Nucleotide Metabolism

1

• Nucleotide Metabolism

2
Pathways in nucleotide metabolism

• De novo and salvage pathways
• Nucleic acid degradation and the importance of
  nucleotide salvage
• PRPP

3
Biosynthetic Routes De novo and salvage pathways

• Most organisms can synthesize purine and
  pyrimidrne nucleotides from low-molecular-weight
  precursors in amounts sufficient for their needs.
  These so-called de novo pathways are essentially
  identical throughout the biological world.
• Salvage pathways involve the utilization of
  preformed purme and pyrimidine compounds that
  would be otherwise lost to biodegradation.
  Salvage pathways represent important sites for
  manipulation of biological systems.

4
Figure 22.1 Overview of nucleotide metabolism.
5
Nucleoside Sugar Base (no phosphate) Nucleotid
e Sugar Base Phosphate
Figure 4.3 Nucleosides and nucleotides
6
purine
pyrimidine
7
Nucleic Acid Degradation and the Importance of
Nucleotide Salvage

• The salvage, or reuse, of purine and pyrimidine
  bases involves molecules released by nucleic acid
  degradation
• Degradation can occur intracellularly, as the
  result of cell death, or, in animals, through
  digestion of nucleic acids ingested in the diet.
• In animals, the extracellular hydrolysis of
  ingested nucleic acids represents the major route
  by which bases and nucleosides become available.
  Catalysis occurs by endonucleases, which function
  to digest nucleic acids in the small intestine.
  The products are mononucleotides.
• If bases or nucleosides are not reused for
  nucleic acid synthesis via salvage pathways, the
  purine and pyrimidine bases are further degraded
  to uric acid or b-ureidopropionate.

8
Endonuclease Phosphodiesterase Nucleotidase
Phosphorylase
Figure 22.2 Reutilization of purine and
pyrimidine bases.
9
Nucleoside phosphorylase
10
PRPP A Central Metabolite in De Novo and Salvage
Pathways

• 5-Phospho-a-D-ribosyl-1-pyrophosphate (PRPP) is
  an activated ribose-5-phosphate derivative used
  in both salvage and de novo pathways.

PRPP synthetase
Phosphoribosyltransferase (HGPRT)
11
De novo biosynthesis of purine nucleotides

• Purine synthesis from PRPP to inosinic acid
• Synthesis of ATP and GTP from inosinic acid
• Utilization of adenine nucleotides in coenzyme
  biosynthesis

12
Glycine 2 Glutamine Asparate 10-formyl-THF CO2
Figure 22.3 Low-molecular-weight precursors to
the purine ring.
13
Purine synthesis from PRPP to inosinic acid

• Purines are synthesized at the nucleotide level,
  starting with PRPP conversion to
  phosphoribosylamine and purine ring assembly on
  the amino group.
• Vertebrate cells have several multifunctional
  enzymes involved in these processes.
• Control over the biosynthesis of inosinic acid is
  provided through feedback regulation of early
  steps in purine nucleotide synthesis. PRPP
  synthetase is inhibited by various purine
  nucleotides, particularly AMP, ADP, and GDP, and
  PRPP amidotransferase is also inhibited by AMP,
  ADP, GMP, and GDP.

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1. PRPP amidotransferase 2. GAR synthetase 3.
GAR transformylase 4. FGAR amidotransferase 5.
FGAM cyclase 6. AIR carboxylase 7. SAICAR
synthetase 8. SAICAR lyase 9. AICAR
transformylase 10. IMP synthase
Figure 22.4 De novo biosynthesis of the purine
ring, from PRPP to inosinic acid.
15
Gln
PRPP amidotransferase
AMP, GMP
Glu, PPi
16
Gly, ATP
GAR synthetase
ADP, Pi
NH2 H2C C O
Glycinamide ribonucleotide (GAR)
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NH2 H2C C O
NH2 H2C C O
10-Formyl- THF
CHO
THF
Glycinamide ribonucleotide (GAR)
Formylglycinamide ribonucleotide (FGAR)
GAR transformylase
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NH2 H2C C O
NH2 H2C C HN
CHO
CHO
Gln, ATP
Glu, ADP, Pi
Formylglycinamide ribonucleotide (FGAR)
Formylglycinamidine ribonucleotide (FGAM)
FGAR amidotransferase
19
Trifunctional enzyme
(AICAR)
(GAR)
(FGAR)
(FAICAR)
Figure 22.5 Transformylation reactions in purine
nucleotide synthesis.
20
Potent inhibitors of purine nucleotide
synthesis -- structural analogs of glutamine --
glutamine amidotransferases
21
Synthesis of ATP and GTP from inosinic acid

