1. FCH 532 Lecture 29 Chapter 28: Nucleotide metabolism
Quiz on Mon (4/16): IMP synthesis-Purine synthesis
Quiz on Wed(4/18): Pyrimidine biosynthesis/regulation
Quiz on Friday(4/20): Ribonucleotide reductase mechanism
Friday (4/20): extra credit seminar, Dr. Jimmy Hougland,
145 Baker, 3-4PM.
ACS exam has been moved to Monday (4/30)
Quiz on Final is scheduled for May 4, 12:45PM-2:45PM, in 111 Marshall

2. Page 1071

3. Page 1074

4. Page 1077

5. Page 1080

6. Synthesis of UTP and CTP
Synthesis of pyrimidine nucleotide triphosphates is similar to purine nucleotide triphosphates.
2 sequential enzymatic reactions catalyzed by nucleoside monophosphate kinase and nucleoside diphosphate kinase respectively:
UMP + ATP  UDP + ADP
UDP + ATP  UTP + ADP

7. Figure 28-10 Synthesis of CTP from UTP.
Page 1080
CTP is formed by amination of UTP by CTP synthetase
In animals, amino group from Gln
In bacteria, amino group from ammonia

8. Regulation of pyrimidine nucleotide synthesis
Bacteria regulated at Reaction 2 (ATCase)
Allosteric activation by ATP
Inhibition by CTP (in E. coli) or UTP (in other bacteria).
In animals pyrimidine biosynthesis is controled by carbamoyl phosphate synthetase II
Inhibited by UDP and UTP
Activated by ATP and PRPP
Mammals have a second control at OMP decarboxylase (competitively inhibited by UMP and CMP)
PRPP also affects rate of OMP production, so, ADP and GDP will inhibit PRPP production.

9. Page 1080

10. Production of deoxyribose derivatives
Derived from corresponding ribonucleotides by reduction of the C2’ position.
Catalyzed by ribonucleotide reductases (RNRs)
dADP
ADP

11. Overview of dNTP biosynthesis
One enzyme, ribonucleotide reductase,
reduces all four ribonucleotides to their
deoxyribose derivatives.
A free radical mechanism is involved
in the ribonucleotide reductase
reaction.
There are three classes of ribonucleotide
reductase enzymes in nature:
Class I: tyrosine radical, uses NDP
Class II: adenosylcobalamin. uses NTPs
(cyanobacteria, some bacteria,
Euglena).
Class III: SAM and Fe-S to generate
radical, uses NTPs.
(anaerobes and fac. anaerobes).

12. Figure 28-12a. Class I ribonucleotide reductase from E. coli
Figure 28-12a Class I ribonucleotide reductase from E. coli. (a) A schematic diagram of its quaternary structure.
Page 1082

13. Proposed mechanism for rNDP reductase

14. Proposed reaction mechanism for ribonucleotide reductase
Free radical abstracts H from C3’
Acid-catalyzed cleavage of the C2’-OH bond
Radical mediates stabilizationof the C2’ cation (unshared electron pair)
Radical-cation intermediate is reduced by redox-active sulhydryl pair-deoxynucleotide radical
3’ radical reabstracts the H atom from the protein to restore the enzyme to the radical state.

15. Thioredoxin and glutaredoxin
Final step in the RNR catalytic cycle is the reduction of disulfide bond to reform the redox-active sulfyhydryl pair).
Thioredoxin-108 residue protein that has redox active Cys (Cys32 and Cys35)-also involved in the Calvin Cycle.
Reduces oxidized RNR and is regenerated via NADPH by thioredoxin reductase.
Glutaredoxin is an 85 residue protein that can also reduce RNR.
Oxidized glutaredoxin is reuced by NADPH using glutredeoxin reductase.

16. Sources of reducing power for rNDP reductase

17. Proposed reaction mechanism for ribonucleotide reductase
Free radical abstracts H from C3’
Acid-catalyzed cleavage of the C2’-OH bond
Radical mediates stabilizationof the C2’ cation (unshared electron pair)
Radical-cation intermediate is reduced by redox-active sulhydryl pair-deoxynucleotide radical
3’ radical reabstracts the H atom from the protein to restore the enzyme to the radical state.

