1. Chapter 26 The Synthesis and Degradation of Nucleotides Biochemistryby
Reginald Garrett and Charles Grisham

2. Outline Can Cells Synthesize Nucleotides?
How Do Cells Synthesize Purines?
Can Cells Salvage Purines?
How Are Purines Degraded?
How Do Cells Synthesize Pyrimidines?
How Are Pyrimidines Degraded?
How Do Cells Form the Deoxyribonucleotides That Are Necessary for DNA Synthesis?
How Are Thymine Nucleotides Synthesized?

3. 26.1 – Can Cells Synthesize Nucleotides?
Nearly all organisms synthesize purines and pyrimidines "de novo biosynthesis pathway"
Many organisms also "salvage" purines and pyrimidines from diet and degradative pathways
Ribose generates energy, but purine and pyrimidine rings do not
Nucleotide synthesis pathways are good targets for anti-cancer/antibacterial strategies

4. 26.2 – How Do Cells Synthesize Purines?
John Buchanan (1948) "traced" the sources of all nine atoms of purine ring
N-1: aspartic acid
N-3, N-9: glutamine
C-2, C-8: N10-formyl-THF - one carbon units
C-4, C-5, N-7: glycine
C-6: CO2

5. Figure 26.1 Nitrogen waste is excreted by birds principally as the purine analog, uric acid.

6. IMP Biosynthesis
The first purine product of this pathway, IMP (inosinic acid or inosine monophosphate)
First step: Ribose-5-phosphate pyrophosphokinase
PRPP synthesis from ribose-5-phosphate and ATP
PRPP is limiting substance for purine synthesis
But PRPP is a branch point so next step is the committed step (fig 26.6)
Second step: Gln PRPP amidotransferase
Form phosphoribosyl-b-amine; Changes C-1 configuration (a→b)
GMP and AMP inhibit this step - but at distinct sites
Azaserine - Gln analog - inhibitor/anti-tumor

7. Figure 26.3 The de novo pathway for purine synthesis.
Step 1: Ribose-5-phosphate pyrophosphokinase.
Step 2: Glutamine phosphoribosyl pyrophosphate amidotransferase.
Step 3: Glycinamide ribonucleotide (GAR) synthetase.
Step 4: GAR transformylase.
Step 5: FGAM synthetase (FGAR amidotransferase).
Step 6: FGAM cyclase (AIR synthetase).
Step 7: AIR carboxylase.
Step 8: SAICAR synthetase.
Step 9: adenylosuccinase.
Step 10: AICAR transformylase.
Step 11: IMP synthase.

8. Figure 26. 4 The structure of azaserine
Figure 26.4  The structure of azaserine. Azaserine acts as an irreversible inhibitor of glutamine-dependent enzymes by covalently attaching to nucleophilic groups in the glutamine-binding site.

9. Step 3: Glycinamide ribonucleotide (GAR) synthetase
Glycine carboxyl condenses with amine in two steps
Glycine carboxyl activated by -P from ATP
Amine attacks glycine carboxyl
Synthesize glycinamide ribonucleotide
Step 4: Glycinamide ribonucleotide (GAR) transformylase
Formyl group of N10-formyl-THF is transferred to free amino group of GAR
Yield N-Formylglycinamide ribonucleotide

10. Step 5: Formylglycinamide ribonucleotide (FGAR) amidotransferase
Formylglycinamidine ribonucleotide (FGAM)
FGAM synthetase
C-4 carbonyl forms a P-ester from ATP and active NH3 attacks C-4 to form imine
Irreversibly inactivated by azaserine

11. Closure of the first ring, carboxylation and attack by aspartate
Step 6: FGAM cyclase (AIR synthetase)
Produce aminoimidazole nucleotide (AIR)
Similar in some ways to step 5. ATP activates the formyl group by phosphorylation, facilitating attack by N.
In avian liver, the enzymes for step 3, 4, and 6 (GAR synthetase, GAR transformylase, and AIR synthetase) reside on a polypeptide

12. Step 8: SAICAR synthetase
Step 7: AIR carboxylase
The product is carboxyaminoimidazole ribonucleotide (CAIR)
Carbon dioxide is added in ATP-dependent reaction
Step 8: SAICAR synthetase
N-succinylo-5-aminoimidazole-4-carboxamide ribonucleotide
Attack by the amino group of aspartate links this amino acid with the carboxyl group
The enzymes for steps 7 and 8 reside on a bifunctional polypeptide in avian

