Lecture 7 (4A) - Pyrimidine Biosynthesis

Question 1

Pyrimidines

Answer 1

thymine and cytosine

1 ring

Question 2

Pyrimidine biosynthesis

Answer 2

2 methods

• de novo - forms uridine monophosphate (before makes T or C)
• salvage pathways

Question 3

de novo biosynthesis

Answer 3

6 steps (purines 9 steps)

Question 4

de novo biosynthesis

steps

Answer 4

1. carbamoyl phosphate synthesized
2. phosphate to aspartate (phosphate displaced by aspartate)
3. ring closure
4. dihydroorotate oxidation
5. sugar added (have ring, then put sugar on)
6. decarboxylation

Question 5

de novo biosynthesis

1. carbamoyl phosphate synthesized

Answer 5

• requires 2 ATP, glutamine, whater, HCO₃^- (bicarbonate)
  
  • glutamine side chain gives up N (ammonia) → glutamate
• 1 ATP for phosphate group
• 1 ATP as energy source
  
  • ATP + H₂O → ADP + Pi
  • glutamine → glutamate
• forms: carbamoyl phosphate (seen in urea cycle in mito, here it cytosol)
• enzyme - carbamoyl phosphate synthetase
  
  • has 3 active sites
  • glutamine hydrolys site ( → ammonia)
  • bicarbonate phosphorylation site
  • carbamic acid phosphorylation site
  • the product of 1 is channelled to become the substrate of the next (nothing escapes except the ultimate products, prevents unwanted byproduct and makes sure you get the product you want)

Question 6

de novo biosynthesis

1. carbamoyl phosphate synthesized

requires

Answer 6

• 2 ATP
  
  • 1 for phosphate group
  • 1 for energy source
    
    • ATP + H2O → ADP + Pi
• glutamine
  
  • side chain gives up N (ammonia) → glutamate
  • glutamine → glutamate
• water
• HCO₃^- (bicarbonate)

Question 7

de novo biosynthesis

1. carbamoyl phosphate synthesized

forms

Answer 7

carbamoyl phosphate

(seen in urea cycle in mitochondria, here in cytosol)

Question 8

de novo biosynthesis

1. carbamoyl phosphate synthesized

enzyme

Answer 8

carbamoyl phosphate synthetase

has 3 active sites

• glutamine hydrolysis site ( → ammonia)
• bicarbonate phosphorylation site
• carbamic acid phosphorylation site
• the product of 1 is channelled to become the substrate of the next
  
  • (nothing escapes except the ultimate products)
  • prevents unwanted byproduct and makes sure you get what you want

Question 9

de novo biosynthesis

2. phosphate to aspartate

Answer 9

phosphate displaced by aspartate

• requires asparate (removes Pi)
• forms carbamoyl aspartate
• important: flux-generating step
  
  • makes production go faster
  • upregulated
  • wante N and C from carbamoyl phosphate
• enzyme asparate transcarbamoylase
  
  • similar to ornithine transcarbamoylase

by the end have the whole pyrimidine ring assembled, just need ring closure

Question 10

de novo biosynthesis

2. phosphate to aspartate

requires

Answer 10

aspartate (removes Pi)

Question 11

de novo biosynthesis

2. phosphate to aspartate

forms

Answer 11

carbamoyl aspartate

whole body of pyrimidine assembled, now just need ring closure

Question 12

de novo biosynthesis

2. phosphate to aspartate

important step…

Answer 12

flux-generating step

• makes production go faster
• upregulated
• want N and C from carbamoyl phosphate

Question 13

de novo biosynthesis

2. phosphate to aspartate

enzyme

Answer 13

aspartate transcarbamoylase

(similar to ornithine transcarbamoylase)

Question 14

de novo biosynthesis

3. ring closure

Answer 14

• condensation reaction (removes water)
• forms dihydroorotate (oxidized to orotate)
• enzyme dihydroorotase

Question 15

de novo biosynthesis

3. ring closure

forms

Answer 15

dihydroorotate

(oxidized to orotate)