18. dNTPs made by phosphorylation of dNDP
Reaction is catalyzed by nucleoside diphosphate kinase (same enzyme that phosphorylates NDPs)
dNDP + ATP  dNTP + ADP
Can use any NTP or dNTP as phosphoryl donor.

19. Thymine synthesis
2 main enzymes: dUTP diphosphohydrolase (dUTPase) and thymidylate synthase
Reaction 1
dTMP is made by methylation of dUMP.
dUMP is made by hydrolysis of dUTP via dUTP diphosphohydrolase (dUTPase)
dUTP + H2O  dUMP+ PPi
Done to minimize the concentration of dUTP-prevents incorporation of uracil into DNA.

20. Thymine synthesis + dUMP + dTMP Reaction 2
dTMP is made from dUMP by thymidylate synthase (TS).
Uses N5, N10-methylene-THF as methyl donor +
dUMP +
dTMP

21. Figure 28-19 Catalytic mechanism of thymidylate synthase.
Enzyme Cys thiolate group attacks C6 of dUMP (nucleophile).
C5 of the enolate ion attacks the CH2 group of the imium cation of N5, N10-methylene-THF.
Enzyme base abstracts the acidic proton at C5, forms methylene group and eliminates THF cofactor
Migration of the N6-H atom of THF to the exocyclic methylene group to form a methyl group and displace the Cys thiolate intermediate.
Page 1090

22. 5-flurodeoxyuridylate (FdUMP)
Antitumor agent.
Irreversible inhibitor of TS
Binds like dUMP but in step 3 of the reaction, F cannot be extracted.
Suicide substrate.
FdUMP F

23. Figure 28-20. The X-ray structure of the E
Figure The X-ray structure of the E. coli thymidylate synthase–FdUMP–THF ternary complex.
Page 1091

24. Thymine synthase oxidizes N5,N10-methyleneTHF
Only enzyme to change the oxidation state of THF.
Regenerated by 2 reactions
DHF is reduced to THF by NADPH by dihydrofolate reductase.
Serine hydroxymethyltransferase transfers the hydroxymethyl group of serine to THF to regenerate N5,N10-methylene-THF and produces glycine.

25. Figure 28-21 Regeneration of N5,N10-methylenetetrahydrofolate.
Page 1091

26. Nucleotide degradation
Nucleic acids can survive the acid of the stomach
Degraded into nucleotides by pancreatic nucleases and intestinal phosphodiesterases in the duodenum.
Components cannot pass through cell membranes, so they are hydrolyzed to nucleosides.
Nucleosides may be directly absorbed by the intestine or undergo further degradation to free bases and ribose or ribose-1-phosphate by nucleosidases and nucloside phosphorylase.
Nucleoside + H2O base + ribose
Nucleoside + Pi base + ribose-1-P
nucleosidase
Nucleoside phosphorylase

27. Purine nucleoside + Pi Purine base + ribose-1-P
Catabolism of purines
All pathways lead to formation of uric acid.
Intermediates could be intercepted into salvage pathways.
1st reaction is the nucleotidase and second is catalyzed by purine nucleoside phosphorylase (PNP)
Ribose-1-phosphate is isomerized by phosphoribomutase to ribose-5-phosphate (precursor to PRPP).
Purine nucleoside + Pi Purine base + ribose-1-P
Adenosine and deoxyadenosine are not degraded by PNP but are deaminated by adenosine deaminase (ADA) and AMP deaminase in mammals
Purine nucleoside phosphorylase

28. Figure 28-23 Major pathways of purine catabolism in animals.
ADA
Genetic defects in ADA kill lymphocytes and result in severe combined immunodeficiencey disese (SCID).
No ADA results in high levels of dATP that inhibit ribonucleotide reductase-no other dNTPs
Page 1093

29. Figure 28-24a. Structure and mechanism of adenosine deaminase
Figure 28-24a Structure and mechanism of adenosine deaminase. (a) A ribbon diagram of murine adenosine deaminase in complex with its transition state analog HDPR.
Page 1094

