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Week 11 Study guide

PURINE/PYRIMIDINE INFORMATION

Purine Nucleotide Cycle


IN: Asp, GTP
OUT: Fumarate, GDP, Pi, NH4

Purine vs. Pyrimidine nucleotide metabolism


Both:
• the committed step in de novo synthesis is also the rate-limiting step
• de novo synthesis is feed-back regulated by end products of the pathway
• the catabolic pathway uses enzymes that dephosphorylate, phosphorolysize and deaminate
• Aspartic acid (aspartate), CO2 and glutamine contribute atoms to the ring structure
• Usage of bifunctional enzyme
Just pyrimidine:
• the majority of salvage synthesis occurs at the nucleoside level
• the nitrogen-containing ring structure is synthesized prior to the addition of ribose and phosphate
Just purine:
• The majority of salvage synthesis occurs at the base level (APRT and HPRT)
• Under steady-state conditions, salvage synthesis consumes more PRPP than does de novo synthesis
• Synthesis of GMP from IMP requires: ATP (energy source), glutamine (amine source) and NAD+

Disorders
Pyrimidine 5’-nucleotidase I deficiency- inherited disorder of nucleotide metabolism that results in elevated
levels of erythrocyte pyrimidine nucleotides and an accompanying anemia
Purine nucleoside phophorylase deficiency- associated with elevated levels of dGTP in cells
UMP synthase deficiency- bifunctional protein, inherited deficiency, pyrimidine metabolism deficiency leading
to anemia, can be rescued by uridine therapy
HPRT deficiency- decreased salvage of hypoxanthine and guanine, increased levels of PRPP, monomerization
of ATase, hyperuricemia  gout, mental retardation with self-mutilation
Patients are able to salvage adenine because they still have HPRT
Thymidine kinase 2 deficiency- increased mutation rates and depletion of mitochondrial genome (mitochondrial
salvage enzyme)
Xanthine dehydrogenase deficiency- xanthinuria, NO uric acid produced

Levels of Synthesis
• Thymidine nucleotides are synthesized directly from deoxyuridine monophosphate using N5,N10-
methylenetetrahydrofolate as the methyl group donor.
o UMP  UDP  dUDP  dUMP  dTMP  dTDP  dTTP
• The de novo synthesis of the cytosine ring structure from the uracil ring structure occurs at the
ribonucleoside triphosphate level
• Hypoxanthine can be converted to adenine (HPRT)
• Cytosine can be converted to uracil during catabolism of pyrimidines
• Adenine can be converted to hypoxanthine during catabolism of purines (AMP deaminase)
• Uracil can be converted to thymine in the thymidylate synthase reaction.
• De novo synthesis of the uracil ring structure occurs at the ribonucleoside monophosphate level (end-
product of pyrimidine biosynthesis is UMP)

If there was an acute decrease in intracellular ATP levels you would expect to see:
• Release of ATase feedback inhibition
• Displacement of the adenylate kinase reaction towards ATP formation (also making AMP)
• Decrease in branchpoint synthesis leading to guanine nucleotides
• Increased AMP deaminase activity
• Increased uric acid production

KNOW the difference between base, nucleoside, nucleotide!


KNOW major mechanism of damage of each type of antimetabolite and mechanisms of resistance for each!

Folate methyl donors used for: purine metabolism and thymine structure formation
o A cell unable to synthesize or obtain tetrahydrofolate would be most likely unable to carry out de novo
synthesis of dTMP
o Methotrexate (antifolate) chemotherapy:
 Effective because consumption of methyl-bearing reduced folates continues in the presence of the drug
and results in an inadequate source of methyl donors for nucleotide synthesis (thymidylate synthase
reaction)
 The reduced folate carrier system is the major route for the intracellular uptake of this compound
 Normal cells may be selectively rescued from methotrexate therapy by providing them with the reduced
folate, leucovorin
• Leucovorin is a downstream metabolite of dihydrofolate and is typically administered to patients
receiving methotrexate chemotherapy
 Structural alterations in the DHFR protein resulting in decreased affinity for MTX can lead to resistance
(gene amplification can also lead to resistance)
 Accumulation of MTX is greatly enhanced by its conversion to a polyglutamate derivative
 Inhibition of DHFR by methotrexate  reduction in thymidine nucleotide synthesis
 Activation is NOT a prerequisite to any chemotherapeutic effect of this drug!

Disorders causing hyperuricemia:


Overexpression of PRPP synthetase
Partial or complete deficiency of HPRT
Myophosphorylase deficiency
** NOT Xanthine dehydrogenase deficiency **

Nucleotide metabolism in tumor cells:


• Purine/pyrimidine de novo pathways are more active than in normal cells (SAME metabolic pathways!!)
• Gene amplification is an antifolate resistance mechanism
• A major target of 5-FU therapy is the thymidylate synthase reaction
• Thiopurines may exert their effects at the DNA and RNA levels

HPRT catalyzes activation step in thiopurine base metabolism (antipurines)


OPRT catalyzes activation step in 5-fluorouracil metabolism (antipyrimidine)

5-FU resistance
• decreased availability of cofactors
• TS gene amplification
• Increased TS gene transcription
• Decreased OPRT activity

ACTIVATED OXYGEN INFORMATION

Hydroxyl radical > singlet oxygen > peroxynitrite > superoxide > hydrogen peroxide

Formation of Oxygen species:


• Hemolytic cleavage of hydrogen peroxide generates 2 molecules of hydroxyl radical.
• Fe2+ can quickly and directly convert hydrogen peroxide to a more toxic species (hydroxyl radical) via the
Fenton reaction
o In the Fenton reaction, Fe2+ reduces hydrogen peroxide to hydroxyl radical and water
• Singlet oxygen is formed from triplet oxygen in the presence of light and pigments
• Superoxide can be formed in RBCs via formation of methemoglobin from oxyhemoglobin.