Chapter 25- Nucleotide biosynthesis Flashcards
Nucleotides are required for which life processes?
- 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
- ATP is an adenine molecule, which acts as a universal source of energy
- Nucleotide derivatives like UDP-glucose participate in biosynthetic processes (like the formation of glycogen)
- Nucleotides like cAMP are essential components of signal-transduction pathways
De novo pathways
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
Salvage pathways
Performed bases are recovered and reconnected as a ribose unit
Nucleoside
A unit consisting of a purine/pyrimidine base bonded to a sugar. There are 4 nucleoside units in RNA and 4 in DNA
Nucleotide
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.
4 nucleoside units in RNA
Adenosine, guanosine, cytidine, and uridine
4 nucleoside units in DNA
Deoxyadenosine, deoxyguanosine, deoxycytidine, and thymidine (thymine nucleosides are rarely found in RNA so by convention the deoxy- prefix is not added)
4 nucleotide units of DNA
The 4 nucleotide units of DNA are deoxyadenylate, deoxyguanylate, deoxycytidylate, and thymidylate
4 nucleotide units of RNA
Adenylate, guanylate, cytidylate, and uridylate
Pyrimidine nucleotide
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
Carbamoyl phosphate synthetase 2
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
Carbamoyl phosphate synthesis
- Bicarbonate is phosphorylated by ATP to form carboxyphosphate and ADP
- Ammonia reacts with carboxyphosphate to form carbamic acid and inorganic phosphate
- Carbamic acid is phosphorylated by another molecule of ATP to form carbamoyl phosphate
ATP-grasp fold
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
What is the main source of ammonia for carbamoyl phosphate synthetase 2?
Glutamine is the main source of ammonia. The small subunit of CPS2 hydrolyzes glutamine to form ammonia and glutamate
Aspartate transcarbamoylase
Second step of de novo pyrimidine synthesis- carbamoyl phosphate reacts with aspartate to form carbamoylaspartate
CAD
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
5-phosphoribosyl-1-pyrophosphate (PRPP)
The compound formed when orotate couples to ribose in pyrimidine synthesis. PRPP is a form of ribose activated to accept nucleotide bases.
5-Phosphoribosyl-1-pyrophosphate synthetase
Synthesizes PRPP by adding a pyrophosphate from ATP to ribose-5-phosphate, which is formed by the pentose phosphate pathway
Orotidylate
A pyrimidine nucleotide formed when orotate reacts with PRPP. This reaction is driven by the hydrolysis of pyrophosphate
Orotate phosphoribosyltransferase
Catalyzes the reaction where orotate and PRPP react to form orotidylate
Orotidylate decarboxylase
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
How is cytidine formed?
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.
Conversion of nucleoside monophosphates to nucleoside triphosphates
Nucleoside monophosphates are converted into diphosphates by specific nucleoside monophosphate kinases that utilize ATP as the phosphoryl group donor
Diphosphate kinase
A nucleoside that interconverts nucleoside diphosphates and triphosphates. This enzyme has broad specificity
Transformation of UTP into CTP
UTP is transformed into CTP by the replacement of a carbonyl group by an amino group
Cytidine triphosphate synthetase
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
Release of thymine from degraded DNA (2 steps)
- Thymine is converted into the nucleoside thymidine by thymidine phosphorylase
- Thymidine is converted into a nucleotide by thymidine kinase.
This is a salvage pathway
De novo purine synthesis
Begins with simple starting materials, like amino acids and bicarbonate. The purine bases are assembled already attached to the ribose ring, unlike in pyrimidines
Glutamine phosphoribosyl amidotransferase
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
De novo purine biosynthesis (9 steps)
- Phosphorylation activates the carboxylate group of a glycine residue. The glycine couples to the amino group of phosphoribosylamine, forms a new amide bond
- N10-formyltetrahydrofolate donates formyl to this amino group, forms formylglycinamide
ribonucleotide - The inner carbonyl group is activated by phosphorylation and then converted into an amidine by the addition of ammonia
- Formylglycinamidine ribonucleotide cyclizes to form the 5 membered imidazole ring found in purines
- Bicarbonate is activated by phosphorylation and attacked by the exocyclic amino group. Bicarbonate is transferred to the imidazole
- The imidazole carboxylate group is phosphorylated again and the phosphate group is displaced by aspartate
- Fumarate is eliminated, aspartate donates an amino group to the imidazole ring
- A formyl group is added to the nitrogen atom to form 5-formaminoimidazole-4-carboxamide ribonucleotide
- 5-Formaminoimidazole-4-carboxamide ribonucleotide cyclizes with the loss of water to form inosinate
Cyclization of the purine ring
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
Thymidylate synthase
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
Ribonucleotide reductase
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.
Lesch-Nyhan syndrome
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
Dihydrofolate reductase
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
When is thymidylate synthesis required?
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
Methotrexate
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
Thymidylate synthase
Converts dUMP to dTMP. Thymidylate synthase requires a monophosphate substrate and will only act on a deoxynucleotide, since thymine is only present in DNA
Fluorouracil
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.
