Pyrimidine Nucleotide Synthesis Flashcards
What are the 3 pyrimidines
- Cytosine
- Thymine
- Uracil
How is the pyrimidine de novo pathway different from the purine de novo pathway (in terms of PRPP)?
Pyrimidine de novo pathway will assemble nitrogenous base FIRST then attach it to PRPP
Describe the salvage pathway in pyrimidine de novo synthesis
Uracil is directly attached to a PRPP molecule by uracil phosphoribosyltransferase to make the UMP precursor
Describe step 1 in the de novo synthesis of pyrimidines
Carbamoyl phosphate is generated in the cytoplasm by CPS II (carbamoyl phosphate synthetase II)
Describe step 2 in the de novo synthesis of pyrimidines
Aspartate trans-carbamoylase takes aspartate and carbamoyl phosphate and synthesizes carbamoylaspartate
Carbamoylaspartate contains all the atoms that will be incorporated into the 6 membered ring
Describe step 3 in the de novo synthesis of pyrimidines
2 steps take place in order to close the ring and form the first pyrimidine intermediate (orotate)
Describe step 4 in the de novo synthesis of pyrimidines
Once you have orotate it gets attached to the pentos sugar which comes from PRPP to form orotidylate
Describe step 5 in the de novo synthesis of pyrimidines
Orotidylate is converted to uridylate (UMP) which is the precursor for all other pyrimidine molecules
Describe step 6 in the de novo synthesis of pyrimidines
Once UMP is formed it can be converted to triphosphate (UTP)
Describe step 7 in the de novo synthesis of pyrimidines
UTP can be aminated to CTP
Why is CTP important in the regulation of the de novo synthesis pathway of pyrimidines
CTP will inhibit the first enzyme involved in this process (aspartate trans-carbamoylase)
How does CTP inhibit aspartate trans-carbamoylase?
- CTP increases the KM of aspartate trans-carbamoylase for aspartate
- If ATP concentrations = high inhibition by CTP is prevented*
How is RNA converted to DNA?
Ribonucleotide reductase
How does ribonucleotide reductase reduce ribonucleotides?
By getting “fed” electrons from NADPH
What are the 2 pathways that NADPH can follow in order to get electrons to ribonucleotide reductase?
- Glutathione reductase pathway
- Thioredoxin reductase pathway
Describe the glutathione reductase pathway
- NADPH + H+
- 2GSH
- Glutaredoxin
- Ribonucleotide reductase
Final step is NDP gets converted to dNDP
Describe the thioredoxin reductase pathway
- NADPH + H+
- FADH2
- Thioredoxin
- Ribonucleotide reductase
Final step is NDP gets converted to dNDP
Why is it important to regulate ribonucleotide reductase?
- Too many dNTP’s = mutagenic
- Not enough dNTP’s = lethal
Describe the structure of ribonucleotide reductase
- Heteroteetramer
- 2 α subunits
- 2 β subunits
- Active site = in between alpha and beta subunits
- 2 regulatory sites
What are the 2 regulatory sites of ribonucleotide reductase?
- Substrate specificity site
- Primary regulation site
Describe the substrate specificity site
- Can bind to any of the 4 nucleotides
- Controls which substrate ribonucleotide reductase will preferentially use
- Maintains balance between deoxy nucleotides
Describe the primary regulation site
- Controls the overall activity of ribonucleotide reductase
- High levels of dATP = inactivated
- High levels of ATP = activated
BRIEFLY Describe the synthesis of Thymidine deoxyribonucleotides (dTPPS)
One carbon transfer reaction done by THF cofactor methylates dUMP to dTMP
Catalyzed by thymidylase synthase
Describe the synthesis of Thymidine deoxyribonucleotides (dTPPS) (be specific)
- Begins with dUTP
- dUTP is converted to dUMP
- dUMP is converted to dTMP
- Catalyzed by thymidylate synthase (requires N5, N10-methylene THF cofactor)
- Can be inhibited by 5-FU (5-Fluorouracil)
How can N5, N10-methylene-THF be used to target different types of cancer cells?
Antioflates such as:
- Aminopterin
- Methotrexate
- Trimethoprim
Inhibit enzymes that produce THF
Remember THF is needed to synthesize dTMP
How does Folic acid deficiency result in reduced dTMP synthesis?
- Reduction in thymidylate synthesis results in dUTP (uracil) getting incorporated into DNA
- Repair mechanisms remove uracil by creating strand breaks that affect the structure and function of DNA
- Symptoms = cancer, heart disease, brain dysfunction etc.
Describe the catabolism of pyrimidines?
- Pyrimidines get converted to free ammonia which then get converted into urea
- Involves nucleotidases and nucleosidases
- Nucleotidases: removes phosphate
- Nucleosidases: removes ribose
- The final products are Malonyl-CoA (CMP and UMP) and Methylmalonyl-CoA (dTMP)
