Pyrimidine Nucleotide Synthesis

Question 1

What are the 3 pyrimidines

Answer 1

• Cytosine
• Thymine
• Uracil

Question 2

How is the pyrimidine de novo pathway different from the purine de novo pathway (in terms of PRPP)?

Answer 2

Pyrimidine de novo pathway will assemble nitrogenous base FIRST then attach it to PRPP

Question 3

Describe the salvage pathway in pyrimidine de novo synthesis

Answer 3

Uracil is directly attached to a PRPP molecule by uracil phosphoribosyltransferase to make the UMP precursor

Question 4

Describe step 1 in the de novo synthesis of pyrimidines

Answer 4

Carbamoyl phosphate is generated in the cytoplasm by CPS II (carbamoyl phosphate synthetase II)

Question 5

Describe step 2 in the de novo synthesis of pyrimidines

Answer 5

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

Question 6

Describe step 3 in the de novo synthesis of pyrimidines

Answer 6

2 steps take place in order to close the ring and form the first pyrimidine intermediate (orotate)

Question 7

Describe step 4 in the de novo synthesis of pyrimidines

Answer 7

Once you have orotate it gets attached to the pentos sugar which comes from PRPP to form orotidylate

Question 8

Describe step 5 in the de novo synthesis of pyrimidines

Answer 8

Orotidylate is converted to uridylate (UMP) which is the precursor for all other pyrimidine molecules

Question 9

Describe step 6 in the de novo synthesis of pyrimidines

Answer 9

Once UMP is formed it can be converted to triphosphate (UTP)

Question 10

Describe step 7 in the de novo synthesis of pyrimidines

Answer 10

UTP can be aminated to CTP

Question 11

Why is CTP important in the regulation of the de novo synthesis pathway of pyrimidines

Answer 11

CTP will inhibit the first enzyme involved in this process (aspartate trans-carbamoylase)

Question 12

How does CTP inhibit aspartate trans-carbamoylase?

Answer 12

• CTP increases the KM of aspartate trans-carbamoylase for aspartate
• If ATP concentrations = high inhibition by CTP is prevented*

Question 13

How is RNA converted to DNA?

Answer 13

Ribonucleotide reductase

Question 14

How does ribonucleotide reductase reduce ribonucleotides?

Answer 14

By getting “fed” electrons from NADPH

Question 15

What are the 2 pathways that NADPH can follow in order to get electrons to ribonucleotide reductase?

Answer 15

1. Glutathione reductase pathway
2. Thioredoxin reductase pathway

Question 16

Describe the glutathione reductase pathway

Answer 16

1. NADPH + H+
2. 2GSH
3. Glutaredoxin
4. Ribonucleotide reductase
   Final step is NDP gets converted to dNDP

Question 17

Describe the thioredoxin reductase pathway

Answer 17

1. NADPH + H+
2. FADH2
3. Thioredoxin
4. Ribonucleotide reductase
   Final step is NDP gets converted to dNDP

Question 18

Why is it important to regulate ribonucleotide reductase?

Answer 18

• Too many dNTP’s = mutagenic
• Not enough dNTP’s = lethal

Question 19

Describe the structure of ribonucleotide reductase

Answer 19

• Heteroteetramer
• 2 α subunits
• 2 β subunits
• Active site = in between alpha and beta subunits
• 2 regulatory sites

Question 20

What are the 2 regulatory sites of ribonucleotide reductase?

Answer 20

1. Substrate specificity site
2. Primary regulation site

Question 21

Describe the substrate specificity site

Answer 21

• Can bind to any of the 4 nucleotides
• Controls which substrate ribonucleotide reductase will preferentially use
• Maintains balance between deoxy nucleotides

Question 22

Describe the primary regulation site

Answer 22

• Controls the overall activity of ribonucleotide reductase
• High levels of dATP = inactivated
• High levels of ATP = activated

Question 23

BRIEFLY Describe the synthesis of Thymidine deoxyribonucleotides (dTPPS)

Answer 23

One carbon transfer reaction done by THF cofactor methylates dUMP to dTMP
Catalyzed by thymidylase synthase

Question 24

Describe the synthesis of Thymidine deoxyribonucleotides (dTPPS) (be specific)

Answer 24

• 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)

Question 25

How can N5, N10-methylene-THF be used to target different types of cancer cells?

Answer 25

Antioflates such as:
- Aminopterin
- Methotrexate
- Trimethoprim
Inhibit enzymes that produce THF
Remember THF is needed to synthesize dTMP

Question 26

How does Folic acid deficiency result in reduced dTMP synthesis?

Answer 26

• 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.

Question 27

Describe the catabolism of pyrimidines?

Answer 27

• 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)