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. Nucleotides and Nucleic Acids
Nucleotides: (nucleoside + phosphate)
Biological molecules that possess a heterocyclic nitrogenous base, a five-carbon sugar (ribose), and phosphate as principal components (chapter 10)
Participate as essential intermediates in cellular metabolisms—NAD, FAD, ATP, cAMP…
The elements of heredity and the agents of genetic information transfer—nucleic acids
Nucleic acids:
Nucleotides are the monomeric units of nucleic acid
Two basic kinds of nucleic acids
Deoxyribonucleic acid (DNA)
Ribonucleic acid (RNA)

4. 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 can be catabolized to generate energy, but nitrogenous bases do not
Nucleotide synthesis pathways are good targets for anti-cancer/antibacterial strategies

5. 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

6. 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.

7. IMP Biosynthesis
IMP (inosinic acid or inosine monophosphate) is the immediate precursor to GMP and AMP
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 - Glutamine analog - inhibitor/anti-tumor

8. Figure 26. 4 The structure of azaserine
Figure 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

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

13. 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

14. 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

16. Step 9: Adenylosuccinase (also see Fig 26.5)
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

18. 6 ATPs are required in the purine biosynthesis from ribose-5-phosphate to IMP, but that this is really 7 ATP equivalents
The dependence of purine biosynthesis on THF (tetrahydrofolate) in two steps means that methotrexate and sulfonamides block purine synthesis

19. Tetrahydrofolate and One-Carbon Units
Folic acid, a B vitamin found in green plants, fresh fruits, yeast, and liver, is named from folium, Latin for “leaf”.
Folates are acceptors and donors of one-carbon units for all oxidation levels of carbon except CO2 (for which biotin is the relevant carrier).
The active form is tetrahydrofolate.

20. Tetrahydrofolate and One-Carbon Units
Folates are acceptors and donors of one-carbon units for all oxidation levels of carbon except CO2 (for which biotin is the relevant carrier).

21. Tetrahydrofolate and One-Carbon Units
Oxidation numbers are calculated by assigning valence bond electrons to the more electronegative atom and then counting the charge on the quasi ion. A carbon assigned four valence electrons would have an oxidation number of 0. The carbon in N5-methyl-THF (top left) is assigned six electrons from the three C-H bonds and thus has a oxidation number of -2.

23. Folate Analogs as Antimicrobial and Anticancer Agents
De novo purine biosynthesis depends on folic acid compounds at steps 4 and 10
For this reason, antagonists of folic acid metabolism indirectly inhibit purine formation and, in turn, nucleic acid synthesis, cell growth, and cell development
Rapidly growing cells, such as infective bacteria and fast-growing tumors, are more susceptible to such agents
Sulfonamides are effective anti-bacterial agents
Methotrexate and aminopterin are folic acid analogs that have been used in cancer chemotherapy

24. 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.

25. AMP and GMP are Synthesized from IMP
Figure 26.5 The synthesis of AMP and GMP from IMP.

26. 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

27. 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

28. 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

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

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

31. 26.3 – Can Cells Salvage Purines?
Salvage pathways
Nucleic acid turnover (synthesis and degradation) is an ongoing metabolic process
mRNA in particular is actively synthesized and degraded
Lead to release of free purines; adenine, guanine, and hypoxanthine (the base in IMP; Fig 26.8)
Salvage pathways exist to recover them in useful form
Involve resynthesis of nucleotides from bases via phosphoribosyltransferases (PRT)

32. 26.3 – Can Cells Salvage Purines?
Base + PRPP
Nucleoside-5’-phosphate + PPi
The purine phosphoribosyltransferases are adenine phosphoribosyltransferases (APRT) and hypoxanthine-guanine phosphoribosyltransferases (HGPRT)
Collect hypoxanthine and guanine and recombine them with PRPP to form nucleotides in the HGPRT reaction (Fig 26.7)
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

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

34. Victims of Lesch-Nyhan syndrome experience severe arthritis due to accumulation of uric acid, as well as retardation, and other neurological symptoms.

