1. Nucleic Acids

Deoxyribonucleotide Synthesis

A good reference for this topic: http://www.ncbi.nlm.nih.gov/books/NBK22368/

Cast of characters (or molecules)

The Ribonucleotide

"Ribonucleotide General" by Binhtruong - Own work. Licensed under CC BY-SA 3.0 via Commons - https://commons.wikimedia.org/wiki/File:Ribonucleotide_General.png#/media/File:Ribonucleotide_General.png
Ribonucleotide

The ribonucleoside diphosphate (NDP), so the above with two phosphates.

The deoxyribonucleoside diphosphate (dNDP), the deoxy version of NDP

The enzyme Ribonucleotide reductase (RNR), the enzyme that catalyzes the formation of deoxyribonucleotides from ribonucleotides.

Precursors of DNA

The precursors of DNA are formed by the reduction of ribonucleotides via the 2-C of the ribose.

NDP —(ribonucleotide reductase)—>dNDP

Requires a reducing agent, which we get from NADPH in the following sequence:

FAD —(+NADPH via thioredoxin reductase)—>FADH2 : releases e- + H+

Those reduce Thioredoxin, which has two S and a disulfide bond. The released electron and proton reduce Ribonucleotide reductase (which also has disulfide bonds).

Lastly, the resulting electron/proton pair reduce NDP -> dNDP + H2O.

This results in the re-oxidation of Ribonucleotide reductase, making it ready for another cycle.

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There’s also an alternate path from NADPH to the reductase that involves glutathione.

NADPH transfers e’s and p’s to glutathione, reducing it (via glutathione reductase). Another pair of e/p is released, reducing Glutaredoxin (with a disulfide bond). The released pairs pickup up on the first sequence at the Ribonucleotide reductase and continues to dNDP.

Ribonucleotide reductase (RNR)

Contains two identical R1 subunits, each with a pair of distinct allosteric regulatory sites. The R1 subunit also contains the SH group that donate e/p to Ribonucleotides.

It also contains a pair of R2 subunits. These have a tyrosyl radical (note: tyrosol is the name for tyrosine as a functional group). Though it doesn’t participate in the enzyme activity, it generates the radicals needed for that activity.

The active site of the RNR is at the interface between the R1 and R2 subunits.

Regulation of RNR

RNR catalyzes the conversion of ADP to dADP. Upon its release, dADP is phosphorylated to dATP (but not by RNR directly). dATP binds to the allosteric site on the R1 subunit to inhibit the process. Conversely, a high concentration of ATP (the substrate) can active the RNR.

  • Activated by ATP
  • Inhibited by dATP

Specificity

The binding of (purines) ATP and dATP to the second regulatory site on R1 promotes the formation of the pyrimidines dUDP and dCDP. Here, RNR favors the reduction of UDP/CDP over ADP.

So dATP inbits RNR while also promoting its activity. But which one occurs depends on the [dATP]. At high levels, dATP shuts down RNR entirely. At moderate levels, it encourages the pyrimidine synthesis to help balance the purines being created.

Similarly, an excess of deoxypyrimidines encourages to an increase in deoxypurine production.

dUDP is converted (post RNR) to dTTP. This binds to one of the allosteric sites on RNR, which in turn encourages the conversion of GDP to dGDP.

dGTP (once post-converted) binds to the same allosteric site and causes RNR to favor ADP conversion.

And on and on…

This provides a balanced pool for the precursors of DNA.

Cancer Treatments

Cancer cells, because they grow more rapidly than normal cells, have a greater demand for nucleotides as precursors for DNA and RNA synthesis. They are also more sensitive to the inhibitors of the nucleotide biosynthesis pathway.

Many chemotherapeutic drugs target steps in the nucleotide biosynthetic pathways, in particular this one:

dUMP –(thymidylate synthase)–> dTMP

The enzyme in the above reaction adds a methyl group to the dUMP, which it gets  from a molecule called  N5, N10-Methylenetetrahydrofolate (THF). Upon donation that molecule is converted into 7,8-dihydrofolate. The original molecule needs to be regenerated to sustain dTMP production. This regeneration process is called a Folate Cycle.

This cycle gets targeted by chemotherapeutic drugs.

Folate Cycle

After the removal of the methyl group, 7,8-dihydrofolate is then reduced to form tetrahydrofolate. This reaction is coupled with the oxidation of NADPH… maybe it’d be better to just show a picture. Note that the Serine is the one donating the methyl group.

Folate Cycle. Image taken from HarvardX/EdX lecture slide. All rights belong to them.
Folate Cycle. Image taken from HarvardX/EdX lecture slide. All rights belong to them.

With this background, it’s time to look at two cancer treatment drugs that target this reaction: Methotrexate and FdUMP.

Methotrexate is a structural analog of 7-8-dihydrofolate. It binds to the enzyme dihydrofolate reductase with a 100x greater affinity.

Methotrexate. Image taken from HarvardX/EdX lecture slide. All rights belong to them.
Methotrexate. Image taken from HarvardX/EdX lecture slide. All rights belong to them.

The drug fluorodeoxyuridine monophosphate (FdUMP) is another cancer treatment drug that targets the dUMP synthesis reaction.

  • FdUMP is called a pro-drug because it only becomes the molecule FdUMP after being administered
  • The administered form is called Fluorouracil. After being administered it gets converted to FdUMP via the salvage pathway.
  • FdUMP is a structural analog of dUMP, but has a fluoride atom where dUMP has a hydrogen.
  • FdUMP inhibits the enzyme thymidylate synthase
also taken from the class lecture slides. All rights belong to them.
also taken from the class lecture slides. All rights belong to them.

Drawback of these drugs

They screw up other highly replicative cells will be targeted and eliminated such blood progenitor cells in the bone marrow, epithelial cells in the intestinal tract, hair follicles. This explains the well-known symptoms of chemo: weakened immune system, nausea, and hair loss, respectively.

SO note that cells that reproduce more often have a greater sensitivity to the inhibitors of the nucleotide cycle.

 

 

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