nucleotide biosynthesis

Question 1

Nucleotide functions

Answer 1

Essential to ALL cells

1) Synthesis of DNA and RNA
2) Carriers of activated intermediates
- adenylation, UDP glucose
3) Components of:
- CoA, FAD, NAD+, NADP+
4) second messengers in signal transduction pathway
- cAMP, cGMP
5) Energy Currency
- ATP

Question 2

Nucleotides are acquired from two sources; What are they?

Answer 2

1) Salvage Pathway
- bases are recovered and attached to a PRPP

2) De Novo Synthesis
- Pyrimidines are assembled first then attached to ribose
- Purines are assembled while attached to ribose

Question 3

What does a nucleotide contains

Answer 3

Nitrogenous Base
Pentose Sugar
Phosphate groups

Question 4

Thymine

Answer 4

Pyrimidine Base

5-methyl-2,4-dioxypyrimidine

Question 5

Cytosine

Answer 5

Pyrimidine Base

2-oxy-4-aminopyrimidine

Question 6

Uracil

Answer 6

Pyrimidine Base

2,4-dioxypyrimidine

Question 7

Adenine

Answer 7

Purine Base

6-aminopurine

Question 8

Guanine

Answer 8

Purine Base

2-amino-6-oxypurine

Question 9

Sugar Phosphate Backbone

Answer 9

Nucleotides are connected 3’ to 5’ phosphodiester bond

1) Imparts uniform negative to DNA/RNA
- negative charge repels nucleophilic species (hydroxyl) thus phosphodiester bonds resist hydrolytic attack
- Cretes 3’ and 5’ end

Question 10

What connects a nitrogenous base to a sugar in nucleotides

Answer 10

B-Glycosidic bond

Question 11

Carbamoyl Phosphate Synthetase II

Answer 11

DeNovo Synthesis of Pyrimidines

HCO3- + Ammonia (NH3+) -> Carbamoyl Phosphate

• located in cytoplasm
• requires 2 ATP
• ammonia is usually provided from hydrolysis of Y amide of glutamine which is converted to Glutamate

Question 12

Aspartate Transcaramoylase

Answer 12

DeNovo Synthesis of Pyrimidines

Carbamoyl Phosphate + Aspartate-> Carbamoyl Aspartate

• Aspartate displaces Phosphorus and attached Alpha Amino to Carbonyl
• Occurs in cytosol of cytoplasm

Question 13

Dihydroorotase

Answer 13

DeNovo Synthesis of Pyrimidines
Hydrolytic Cyclization

Carbamoyl Aspartate-> Dihydroorotate

• loss of H2O
• occurs in cytosol of cytoplasm

Question 14

Dihydroorotate Dehydrogenase

Answer 14

DeNovo Synthesis of Pyrimidines

Dihyrdoorotate Oxidized to Orotate

• NAD+ to NADH
• enzyme found in inner mitochondrial membrane

Question 15

Pyrimidine Phosphoribosyltransferase

Answer 15

Rotate + PRPP-> Orotidylate (OMP)

-displaces PPi

Question 16

Orotidylate Decarboxylase

Answer 16

Conversion of Orotidylate(OMP) to Uridylate (UMP)
-Signification rate enhancement (VERY SLOW); catalyzed once per second

OMP-> UMP
-lose CO2

Question 17

UMP synthase

Answer 17

single peptide containing the Pyrimidine Phosphoribosyltransferase activity and the Orotidylate Decarboxylase activity

Question 18

Oritic Aciduria

Answer 18

Rare genetic defect caused by a deficiency of one of the activities of UMP synthase

Symptoms:

• poor growth
• megaloblastic anemia
• excretion of orotate urine

Question 19

CTP synthetase

Answer 19

UTP-> CTP
-ATP-> ADP
Glutamine provides the amino group

Question 20

Thymidylate Synthase

Answer 20

dUMP->dTMP
-N5,N10 methylene THF->DHF

inhibited by 5-fluorouracil
-nucleotide analog becomes irreversibly attached to the enzyme

Question 21

Dihydrofolate Reductase

Answer 21

DHF-> THF

• reduces DHF to THF
• NADPH to NADP+
• inhibited by methotextrate

Question 22

Anticancer drugs

Answer 22

Cancer cells require an abundant supply of thymidylate for synthesis
-rapidly dividing

Thymidylate synthase inhibitors:
Fluoruracil
-Nucleotide analog inhibits thymidylate
-suicide inhibitor

Dihydrofolate reductase inhibitors:

• Aminopterin-analog of dihydrofolate
• Methotrexate-competitive inhibitor

Question 23

What is the common theme in purine synthesis

Answer 23

a carbonyl is activitated by phosphorylation

-the phosphate group is displaced by a nucleophile which becomes covalently attached

Question 24

Glutamine Phosphoribosyl amidotransferase

Answer 24

Purine de novo synthesis

Amide group of glutamine replaces the pyrophosphate group on ribose

PRPP-> PR-NH2 + PPi
-Gln + H2O-> NH3+ + Glutamate (provides NH3+)

Pyrophosphorylase-Hydrolyzes Pyrophosphate to 2Pi proving energy

Inhibited by IMP-Inosine Monophosphate
-feedback inhibition

Controlled by the concentration of PRPP

• High Concentration=fast rate
• low concentration=slow rate

Question 25

Methotrexate

Answer 25

Inhibits ALL enzymes using THF as prosthetic group
-limits the conversion of DHF to THF, which limits the synthesis of purines and inhibits replication and transcription in rapidly dividing cells (Cancer cells)

Question 26

Carboxylase

Answer 26

Purine de novo synthesis-> sixth step

• activation of bicarbonate by Phosphorylation leads to the formation of N-C bond
• Bicarbonate then JUMPS

Question 27

Adenylsuccinate Lyase

Answer 27

-cleavage of C-N bond releases Fumurate (comes from Aspartate)

Question 28

Cyclohydrolase

Answer 28

Purine de novo synthesis (final step

-clyclizes by dehydration to get Inosinate

Question 29

How to form AMP

Answer 29

Inosinate (IMP) is converted into AMP

Adenylosuccinate Synthase (catalyzes both reactions)
-substitution of Amino group at carbonyl provided by Aspartate 

1) IMP + GTP + Asp->Asp attached to Carboxyl
2) Loss of fumurate to form amino group=AMP

Question 30

How to form GMP

Answer 30

GMP synthetase

1) inosinate is oxidized to xanthylate
- -NAD+ reduced to NADH
2) Keto group is aminated by ammonia provided by Gln

Question 31

How are deoxyribonucleotides synthesized?

Answer 31

Synthesized from ribonucleotides by reduction of Hydroxyl on 2’ carbon

Ribonucleotide reductase

• has to be in diphosphate form not monophosphate
• NADPH oxidized to NADP+
• product=dNDPs which can then be phosphorylated to dNTPs