11-12. Nucleotide synthesis

Question 1

What are the three main components of a nucleotide?

Answer 1

1. inorganic phosphate
2. ribose sugar (5C sugar)
3. nitrogenous base (purine / pyrimidine)

Question 2

What are the chemical structures for :
- Purines
- Pyrimidines

Hint : rings

Answer 2

Purines : 5 membered ring fused to a 6 membered ring
Pyrimidine : 6 membered ring

Question 3

What is the difference between a nucleoside and a nucleotide?

Answer 3

Nucleotide : inorganic phosphate + ribose sugar + nitrogenous base

Nucleoside : ribose sugar + nitrogenous base

Question 4

Which nitrogenous bases are purines?

Answer 4

Adenosine, guanine

Question 5

Which nitrogenous bases are pyrimidines?

Answer 5

Cytosine
Thymine (DNA)
Uracil (RNA)

Question 6

Why is RNA more abundant in cells than DNA?

Answer 6

RNA is constantly synthesized (to synthesise proteins), while DNA is only synthesized during cell division.

Question 7

Nucleotides are not a significant source of metabolic energy.
What are some key roles of nucleotides in cellular metabolism then? [3]

Answer 7

Nucleotides serve as…
1. Building blocks of nucleic acids (DNA, RNA)
2. Energy carriers - ATP
3. Coenzymes - FAD, NAD⁺

Question 8

Purines are synthesized directly. True or False?

Answer 8

False, purines are directly synthesized on ribose-5-phosphate. (Check : shld b PRPP)

Question 9

Synthesis of purine nucleotides

What is the precursor for purine nucleotide synthesis (from another metabolic pathway) and is also used in the pathway for pyrimidine synthesis (step 4) ?

Answer 9

Ribose-5-phosphate (R5P) from the pentose phosphate pathway,, which is then converted into PRPP, phosphoribosyyrophosphate (additional PPi on R5P)

Question 10

Synthesis of purine nucleotides

What is the first nucleotide formed in purine biosynthesis?

Answer 10

IMP

Question 11

Synthesis of purine nucleotides

What are the two key end products of purine synthesis?

Answer 11

AMP, GMP

Question 12

Regulation of purine synthesis

What are the 2 regulatory strategies of purine synthesis?

Answer 12

1. Balanced production of products (AMP,GMP) : cross-dependency of ATP for GMP synthesis and GTP for AMP synthesis.
2. Control of flux through purine synthesis pathway and enzymatic activity through feedback inhibition and feedforward activation

Question 13

Regulation of purine synthesis

IMP → adenylo-succinate (catalysed by adenylosuccinate synthetase)
IMP → XMP (catalysed by IMP dehydrogenase).

When AMP and GMP (products) concentrations are high, AMP and GMP inhibit the enzymes adenylosuccinate synthetase and IMP dehydrogenase via binding to allosteric sites . True or False?

Answer 13

False, AMP and GMP are structurally similar to IMP (substrate), thus they bind to the active site of the enzymes to inhibit further conversion of IMP into adenylo-succinate / XMP (which is further converted into AMP/GMP).

Competitive inhibitors

Question 14

What is Lesch-Nyhan Syndrome?

Answer 14

A genetic disorder caused by HGPRT (Hypoxanthine-guanine phosphoribosyl transferase) deficiency, where purines cannot be salvaged, leading to excessive uric acid production and neurological symptoms.

Question 15

Pyrimidine synthesis (anabolism)

Pyrimidines are synthesised directly onto ribose-5-phosphate. True or False?

Answer 15

False. Pyrimidines are synthesized first, before being added onto ribose-5-phosphate (PRPP)

Question 16

Pyrimidine synthesis (anabolism)

After the 6 enzymatic reactions, what is the precursor that is used in synthesising the ribonucleotides UTP and CTP?

Answer 16

UMP, uridine monophosphate

Question 17

Pyrimidine synthesis (anabolism)

What are the precursors for pyrimidines? [3]

Answer 17

1. HCO₃^- → C2 in 6 membered ring
2. Glutamine → provides ammonia and form N3 in the 6 membered ring
3. Aspartate → forms most of the pyrimdine ring (N1, C4,5,6)

Question 18

Pyrimidine synthesis (anabolism)

First step of pyrimidine synthesis :
HCO₃^- + 2 ATP + glutamine + H₂O → carbamoyl phosphate + 2ADP + P_i + glutamate

What enzyme catalyses this reaction?

Answer 18

Carbamoyl phosphate synthethase II

Question 19

Pyrimidine synthesis (anabolism)

First step of pyrimidine synthesis :
HCO₃^- + 2 ATP + glutamine + H₂O → carbamoyl phosphate + 2ADP + P_i + glutamate

What is the difference between CPS II and CPS I (in urea cycle)?

Answer 19

CPS I utilises free ammonia to form carbamoyl phosphate (enzyme in liver, where there is free ammonia)

However, CPS II needs to extract the ammonia from glutamine (in cytosol of all other organs, where there is no free ammonia)

CPS II found in all organs because all cells need to regularly synthesise RNA

Question 20

Pyrimidine synthesis (anabolism)

What is the first comitted step in pyrimidine synthesis and what enzyme catalyses this reaction?

Answer 20

Aspartate + carbamoyl phosphate → carbamoyl aspartate ;;
catalysed by aspartate transcarbamolyase (ATCase)

Question 21

Pyrimidine synthesis (anabolism)

What are the 2 regulatory strategies for carbamoyl phosphate synthethase II?

