20: Nucleotide Metabolism Flashcards
What are nucleotides, purines and pyrimidines?
Purines: Adenine, Guanine, Hypoxanthine, Xanthine
Pure As Gold
Pyrimidines: Cytosine, Uracil, Thymine
CUT the Pye
Nucleotides are organic molecules that serve as the monomers, or subunits, of nucleic acids like DNA and RNA. The building blocks of nucleic acids, nucleotides are composed of a nitrogenous base, a five-carbon sugar (ribose or deoxyribose), and at least one phosphate group. Nucleotides also serve to carry packets of energy within the cell (ATP).
What is PRPP and why is it important?
5’-Phosphoribosyl-1-pyrophosphate (PRPP) is an intermediate of major significance in
nucleotide metabolism.
PRPP is important because it is required in:
a. The de novo synthesis of pyrimidine and purine nucleotides.
b. The salvage pathways for purine nucleotides.
c. The biosynthesis of nucleotide coenzymes, NAD and FAD.
PRPP is formed from ribose 5-phosphate and adenosine triphosphate (ATP). Catalyzed by PRPP synthetase.
PRPP is generated in:
a. Glucose metabolism, pentose phosphate shunt.
b. Nucleoside degradation generates ribose 1-phosphate, which can be converted to
ribose 5-phosphate.
How do sulfinamides and azaserine inhibit nucleotide synthesis?
These drugs inhibit IMP synthesis @ different steps:
Azaserine (an analog of glutamine) blocks amide transfer from GLUTAMINE.
Sulfonamides (analogs of p-aminobenzoic acid) block the biosynthesis of FOLIC ACID in bacteria and prevent its formation. This blocks nucleotide synthesis.
What are salvage pathways?
A salvage pathway is a pathway in which nucleotides (purine and pyrimidine) are synthesized from intermediates in the degradative pathway for nucleotides.
Salvage pathways are used to recover bases and nucleosides that are formed during degradation of RNA and DNA. This is important in some organs because some tissues cannot undergo de novo synthesis.
Purine salvage pathways are done by transferases:
Hypoxanthine-guanine phosphoribosyl transferase (HGPRT)–important in gout.
Adenine phosphoribosyl transferase (APRT)
Pyrimidine salvage pathways:
The free pyrimidine bases that result from nucleotide degradation are uracil and thymine.
Uracil is acted on by uridine phosporylase & then uridine kinase to become UMP.
Cytidine can also be phosphorylated by uridine kinase.
In the salvage pathway for thymine, thymine phosphorylase converts thymine to thymidine & thymidine kinase converts thymidine to dTMP.
When are deoxynucleotides made and how are they made?
Deoxyribonucleosides are found in DNA. The cellular levels of deoxyribonucleotides are ordinarily low, and they increase at the time of DNA REPLICATION (S phase = SYNTHESIS PHASE).
NOTE: 99.9% of the cells in your body are not making deoxyribonucleotides. Only cells that are actively dividing and in S phase make them.
The enzyme used to synthesize deoxyribonucleosides is ribonucleotide reductase.
Ribonucleotide reductase is regulated by ribo- and deoxyribonucleotides in a complex fashion. The pattern of regulation ensures that the proper ratios and levels of deoxyribonucleotides are present for the synthesis of DNA.
The products of ribonucleotide reductase include deoxyadenosine diphosphate (dADP), deoxycytidine diphosphate (dCDP), deoxyguanosine diphosphate (dGDP), and deoxyuridine diphosphate (dUDP)–Cells do not want to incorporate deoxy-U into DNA, so this molecule is efficiently dephosphorylated to the dUMP state.
dTMP is formed from dUMP in a reaction that is catalyzed by thymidylate synthase. In this reaction, a methylene group from N5,N10-methylenetetrahydrofolate is transferred
and reduced to a methyl group. N5,N10-methylenetetrahydrofolate is thereby oxidized to dihydrofolate.Tetrahydrofolate is regenerated by dihydrofolate reductase in a reaction that requires NADPH.
- The methyl group in the reaction is coming from the conversion of Serine to Glycine which generates the N5, N10 Methylene-THF. The Serine can be easily synthesized from intermediates of glycolosis. Hence the one-carbon groups can be funneled right out of glycolosis and onto dUMP to make dTMP.
What is thymidylate synthase and ribonucleotide reductase, how do these
enzymes work and why are inhibitors against them important chemotherapy
agents?