13. Step 9: adenylosuccinase
The product is 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR); remove fumarate
AICAR is also an intermediate in the histidine biosynthetic pathway
Step 10: AICAR transformylase
N-formylaminoimidazole-4-carboxamide ribonucleotide (FAICAR)
Another 1-C addition (N10-formyl-THF)
Step 11: IMP synthase (IMP cyclohydrolase)
Amino group attacks formyl group to close the second ring
The enzymes for steps 10 and 11 reside on a bifunctional polypeptide in avian

14. 6 ATPs, but that this is really 7 ATP equivalents
The dependence of purine biosynthesis on THF in two steps means that sulfonamides block purine synthesis in bacteria

16. Sulfa drugs, or sulfonamides, owe their antibiotic properties to their similarity to p-aminobenzoate (PABA),an important precursor in folic acid synthesis. Sulfonamides block folic acid formation by competing with PABA.

17. AMP and GMP are synthesized from IMP
IMP is the precursor to both AMP and GMP
AMP synthesis
Step 1: adenylosuccinate synthetase
the 6-O of inosine is displaced by aspartate to yield adenylosuccinate
GTP is the energy input for AMP synthesis, whereas ATP is energy input for GMP
Step 2: adenylosuccinase (adenylosuccinate lyase)
carries out the nonhydrolytic removal of fumarate from adenylosuccinate, leaving AMP.
the same enzyme catalyzing Step 9 in the purine pathway

18. Starting from ribose-5-phosphate
GTP synthesis
Step 1: IMP dehydrogenase
Oxidation at C-2
NAD+-dependent oxidation
xanthosine monophosphate (XMP)
Step 2: GMP synthetase
Replacement of the O by N (from Gln)
ATP-dependent reaction; PPi
Starting from ribose-5-phosphate
8 ATP equivalents are consumed in the AMP synthesis
9 ATP equivalents in GMP synthesis

19. Figure 26.5 The synthesis of AMP and GMP from IMP.

20. The regulation of purine synthesis
Reciprocal control occurs in two ways
IMP synthesis:
Allosterically regulated at the first two steps
R-5-P pyrophosphokinase:
ADP & GDP
phosphoribosyl pyrophosphate amidotransferase
A “series”: AMP, ADP, and ATP
G “series”: GMP, GDP, and GTP
PRPP is “feed-forward” activator

21. AMP synthesis:
adenylosuccinate synthetase is feedback-inhibited by AMP
GMP synthesis:
IMP dehydrogenase is feedback-inhibited by GMP

22. Nucleoside diphosphate and triphosphate
Nucleoside diphosphate: ATP-dependent kinase
Adenylate kinase: AMP +ATP → ADP +ADP
Guanylate kinase: GMP +ATP → GDP +ADP
Nucleotide triphosphate: non-specific enzyme
Nucleoside diphosphate kinase
GDP +ATP  GTP +ADP
NDP +ATP  NTP +ADP (N=G, C, U, and T)

23. 26.3 – Can Cells Salvage Purines?
Salvage pathways
Recover them in useful form
Collect hypoxanthine and guanine and recombine them with PRPP to form nucleotides in the HGPRT reaction
Absence of HGPRT is cause of Lesch-Nyhan syndrome (sex-linked); In Lesch-Nyhan, purine synthesis is increased 200-fold and uric acid is elevated in blood
HGPRT & APRT

24. Hyperxanthine-Guanine PhosphoRibosylTransferase
Figure Purine salvage by the HGPRT reaction.

26. 26.4 – How Are Purines Degraded?
Purine catabolism leads to uric acid
Nucleotidases and nucleosidases release ribose and phosphates and leave free bases
Nucleotidase: NMP + H2O → nucleoside + Pi
Nucleosidase: nucleoside + H2O → base + ribose
PNP: nucleoside + Pi → base + ribose-P
The PNP products are converted to xanthine by xanthine oxidase and guanine deaminase
Xanthine oxidase converts xanthine to uric acid
Note that xanthine oxidase can oxidize two different sites on the purine ring system
Neither adenosine nor deoxyadenosine is a substrate for PNP
Converted to inosine by adenosine deaminase (ADA)

27. Figure 26. 8 The major pathways for purine catabolism in animals
Figure The major pathways for purine catabolism in animals. Catabolism of the different purine nucleotides converges in the formation of uric acid.

28. Severe combined immunodeficiency syndrome (SCID)
The effect of elevated levels of deoxyadenosine on purine metabolism. If ADA is deficient or absent, deoxyadenosine is not converted into deoxyinosine as normal (see Figure 26.8). Instead, it is salvaged by a nucleoside kinase, which converts it to dAMP, leading to accumulation of dATP and inhibition of deoxynucleotide synthesis (see Figure 26.24). Thus, DNA replication is stalled.

