Deoxynucleotide Synthesis

The enzyme DNA polymerase, which uses deoxynucleoside triphosphates as substrates, makes DNA. To ensure enough precursors for DNA synthesis, two reactions must occur. First, the 2′ position of the ribose ring of ribonucleotides must be reduced from a C‐OH to a C‐H before the nucleotides can be used for DNA synthesis. Secondly, the thymine ring must be made by addition of a methyl group to uridine.

Ribonucleotide reductase uses ribonucleoside diphosphates (ADP, GDP, CDP, and UDP) as substrates and reduces the 2′ position of ribose.

The small protein thioredoxin supplies reducing equivalents to ribonucleotide reductase for the ribose ring reduction. Thioredoxin is itself reduced by another protein, thioredoxin reductase, a flavoprotein. Reduced glutathione can also carry reducing equivalents to ribonucleotide reductase. In both cases, the ultimate source of reducing equivalents is NADPH.

The regulation of ribonucleotide reductase is complex, with many feedback reactions used to keep the supplies of deoxynucleotides in balance. For example, dGTP and dTTP are feedback inhibitors of their own formation. Each is also an activator of the synthesis of the complementary nucleotide (dCDP or dADP), while dATP is an inhibitor of the reductions to make dADP, dCDP, dGDP, and dUDP. These control functions keep the supply of deoxynucleotides in balance, so that a roughly equivalent amount of each remains available for DNA synthesis.

Thymidylate synthase

DNA contains thymidine, while RNA contains uridine. The formation of thymidine must be controlled and, more crucially, the formation of dUTP and its incorporation into DNA must be prevented. (Deoxyuridine in DNA can lead to mutations in the sequence and possible genetic defects.)

The thymidylate synthase reaction involves the methylation of deoxy‐UMP to deoxy‐TMP (thymidylate). Deoxy‐UMP is the result of dephosphorylation of the product of the ribonucleotide reductase reaction, dUDP. The conversion of the diphosphate nucleotide to the monophosphate nucleotides helps channel deoxyuridine to thymidylate synthase rather than directly to DNA. N 5,N 10‐methylene tetrahydrofolate donates the methyl.





                                  Figure 1

The donation of the methyl group from N 5,N 10‐methylene tetrahydrofolate leads to the oxidation of the cofactor to dihydrofolate. This points to the importance of dihydrofolate reductase (DHFR) in the functioning of thymidylate synthase. Thus, synthesis of TMP requires a supply of both methyl groups—for example, from serine—and reducing equivalents.

Chemotherapy

Both dihydrofolate reductase and thymidylate synthase reactions are targets for anticancer chemotherapy. Cancer is basically a disease of uncontrolled cell replication, and an essential part of cell replication is DNA synthesis. This means that a requirement exists for deoxynucleotide synthesis for growth. Inhibition of deoxynucleotide synthesis should inhibit the growth of cancer cells.

The compound 5‐fluorouridine targets thymidylate synthase. After a nucleoside kinase phosphorylates it, resembles the natural substrate for the enzyme, except that it contains a fluorine where dUMP has a hydrogen. The fluorine isn't removed from the ring by thymidylate synthase, and this causes the ring to remain covalently bound to the enzyme, which means that the enzyme is irreversibly inactivated. The 5‐fluorouridine monophosphate is an example of a “ suicide substrate”—a compound whose reaction with an enzyme causes the enzyme to no longer function.






                               Figure 2

Another way to reduce the supply of deoxynucleotides for cell replication is to target the reduction of dihydrofolate to tetrahydrofolate. Folate antagonists are used in antimicrobial and anticancer chemotherapy. These compounds are competitive inhibitors of dihydrofolate reductase because they resemble the natural substrate. For example, methotrexate is used in antitumor therapy.



                 
                                Figure 3
   

Folate antagonists can be overcome by increasing the amount of dihydrofolate reductase in the tumor cells. These resistant cells increase the amount of the enzyme by amplifying the DNA sequence encoding the enzyme. More copies of the gene make more molecules of enzyme. You can understand this by remembering that the Michaelis‐Menten equation gives the velocity of an enzyme.

 




The velocity of a reaction is therefore proportional to the total amount of enzyme (E t) in the previous reaction. If the activity of dihydrofolate reductase were to be inhibited tenfold by methotrexate, increasing E t tenfold can restore the velocity (amount of tetrahydrofolate made per time).