The enzyme used to synthesize deoxyribonucleosides is ribonucleotide reductase. Ribonuclotide reductase is inhibited by hydroxyurea a potent chemotherapy agent.
dTMP is formed from dUMP in a reaction that is catalyzed by thymidylate synthase.
What is 5-fluorouracil and methotrexate and how are they related to THF?
Methotrexate is an analog of folic acid that inhibits dihydrofolate reductase. Therefore, the regeneration of tetrahydrofolate is blocked, and synthesis of dTMP is inhibited.
5-Fluorouracil is a drug that is a pyrimidine analog which is used in the treatment of cancer.
5-Fluorouracil undergoes conversion to the nucleoside 5’-monophosphate F-UMP in cells. F-UMP can be phosphorylated to the nucleoside diphosphate. In this form, it is reduced to the deoxyribonucleotide.
- The reduced nucleoside diphosphate is dephosphorylated to 5-fluorodeoxyuridine 5’-monophosphate (F-dUMP), which is the critical form for antineoplastic activity.
- F-dUMP interacts with thymidylate synthase and N5,N10-methylenetetrahydrofolate. This complex resembles the transition state formed during the conversion of dUMP to dTMP .
- Inhibition. The transfer of a methylene group to the pyrimidine ring is blocked by the fluorine atom. Therefore, thymidylate synthase is trapped in a complex with F-dUMP, and synthesis of dTMP is inhibited.
What is the final end product of purine degradation, and why is it a problem
in gout? What is allopurinol?
Uric acid is the product of purine degradation.
Xanthine is oxidized to uric acid by xanthine oxidase.
Gout is caused by the precipitation of sodium urate crystals in the joints and kidneys. Sodium urate crystals precipitate because the serum levels of urate exceed its solubility limit. Elevated uric acid levels may be due to one of several disorders.
In some patients, PRPP synthetase is abnormal and is not responsive to feedback
inhibition by purine nucleoside diphosphates. A partial deficiency of HGPRT leads to increased cellular levels of PRPP, which
leads to increased de novo synthesis of purines.
Inflammation and erosion of the joints occur when leukocytes engulf the deposited crystals and consequently rupture, releasing lysosomal enzymes. Sodium urate crystals in the urinary tract impair renal function.
ALLOPURINOL INHIBITS XANTHINE OXIDASE & blocks the production of uric acid.
Allopurinol is often given as a pretreatment for chemotherapy. During chemotherapy, large quantities of nucleotides are released from killed cancer cells. These often lead to uricemia and gout.
How are purines put together?
Base: The purine ring is built on a molecule of PRPP.
The precursors of the ring are glutamine, glycine, carbon dioxide (CO2), aspartate, and two one-carbon fragments from the one-carbon folate pool.
Rxns:
- Synthesis of inosine 5’-monophosphate (IMP).
- The formation of IMP is a ten-step process that uses six high-energy phosphate bonds. It is an energetically expensive process.
- The first step is the rate limiting and regulated step.
- Two steps require folate, and are blocked by drugs that block folate biosynthesis in bacteria (sulfonamides).
- The nucleotide ring is made from glutamine, glycine, carbon dioxide, aspartate, and two one-carbon fragments from the one-carbon folate pool.
- Two steps require glutamine amino transfer reactions that are inhibited by azaserine.
- This is an expensive process; thus, 90% of our nucleotides are salvaged. - Synthesis of AMP & GMP from IMP. GTP is cleaved in the synthesis of AMP & ATP is cleaved in the synthesis of GMP (this balances adenine & guanine levels).
2a. IMP to AMP: aspartate + IMP gives adenylosuccinate. Fumarate then leaves adenylosuccinate & makes AMP.
2b. IMP to GMP: IMP is oxidized to xanthosine 5’-monophosphate. An amino group from glutamine is then added to XMP to make GMP.
Purine biosynthetic machinery localizes to the purinosome. Presence of this organelle is regulated by purine abundance. All ten steps catalyzed by six different enzymes localize in these organelles along with the purine salvage pathway enzymes.
How is purine synthesis regulated?