29. Purines nucleotide cycle
Serve as an anaplerotic pathway in skeletal muscle
AMP deaminase
Adenylosuccinate synthetase
Adenylosuccinate lyase

30. Figure The purine nucleoside cycle for anaplerotic replenishment of citric acid cycle intermediates in skeletal muscle.

31. Xanthine Oxidase and Gout
Xanthine Oxidase in liver, intestines mucosa, and milk can oxidize hypoxanthine to xanthine and xanthine to uric acid
Humans and other primates excrete uric acid in the urine, but most N goes out as urea
Birds, reptiles and insects excrete uric acid and for them it is the major nitrogen excretory compound
Gout occurs from accumulation of uric acid crystals in the extremities
Allopurinol, which inhibits XO, is a treatment

32. Figure 26.11 Allopurinol, an analog of hypoxanthine, is a potent inhibitor of xanthine oxidase.
Figure Xanthine oxidase catalyzes a hydroxylase-type reaction.

33. Animals other than humans oxidize uric acid to form excretory products
Urate oxidase: Allantoin
Allantoinase: Allantoic acid
Allantoicase: Urea
Urease: Ammonia
Figure The catabolism of uric acid to allantoin, allantoic acid, urea, or ammonia in various animals.

34. 26.5 – How Do Cells Synthesize Pyrimidines?
In contrast to purines, pyrimidines are not synthesized as nucleotides
The pyrimidine ring is completed before a ribose-5-P is added
Carbamoyl-P and aspartate are the precursors of the six atoms of the pyrimidine ring

35. Figure 26.15 The de novo pyrimidine biosynthetic pathway.

36. de novo Pyrimidine Synthesis
Step 1: Carbamoyl Phosphate synthesis
Carbamoyl phosphate for pyrimidine synthesis is made by carbamoyl phosphate synthetase II (CPS II)
This is a cytosolic enzyme (whereas CPS I is mitochondrial and used for the urea cycle)
Substrates are HCO3-, glutamine (not NH4+), 2 ATP
In mammals, CPS-II can be viewed as the committed step in pyrimidine synthesis
Bacteria have but one CPS; thus, the committed step is the next reaction, which is mediated by aspartate transcarbamoylase (ATCase)

37. (also called carbonyl-phosphate)
Figure The reaction catalyzed by carbamoyl phosphate synthetase II (CPS II).

38. Step 2: Aspartate transcarbamoylase (ATCase)
catalyzes the condensation of carbamoyl phosphate with aspartate to form carbamoyl-aspartate
carbamoyl phosphate represents an ‘activated’ carbamoyl group
Step 3: dihydroorotase
ring closure and dehydration via intramolecular condensation
Produce dihydroorotate

39. Step 4: dihydroorotate dehydrogenase
Synthesis of a true pyrimidine (orotate)
Step 5: orotate phosphoribosyltransferase
Orotate is joined with a ribose-P to form orotidine-5’-phosphate (OMP)
The ribose-P donor is PRPP
Step 6: OMP decarboxylase
OMP decarboxylase makes UMP (uridine-5’-monophposphate, uridylic acid)

40. Metabolic channeling In bacteria, the six enzymes are distinct
Eukaryotic pyrimidine synthesis involves channeling and multifunctional polypeptides
CPS-II, ATCase, and dihydroorotase are on a cytosolic polypeptide
Orotate PRT and OMP decarboxylase on the other cytosolic polypeptide (UMP synthase)
The metabolic channeling is more efficient

41. UTP and CTP Nucleoside monophosphate kinase
UMP + ATP → UDP + ADP
Nucleoside diphosphate kinase
UDP + ATP → UTP + ADP
CTP sythetase forms CTP from UTP and ATP

42. Regulation of pyrimidine biosynthesis
In bacteria
allosterically inhibited at ATCase by CTP (or UTP)
allosterically activated at ATCase by ATP (compete with CTP)
In animals
UDP and UTP are feedback inhibitors of CPS II
PRPP and ATP are allosteric activators

43. Figure A comparison of the regulatory circuits that control pyrimidine synthesis in E. coli and animals.

44. 26.6 – How Are Pyrimidines Degraded?
In humans, pyrimidines are recycled from nucleosides (via phosphoribosyltransferase), but free pyrimidine bases are not salvaged
Catabolism of cytosine and uracil yields -alanine, ammonium, and CO2
-alanine can be recycled into the synthesis of coenzyme A
Catabolism of thymine yields -aminoisobutyric acid, ammonium, and CO2

45. Figure 26. 18 Pyrimidine degradation
Figure Pyrimidine degradation. Carbons 4, 5, and 6 plus N-1 are released as b-alanine, N-3 as NH4+, and C-2 as CO2. (The pyrimidine thymine yields b-aminoisobutyric acid.) Recall that aspartate was the source of N-1 and C-4, -5, and -6, while C-2 came from CO2 and N-3 from NH4+ via glutamine.

