Deoxyribonucleotide Synthesis and Ribonucleotide Reductase

by Kevin Ahern, PhD

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    00:00 Now, I want to turn our attention to deoxyribonucleotides and how they are made because we've now gone through all of the de novo and salvage synthesis pathways for the ribonucleotides and also the catabolic processes that break them down. The deoxyribonucleotides are made from ribonucleoside diphosphates. These are ADP, CDP, GDP, and UDP and the enzyme responsible for this catalyzes the conversion of all of them. That enzyme is ribonucleotide reductase and we’ll see how that operates in just a second. The schematic representation of the reaction catalyzed by ribonucleotide reductase is shown here. In the reaction NDP, the N stands for any of the bases A, G, C, or U. NDP is converted to the deoxy equivalent of those (dGDP, dADP, dCDP, and dUDP) by this enzyme. This involves loss of a water and we’ll see how that happens mechanistically in just a bit. There is the enzyme that catalyze it as I just said. So, for example, if we look at CDP, what happens in the catalysis by this enzyme is the production of dCDP and what has happened is the hydroxyl at position 2 on the sugar has been removed. We see that it is gone in the molecule on the right. That’s the deoxy part of the deoxyribonucleotides that oxygen is gone. Now, ribonucleotide reductase gets changed in the process of that. It starts out in a reduced state and it ends up being oxydized. Well we know when an enzyme changes is it somehow got to be changed back because if we don’t change it back, we get to a dead end where we have a dead enzyme and we can’t have that enzyme being dead because it’s necessary for making other deoxyribonucleotides. Well, it’s converted back to the reduced form by action of a molecule called thioredoxin. Thioredoxin can reduce and oxydize ribonucleotide reductase and in the process, it becomes oxydized. Well, you start thinking do we have to recycle that? The answer is yes you do. To replenish the thioredoxin in a reduced form, electrons are donated ultimately from any DPH. They get to the thioredoxin by several steps, and again, those steps aren’t important but what matters is that the thioredoxin is regenerated so that they can regenerate the ribonucleotide reductase as you see here. Now ribonucleotide reductase is a fascinating enzyme in terms of the number of things that it balances and does for being a relatively small protein.

    02:45 It has 2 subunits, a large subunit and a small subunit. The large subunit is called R1 and it has 2 allosteric sites. The allosteric sites, of course, are sites that bind other molecules and those molecules affect the enzyme’s activity and the active site is at least partly in the large subunits kind of shared between the large and small subunits. The small subunit’s primary function is that it has a tyrosine amino acid within it that gets radicalized and that radicalizaton of the tyrosine is necessary for the reaction mechanism that produces the deoxyribonucleotides. RNR, ribonucleotide reductase, as it's called, controls the balance of all the deoxyribonucleotides and it does it with complex allosteric controls as we shall see. The reaction mechanism for ribonucleotide reductase is depicted on this screen. Now, it’s a complicated process but it’s important to go through so that you understand how the enzyme is accomplishing what it accomplishes. We remember that ribonucleotide reductase starts out in a reduced form as shown here, and by the end of the reaction, it’s in an oxydized form. Remember that I said that the ribonucleotide reductase in the oxydized form has to be converted back to the reduced form and that happens as a result of action of a thioredoxin. Well, how this does these all thing occur? Remember that I said also that the process starts with the tyrosyl radical into small subunit of the ribonucleotide reductase. We can see that radical right here. That radical actually pulls proton off of the ring of carbon #3 on the ribose. As you can see with that arrow moving towards the top there. So tyrosine is pulling that hydrogen off and in the process of doing that it takes the electron with it leaving behind a radical that’s on the ring. That radical in the ring is shown in the next molecule with a little dot above that carbon #3. Well, that creates some instability on the ribose and that instability on the ribose causes the hydroxyl position to pull a hydrogen off of the sulfhydryl of ribonucleotide reductase. You’ll notice that that creates an H2O at that point as shown in the 3rd molecule. Well we can see this process happening and that H2O is unstable.

    05:06 That H2O is lost, so the loss of the H2O results in now a ribose that has had the radical transferred to another position, that below that of position 2. Well, that radical is very much seeking a hydrogen, and we can see that that hydrogen is lost here from the other sulfur of the sulfhydryl on the ribonucleotide reductase to stabilize the overall sugar. Well, at this point, we have now made the deoxyribose sugar. The radical has to be regenerated and that radical is regenerated by fixing the other part of the radical on the deoxyribose sugar that happens here, and as a result, the ribonucleotide reductase enzyme is completely regenerated into the radical state and simply has to be reduced by thioredoxin.

    About the Lecture

    The lecture Deoxyribonucleotide Synthesis and Ribonucleotide Reductase by Kevin Ahern, PhD is from the course Purine and Pyrimidine Metabolism. It contains the following chapters:

    • Deoxyribonucleotide Synthesis
    • Ribonucleotide Reductase
    • RNR Reaction Mechanism

    Included Quiz Questions

    1. It requires ribonucleotide reductase.
    2. It starts with ribonucleoside monophosphates.
    3. It produces dTTP from dCTP.
    4. Ribonucleotide reductase requires one water molecule to convert from its reduced form to an oxidized form.
    5. It requires the enzyme ribonucleotide hydroxylase.
    1. It has allosteric sites on the large subunit.
    2. It has a small subunit called R1.
    3. The small subunit has three allosteric sites.
    4. The large subunit has three allosteric sites.
    5. The large subunit has a tyrosine which is important in the reaction mechanism.
    1. It involves the transfer of electrons through sulfhydryls.
    2. It requires a phenylalanine radical.
    3. It involves the donation of electrons to the enzyme from the ribonucleotide.
    4. The reduced form of RNR has a hydroxyl radical that is important for the reaction mechanism.
    5. The reduction of sulfhydryl is necessary to generate the oxidized form of RNR.
    1. Reduced thioredoxin
    2. Oxidized thioredoxin
    3. Radicalized tyrosine
    4. Oxidized tyrosyl radicals
    5. Reduced tyrosyl radicals

    Author of lecture Deoxyribonucleotide Synthesis and Ribonucleotide Reductase

     Kevin Ahern, PhD

    Kevin Ahern, PhD

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