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Competitive Reversible Enzyme Inhibition – Enzyme Inhibitors

by Kevin Ahern, PhD
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    00:01 Understanding how enzymes are inhibited has important implications both for our understanding of the mechanism of enzymatic action and with medical considerations.

    00:09 In this lecture I will talk about two primary things.

    00:12 Reversible enzyme inhibitors and also irreversible enzyme inhibitors.

    00:18 Cells of course rely on enzymes to catalyze reactions and that reliance on enzymes allow us to be able to control cells if we can control enzymes. Then it is a consideration particularly if we have a bacterium, for example, that we want to stop from infecting something, or a cancer cell that we want to stop from spreading.

    00:38 So inhibiting enzymes is an important consideration for us for health purposes.

    00:43 I want to spend some time talking about three different types of inhibition of enzymes.

    00:48 And the first of these that I will talk about is called competitive inhibition.

    00:52 You can see this is shown schematically on the screen. The enzyme with its normal substrate is shown on the left.

    00:57 The enzyme binds to the substrate and converts the substrate into product.

    01:02 On the right we see that same enzyme that is the target of an inhibitor of it.

    01:07 And in this case the target inhibitor looks like the original substrate.

    01:12 It fits in the active site of the enzyme.

    01:15 The same way that the normal substrate did.

    01:18 But there is something about the inhibitor that the enzyme can't manipulate. It can't do anything with it.

    01:24 And that causes the enzyme to sort of sit and spin it's wheels while it's bound to that inhibitor.

    01:30 That inhibitor is called the competitive inhibitor and the competitive inhibitor has the properties I have shown here.

    01:36 That it looks like the substrate and binds to the active site.

    01:40 Now on the screen here you can see a couple of different molecules.

    01:45 The bottom molecule is a molecule that is used by an enzyme called dihydrofolate reductase.

    01:51 The enzyme dihydrofolate reductase uses this molecule and converts it into a product where the product is used to make nucleotides, very important for nucleotides.

    02:02 The molecule above that is called methotrexate. And methotrexate is very similar to dihydrofolate.

    02:10 However, there is an important difference to it. And the difference prohibits the enzyme dihydrofolate reductase from acting.

    02:17 Well methotrexate is an inhibitor that enzyme and by inhibiting an enzyme that makes nucleotides that's specific for a cell one could imagine that one could stop that cell from dividing.

    02:27 And that's exactly what this inhibitor is used for.

    02:31 Now let's study the effects of that competitive inhibitor on an enzyme.

    02:35 If we take an enzyme and we compare the V versus S plot of an uninhibited reaction with an inhibited reaction, we will get something like what we see on the screen here.

    02:45 Now I need to explain how this was done.

    02:47 I have described how we used say 20 tubes to generate the data that's used to make the first line that is the enzyme plus varying about of substrate, each tube has a different amount of substrate, and a buffer are used and we measure the velocity by measuring the the concentration of products produced over time.

    03:06 If we want to study the inhibitor reaction, we want to remember that we wanna have one variable.

    03:14 And the one variable we said we have is substrate concentration.

    03:17 That means that we can't vary the amount of inhibitor.

    03:21 So when we do the second set of reactions, we have the same amount of enzyme, we have the same buffer and we have the same amount of inhibitor in each tube.

    03:29 But we have varying amounts of substrate.

    03:32 What happens when we do that? Well when we do that we see that the reaction starts off and it's at a lower rate.

    03:38 That's not too surprising; because, there is an inhibitor their that's inhibiting enzyme. The velocity is lower.

    03:43 But as we go to increase in amount of substrate, we see that the inhibitor keeps rising, and rising and rising and by the end it's actually rising fast enough that it is getting in the range of the velocity of the uninhibited reaction, okay? We see that the difference between two curves is decreasing.

    04:01 Now I will cut to the chase here and cutting to the chase, I will tell you that if we go to very very large amounts of substrate we will discover that the two enzymes have the same Vmax.

    04:12 Now why is that the case? Why does a competitively inhibited reaction has a same Vmax as no inhibitor what's so ever? The answer is do it the way that the experiment was setup.

    04:23 I said that we had a fixed amount of inhibitor.

    04:26 It gigantic concentrations of substrate what happens Well the substrate is much more likely the substrate would be found by the enzyme than the inhibitor would be found by the enzymes.

    04:36 At low concentrations they compete pretty well.

    04:40 But at high concentrations where I might have a million times as much substrate as I have inhibitor.

    04:45 The difference between the uninhibited and the inhibited is difficult for me to see.

    04:50 In addition to the Vmax not changing for a competitively inhibited reaction, something does change in the reaction and the thing that changes is the Km.

    04:59 Since the two reactions that is the unhibited and the inhibited reaction have the same Vmax, they have the same Vmax/2.

    05:07 So if we plot on each curve the Km value which we get from Vmax/2 we discover that the Km value for the uninhibited reaction is as we would expect.

    05:17 But the Km for the competitive inhibited reaction increases.

    05:22 Now that increase is indicating an apparent change in the affinity of the enzyme for the substrate.

    05:27 Now that I say apparent; because, it doesn't actually change the affinity of the enzyme for the substrate. That's a deeper topic than I talk about here. But the apparent Km increases making it seem that the enzyme is loosing its affinity for its substrate.

    05:43 This is shown graphically in another way using a Lineweaver Burk Plot.

    05:47 So remember with the Lineweaver Burk, we take the same data that we had for the V versus S plot and we invert all the data and then plot that on an inverted plot, as you see here 1/Vo versus 1 over the concentration of S.

    05:59 When we do that, we see that, not surprisingly the V versus S data comes to a line as shown in green with the y-intercept corresponding to 1/Vmax and an x-intercept corresponding to -1/Km.

    06:13 When we plot the competitive inhibitor, we see exactly what we learned in our last plot which was the Vmax is the same and the two lines cross at the y-axis.

    06:22 And since the Km value increased for the competitive inhibition what we see then is that the -1/Km gets closer to 0.

    06:31 The Lineweaver Burk plot shows us very graphically what's happening with that inhibition.


    About the Lecture

    The lecture Competitive Reversible Enzyme Inhibition – Enzyme Inhibitors by Kevin Ahern, PhD is from the course Enzymes and Enzyme Kinetics.


    Included Quiz Questions

    1. It increases
    2. It decreases
    3. It remains unaffected
    4. It decreases to half
    5. It decreases by 10%
    1. In competitive reversible enzyme inhibition, the enzyme participates in a new chemical reaction involving the conversion of the inhibitor molecule to the product instead of using the original substrate
    2. In a competitive reversible enzyme inhibition, the Vmax and 1/Vmax values do not change in the presence of an inhibitor during an enzymatic reaction
    3. At very low substrate concentration, the competitive inhibitor has a more pronounced effect on the rate of enzymatic reaction than at higher substrate concentrations
    4. The competitive inhibitor of enzymes plays a magnificent role in controlling the bacterial diseases and cancerous growths
    5. The competitive inhibitor resembles the original substrate and hence easily gets fit to the active site of the enzyme
    1. Methotrexate
    2. Dihydrofolate
    3. Nucleotides
    4. Penicillin
    5. Isoleucine

    Author of lecture Competitive Reversible Enzyme Inhibition – Enzyme Inhibitors

     Kevin Ahern, PhD

    Kevin Ahern, PhD


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