46. 26.7 – How Do Cells Form the Deoxyribonucleotides That Are Necessary for DNA Synthesis?
Reduction at 2’-position of ribose ring
Serve as precursor for DNA synthesis
Replacement of 2’-OH with hydride is catalyzed by ribonucleotide reductase
An 22-type enzyme - subunits R1 (86 kD) and R2 (43.5 kD)
R1 has two regulatory sites, a specificity site and an overall activity site

47. Figure Deoxyribonucleotide synthesis involves reduction at the 2'-position of the ribose ring of nucleoside diphosphates.

48. Ribonucleotide Reductase
The enzyme system consists of 4 proteins
Two of which constitute the Ribonucleotide Reductase (a2b2)
Thioredoxin and thioredoxin reductase deliver reducing equivalents
Has three different nucleotide-binding sites
Substrate: NDPs
Activity-determining: ATP & dATP
Specificity-determining: ATP, dTTP, dGTP, and dATP

49. Figure 26.20 E. coli ribonucleotide reductase: its binding sites and subunit organization.

50. Activity depends on Cys439, Cys225, and Cys462 on R1 and on Tyr122 on R2 (generate free radical)
Tyr122 free radical on R2 leads to removal of the Ha hydrogen (Cys439) and creation of a C-3‘ radical
dehydration via removal of Hb together with the C-2‘-OH group and restoration of Ha to C-3' forms the dNDP product
accompanied with disulfide formation between Cys225 and Cys462

51. Thioredoxin provides the reducing power for ribonucleotide reductase
NADPH is the ultimate source
Sulfide : sulfhydryl transition
Figure The (¾S¾S¾)/(¾SH HS¾) oxidation-reduction cycle involving ribonucleotide reductase, thioredoxin, thioredoxin reductase, and NADPH.

52. Glutaredoxin can also function in ribonucleotide reductase
Oxidized Glutaredoxin is reduced by 2 Glutathione
Figure The structure of  glutathione.

53. Regulation of dNTP Synthesis
The overall activity of ribonucleotide reductase must be regulated
ATP activates, dATP inhibits at the overall activity site
Balance of the four deoxynucleotides must be controlled
ATP, dATP, dTTP and dGTP bind at the specificity site to regulate the selection of substrates and the products made

54. Figure Regulation of deoxynucleotide biosynthesis: The rationale for the various affinities displayed by the two nucleotide-binding regulatory sites on ribonucleotide reductase.

55. 26.8 – How Are Thymine Nucleotides Synthesized?
Thymine nucleotides are made from dUMP, which derives from dUDP, dCDP
Thymidylate synthase methylates dUMP at 5-position to make dTMP
N5,N10-methylene THF is 1-C donor
If the dCDP pathway is traced from the common pyrimidine precursor, UMP, it will proceed as follows:
UMP ® UDP ® UTP ® CTP ® CDP ® dCDP ® dCMP ® dUMP ® dTMP

56. Figure 26.26 The dCMP deaminase reaction.

57. Figure 26. 27 The thymidylate synthase reaction
Figure The thymidylate synthase reaction. The 5-CH3 group is ultimately derived from the b-carbon of serine.

58. Figure Precursors and analogs of folic acid employed as antimetabolites: sulfonamides (see Human Biochemistry box on page 858), as well as methotrexate, aminopterin, and trimethoprim, whose structures are shown here.
These compounds shown here bind to dihydrofolate reductase (DHFR) with about 1000-fold greater affinity than DHF and thus act as virtually irreversible inhibitors.

59. Fluoro-substituted analogs as therapeutic agents
The effect of the 5-fluoro substitution on the mechanism of action of thymidylate synthase. An enzyme thiol group (from a Cys side chain) ordinarily attacks the 6-position of dUMP so that C-5 can react as a carbanion with N5,N10-methylene-THF. Normally, free enzyme is regenerated following release of the hydrogen at C-5 as a proton. Because release of fluorine as F+ cannot occur, the ternary (three-part) complex of [enzyme: flourouridylate:methylene-THF] is stable and persists, preventing enzyme turnover. (The N5,N10-methylene-THF structure is given in abbreviated form.)

60. HB page 876
The structures of 5-fluorouracil (5-FU),5-fluorocytosine, and 5-fluoroorotate.