• IMP is the first fully formed purine nucleotide
  and is a branch point between adenine and guanine
  nucleotide biosynthesis.
• The energy to drive the aspartate transfer
  reaction comes not from ATP but from GTP.
• GTP accumulation would tend to promoter the
  pathway toward adenine nucleotide. Also
  accumulation of ATP could promote guanine
  nucleotide synthesis.
• The enzyme catalyzing the pathway to make AMP is
  inhibited by AMP and the enzyme catalyzing the
  pathway to make GMP is inhibited by GMP

22
IMP dehydrogenase
AMP
GMP
Inosine Monophosphate (IMP)
Adenylosuccinate synthetase
H
XMP aminase
Adenylosuccinate lyase
Figure 22.6 Pathways from inosinic acid to GMP
and AMP.
23

• Nucleotides are active in metabolism primarily as
  the nucleoside triphosphates. GMP and AMP are
  converted to their corresponding triphosphates
  through two successive phosphorylation reactions.
  Conversion to the diphosphates involves specific
  ATP-dependent kinases.

Guanylate kinase
GMP ATP GDP
ADP AMP ATP 2ADP
Adenylate kinase
24

• Phosphorylation of ADP to ATP occurs through
  energy metabolism, by oxidative phosphorylation,
  or by substrate-level phosphorylations. ATP can
  also be formed from ADP through the action of
  adenylate kinase, acting in the reverse of the
  direction.
• ATP is the phosphate donor for conversion of GDP
  (and other nucleotide diphosphate) to the
  triphosphate level through the action of
  nudeoside diphosphokinase.
• GDP ATP GTP ADP
• Nucleoside diphosphokinase is an
  equilibrium-driven enzyme that transfers
  phosphate from ATP in the synthesis of all other
  nucleoside triphosphates.

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Purine degradation and clinical disorders of
purine metabolism

• Formation uric acid
• All purine nucleotide catabolism yields uric
  acid.
• Purine catabolism in primates ends with uric
  acid, which is excreted. Most other animals
  further oxidize the purine ring, to allantoin and
  then to allantoic acid, which is either excreted
  or further catabolized to urea or ammonia.

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PNP Purine nucleoside phosphorylase
ADA Adenosine deaminase
(Muscle)
Nucleotidase
Nucleotidase
ADA
PNP
Guanine deaminase
PNP
Xanthine oxidase
Xanthine oxidase
Hypoxanthine
Xanthine
Uric acid
Figure 22.7 Catabolism of purine nucleotides to
uric acid.
27
Figure 22.8 Catabolism of uric acid to ammonia
and CO2
28
Excessive accumulation of uric acid gout

• Uric acid and its urate salts are very insoluble.
  This is an advantage to egg-laying animals,
  because it provides a route for disposition of
  excess nitrogen in a closed environment.
• Insolubility of urates can present difficulties
  in mammalian metabolism. About 3 humans in 1000
  suffer from hyperuricemia, which is chronic
  elevation of blood uric acid levels well beyond
  normal levels. The biochemical reasons for this
  vary, but the condition goes by a single clinical
  name, which is gout.

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• Prolonged or acute elevation of blood urate leads
  to precipitation, as crystals of sodium urate, in
  the synovial fluid of joints. These precipitates
  cause inflammation, resulting in painful
  arthritis, which can lead to severe degeneration
  of the joints.
• Gout results from overproduction of purine
  nucleotides, leading to excessive uric acid
  synthesis, or from impaired uric acid excretion
  through the kidney
• Several known genetic alterations in purine
  metabolism lead to purine oversynthesis, uric
  acid overproduction, and gout. Gout can also
  result from mutations in PRPP amidotransferase
  that render it less sensitive to feedback
  inhibition by purine nucleotides. Another cause
  of gout is a deficiency of the salvage enzyme
  hypoxanthine-Guanine phosphoribosyltransferase
  (HGPRT).

30

• Many cases of gout are successfully treated by
  the antimetabolite allopurinol, a structural
  analog of hypoxanthine that strongly inhibits
  xanthine oxidase.
• This inhibition causes accumulation of
  hypoxanthine and xanthine, both of which are more
  soluble and more readily excreted than uric acid.

