Thymidylate Synthase and Recycling of Folates

by Kevin Ahern, PhD

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    00:00 Now let’s take a closer look at the reaction catalyzed by thymidylate synthase to convert dUMP to dTMP. This reaction involves addition of a methyl group at this position on the dUMP to make the dTMP. That’s the only difference between these 2 molecules. This reaction requires a methyl group that comes from 5, 10-methylenetetrahydrofolate. We talked about this actually when I talked about the folate metabolism in another lecture. The product of the donation of this carbon coming from the 5, 10-methylenetetrahydrofolate causes that molecule to become dihydrofolate, and the structure of the molecule on the left, whose name I won’t say again, is shown below and the dihydrofolate is shown there. The difference between those two is shown with a green box. The loss of that carbon there is apparent right here. So, that large molecule is used to donate that single carbon but that’s a pretty important reaction because without it the cell can’t make dTMP. So I want to say a few words about the ways in which we control this enzyme for medical purposes. Now, at the top of the screen you see 5-fluorouracil. 5-fluorouracil is a treatment used to treat cancer cells. Cancer cells that are rapidly dividing need to be making plenty of thymidine nucleotides, and this is the only way that the cell can make thymidine nucleotides. So if you inhibit this enzyme, you inhibit the ability of the cancer cell to produce the nucleotides that makes a cell division. So 5-fluorouracil is what we know as what we referred to as a suicide inhibitor. Now, suicide inhibitor is typically a molecule that looks like the normal substrate for the enzyme and the enzyme grabs that inhibitor, but after it grabs the inhibitor, the inhibitor makes a covalent bond with the active site of the enzyme and so the enzyme commits suicide without realizing it by grabbing the wrong thing. As a consequence, the enzyme is taken out of action and the production of thymidine nucleotides is stopped. The cancer can be stopped using a molecule like this. So, as I said, this is used as anti-cancer treatment. Another consideration in the synthesis of thymidine nucleotides are these molecules that I showed that involved the donation of that single carbon. 5, 10-methylenetetrahydrofolate on the left is what donates the carbon and becomes dihydrofolate on the right. The loss of that carbon, as I showed earlier, is right there. The problem with this reaction as far as the cell is concerned is that dihydrofolate is a dead end in the reaction of all the folates. It has to be converted into one of the other folates in order for the folates to be available for other reactions. If dihydrofolate is not converted to the other folates, then all of the folates will end up in the dihydrofolate form and none will be available to donate carbons to make thymidine nucleotides.

    03:09 This conversion of dihydrofolate to tetrahydrofolate is therefore very important reaction and we saw this in the recycling of folates in the folate lecture. Dihydrofolate reductase catalyzes this reduction as you can see, and tetrahydrofolate which results can then go back into the folate pool and make other folates as necessary for the cell. This dihydrofolate reductase is therefore an important enzyme. The serine hydroxymethyltransferase is the enzyme that catalyzes the conversion of the tetrahydrofolates back to the other folate forms using a carbon from serine to make glycine. That happens here and you can see the addition of that carbon back here. So now we’ve completed the circle. We started with originally the 5, 10-methylenetetrahydrofolate.

    03:58 The thymidylate synthase produced the dihydrofolate, the dihydrofolate reductase produced a tetrahydrofolate and the serine hydroxymethyltransferase reproduced the 5, 10-methylenetetrahydrofolate Now, one of the interesting things about that last enzyme, the serine hydroxymethyltransferase, is that the plasmodium enzyme is different from the human enzyme. Plasmodium, of course, is involved in causing malaria. So the plasmodium enzyme is a target for anti-malarial studies and may be part of an effective treatment against malaria. Now, on the other side and I’ve mentioned this in the folate lecture as well, that if we can inhibit the dihydrofolate reductase, we can inhibit the thymidylate synthetase reaction indirectly. Remember that 5-fluorouracil was a direct inhibitor by suicide inhibition and the indirect inhibition of that enzyme comes by using methotrexate to treat cells. That will inhibit the dihydrofolate reductase. If that occurs, then of course the cell cannot go through this recycling process that I’ve just described and therefore there will be no 5, 10-methylenetetrahydrofolate for the thymidylate synthase and thymidylate synthesis will stop. The recycling of folates is inhibited as I said and we can see why this methotrexate is very good at the inhibition. It looks an awful lot like the dihydrofolate. It binds therefore to the active site of the dihydrofolate reductase and competes with the dihydrofolate for action of the enzyme. Methotrexate is used in chemotherapy and for the same reason that we use 5-fluorouracil.

    05:41 If we can inhibit thymidylate synthesis, then we can inhibit the ability of a tumor cell to grow and divide. This type of treatment is effective against rapidly dividing cells because remember for any of these treatments that we’re giving to an individual, they can get into a normal cell as well and they can have some of the same effects on a normal cell. So if we have a rapidly dividing cell and we do this treatment, then we’re much more likely to kill the rapidly dividing cell than we are the more slowly dividing normal cell.

    About the Lecture

    The lecture Thymidylate Synthase and Recycling of Folates by Kevin Ahern, PhD is from the course Purine and Pyrimidine Metabolism. It contains the following chapters:

    • Thymidylate Synthase
    • Recycling of Folates

    Included Quiz Questions

    1. It transfers a carbon from a folate to make dTMP.
    2. It catalyzes the removal of a methyl group from dUMP to form dTMP.
    3. It requires 5-fluorouracil to function.
    4. All of the answers are true.
    5. None of the answers are true.
    1. It is necessary for folate recycling.
    2. It is inhibited by dihydrofolate.
    3. It is inhibited by tetrahydrofolate.
    4. All of the answers are true.
    5. None of the answers are true.
    1. All of the answers are true.
    2. It is an inhibitor of dihydrofolate reductase.
    3. None of the answers are true.
    4. It is used in chemotherapy.
    5. It resembles dihydrofolate.
    1. The suicide inhibitor causes the activation of cancer-causing enzymes and leads to the formation of malignant tissues.
    2. The suicide inhibitor binds to the enzyme by covalent bonding and causes irreversible enzyme inhibition.
    3. The suicide inhibitor is usually a substrate analog.
    4. 5-fluorouracil is a suicide inhibitor of the thymidylate synthase enzyme.
    5. Due to its suicide inhibitor nature, 5-fluorouracil is used in the treatment of cancerous tissues.
    1. 5,10-Methylenetetrahydrofolate - donates a methyl group to convert dUMP to dTMP
    2. Methotrexate - stimulates cell division by participating in regeneration of 5,10-methylenetetrahydrofolate
    3. Thymidylate synthase - converts dTMP to dUMP
    4. 5-fluorouracil - promotes the conversion of dUMP to dTMP
    5. Serine hydroxymethyltransferase - promotes the conversion of dihydrofolate into tetrahydrofolate

    Author of lecture Thymidylate Synthase and Recycling of Folates

     Kevin Ahern, PhD

    Kevin Ahern, PhD

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