35. 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)

36. 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.

37. 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.

38. Convert aspartate to fumarate plus NH4+
The purine nucleoside cycle in skeletal muscle Serve as an anaplerotic pathway
Convert aspartate to fumarate plus NH4+
Figure 26.9 The purine nucleoside cycle for anaplerotic replenishment of citric acid cycle intermediates in skeletal muscle.

39. 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 xanthine oxidase , is a treatment

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

41. 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.

42. 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

43. Figure 26.15 The de novo pyrimidine biosynthetic pathway.

44. 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)

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

46. 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

47. 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)

48. 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

49. 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

50. 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

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

52. 26.6 – How Are Pyrimidines Degraded?
In some organisms, free pyrimidines are salvaged and recycled to form nucleotides
In humans, pyrimidines are recycled from nucleosides, 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

53. Figure 26.18 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.

54. 26.7 – How Do Cells Form the Deoxyribonucleotides That Are Necessary for DNA Synthesis?
90% of the total nucleic acid in cells is RNA, with the remainder being DNA
The deoxynucleotides have only one metabolic purpose: to serve as precursor for DNA synthesis
NDPs are the substrate for deoxynucleotides formation
Reduction at 2’-position of ribose ring in NDPs produces 2’-deoxy forms of these nucleotides

55. Replacement of 2’-OH with hydride is catalyzed by ribonucleotide reductase
Figure Deoxyribonucleotide synthesis involves reduction at the 2'-position of the ribose ring of nucleoside diphosphates.

56. An 22-type enzyme has subunits R1 (2, 86 kD) and R2 (b2, 43.5 kD)
R1 has two regulatory sites, a substrate specificity site (S) and an overall activity site (A)
Figure E. coli ribonucleotide reductase.

57. 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 (active site)
Activity-determining: ATP & dATP (overall actiyity site)
Specificity-determining: ATP, dTTP, dGTP, and dATP (sbustrate specific site)

58. 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 follows with disulfide formation between Cys225, and Cys462 and forms the dNDP product

59. Thioredoxin provides the reducing power for ribonucleotide reductase
NADPH is the ultimate source
Reversible Sulfide : sulfhydryl transition
Figure The (—S—S—)/(—SH HS—) oxidation-reduction cycle involving ribonucleotide reductase, thioredoxin, thioredoxin reductase, and NADPH.

60. 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 substrate specificity site to regulate the selection of substrates and the products made

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

62. 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:
dUTPase
dCMP deaminase
UMP → UDP→ UTP → CTP → CDP → dCDP → dCMP → dUMP → dTMP

63. Figure 26. 25 (a) The dCMP deaminase reaction
Figure (a) The dCMP deaminase reaction. An alternative route to dUMP is provided by dCDP, which is dephosphorylated to dCMP and then deaminated by dCMP deaminase

64. Synthesis of dTMP from dUMP is catalyzed by thymidylate synthase
This enzyme methylates dUMP at the 5-position to create dTMP
The methyl donor is the one-carbon folic acid derivative N5, N10-methylene-THF
The reaction is a reductive methylation; the one-carbon unit is transferred at the methylene level of reduction and then reduced to the methyl level
The THF cofactor is oxidized to yield DHF
DHFR reduces DHF back to THF for serving again
dTMP synthesis has become a preferred target for inhibitors designed to disrupt DNA synthesis

65. Figure 26.26 The thymidylate synthase reaction.

66. Precursors and analogs of folic acid employed as antimetabolites: sulfonamides (see Human Biochemistry box on page 896), 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.

67. 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.)

68. 5-fluorouracil (5-FU) is used as a chemotherapeutic agent in the treatment of human cancers
5-fluorocytosine is used as an antifungal drug
5-fluoroorotate is an effective antimalarial drug