30. Figure 28-24b (b) The proposed catalytic mechanism of adenosine deaminase.
Zn2+ polarized H2O molecule nucleophilically attacks C6 of the adenosine. His is general base catalyst, Glu is general acid, and Asp orients water.
Results in tetrahedral intermediate which decomposes by elimination of ammonia.
Product is inosine in enol form (assumes dominant keto form upon release from enzyme).
Page 1094

31. Purine nucleotide cycle
Deamination of AMP to IMP combined with synthesis of AMP from IMP results in deaminating Asp to yield fumarate.
Important role in skeletal muscle-increased activity requires increased activity in the citric acid cycle.
Muscle replenishes citric acid cycle intermediates through the purine nucleotide cycle.

32. Figure 28-25 The purine nucleotide cycle.
Page 1095

33. Xanthine oxidase
Xanthine oxidse (XO) converts hypoxanthine to xanthine, and xanthine to uric acid.
In mammals, found in the liver and small intestine mucosa
XO is a homodimer with FAD, two [2Fe-2S] clusters and a molybdopterin complex (Mo-pt) that cycles between Mol (VI) and Mol (IV) oxidation states.
Final electron acceptor is O2 which is converted to H2O2
XO is cleaved into 3 segments. The uncleaved enzyme is known as xanthine dehydrogenase (uses NAD+ as an electron acceptor where XO does not).
XO hydroxylates hypoxanthine at its C2 position and xanthine at the C8 positon to produce uric acid in the enol form.

34. N-terminal domain is cyan
Figure 28-26a X-Ray structure of xanthine oxidase from cow’s milk in complex with salicylic acid.
N-terminal domain is cyan
Central domain is gold
C-terminal domain is lavender
Page 1095

35. Mechanism for XO
Reaction initiated by attack of enzyme nucleophile on the C8 position of xanthine.
The C8-H atom is eliminated as a hydride ion that combines with Mo (VI) complex, reducing it to Mo (IV).
Water displaces the enzyme nucleophile producing uric acid.

36. Figure 28-27 Mechanism of xanthine oxidase.
Page 1096

37. Figure 28-23 Major pathways of purine catabolism in animals.
ADA
Genetic defects in ADA kill lymphocytes and result in severe combined immunodeficiencey disese (SCID).
No ADA results in high levels of dATP that inhibit ribonucleotide reductase-no other dNTPs
Page 1093

38. Goal is the conservation of water.
Purine degredation in other animals
Primates, birds, reptiles, insects-final degradation product id uric acid which is excreted in urine.
Goal is the conservation of water.

39. Figure 28-29 The Gout, a cartoon by James Gilroy (1799).
Page 1097
Gout is a disease characterized by elevated levels of uric acid in body fluids. Caused by deposition of nearly insoluble crystals of sodium urate or uric acid.

40. Clinical disorders of purine metabolism
Excessive accumulation of uric acid: Gout
The three defects shown each result in elevated de novo purine biosynthesis

41. Common treatment for gout: allopurinol
Allopurinol is an analogue of hypoxanthine that strongly inhibits
xanthine oxidase. Xanthine and hypoxanthine, which are soluble, are accumulated and excreted.

42. Catabolism of pyrimidines
Animal cells degrade pyrimidines to their component bases.
Happen through dephosphorylation, deamination, and glycosidic bond cleavage.
Uracil and thymine broken down by reduction (vs. oxidation in purine catabolism).

43. Page 1098

44. Biosynthesis of of NAD and NADP+
Produced from vitamin precursors Nicotinate and Nicotinamide and from quinolinate, a Trp degradation product
Page 1099

45. Biosynthesis of FMN and FAD from riboflavin
FAD is synthesized from riboflavin in a two-reaction pathway.
Flavokinase phosphorylates the 5’OH group to give FMN
FAD pyrophosphorylase catalyzes the next step (coupling of FMN to ADP).
Page 1100

46. Biosynthesis of CoA from pantothenate
Page 1101