Chapter 25- Nucleotide biosynthesis

Question 1

Nucleotides are required for which life processes?

Answer 1

1. They are activated precursors of nucleic acids that are necessary for the replication of the genome and the transcription of the genetic information into RNA
2. ATP is an adenine molecule, which acts as a universal source of energy
3. Nucleotide derivatives like UDP-glucose participate in biosynthetic processes (like the formation of glycogen)
4. Nucleotides like cAMP are essential components of signal-transduction pathways

Question 2

De novo pathways

Answer 2

From scratch- the nucleotide bases are assembled from simpler compounds, including amino acids. ATP hydrolysis is required for de novo synthesis. De novo pathways lead to the synthesis of ribonucleotides. All deoxyribonucleotides are synthesized from the corresponding ribonucleotides

Question 3

Salvage pathways

Answer 3

Performed bases are recovered and reconnected as a ribose unit

Question 4

Nucleoside

Answer 4

A unit consisting of a purine/pyrimidine base bonded to a sugar. There are 4 nucleoside units in RNA and 4 in DNA

Question 5

Nucleotide

Answer 5

The monomer unit in the nucleic acid polymer. Nucleotides consists of 3 components- a sugar, a phosphate, and a base. It is a nucleoside joined to one or more phosphoryl groups by an ester linkage.

Question 6

4 nucleoside units in RNA

Answer 6

Adenosine, guanosine, cytidine, and uridine

Question 7

4 nucleoside units in DNA

Answer 7

Deoxyadenosine, deoxyguanosine, deoxycytidine, and thymidine (thymine nucleosides are rarely found in RNA so by convention the deoxy- prefix is not added)

Question 8

4 nucleotide units of DNA

Answer 8

The 4 nucleotide units of DNA are deoxyadenylate, deoxyguanylate, deoxycytidylate, and thymidylate

Question 9

4 nucleotide units of RNA

Answer 9

Adenylate, guanylate, cytidylate, and uridylate

Question 10

Pyrimidine nucleotide

Answer 10

Cytosine (C) and Thymine (T) are pyrimidine derivatives. Uracil in RNA is also a pyrimidine. They have a single ring structure. The first nitrogen at the bottom of the ring forms a bond with sugar. The ring is attached to a ribose phosphate to form the pyrimidine. The rings are assembled from bicarbonate, aspartate, and ammonia

Question 11

Carbamoyl phosphate synthetase 2

Answer 11

Catalyzes the first step of de novo pyrimidine biosynthesis, where carbamoyl phosphate is synthesized from bicarbonate and ammonia. This reaction requires the cleavage of 2 molecules of ATP. This enzyme requires 3 different active sites. Intermediates generated at one site move to the next through substrate channeling, without leaving the enzyme. This prevents loss of the substrates and prevents intermediates from being hydrolyzed

Question 12

Carbamoyl phosphate synthesis

Answer 12

1. Bicarbonate is phosphorylated by ATP to form carboxyphosphate and ADP
2. Ammonia reacts with carboxyphosphate to form carbamic acid and inorganic phosphate
3. Carbamic acid is phosphorylated by another molecule of ATP to form carbamoyl phosphate

Question 13

ATP-grasp fold

Answer 13

The active site of CPS 2- the domain forms a structure that surrounds ATP and holds it in an orientation suitable for nucleophilic attack. ATP grasp folds are widely used in nucleotide biosynthesis

Question 14

What is the main source of ammonia for carbamoyl phosphate synthetase 2?

Answer 14

Glutamine is the main source of ammonia. The small subunit of CPS2 hydrolyzes glutamine to form ammonia and glutamate

Question 15

Aspartate transcarbamoylase

Answer 15

Second step of de novo pyrimidine synthesis- carbamoyl phosphate reacts with aspartate to form carbamoylaspartate

Question 16

CAD

Answer 16

A large polypeptide chain- carbamoyl phosphate synthase,
aspartate transcarbamoylase and dihydroorotase. Carbamoylaspartate cyclizes to form dihydroorotate, which is then oxidized by NAD+ to form orotate

Question 17

5-phosphoribosyl-1-pyrophosphate (PRPP)

Answer 17

The compound formed when orotate couples to ribose in pyrimidine synthesis. PRPP is a form of ribose activated to accept nucleotide bases.

Question 18

5-Phosphoribosyl-1-pyrophosphate synthetase

Answer 18

Synthesizes PRPP by adding a pyrophosphate from ATP to ribose-5-phosphate, which is formed by the pentose phosphate pathway

Question 19

Orotidylate

Answer 19

A pyrimidine nucleotide formed when orotate reacts with PRPP. This reaction is driven by the hydrolysis of pyrophosphate

Question 20

Orotate phosphoribosyltransferase

Answer 20

Catalyzes the reaction where orotate and PRPP react to form orotidylate

Question 21

Orotidylate decarboxylase

Answer 21

Catalyzes the reaction where orotidylate is decarboxylated to form uridylate (UMP)- this is a major pyrimidine nucleotide that is a precursor to RNA. Orotidylate decarboxylase is one of the most proficient enzymes, as decarboxylation would be extremely slow without it

Question 22

How is cytidine formed?

Answer 22

It is synthesized from the uracil base of UMP. However, synthesis can take place only after UMP has been converted into UTP. Diphosphates and triphosphates are the active forms of nucleotides.

