1. Nucleic Acids Metabolism

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: Nucleotides:
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. 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

14. 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

15. Purine Nucleotide Synthesis

16. 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)

17. Coupling of Reactions
Hydrolyzing a phosphate from ATP is relatively easy
G°’= kJ/mol
If exergonic reaction released energy into cell as heat energy, wouldn’t be useful
Must be coupled to an endergonic 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)

18. 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

19. IMP Conversion to AMP

20. IMP Conversion to GMP

21. 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

22. 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

23. Purine Catabolism and Salvage
All purine degradation leads to uric acid
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.

24. 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)

25. 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

26. Adenosine Degradation

27. 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

28. 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

29. 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

30. 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

31. Purine Salvage Adenine + PRPP  AMP + PPi
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 !

32. 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

33. Pyrimidine Synthesis

34. 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

35. UMP  UTP and CTP
Nucleoside monophosphate kinase catalyzes transfer of Pi to UMP to form UDP; nucleoside diphosphate kinase catalyzes transfer of Pi from ATP to UDP to form UTP
CTP formed from UTP via CTP Synthetase driven by ATP hydrolysis
Glutamine provides amide nitrogen for C4 in animals

37. Regulatory Control of Pyrimidine Synthesis
Differs between bacteria and animals
Bacteria – regulation at ATCase rxn
Animals – regulation at carbamoyl phosphate synthetase II
UDP and UTP inhibit enzyme; ATP and PRPP activate it
UMP and CMP competitively inhibit OMP Decarboxylase
*Purine synthesis inhibited by ADP and GDP at ribose phosphate pyrophosphokinase step, controlling level of PRPP  also regulates pyrimidines

38. Degradation of Pyrimidines
CMP and UMP degraded to bases similarly to purines
Dephosphorylation
Deamination
Glycosidic bond cleavage
Uracil reduced in liver, forming b-alanine
Converted to malonyl-CoA  fatty acid synthesis for energy metabolism

39. Deoxyribonucleotide Formation
Purine/Pyrimidine degradation are the same for ribonucleotides and deoxyribonucleotides
Biosynthetic pathways are only for ribonucleotide production
Deoxyribonucleotides are synthesized from corresponding ribonucleotides

40. DNA vs. RNA: REVIEW DNA composed of deoxyribonucleotides
Ribose sugar in DNA lacks hydroxyl group at 2’ Carbon
Uracil doesn’t (normally) appear in DNA
Thymine (5-methyluracil) appears instead

41. Formation of Deoxyribonucleotides
Reduction of 2’ carbon done via a free radical mechanism catalyzed by “Ribonucleotide Reductases”
E. coli RNR reduces ribonucleoside diphosphates (NDPs) to deoxyribonucleoside diphosphates (dNDPs)
Two subunits: R1 and R2
A Heterotetramer: (R1)2 and (R2)2

42. Thymine Formation
Formed by methylating deoxyuridine monophosphate (dUMP)
UTP is needed for RNA production, but dUTP not needed for DNA
If dUTP produced excessively, would cause substitution errors (dUTP for dTTP)
dUTP hydrolyzed by dUTPase
(dUTP diphosphohydrolase) to dUMP  methylated at C5 to form dTMP rephosphorylate to form dTTP