Question 16

de novo biosynthesis

3. ring closure

enzyme

Answer 16

dihrydoorotase

Question 17

de novo biosynthesis

4. dihydroorotate oxidation

Answer 17

• NAD+ → NADH
  
  • mammals FAD and FADH
• requires a flavin-cotainiing enzyme
  
  • flavin in mitochondrial membrane
• quinone → reduced quinone
• forms orotate
• enzyme - dihydroorotate oxidase

Question 18

de novo biosynthesis

4. dihydroorotate oxidation

requires

Answer 18

a flavin-containing enzyme

(in inner mitochondrial membrane)

Question 19

de novo biosynthesis

4. dihydroorotate oxidation

forms

Answer 19

orotate

Question 20

de novo biosynthesis

4. dihydroorotate oxidation

enzyme

Answer 20

dihydoorotate oxidase

Question 21

de novo biosynthesis

5. sugar added

Answer 21

• have ring then put sugar on
• compare with purines - purines built onto sugar
• requies sugar PRPP
  
  • formed in ourine biosynthesis (primer)
  • hydrolyzed → energy
• hydrolysis of PRPP (sugar) drives action
• PPi lost
• forms orotidine monophosphate (OMP) aka oroditylate
• enzyme pyrimidine phosphoribosyl transferase
  
  • inversion of β to α form (nucleophilic attack
  • enzyme has 2 functions - named separately (see next step)

Question 22

de novo biosynthesis

6. decarboxylation

Answer 22

• unusual step because requires no cofactors
  
  • enzyme itself has the capacity to carry out reaction
• loses CO₂
• forms: uridine monophosphate (UMP) or uridylate
• enzyme: orotidylate decarboxylation
  
  • 2nd half of the reaction step 5 enzyme

Question 23

de novo biosynthesis

6. decarboxylation

unusual step because

Answer 23

requires no cofactors

the enzyme itself has the capacity to carry out the reaction

Question 24

de novo biosynthesis

6. decarboxylation

forms

Answer 24

uridine monophosphate (UMP) or uridylate

(loses CO2)

Question 25

de novo biosynthesis

6. decarboxylation

enzyme

Answer 25

orotidylate decarboxylase

(second half of step 5 enzyme)

Question 26

CTP formation

(cytosine triphosphate)

Answer 26

• phosphate addition first, activates bond, nucleophilic attack
• formed from amination of UTP
• requiers UTP to be formed first

UMP → UTP → CTP

• enzyme CTP synthetase

Question 27

Pyrimidine biosynthesis regulation

Answer 27

regulation is species dependent

Question 28

Pyrimidine biosynthesis regulation

bacteria

Answer 28

• primary regulation is at step 2
  
  • phosphate displaced by aspartate
  • flux regulating - up/down regulated - qunatity of production, not rate
• inhibition by CTP
• activation by ATP
• feedback inhibition/feed forward activation
  
  • lots of ATP → make more pyrimidines
  • inhibition by pyrimidines

Question 29

Pyrimidine biosynthesis regulation

bacteria

primary regulation is at

Answer 29

step 2

• phosphate dispalced by aspartate
• flux regulating
  
  • up/down regulated
  • quantity of production, not rate

Question 30

Pyrimidine biosythesis regulation

bacteria

inhibition by…

activation by…

Answer 30

• inhibition by CTP
• activation by ATP
• feedback inhibition/feed forward activation
  
  • lots of ATP → make more pyrimidines
  • inibition by pyrimidines

Question 31

Pyrimidine biosynthesis regulation

animals

Answer 31

• more complex, more regulation steps
• regulation is through control of carbamoyl phosphate synthetase
  
  • product inhibition feedback
• inhibition by UPT and CTP
• activation by ATP and PRPP

second regulation

• competitive inhibition of orotidylate decarboxylate by UMP (product inhibition - slows down)
• and CMP (competitive)
  
  • may sit in active site and block substrate

Question 32

Salvage pathways

Answer 32

addition of ribose (sugar) ring to pyrimidine rings

base + ribose-1-phosphate → nucleoside + Pi

• get bases from breakdown in gut
• enzyme nucleoside phosphorylase

Question 33

Salvage pathways

enzyme

Answer 33

nucleoside phosphorylase

Question 34

Disease

Answer 34

orotic aciduria

• severe deficiency of pyrimidine phosphoribosyl transferase / orotidylate decarboxylase
• admisnister precursors of bases → reduce orotic acid