31
Elevated levels
Loss of feedback inhibition
Decreased levels
Figure 22.9 Enzymatic abnormalities in three
types of gout.
HGPRThypoxanthine-guanine phosphoribosyltransfera
se APRT adenine phosphoribosyltransferase
32
Lesch-Nyhan syndrome HGPRT defficiency

• Lesch-Nyhan syndrome is a sex-linked trait,
  because the structural gene for HGPRT is located
  on the X chromosome.
• Patients with this condition display a severe
  gouty arthritis, but they also have dramatic
  malfunction of the nervous system, manifested as
  behavioral disorders, learning disabilities, and
  hostile or aggressive behavior, often
  self-directed.
• At present, there is no successful treatment, and
  afflicted individuals rarely live beyond 20
  years.

33
Severe combined immune deficiency (SCID)

• Patients with a hereditary condition called
  severe combined immunodeficiency syndrome are
  susceptible, often fatally, to infectious
  diseases because of an inability to mount an
  immune response to antigenic chanllenge.
• In this condition, both B and T lymphocytes are
  affected. Neither class of cells can proliferate
  as they must if antibodies are to be synthesized.
  In many cases the condition is caused from a
  heritable lack of the degradative enzyme
  adenosine deaminase (ADA).
• The deficiency of ADA leads to
• accumulation of dATP which is
• known to be a potent inhibitor of
• DNA replication.

34

• A less severe immunodeficiency results from the
  lack of another purine degradative enzyme, purine
  nuceloside phosphorylase (PNP). Decreased
  activity of this enzyme leads to accumulation
  primarily of dGTP. This accumulation also affects
  DNA replication, but less severely than does
  excessive dATP.
• Interestingly, the phosphorylase deficiency
  destroys only the T class of lymphocytes and not
  the B cells.

35
Pyrimidine nucleotide metabolism

• Pyrimidine nucleotide synthesis occurs primarily
  at the free base level, with conversion to a
  nucleotide occurring later in the unbranched
  pathway.
• Pyrimidine synthesis begins with formation of
  carbamoyl phosphate. The first reaction committed
  solely to pyrimidine synthesis is the formation
  of carbamoyl aspartate from carbamoyl phosphate
  and aspartate, catalyzed by aspartate
  transcarbamoylase, or ATCase.
• In enteric bacteria, this enzyme represents an
  example of feedback control. The enzyme is
  inhibited by the end product CTP and activated by
  ATP.

36
Aspartate Cambamoyl phosphate PRPP (glutamine)
Cambamoyl phosphate
Aspartate
PRPP
Aspartate transcarbamoylase
CTP synthetase
Figure 22.10 De novo synthesis of pyrimidine
nucleotides.
37
Multifunctional Enzymes in Eukaryotic Pyrimidine
Synthesis

• Aspartate transcarbamoylase in eukaryotes is
  strikingly different from the E. coli enzyme. In
  eukaryotes, the first three reactions of
  pyrimidine synthesis are catalyzed by a
  multifunctional enzyme, the CAD protein
  (Carbamoyl phosphate synthetase, Aspartate
  transcarbamoylase, and Dihydroorotase).
• In mammalian cells, reactions 5 and 6 (see Figure
  22.10) are also catalyzed by a single protein,
  which is called UMP synthase.
• A site for control of pyrimidine nucleotide
  synthesis is the amidotransferase, CTP
  synthetase, which converts UTP to CTP.

38
Salvage Synthesis and Pyrimidine Catabolism

• Pyrimidine nucleotides are also synthesized by
  salvage pathways involving phosphorylases and
  kinases, comparable to those already discussed
  for purines.
• The catabolic pathways for pyrimidines are
  simpler than those for purines. Because the
  intermediates are relatively soluble, there are
  few known derangements of pyrimidine breakdown.
• b-alanine is used in the biosynthesis of
  coenzyme A

39
Figure 22.11 Catabolic pathways in pyrimidine
nucleotide metabolism.
40
Deoxyribonucleotide biosynthesis and metabolism

• Most cells contain 5 to 10 times as much RNA as
  DNA.
• The small fraction that is diverted to the
  synthesis of deoxyribonucleoside triphosphates
  (dNTPs) is of paramount importance to the cell.
• dNTPs are used almost exclusively in the
  biosynthesis of DNA. There are very close
  regulatory relationships between DNA synthesis
  and dNTP metabolism.
• DNA differs chemically from RNA in the nature of
  the sugar and in the identity of one of the
  pyrimidine bases.
• The deoxyribonucleotide biosynthesis on two
  specific processes the conversion of ribose to
  deoxyribose, and the conversion of uracil to
  thymine. Both processes occur at the nucleotide
  level.