Answer 21

1) Substrate chanelling
- Domain I : glutaminase domain (glutamine → glutamate + NH₃)
- Domain II : synthethase domain : NH₃ from domain I is channeled to domain II to form CP from ammonia, HCO₃^- and H₂O

2) Allosteric regulation
- Feedforward activation by PRPP and product inhibition by UMP / UDP / UTP (allosteric regulation)

Question 22

Pyrimidine synthesis (anabolism)

Step 3 : Carbamoyl aspartate → dihydroorotate.

What enzyme catalyses this reaction and what is this reaction about?

Answer 22

Dihydroorotase
It involves ring closure

Question 23

Pyrimidine synthesis (anabolism)

Step 4 is one of another key steps of regulation as it is irreversible. State the equation and enzyme catalysing it.

What does this reaction do?

Answer 23

Dihydroorotate → orotate (orotic acid) ;; catalysed by dihydroorotate dehydrogenase
- This reaction oxidises dihydroorotate, forming a C=C bond between C5 and C6

Question 24

Pyrimidine synthesis (anabolism)

In the 6 steps of UMP synthesis of UMP, what is the first pyrimidine base formed?

Answer 24

Orotate
- it has 3 C=C bonds, conjugated ring (structure of pyrimidines)

Question 25

# **Pyrimidine synthesis (anabolism)** What does step 5 of UMP synthesis involve? (Name the product formed) | ***Hint : orotate is a reactant***

Answer 25

It involves addition of the base, orotate to PRPP (activated version of ribose-5-phosphate) - PRPP + orotate → orotidine-5-monophospate (OMP) ;; catalysed by orotate phosphoribosyltransferase.

Question 26

# **Pyrimidine synthesis (anabolism)** How is UMP converted into CTP?

Answer 26

The carbonyl group in the uracil base (in UTP) is replaced with -NH₂ to form cytosine (in CTP)

Question 27

# **Pyrimidine synthesis (anabolism)** State the last step (step 6) in UMP synthesis and the enzyme involved.

Answer 27

Orotidine-5-monophosphate → uridine-5-monophosphate (UMP); catalysed by **OMP decarboxylase**

Question 28

# **Pyrimidine synthesis (anabolism)** What enzyme catalyses the conversion of UTP to CTP, and how is it regulated?

Answer 28

CTP synthetase It is regulated via substrate channeling, where NH₃ from glutaminase domain (extraction of NH₃ from glutamine) is channeled to the synthetase domain (UTP and NH₃reacts) ## Footnote **Recall: 1. Glutaminase domain : glutamine → NH₃ + glutamate 2. Synthetase domain : UTP reacts with NH₃ to yield CTP**

Question 29

# **Pyrimidine synthesis (anabolism)** Which are the 2 key steps of regulation in pyrimidine synthesis and how are they all regulated?

Answer 29

All regulated by allosteric regulation 1. ** Step 1 : Formation of carbomoyl phosphate**: feedforward activation by PRPP, ATP and product inhibition by UDP, UTP 2. **Step 6 : OMP → UMP**, where OMP decarboxylase is allosterically inhibited by UMP (product inhibition)

Question 30

# **Deoxyribonucleotide synthesis** To synthesise deoxyribonucleotides, the ribose sugar is reduced, then the whole process of synthesis follows the pathway similar to ribonucleotide synthesis. True or False?

Answer 30

False. Formation of deoxyribonucleotides occurs through the reduction at the nucleotide level (Nucleotide diphosphates) - e.g. UTP reduced into dUTP (d = deoxy)

Question 31

# **Deoxyribonucleotide synthesis** What is the enzyme that is responsible for reducing nucleotide diphosphates (NDP) into deoxyribonucleotides?

Answer 31

Ribonucleotide reductase, RNR

Question 32

# **Deoxyribonucleotide synthesis** Describe how ribonucleotide reductase (RNR) reduces ribonucleotides into deoxyribonucleotides.

Answer 32

1. Radical on tyrosine residue in R2 dimer attacks the ribonucleotide and abstracting H atom, making the nucleotide more reactive. 2. The cysteine residue (-SH) in the R1 dimer of RNR gets oxidised into a disulfide bond (-S-S) while the hydroxyl group on C2 gets reduced. 3. Cysteine residues are regenerated when the disulfide bond is reduced by NADPH.

Question 33

# **Deoxyribonucleotide synthesis** How is ribonucleotide reductase (RNR) regulated?

Answer 33

1. Allosteric regulation, affecting the enzyme's catalytic activity 2. Achieving balanced production of dNDPs (specificity in synthesising specific dNDPs).

Question 34

# **Deoxyribonucleotide synthesis** What 3 types of allosteric sites are there in RNR? Which dimer is it present in?

Answer 34

Allosteric sites are present in the R1 dimer. - Specificity site - all dNDPs bind - Hexamerisation site - ATP - Activity site - ATP, dATP

Question 35

# **Deoxyribonucleotide synthesis** Effector-induced oligomerisation controls overall catalytic activity of RNR. Explain what this means.

Answer 35

When specific effectors bind to allosteric sites (specificity, activity and hexamerisation sites), they cause RNR to change conformation and assemble into higher-order oligomeric complexes (tetramer, hexamer), which further regulates enzymatic activity.

Question 36

# **Deoxyribonucleotide synthesis** ATP concentrations in cells are generally high (1-10 mM). Thus, what form (oligomeric form) is RNR typically in?

Answer 36

Tetramer / hexamer form

Question 37

# **Deoxyribonucleotide synthesis** Production of balanced quantities of dNTPs is determined by effector binding to the ____ site.

Answer 37

Specificity