Question 23

Conversion of nucleoside monophosphates to nucleoside triphosphates

Answer 23

Nucleoside monophosphates are converted into diphosphates by specific nucleoside monophosphate kinases that utilize ATP as the phosphoryl group donor

Question 24

Diphosphate kinase

Answer 24

A nucleoside that interconverts nucleoside diphosphates and triphosphates. This enzyme has broad specificity

Question 25

Transformation of UTP into CTP

Answer 25

UTP is transformed into CTP by the replacement of a carbonyl group by an amino group

Question 26

Cytidine triphosphate synthetase

Answer 26

Transforms UTP into CTP by the replacement of a carbonyl group by an amino group. This reaction requires ATP and uses glutamine as the source of the amino group

Question 27

Release of thymine from degraded DNA (2 steps)

Answer 27

1. Thymine is converted into the nucleoside thymidine by thymidine phosphorylase
2. Thymidine is converted into a nucleotide by thymidine kinase.
   This is a salvage pathway

Question 28

De novo purine synthesis

Answer 28

Begins with simple starting materials, like amino acids and bicarbonate. The purine bases are assembled already attached to the ribose ring, unlike in pyrimidines

Question 29

Glutamine phosphoribosyl amidotransferase

Answer 29

Catalyzes the committed step in purine regulation. PRPP is used and provides the foundation on which the bases are constructed step by step. The first step is the displacement of pyrophosphate by ammonia to produce 5-phosphoribosyl-1-amine. A cysteine residue located at the amino terminus of the enzyme facilitates glutamine hydrolysis

Question 30

De novo purine biosynthesis (9 steps)

Answer 30

1. Phosphorylation activates the carboxylate group of a glycine residue. The glycine couples to the amino group of phosphoribosylamine, forms a new amide bond
2. N10-formyltetrahydrofolate donates formyl to this amino group, forms formylglycinamide
   ribonucleotide
3. The inner carbonyl group is activated by phosphorylation and then converted into an amidine by the addition of ammonia
4. Formylglycinamidine ribonucleotide cyclizes to form the 5 membered imidazole ring found in purines
5. Bicarbonate is activated by phosphorylation and attacked by the exocyclic amino group. Bicarbonate is transferred to the imidazole
6. The imidazole carboxylate group is phosphorylated again and the phosphate group is displaced by aspartate
7. Fumarate is eliminated, aspartate donates an amino group to the imidazole ring
8. A formyl group is added to the nitrogen atom to form 5-formaminoimidazole-4-carboxamide ribonucleotide
9. 5-Formaminoimidazole-4-carboxamide ribonucleotide cyclizes with the loss of water to form inosinate

Question 31

Cyclization of the purine ring

Answer 31

Cyclization forms a 5 membered imidazole ring. This reaction is favorable thermodynamically, but a molecule of ATP is consumed to make it irreversible. Cyclization is an intramolecular reaction where the nucleophile and phosphate activated carbon atom are present within the same molecule

Question 32

Thymidylate synthase

Answer 32

Deoxyuridylate (dUMP) is methylated to thymidylate (TMP). This is a finishing step in generating thymidylate from uracil. The methylation of this nucleotide marks sites of DNA damage for repair and therefore helps to preserve the integrity of the genetic information stored in DNA

Question 33

Ribonucleotide reductase

Answer 33

Responsible for the reduction reaction for all 4 ribonucleotides. Ribonucleotide reductase reduces ribonucleotides to deoxyribonucleotides in its active site, which contains three
key cysteine residues and one glutamate residue. Each R2 subunit contains a
tyrosyl radical that accepts an electron from one of the cysteine residues in the
active site to initiate the reduction reaction. Two R1 subunits come together to
form a dimer as do two R2 subunits.

Question 34

Lesch-Nyhan syndrome

Answer 34

X linked recessive condition that mainly affects males. It is caused by defective purine salvage due to absent HGPRT. Symptoms- intellectual disability, compulsive self destructive behavior, hyperuricemia, gout, tense muscle (dystonia), infants may have red/orange crystals in their diaper

Question 35

Dihydrofolate reductase

Answer 35

Catalyzes the reaction where tetrahydrofolate is regenerated from the dihydrofolate that is produced in the synthesis of thymidylate. NADPH is used as a reductant. This enzyme is a target for cancer drugs- the synthesis of TMP can also be blocked by inhibiting the
regeneration of tetrahydrofolate, which is done by inhibiting dihydrofolate reductase

Question 36

When is thymidylate synthesis required?

Answer 36

In rapidly dividing cells that need to maintain high rates of DNA synthesis. Because cancer cells divide quickly, some thymidylate synthesis inhibitor drugs are used to treat cancer

Question 37

Methotrexate

Answer 37

A folate analog that inhibits the enzyme dihydrofolate reductase, preventing regeneration of N5,N10 methylenetrahydrofolate, which is required for the thymidylate synthase reaction. Aminopterin has a similar mechanism. Methotrexate is used for fast growing tumors like acute leukemia and choriocarcinoma

Question 38

Thymidylate synthase

Answer 38

Converts dUMP to dTMP. Thymidylate synthase requires a monophosphate substrate and will only act on a deoxynucleotide, since thymine is only present in DNA

Question 39

Fluorouracil

Answer 39

An anticancer drug that is converted into fluorodeoxyuridylate (f-dUMP) in vivo. This analog of dUMP inhibits thymidylate synthase after acting as a normal substrate through part of the catalytic cycle. The enzyme can’t get rid of the F from F-dUMP, so catalysis is blocked at this stage. This is an example of suicide inhibition- F-dUMP traps thymidylate synthase in a form that cannot proceed down
the reaction pathway.