De novo synthesis of purine nucleotides is regulated by feedback inhibition.
a. The enzymes that catalyze the first two steps of IMP synthesis–PRPP synthetase and PRPP amidotransferase–are both inhibited by IMP, GMP, and AMP. This is classical feedback inhibition of the first steps in a long biosynthetic pathway.
b. PRPP amidotransferase has two allosteric sites, one for IMP or GMP and one for AMP. If both sites are occupied, then inhibition is synergistic.
c. Inhibition. The synthesis of adenylosuccinate from IMP is inhibited by AMP, and the synthesis of XMP is inhibited by GMP.
How are pyrimidines assembled?
The pyrimidine ring is formed, and then it reacts with PRPP to form the nucleotide.
The precursors of the ring are carbamoylphosphate and aspartate.
Carbamoylphosphate is synthesized in the cytosol from glutamine and carbon
dioxide.
uridine 5’-monophosphate (UMP) is made from carbamoylphosphate.
- Patients with a urea cycle OTC deficiency build up high levels of pyrimidines in there serum and urine. The first step is the rate limiting step. The excess carbamoylphosphate that accumulates in these patients is converted into pyrimidines.
UMP becomes UTP. 2 rxns; rxn 1 uses UMP kinase & rxn 2 uses nucleoside diphosphatate kinase.
An amino group from glutamine is donated to uridine triphosphate (UTP) to form cytidine triphosphate (CTP). This is another example of a glutamine amino-transferase reaction.
How is pyrimidine synthesis regulated?
- Regulate the enzyme
- Pyrimidine nucleotides decrease the aspartate transcarbamoylase and dihydroorotase activities of the multifunctional enzyme
How are purines and pyrimidines degraded? What is Xanthine Oxidase?
- Nucleotidases remove the 5’-phosphates from purine and pyrimidine ribo- and deoxyribonucleotides, converting them to ribo- and deoxyribonucleosides.
- Nucleoside phosphorylases catalyze the phosphorolysis of nucleosides to free bases and ribose 1-phosphate or deoxyribose 1-phosphate.
Pyrimidine degradation: The final products of pyrimidine degradation are β-alanine (from uracil) and β-aminoisobutyrate (from thymine).
Purine Degradation The final product of purine degradation is uric acid:
1. Hypoxanthine, from the breakdown of AMP, is oxidized to xanthine by the enzyme xanthine oxidase. 2. Guanine, from the breakdown of GMP, is deaminated to xanthine. 3. Xanthine is oxidized to uric acid by xanthine oxidase. ⁃ Oxygen (O ) is required, and superoxide is formed. The O - is converted to 22 hydrogen peroxide (H2O2) by superoxide dismutase. ⁃ Xanthine oxidase contains molybdenum, which is why this element is required in trace amounts in humans. This enzyme also contains iron and sulfur.
What is Lesch-Nyhan Syndrome?
Lesch-Nyhan syndrome is a hereditary X-linked recessive condition that is due to a severe or complete deficiency of HGPRT activity.
Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) catalyzes the formation of nucleotides from either hypoxanthine or guanine for purine salvage.
There is increased synthesis of purines. Because there is little or no HGPRT activity in people affected with Lesch-Nyhan syndrome, hypoxanthine and guanine are not salvaged. Also, the intracellular levels of PRPP increase, while those of IMP and GMP decrease. This leads to increased de novo synthesis of purines.
Several forms of HGPRT deficiency have been identified in Lesch-Nyhan syndrome:
(1) In one form, patients have normal levels of HGPRT protein, but the enzyme is
inactive.
(2) Some patients have an enzyme that is apparently unstable; its activity is higher in
young red blood cells than in old.
The symptoms include hyperuricemia, gout, urinary tract stones, and the neurological symptoms of mental retardation, spasticity, and self-mutilation. Brain cells normally have much higher levels of purine salvage enzymes than other cells and may normally use salvage pathways to a greater extent.
Treatment with allopurinol reduces the uric acid formation but does not alleviate the neurologic symptoms.
What is adenosine deaminase deficicency?
Cause: ADA deficiency is due to a lack of the enzyme adenosine deaminase. This deficiency results in an accumulation of deoxyadenosine, which, in turn, leads to a build up of dATP in all cells, which inhibits ribonucleotide reductase and prevents DNA synthesis, so cells are unable to divide. Since developing T cells and B cells are some of the most mitotically active cells, they are highly susceptible to this condition.
Treatment: Bone marrow transplantation and enzyme replacement have been used to treat SCID. Gene therapy is an experimental treatment.
In SCID both B & T cells are affected & deficient.
