Lecture 51

Question 1

What are four pyrimidines?

Answer 1

Cytosine (2 oxy, 4 amino)
Thymine (2,4 dioxy 5-methyl)
Uracil (2,4 dioxy)
Orotic Acid (2,4 dioxy 6 carboxy)

Question 2

What are the three forms of pyrimidines?

Answer 2

1) Base [cytosine]
2) Nucleoside (Base + Ribose) [cytidine]
3) Nucleotide (Base + Ribose + PO4 ester) [cytidine monophosphate]

Question 3

What is the 1st step in pyrimidine synthesis de novo?

Answer 3

1) Glutamine + HCO3- + 2ATP → Glutamate + Carbamoyl phosphate + 2 ADP + Pi
2) Uses Carbamoyl phosphate synthetase 2
3) Occurs in the cytosol

Question 4

What are activators and inhibitors of the 1st step of pyrimidine synthesis (of Carbamoyl phosphate synthetase 2 in the cytosol)?

Answer 4

1) Inhibited by UTP
2) Activated by ATP
3) Activated by PRPP (5-phosphoribosylpyrophosphate)

Question 5

Describe the reactions catalyzed by CAD

Answer 5

1) Glutamine + 2 ATP + CO2 → Carbamoyl phosphate + 2 ADP + Pi + Glutamate (uses Carbamoyl phosphate synthase 2)
2) Carbamoyl phosphate + Aspartate → Carbamoyl aspartate + Pi (uses aspartate transcarbamylase)
3) Carbamoyl aspartate → Dihydroorotate + H2O (uses Dihydroorotase)
4) CAD = Carbamoyl phosphate synthase 2 + Aspartate transcarbamylase + Dihydroorotase (1 polypeptide, 3 domains, 3 activities)

Question 6

What is different between pyrimidine and purine synthesis?

Answer 6

Pyrimidines are synthesized as a free ring while purines are not

Question 7

What happens to Dihydroorotate once it is formed?

Answer 7

1) It is oxidized to Orotate by reducing NAD+ to NADH

2) Uses Dihydroorotate dehydrogenase

Question 8

What contributes to the different parts of a pyrimidine base?

Answer 8

1) Glutamine donates N
2) Aspartate donates 2/3rds of the ring with 3 Cs and 1 N
3) CO2 donates a C

Question 9

What happens to Orotate once it is formed?

Answer 9

1) Orotate + PRPP → Orotidine 5’-monophosphate (OMP) + PPi (uses Orotate phosphoribosyl transferase
2) OMP → Uridine 5’-monophosphate (UMP) + CO2 (uses OMP decarboxylase)

Question 10

What does UMP synthase consist of?

Answer 10

1) Orotate phosphoribosyl transferase
2) OMP decarboxylase
3) 1 polypeptide, 2 domains, 2 activities

Question 11

What is low UMP synthase activity indicative of?

Answer 11

Orotic aciduria (abnormal growth, megaloblastic anemia, & treated with uridine-rich diet)

Question 12

How is CTP made from UTP?

Answer 12

UTP + ATP + Glutamine → CTP + ADP + Pi + Glutamate

Question 13

How are ribonucleotides converted to deoxyribonucleotides?

Answer 13

1) Ribonucleoside diphosphate + Thioredoin (2 SH) (reduced) → Deoxyribonucleoside diphosphate + H2O + Thioredoxin (S-S) (oxidized) (uses ribonucleotide reductase)
2) Thioredoxin (S-S) (oxidized) + NADPH + H+ → Thioredoxin (2SH) (reduced) + NADP+ (uses Thioredoxin reductase)

Question 14

What inhibits & activates the conversion of ribonucleotides to deoxyribonucleotides?

Answer 14

1) Inhibited by dATP

2) Activated by ATP

Question 15

How does the rate limiting step differ between prokaryotes and eukaryotes in pyrimidine synthesis?

Answer 15

1) In Eukaryotes: The Rate limiting step is the reaction catalyzed by Carbamoyl phosphate synthetase 2
2) In Prokarotes: The rate limiting step is the reaction catalyzed by aspartate transcarbamylase

Question 16

What inhibits and activates the rate limiting steps in pyrimidine synthesis?

Answer 16

1) UTP inhibits the rate limiting step in eukaryotes (using carbamoyl phosphate synthetase 2)
2) UTP inhibits the rate limiting step in prokaryotes (using aspartate transcarbamylase)
3) ATP activates CPS2
4) PRPP activates CPS2

Question 17

What inhibits Aspartate transcarbamylase (ATCase)?

Answer 17

The endproducts of pyrimidine biosynthesis inhibit ATCase (e.g. CTP)

Question 18

What is ADA deficiency?

Answer 18

ADA deficiency is due to a lack of the enzyme adenosine deaminase. This deficiency results in an accumulation of deoxyadenosine,[5] which, in turn, leads to a buildup 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.

Question 19

What reaction does ribonucleotide reductase catalyze?

Answer 19

1) ADP → dADP → dATP
2) GDP → dGDP → dGTP
3) CDP → dCDP → dCTP
4) UDP →dUDP → dUTP → dTTP

Question 20

How is thymidine produced?

Answer 20

1) dUMP + reduced N5,N10-methylene-tetrahydrofolate → TMP + oxidized Dihydrofolate (Thymidylate synthase)
2) Dihydrofolate + NADPH → Tetrahydrofolate + NADP+ (Dihydrofolate reductase)
3) Tetrahydrofolate + Serine → N5,N10-methylene-tetrahydrofolate + Glycine (Serine transhydroxymethylase)

Question 21

What is the salvage reaction for a pyrimidine?

Answer 21

Uridine-cytidine kinase (deoxycytidine kinase; thymidine kinase) converts nucleosides to nucleotides

Question 22

What is the degradation reaction for a pyrimidine?

Answer 22

Pyrimidine rings are cleaved and degraded to soluble structures (contrast to purines)

Question 23

What is an example of drug therapy to reduce over producton of thymidine (TMP)?

Answer 23

1) Fluorodeoxyuridylate (5-FU) down regulates Thymidylate synthase. 5FU is a simple derivative of Uracil (5-Fluoro-Uracil)
2) Methotrexate & Aminopterin down regulates Dihydrofolate reductase

Question 24

What is the function of AZT?

Answer 24

AZT inhibits HIV reverse transcriptase (RNA-dependent DNA polymerase). This class of compounds (chemotherapeutics, viral inhibitors, etc.) are called nucleoside analogs

Question 25

What are common side effects of DNA inhibitor chemotherapeutics?

Answer 25

1) Diarrhea
2) Skin & eye sensitivity to sunlight
3) Abnormal liver function tests
4) Hair loss
5) Immuno-suppression
6) Skin rashes
7) Fatigue
8) Headache, backache
9) Spinal cord irritation
10) Peripheral neuropathies