41
Ribonucleoside diphosphate reductases (rNDP
reductase) One enzyme reduces all four
ribonucleotide to their deoxyriboderivatives
dUMP
Thymidylate synthase
dTMP
Figure 22.12 Overview of deoxyribonucleoside
triphosphate (dNTP) biosynthesis.
42
Ribonucleoside diphosphate reductase

• The enzyme catalyzing the synthesis of dNDPs from
  rNDPs, reduces the hydroxyl at carbon 2 to a
  hydrogen via a free radical mechanism.
• Ribonucleotide reductase contains catalytic
  residues on each of its subunits-redox-active
  thiols and a tyrosine free radical stabilized by
  an iron-Oxygen complex.
• Hydroxyurea, an inhibitor of ribonucleotide
  reductase,
• destroys the free radical.

43
rNDP reductase a2b2, tetramer a-subunits form
R1 containing the active site. b-subunits
make up R2 containing the free radical. A
clue to the mechanism of action of the
enzyme (tyrosine free radical)
Figure 22.13 Structure of E. coli ribonucleoside
diphosphate reductase.
44
Figure 22.15 Reduction of a ribonucleoside
diphosphate by rNDP reductase.
45
Source of Electrons for rNDP Reduction

• Electrons for the reduction of ribonucleotides
  come ultimately from NADPH, but they are shuttled
  to rNDP reductase by a coenzyme that is unusual
  because it is itself a protein.
• Ribonucleotide reductase uses a protein cofactor,
  thioredoxin or glutaredoxin, to provide electrons
  for reduction of the ribonucleotide substrate.

46
Figure 22.16 Reductive electron transport
sequences in the action of rNDP reductase.
47
Table 22.1 Biological activities of thioredoxin
48
Regulation of Ribonucleotide Reductase Activity

• Ribonucleotide reductase has two classes of
  allosteric sites. Activity sites influence
  catalytic efficiency, and specificity sites
  determine specificity for one or more of the four
  substrates.
• The activity sites bind either ATP or dATP with
  relatively low affinity, whereas the specificity
  sites bind ATP, dATP, dGTP, or dTTP with
  relatively high affinity.
• Inhibition of DNA synthesis by thymidine or
  deoxyadenosine involves allosteric inhibition of
  ribonucleotide reductase by dTTP or dATP,
  respectively

49
Table 22.2 Regulation of Ribonucleotide Reductase
Activity
50
Biosynthesis of Thymine Deoxyribonucleotides

• dUMP, the substrate for thymidylate synthesis,
  can arise either from UDP reduction and
  dephosphorylation or from deammation of a
  deoxycytidine nucleotide (dCMP).
• In the reaction catalyzed by thymidylate
  synthase, 5,10-methylenetetrahydrofolate donates
  both a single-carbon group and an electron pair
  to reduce that group to the methyl level.

51
Deoxycytidine kinase
dUTPase
dCMP deaminase
Tymidine kinase
Thymidylate synthase
Figure 22.17 Salvage and de novo synthetic
pathways to thymine nucleotides.
Tymidine kinase
52
Thymidylate synthase
Dihydrofolate reductase
Serine transhydroxymethylase
Figure 22.18 Relationship between thymidylate
synthase and enzymes of tetrahydrofolate
metabolism.
53
Deoxyuridine Nucleotide Metabolism

• In addition to the biosynthetic function of
  dUTPase in forming dUMP for thymine nucleotide
  formation, the enzyme plays an important role in
  excluding uracil from DNA.
• dUMP residues can arise in DNA not only by dUTP
  incorporation but also by the spontaneous
  deamination of dCMP residues.

54
Salvage Routes to Deoxyribonucleotide Synthesis

• Of the various deoxyribonucleoside kinases, one
  that merits special mention is thymidine kinase
  (TK). This enzyme is allosterically inhibited by
  dTTP. Activity of thymidine kinase in a given
  cell is closely related to the proliferative
  state of that cell. During the cell cycle,
  activity of TK rises dramatically as cells enter
  S phase. In general, rapidly dividing cells have
  high levels of this enzyme.
• The salvage pathway to dTTP competes very
  efficiently with the thymidylate
  synthase-mediated de novo pathway.
• Deoxycytidine kinase is also a salvage enzyme
  that is feedback inhibited by dCTP. This enzyme
  also functions as a deoxyadenosine kinase and a
  deoxyguanosine kinase. Unlike thymidine kinase,
  whose activity fluctuates over the course of the
  cell cycle, the activity of deoxycytidine kinase
  stays relatively constant.

