROFICIENT ENZYME • FINAL REACTION OF PYRIMIDINE PATHWAY • ANOTHER MECHANISM FOR DECARBOXYLATION • A HIGH ENERGY CARBANION INTERMEDIATE NOT NEEDED • NO COFACTORS NEEDED ! • SOME OF THE BINDING ENERGY BETWEEN OMP AND THE ACTIVE SITE IS USED TO STABILIZE THE TRANSITION STATE • “PREFERENTIAL TRANSITION STATE BINDING”