Nucleotide metabolism II

Question 1

Draw the structures of the 2 purine bases.

Answer 1

Question 2

Draw the structures of the 3 pyrimidine bases.

Answer 2

Question 3

Describe the basic scheme of the de novo synthesis of the 2 pyrimidine nucleotides.

Answer 3

use UMP as common precursor

1. de novo synthesis of UMP
2. … → UTP
3. … → CTP

Question 4

Which enzymes are involved in the de novo synthesis of UMP?

Cellular location?

Answer 4

3 enzymes

• CAD complex: multifunctional enzyme, step 1-3
  → in cytosol
• dihydroorotate dehydrogenase: step 4
  → in inner mitochondrial membrane
• UMP synthase: multifunctional enzyme, step 5-6
  ​→ in cytosol

Question 5

The CAD complex is a multifunctional enzyme.

What are its 3 activities?

Answer 5

• carbamoyl-P synthetase II
• Asp transcarbamoylase
• dihydroorotase

_{<u><strong>​</strong>NOTE:</u> in urea cycle step 1 and 2 catalyzed by carbamoyl-P synthetase I and ornithine transcarbamoylase}

Question 6

UMP synthase is also a multifunctional enzyme.

What are its 2 activities?

Answer 6

• orotate phosphoribosyl transferase (OPRT)
• oritidylate (OMP) decarboxylase

Question 7

Which structures form the pyrimidine ring system?

Answer 7

• carbamoyl-phosphate
• Asp

Question 8

The enzyme activity of which enzyme is needed to catalyze the first step of pyrimidine nucleotide synthesis?

Reaction.

Where does it happen?

Answer 8

Carbamoyl-P synthetase II (CPS II) (CAD)
in cytosol

CO₂ + Gln + 2ATP + H₂O
→ Glu + 2ADP + P_i + carbamoyl-P

Question 9

What happens to carbamoyl-P during the de novo synthesis of pyrimidine nucleotides?

Enzyme + reaction.

Where does it happen?

Answer 9

Asp transcarbamoylase (CAD)
in cytosol

carbamoyl-P + Asp → carbamoyl-Asp + P_i

Question 10

What happens to carbamoyl-Asp during the de novo synthesis of pyrimidine nucleotides?

Enzyme + reaction.

Where does it happen?

Answer 10

dihydroorotase (CAD)
in cytosol

carbamoyl-Asp → dihydroorotate + H₂O

Question 11

What happens to dihydroorotate during the de novo synthesis of pyrimidine nucleotides?

Enzyme + reaction.

Where does it happen?

Answer 11

dihydroorotate dehydrogenase
on inner mitochondrial membrane

dihydroorotate + NAD⁺ → orotate + NADH

_{<u>NOTE:</u> ubiquinone used as electron acceptor}

Question 12

What happens to orotate during the de novo synthesis of pyrimidine nucleotides?

Enzyme + reaction.

Where does it happen?

Answer 12

orotate phosphoribosyl transferase (UMP-synthase)
in cytosol

orotate + PRPP → orotidylate (OMP) + PP_i

_{<u>NOTE:</u> same concept as for purines:<br></br>base + PRPP → nucleotide + PPi}

Question 13

What happens to orotidylate during the de novo synthesis of pyrimidine nucleotides?

Enzyme + reaction.

Where does it happen?

Answer 13

oritidylate (OMP) decarboxylase (UMP-S)
in cytosol

OMP → CO₂ + uridine monophosphate (UMP)

Question 14

How is UTP formed?

Which enzymes catalyze those reactions?

Answer 14

in 2 steps

1. UMP kinase:
   UMP + ATP → UDP + ADP
2. UDP kinase:
   UDP + ATP → UTP + ADP

Question 15

How is CTP formed?

Enzyme + reaction.

Answer 15

CTP synthetase

UTP + ATP + Gln + H₂O → ADP + P_i + Glu + CTP

Question 16

Which enzyme catalyzes the rate-limiting step of de novo pyrimidine synthesis?

Answer 16

carbamoyl-P synthetase 2

Question 17

Which substances regulate the activity of the CAD complex?

Answer 17

​allosteric regulation

• inhibited by UTP (product)
• activated by PRPP (also substrate of purine synthesis → if we have many purines, we also need pyrimidines for base pairing)

Question 18

Which substances are able to regulate the activity of UMP synthase?

Answer 18

inhibited by UMP (product)

Question 19

What is the consequence of hereditary UMP-S deficiency?

