Biosynthesis of nucleotides

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Biosynthesis of nucleotides
Phar 6152
Spring 2004
Natalia Tretyakova, Ph.D.
Required reading Stryers Biochemistry 5th
edition, p. 262-268, 693-712 (or Stryers
Biochemistry 4th edition p. 238-244, 739-759)
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Tentative Lecture plan Biosynthesis of
Nucleotides
03-31 Introduction. Biological functions and
sources of nucleotides. Nucleotide
metabolism. 04-02 Biosynthesis of pyrimidine
ribonucleotides. 04-05 Biosynthesis of purine
ribonucleotides 04-07 Biosynthesis of
deoxyribonucleotides. Inhibitors of nucleotide
metabolism as drugs. 04-09 Review 04-12 Exam
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Biological functions and sources of nucleotides.
Nucleotide metabolism
Required reading Stryers Biochemistry 5th Ed.,
p. 693-694, 709-711
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Biological functions of nucleotides 1.     
Building blocks of nucleic acids (DNA and
RNA). 2. Involved in energy storage, muscle
contraction, active transport, maintenance of
ion gradients. 3.      Activated intermediates in
biosynthesis (e.g. UDP-glucose,
S-adenosylmethionine). 4.      Components of
coenzymes (NAD, NADP, FAD, FMN, and
CoA) 5.      Metabolic regulators a.      Second
messengers (cAMP, cGMP) b.      Phosphate donors
in signal transduction (ATP) c.       Regulation
of some enzymes via adenylation and
uridylylation
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Nucleotides
b-glycosidic bond
RNA- ribose (R) DNA deoxyribose (dR)
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Nucleobase structures
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Hypoxanthine Inosine Inosinate
(IMP) Xanthine Xanthosine Xanthylate (XMP)
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Two major routes for nucleotide biosynthesis
dNTPs
dNTPs
Stryer Fig. 25.1
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Nucleobase Products of Intracellular or
dietary/intestinal degradation can be recycled
via salvage pathways 1 and 2 (red)
1
2
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Phosphoribosyl transferases involved in salvage
pathway convert free bases to nucleotides
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(HGPRT)

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Biodegradation of Nucleotides
(Stryer p. 709-711)
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Nucleobase Products of Intracellular or
dietary/intestinal degradation can be recycled
via salvage pathways 1 and 2 (red)
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2
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Purine biodegradation in humans leads to uric
acid
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AMP is deaminated to IMP
AMP deaminase
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IMP is deribosylated to hypoxanthine
phosphorylase
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Hypoxanthine is oxidized to xanthine
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Guanine can be deaminated to give xanthine
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Uric acid is the final product of purine
degradation in mammals
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Uric acid is excreted as urate
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Deleterious consequences of defective purine
metabolism

• Gout (excess accumulation of uric acid)
• Lesch-Nyhan syndrome (HGPRT null)
• Immunodeficiency

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Gout

• Precipitation and deposition of uric acid causes
  arthritic pain and kidney stones
• Causes impaired excretion of uric acid and
  deficiencies in HGPRT

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Lesch-Nyhan Syndrome

• Caused by a severe deficiency in HGPRT activity
• Symptoms are gouty arthritis due to uric acid
  accumulation and severe neurological malfunctions
  including mental retardation, aggressiveness, and
  self-mutilation
• Sex-linked trait occurring mostly in males

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Lack of HGPRT activity in Lesch-Nyhan Syndrome
causes a buildup of PRPP, which activates the
synthesis of purine nucleotides
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• Excessive uric acid forms as a degradation
  product of purine nucleotides
• Basis of neurological aberrations is unknown

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Immunodeficiency induced by Adenosine Deaminase
defects
AMP deaminase

• Defects in AMP deaminase prevent biodegradation
  of AMP
• AMP is converted into dATP
• dATP inhibits the synthesis of deoxyribonucleotide
  s by ribonucleotide reductase, causing problems
  with the immune
• system (death of lymphocytes, immunodeficiency
  disease)

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Summary

• Nucleotides have many important functions in a
  cell.
• Two major sources of nucleotides are salvage
  pathway and de novo biosynthesis
• Purine nucleotides are biodegraded by
  nucleotidases,
• nucleotide phosphorylases, deaminases, and
• xanthine oxidase.
• Uric acid is the final product of purine
  biodegradation in mammals
• Defective purine metabolism leads to clinical
• disease.