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Thymidylate synthase a target enzyme for
chemotherapy

• A goal of chemotherapy is to exploit a
  biochemical difference between the disease
  process and the host tissue in order to interfere
  selectively with the disease process.
• Many chemotherapeutic agents were discovered by
  chance, through testing of analogs of normal
  metabolites. Most of these agents are limited in
  their effectiveness by unanticipated side
  effects, incomplete selectivity, and the
  development of resistance to the agent.
• One of the most exciting areas of modern
  biochemical pharmacology is drug architecture
  the design of specific inhibitors based on
  knowledge of the molecular structure of the site
  to which the inhibitor will bind and the
  mechanism of action of the target molecule.

56

• Inhibition of thymidylate synthase is an approach
  to cancer chemotherapy, by causing specific
  inhibition of DNA synthesis. Cells that are not
  rapidly proliferating should be relatively immune
  to such agents. Thus, cancer and a wide range of
  infectious diseases should be amenable to
  treatment by this approach.
• Knowing the active site structure and reaction
  mechanism of an enzyme allows the design of novel
  inhibitors, an approach used for thymidylate
  synthase, but applicable to many other drugs.

57

• 5-fluorouracil (FUra) and its deoxyribonucleoside,
  5-fluorodeoxyuridine (FdUrd) were found to be
  potent inhibitors of DNA synthesis, and both are
  used in cancer treatment. They are not completely
  selective in their effects.
• 5-fluorodeoxyuridine monophosphate (FdUMP) is a
  dUMP analog that acts as an irreversible
  inhibitor of thymidylate synthase.
• FdUMP is a true mechanism-based inhibitor, in
  that irreversible binding occurs only in the
  presence of 5,10-methylenetetrahydrofolate.

58
Figure 22.20 Mechanism for the reaction
catalyzed by thymidylate synthase.
59
Figure 22.22 Orientation of substrate and
coenzyme in the active site of thymidylate
synthase.
60
(No Transcript)
61
Virus-Directed Alterations of Nucleotide
Metabolism

• The T-even bacteriophages that viruses can
  redirect the metabolism of their host cells.

62
Figure 22.23 Metabolic pathways leading to
nucleotide modifications in T-even phage-infected
E. coli.
Glycosylated DNA
63
Other modifications made by viruses include the
following 1. Some Bacillus subtilis phages
substitute uracil for thymine in their DNA 2.
Some Bacillus subtilis phages contain
5-hydroxymethyluracil in place of thymine. 3. A
phage of Xanthomonas oryzae substitutes
5-methylcytosine for every one of the cytosines
in its DNA
64
Biological and Medical Importance of Other
Nucleotide Analogs

• Nucleotide Analogs as Chemotherapeutic Agents
• Antiviral Nucleoside Analogs
• Purine Salvage as a Target
• Folate Antagonists (Figure 22.18)
• Nucleotide Analogs and Mutagenesis (Figure 22.24)
• Nucleotide-Metabolizing Enzymes as Selectable
  Genetic Markers

65
Nucleotide Analogs as Chemotherapeutic Agents

• The enzymes of nucleotide synthesis have been
  widely studied as target sites for the action of
  antiviral or antimicrobial drugs.
• Other analogs receiving considerable attention
  are those being used to combat acquired immune
  deficiency syndrome (AIDS). One such analog is
  AZT, the first drug approved in the United States
  for the treatment of HIV infections.

66

67
Figure 22.24 Mechanisms of mutagenesis by
nucleotide analogs.
68
Selectable genetic markers

• Because most cells can synthesize nucleotides de
  novo, the enzymes of salvage synthesis are
  usually nonessential for cell viability. This
  means that nucleotidemetabolizing enzymes and
  the genes that encode them provide selectable
  genetic markers, which have a variety of uses.
• Separate salvage and de novo pathways allow
  selection for survival or death of cells with
  particular metabolic traits.

69

• Modified nucleotides can be used to select cells
  containing or lacking specific enzymes. Examples
  include the following 6-Thioguanine selects for
  cells lacking an active hypoxanthine-guanine
  phosphoribosyltransferase (HGPRT).
• Cells containing an active enzyme convert
  6-thioguanine to a toxic compound.
• 5-Bromodeoxyuridine (BrdUrd) can be used to
  select cells lacking thymidine kinase, which is
  needed to metabolize BrdUrd to a toxic metabolite.

70

• HAT Selection - The compounds hypoxanthine,
  aminopterin, and thymidine (H,A, and T,
  respectively) can be used to select for cells
  having functional salvage pathways.
• Aminopterin inhibits dihydrofolate reductase,
  which blocks de novo purine and thymidine
  synthesis. Only cells which can utilize thymidine
  (pyrimidine salvage) and hypoxanthine (purine
  salvage) can grow in this medium.