How is it treated?

Symptoms?

Answer 19

causes oritic aciduria
no formation of UTP, but orotate accumulates → disinhibition of CAD → incr. orotate production, eventually excreted via urine

• treatment: oral uridine
• symptoms: megaloblastic anemia, delayed physical/intellectual development, leukopenia (↓ leukocytes), early death

Question 20

Which substances are inhibitors of UMP-S?

Consequence?

Answer 20

• allopurinol: used to treat gout
• 6-azauridine: synthetic nucleotide analog, used in cancer treatment

→ inhibition of UMP-S leads to orotic aciduria (same mechanism as UMP-S deficiency)

Question 21

What is the consequence of an ornithine transcarbamoylase deficiency?

Mechanism.

Answer 21

reason for hyperammonemia type II
+ also causes orotic aciduria

carbamoyl-P not used in urea cycle, accumulates → used by CAD instead → incr. orotate formation + excretion

Question 22

What is the difference btw purine and pyrimidine salvage reactions?

Answer 22

• purine salvage reactions:
  
  • purine bases → nucleotides
  • esp. important in extrahepatic tissues b/c are unable to undergo de novo synthesis
• pyrimidine salvage reactions:
  
  • pyrimidine nucleosides → nucleotides
  • not as important as purine salvage RXNs b/c all tissues can do de novo synthesis

⇒ pyrimidine nucleosides only require phosphorylation

Question 23

Describe the mechanism of pyrimidine salvage reactions.

Answer 23

• UMP/CMP can be recovered directly
  urdine/cytidine kinase
  uridine/cytidine + ATP → UMP/CMP + ADP
• CMP can also be converted to UMP
  cytidilate deaminase
  CMP + H₂O → UMP + NH₃

Question 24

Describe the general scheme of pyrimidine nucleotide catabolism.

Answer 24

1. pyrimidine nucleotides → pyrimidine nucleosides
2. transported via blood to liver/kidney
3. … → pyrimidine bases
4. … multiple steps → CO₂, NH₃, _β-_Ala

Question 25

Which groups of enzymes catalyzes the catabolism of pyrimidine nucleotides? Reaction scheme. Individual reaction products.

Answer 25

**_5' nucleotidase_** specific for each nucleotide **monophosphate nucleotide** → **nucleoside + P_i** * UMP → uridine * dTMP → thymidine * CMP → cytidine _{just like purine catabolism}

Question 26

Which groups of enzymes catalyzes the catabolism of pyrimidine nucleosides? Reaction scheme. Individual reaction products.

Answer 26

**_pyrimidine nucleotide phosphorylases_** specific for each nucleoside **nucleoside + P_i → pyrimidine base + sugar** * uridine → uracil + ribose-1P * thymidine → thymine + **d**ribose-1P _NOTE:_ since thymin only exists in DNA, there is no oxy-version besides that same mechanism as for purines

Question 27

One pyrimidine nucleoside is not catabolized as described earlier. Which one? Enzyme + reaction.

Answer 27

**_cytidine deaminase_** **cytidine + H₂O → uridine + NH₃**

Question 28

Which C atom of deoxynucleotides is the deoxygenated one? Consequence?

Answer 28

C2 ⇒ **DNA is less prone to hydrolysis** due to missing -OH group (ergo more stable)

Question 29

How are deoxyribonucleotides produced? Enzyme + reaction. What would be a prerequisite for this reaction to happen?

Answer 29

**_ribonucleotide reductase_** **NDP → dNDP + H₂O** _BUT:_ has to be _reduced firs_t, by either of the 2 mechanisms: * **tioredoxin** * **glutaredoxin**

Question 30

Describe the structure of ribonucleotide reductase. When is it active?

Answer 30

**_ribonucleotide reductase (RR)_** dimer * **R1-subunit**: catalytic subunit, const. expressed * **R2-subunit:** regulatory subunit, _induced by E2F_ transcription factor ⇒ only active during _S phase_ of cell cycle (dividing cells)

Question 31

How does tioredoxin help to reduce ribonucleotide reductase? Describe the pathway.

Answer 31

1. **NADPH** used to reduce FAD of tioredoxine reductase to **FADH₂** 2. **_tioredoxine reductase:_** used to reduce Trx-S₂ to **Trx(SH)₂** 3. used to reduce ribonucleotide reductase-S to **RR(SH)₂** ⇒ ribonucleotide reductase then catalyzes dNDP formation

Question 32

How does glutaredoxin help to reduce ribonucleotide reductase? Describe the pathway.