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Key concepts in Biosynthesis Review

• Committed step
• Regulated step
• Allosteric inhibitor
• Feedback inhibition

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De novo Biosynthesis of Pyrimidines
Required reading Stryers Biochemistry 5th Ed.,
p. 262-267, 694-698
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De novo Biosynthesis of Pyrimidines
dTTP
Stryer Fig. 25.2
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Part 1. The formation of carbamoyl phosphate
Enzyme carbamoyl phosphate synthetase II
(CPS) This is the regulated step in pyrimidine
biosynthesis
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Bicarbonate is phosphorylated
CPS
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Phosphate is displaced by ammonia

CPS
General strategy for making C-N bonds C-OH
is phosphorylated to generate a good leaving
group (phosphate)
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General Mechanism for making C-N bonds  
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Ammonia necessary for the formation of carbamic
acid originates from glutamine
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Structure of Carbamoyl phosphate synthetase II
Stryer Fig. 25.3
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The active site for glutamine hydrolysis to
ammonia contains a catalytic dyad of Cys and His
residues
Stryer Fig. 25.4
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Carbamic acid is phosphorylated
CPS
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Substrate channeling in CPS
Stryer Fig. 25.5
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Carbamoyl phosphate supplies the C-2 and the N-3
of the pyrimidine ring
dTTP
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Part 2. The formation of orotate.
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Aspartate is coupled to carbamoyl phosphate
Enzyme aspartate transcarbamoylase
This is the committed step in pyrimidine
biosynthesis
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Aspartate transcarbamoylase is allosterically
inhibited by CTP
Stryer Fig. 10.2
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Allosteric regulation of Aspartate
Transcarbamoylase
Stryer Fig. 10.5
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PALA is a bisubstrate analog that mimics the
reaction intermediate on the way to carbamoyl
aspartate
Bisubstrate analog
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PALA binds to the active site within catalytic
subunit
Stryer Fig. 10.7
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Substrate binding to Aspartate Transcabamoylase
induces a large change in ATC quaternary structure
Stryer Fig. 10.8
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CTP binding prevents ATC transition to the active
R state
Stryer Fig. 10.9
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Allosteric regulation of Aspartate
Transcabamoylase
Stryer Fig. 10.10
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N-Carbamoylaspartate cyclizes to dihydroorotate
- H2O
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Dihydroorotate is oxidized to orotate
Dihydroorotate dehydrogenase
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Part 3. The formation of UMP
a. Orotate is phosphoribosylated to OMP
Pyrimidine phosphoribosyl transferase
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b. OMP is decarboxylated to form UMP
OMP decarboxylase (UMP synthetase)
(OMP)
(UMP)
Note phosphoribosyl transfer and decarboxylase
activities are co-localized in UMP synthetase
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c.Phosphorylation of UMP gives rise to UDP and
UTP      
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CTP is produced by replacing the 4-keto group of
UTP with NH2
CTP synthetase
Note TTP for DNA synthesis is produced via
methylation of CTP (will discuss later)
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Regulation of pyrimidine nucleotide biosynthesis
Carbamoyl phosphate synthetase
Regulated step
Aspartate transcarbamoylase
Committed step
OMP decarboxylase (UMP synthetase)
CTP synthetase
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Defects in de novo pyrimidine biosynthesis lead
to clinical disease

• Orotic acidurea
• Symptoms anemia, growth retardation, orotic acid
  excretion
• Causes a defect in phosphoribosyl transferase or
  orotidine decarboxylase
• Treatment patients are fed uridine
• U ? UMP ? UDP ? UTP
• UTP inhibits carbamoyl phosphate synthase II,
  preventing the biosynthesis and accumulation of
  orotic acid

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UTP inhibits carbamoyl phosphate synthase II,
preventing the biosynthesis and accumulation of
orotic acid
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Carbamoyl phosphate synthetase
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Drug inhibitors of pyrimidine biosynthesis
Inhibitors of PRPP synthetase
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Inhibitors of dihydroorotase
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Pyrimidine biosynthesis take home message

• Pyrimidines are synthesized by de novo and
  salvage pathways.
• 2. The pyrimidine ring is synthesized from
  pre-assembled ingredients (carbamoyl phosphate
  and aspartate) and then attached to the ribose.
• 3. Pyrimidine biosynthesis is tightly regulated
  via feedback inhibition (CTP synthetase,
  carbamoyl phosphate synthetase, aspartate
  transcarbamoylase) and transcriptional regulation
  (ATCase).
• 4. The mammalian enzymes are multifunctional
  (e.g. carbamoyl phosphate synthetase, UMP
  synthetase) and form multienzyme complexes to
  increase efficiency.
• 5. Drug inhibitors of pyrimidine biosynthesis are
  under development as potential antimicrobial and
  anticancer agents.