Nucleotide Metabolism and Thymidine Metabolism

by Kevin Ahern, PhD

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    00:00 Well now that we’ve gone through the mechanism by which ribonucleotide reductase catalyzes its reactions, it’s appropriate we spend some time talking about how it is that the enzyme balances the relative amounts of deoxyribonucleotides that it makes. It accomplishes this magic as it were as a result of action of 2 different sites on the enzyme in addition to the catalytic site. The 2 sites of the allosteric sites that you can see on the left side of the figure here. The first of these that we will consider is the activity site. The activity site is bound by 2 different molecules, dATP or ATP. If dATP binds to the activity site, it inactivates the enzyme. On the other hand, if ATP binds to the activity site, it activates the enzyme. So this is the on/off switch for the enzyme. That on/off switch is important because if the cell does not want to be making deoxyribonucleotides if it has abundant deoxyribonucleotides and dATP is the measure of that. If dATP is abundant, it binds to the activity site and turns the enzyme off. On the other hand, if ATP is more abundant that means that the cell has plenty of energy, and when the cell has plenty of energy, it wants to go do things like divide, which takes deoxyribonucleotides. So the decision to make or not make deoxyribonucleotides is made right here at the activity site.

    01:32 The other allosteric site that’s important for consideration is that of the substrate specificity site. The substrate specificity site can be bound by several of the different nucleotides and deoxyribonucleotides as you can see and its regulation is complex but there is some pattern to the way in which it controls the molecules being made. So, let me step you through this process. Let’s consider first of all the binding of dATP to the substrate specificity site. I’d use color to indicate the molecules that are being affected on the binding at the active site. The active site is the place where the reaction is being catalyzed. Binding of dATP favors the binding of CDP at the active site. Well, remember that the active site is going to convert CDP into dCDP, and dATP is a purine. CDP is a pyrimidine. Remember the pairing of purines and pyrimidines. So if we have more purines appearing in the cell and they bind to the substrate specificity site, they favor the production of pyrimidine deoxyribonucleotides through the active site. We see that the other nucleotide whose synthesis is favored at the active site is UDP, the other pyrimidine.

    02:59 Notice in addition that binding of ATP at the substrate specificity site has exactly the same effect. It is an indicator of purine richness that is favoring the producton of pyrimidine deoxyribonucleotides. Similar thing happens with the binding of dTTP at the allosteric site. That favors the production of GDP at the active site. Well, the comparison isn’t perfect and the enzyme has some complex controls including some inhibitions that I won’t talk about here but you can see that binding of dGTP favors the binding of ADP at the active site, and I won’t go into the regulation of that at this time. Now, the last deoxyribonucleotide metabolism we need to consider is that of thymidine. We haven’t talked about it so far. Remember that the dNDPs are converted to dNTPs by the enzyme NDPK, it does all of the nucleoside diphosphates and the triphosphates and that includes the deoxyribonucleoside diphosphates as well. Thymidine nucleotides are made in a little bit different way and they’re made from the uridine nucleotides, so we have to think about them and talk about them separately from the other ones. DUTP is actually made in the synthesis of thymidine and it’s a problem in the synthesis of thymidine because dUTP can actually be used by DNA polymerase to make DNA. So the cell has to do a little 2-step to keep from getting too much dUTP while at the same time having sufficient thymidine nucleotides to make.

    04:35 Let’s see how that goes. The synthesis of thymidine nucleotides starts with UDP. UDP is converted to dUDP in the ribonucleotide reductase. DUDP is converted to dUTP by the enzyme NDPK. There we go again. Now, dUTP ultimately is not going to end up in DNA and it’s important that the cell not get that into DNA because it can ultimately cause some problems with stability of the DNA and possibly mutation ultimately as well. So what the cell does with that dUTP is the first thing it does as soon as soon as it’s made. It uses an enzyme known as dUTPase to break down dUTP and make the monophosphate derivative of dUTP known as dUMP or dUMP. DUMPs cannot be used by DNA polymerase, and it’s dUMP that is used to make dTMP. We might wonder why in the world does it go to make dUTP and then break it down. Well, the answer to that question is that NDPK grabs everything. It grabs all the diphosphates and so dUTP automatically appears because of the widespread action of NDPK. Because of this, it’s necessary that the cell have abundant quantities of dUTPase. Well production of dTMP from dUMP, is the result of catalysis of an enzyme known as thymidylate synthase. Thymidylate synthase is an interesting enzyme whose regulation and whose activity we’ll talk about in just a second. To go from dTMP to dTDP requires energy from ATP and requires a kinase known as dTMP kinase and I’m sure you can guess the next reaction in going from dTDP to dTTP, that reaction requires our friend, NDPK.

    About the Lecture

    The lecture Nucleotide Metabolism and Thymidine Metabolism by Kevin Ahern, PhD is from the course Purine and Pyrimidine Metabolism. It contains the following chapters:

    • Nucleotide Metabolism
    • Thymidine Metabolism

    Included Quiz Questions

    1. ...binding of dGTP at the allosteric site favors binding of ADP at the active site.
    2. ...binding of dATP at the active site inactivates the enzyme.
    3. ...binding of dTTP at the allosteric site favors binding of UDP at the active site.
    4. All of the answers are true.
    5. None of the answers are true.
    1. it favors the production of GDP
    2. dTDP is made from dUDP.
    3. dTDP is made from UDP.
    4. All of the answers are true.
    5. None of the answers are true.

    Author of lecture Nucleotide Metabolism and Thymidine Metabolism

     Kevin Ahern, PhD

    Kevin Ahern, PhD

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