Answer 32

1. **NADPH** used to reduce **glutatione** 2. **_glutathione reductase:_** used to reduce glutaredoxin-S₂ to **Grx(SH)₂** 3. used to reduce ribonucleotide reductase-S to **RR(SH)₂** ⇒ ribonucleotide reductase then catalyzes dNDP formation

Question 33

Describe the allosteric regulation of RR.

Answer 33

2 types of regulation: * complete inhibition: by **dATP** * change of ratio of dNTP synthesis * * **dTTP** = ↑dGTP, ↓dTTP/dCTP * **dGTP** = ↑dATP, ↓dGTP/dTTP/dCTP * → _NO_ formation of any dNTPs

Question 34

The ribonucleotide reductase forms dNDPs. But how do they become dNTPs? List the individual products.

Answer 34

phosphorylated by **_kinases_** **dNDP + ATP → dNTP + ADP** * dADP **→** dATP * dGDP **→** dGTP * dCDP **→** dCTP

Question 35

There is one dNTP which is not formed by phosphorylation from its dNDP. How is it formed instead?

Answer 35

1. **dUDP → dUMP** (otherwise higher risk for accidental incorporation into DNA) to 2. **_thymidylate synthase_** dUMP + methylene H₄F → dTMP + H₂F 3. eventually phosphorylated twice by kinases dTMP →→ **dTTP**

Question 36

What does 5-fluoro-dUMP do?

Answer 36

**synthetic nucleotide analog:** cannot be converted to dTMP, hence _inhibiting thymidilate synthase_ → treatment of colon cancer

Question 37

How is CH₂-H₄F restored after synthesis of dTMP? Enzyme + reactions.

Answer 37

_folate cycle_ 1. **_dihydrofolate reductase_** H₂F + NADPH → H₄F + NADP⁺ 2. **CH-H₄F + Ser/Gly → CH₂-H₄F + H₂O** ⇒ eventually methylene-H₄F​ can be used again for dTMP synthesis or continue further in folate cycle

Question 38

What does **methotrexate** do?

Answer 38

**folate antagonists** _inhibit dihydrofolate reductase_ which is required to restore H₄F, hence dTMP cannot be generated → used in tumor therapy

Question 39

Explain the function of sulphonamides.

Answer 39

* humans: acquire essential vitamine folate through diet * bacteria: synthesize folate ⇒ **sulphonamides** inhibit synthesis of folate → inhibit growth/proliferation of bacteria = _antibacterial effect_

Question 40

There are also salvage reactions for **_deoxy_**nucleotides. Any similarites to the salvage reactions of purine/pyrimidine nucleotides? Individual products.

Answer 40

_same mechanism as pyrimidine salvage reactions:_ **deoxynucleoside + ATP → dNMP + ADP** * dcytidine **→** dCMP * dadenine **→** dAMP * dguanine **→** dGMP * thymidine **→** dTMP

Question 41

Which enzymes catalyze those deoxy salvage reactions?

Answer 41

* **deoxycytidine kinase (dCK)** * **thymidine kinase (dTK)**

Question 42

Which reaction is catalyzed by dCK? Characteristics.

Answer 42

**_deoxycytidine kinase (dCK)_** **deoxycytidine + ATP → dCMP + ADP** * lymphoid specific * _broad substrate specificity_ * *dAde, dGua, dCyt**

Question 43

Explain the function of antileucemic agents acting on deoxycitidine kinase Examples.

Answer 43

**= deoxynucleotide (substrates for dCK) analogues →**cannot be converted to physiological dNMPs * arabinosyl-cytosine * 2-chloro-deoxyadenosine

Question 44

Which reaction is catalyzed by dTK? Characteristics.

Answer 44

**_thymidine kinase (dTK)_** **thymidine + ATP → ADP + dTMP** * S-phase dependent enzyme * substrate specific

Question 45

What is an antiviral medication for the herpes simplex virus? Why?

Answer 45

* human dTK: substrate specific * HSV dTK: wide substrate specificity → selective inhibition w/ nucleoside analogues (**aciclovir**)

Question 46

How can the earlier described dTK version of the herpes simplex virus be clinically useful?

Answer 46

can be transfected into T lymphocytes, then **ganciclovir** administered to selectively kill them → used for treatment of acquired immunodeficiencies (e.g. AIDS)