BB 451 Nucleotide Metabolism

Why is it important to balance amounts of nucleotides?

Imbalances can lead to mutation.

Contrast nucleoside with nucleotide vs nucleic acid.

Nucleoside has sugar and a base; nucleotide has a base, sugar, and at least one phosphate; nucleic acid is made up of many nucleotides.

What is a 'base'?

A nitrogen-containing organic molecule, specifically a purine or pyrimidine.

What are the sugars in DNA and RNA?

RNA: ribose; DNA: Deoxyribose.

What is meant by de novo and salvage?

De novo: ribonucleotides are made from scratch. Salvage: ribonucleotides are made from recycled parts.

Which are the purines? Pyrimidines?

Purines: Adenine (A) and Guanine (G); Pyrimidines: Cytosine (C), Thymine (T), and Uracil (U).

What are the starting components for the bases?

Amino acids, one carbon donors, and CO2.

Is it surprising that DNA and RNA formed on early Earth? Why or why not?

No, building blocks were readily available.

What is important about ribose-5-phosphate?

It is a starting molecule for the synthesis of purines.

What is PRPP? Is it for purines or pyrimidines or both?

PRPP is used in both; it's ribose with one phosphate on one end and two on the other.

What is the significance of inosine monophosphate?

IMP is a branch molecule that splits into either adenine-containing or guanine-containing.

How is de novo synthesis of AMP and GMP balanced?

Through negative feedback, they inhibit PRPP amidotransferase. (need both) also inhibit their own production pathways.

How is PRPP amidotransferase regulated?

It's turned off by both AMP and GMP binding to it.

How is PRPP synthase inhibited?

By high phosphate and ADP. (low ATP means shouldn’t store fats).

Are phosphates put on the bases one at a time or all three at once?

One at a time.

What is the source of energy to make guanine nucleotides? How about vice versa? Why does this work?

ATP for guanine and GTP for adenine; using different sources prevents depletion.

When converting a nucleoside diphosphate to a triphosphate, what is the enzyme?

NDPK (nucleoside diphosphate kinase).

Why do we need the nucleosides to be triphosphates to incorporate into nucleic acid?

Because polymerases will only use those as starting material.

Can you describe the various regulatory roles that AMP and GMP have on their synthesis pathway?

AMP inhibits adenylosuccinate synthetase; GMP inhibits IMP dehydrogenase.

What are the starting materials to make them?

Bicarbonate, glutamine, ATP, and water.

What pyrimidine is made initially?

UMP.

What is the role of NDPK? Is it similar to the above use?

Phosphorylates UDP to UTP; yes, it's similar.

What does ATCase do?

Catalyzes a key step in the production of pyrimidine nucleotides.

How is ATCase regulated? What activates it and what inhibits it?

Activates: ATP and aspartic acid; Inhibits: CTP.

How is this pathway related to the citric acid cycle?

Aspartic acid is readily made from oxaloacetate from the CAC.

Why is CTP synthetase regulated by GTP?

Gs and Cs are paired; high Gs indicate a need for Cs.

When nucleotides are catabolized, what are they disassembled into?

Bases and sugars.

What is salvage?

Reassembly of bases and sugars.

Which is more permanent, DNA or RNA?

DNA is more permanent; RNA is more transient.

How long does RNA last in the cell?

Minutes to hours.

What are the steps in taking apart nucleosides?

RNA→ nucleoside monophosphate→ nucleoside→ base+sugar.

From what nucleoside could we get inosine or hypoxanthine?

Adenosine.

Why would we get excess uric acid and what disease is caused by this?

It's not water soluble, forming crystals in nerves and causes gout.

Why is allopurinol a competitive inhibitor of xanthine oxidase and what does it treat?

It inhibits uric acid formation, used to treat gout.

What is the enzyme HGPRT and what does it do?

Recycles purine bases hypoxanthine and guanine into nucleotides.

How are deoxyribonucleotides made?

By reduction of nucleoside diphosphates.

What does ribonucleotide reductase (RNR) do?

Catalyzes the conversion of ribonucleoside diphosphates to deoxyriboside diphosphates.

How is RNR regulated? What is the active form?

Needs to be reduced; reduced form is active.

What is thioredoxin and how is it activated? What molecule provides the electrons?

Reduces RNR to activate it; electrons come from NADH.

What are the regulatory sites of RNR?

Specificity site and activity site.

How do these sites work?

Activity: binding of ATP (stimulatory) or dATP (inhibitory); Specificity: influences substrate binding.

Which nucleotide triphosphate tends to exist at artificially high levels in the cell? Why?

ATP, because cells use it as fuel.

Is there any reason to turn off NDPK?

No.

How are thymine nucleotides made?

From Uridine nucleotides through multiple steps of conversion.

What is the role of folic acid here?

Donates a methyl group for one carbon metabolism.

How does the chemotherapeutic drug 5-fluorouracil work?

Suicide inhibitor of thymidylate synthase, preventing DNA synthesis.

How is Folic acid regenerated?

By recycling via DHFR.

How does the chemotherapeutic drug methotrexate work?

Competitive inhibitor of DHFR, preventing recycling of folic acid.

What are synthetic nucleotide analogs used for, medically? How do they work?

Treat cancers and viral infections by fooling polymerases or acting as chain terminators.