Uncompetitive Inhibition: Videos & Practice Problems

Uncompetitive inhibition occurs when an inhibitor binds only to the enzyme-substrate complex, forming the ESI complex, which decreases both the apparent Km and Vmax of the enzyme. This results in a stronger affinity for the substrate, indicated by a lower Km, while the Vmax is reduced due to the inhibitor's presence. In Michaelis-Menten plots, the curves shift downwards, and in Lineweaver-Burk plots, the slope remains unchanged, reflecting the proportional decrease in both Km and Vmax. Understanding these dynamics is crucial for grasping enzyme kinetics and inhibition mechanisms.

Uncompetitive Inhibition

In this video, we're going to talk about our second type of reversible inhibition, which is uncompetitive inhibition. And so, of course, uncompetitive inhibition is going to be caused by uncompetitive enzyme inhibitors. And unlike competitive enzyme inhibitors, uncompetitive enzyme inhibitors will only bind to the enzyme-substrate complex and they do not bind to the free enzyme like, again, competitive inhibitors do. Of course, when the uncompetitive inhibitor binds to the enzyme substrate complex, it's going to form the ESI complex. We already know that all enzyme inhibitors, regardless of what type they are, are going to decrease the initial reaction velocity \( v_0 \) of the enzyme-catalyzed reaction. So, no surprise here. Now, what's important to know, as is already anticipated, uncompetitive inhibitors do not compete. So there is no competition when it comes to uncompetitive enzyme inhibitors. What this means is that the uncompetitive inhibitor does not compete with the substrate for the same binding site. Instead, uncompetitive inhibitors' binding site is only created after the substrate binds to the free enzyme to form the enzyme-substrate complex. We'll be able to see this a little bit better down below in our image.

Also note that the binding of an uncompetitive inhibitor to the enzyme-substrate complex is going to prevent the conversion of the substrate into the product. That is the nature of inhibitors as a whole. When they are bound to the enzyme, they're going to somehow prevent the reaction from taking place. If we take a look at our example image down below of uncompetitive inhibition, notice on the left-hand side, we have an image that we have seen before in our previous lesson videos. You can see that we have the same exact enzyme-catalyzed reaction that we've seen in our previous lesson videos and notice that the uncompetitive inhibitor here is not affecting the free enzyme like the competitive inhibitor did in our previous lesson video. Instead, the competitive inhibitor is only binding to the enzyme substrate complex to form the ESI complex. Of course, whenever the inhibitor is bound to the enzyme that's going to prevent the reaction. So, there will be no reaction when the inhibitor is bound to the enzyme. Over here on the right-hand side, we essentially have the same exact image of the left-hand side, except just a different visual representation. Uncompetitive inhibitor here is not interacting with the free enzyme specifically because there's no binding site for the uncompetitive inhibitor on the free enzyme. Notice that this allows the substrate to bind to the free enzyme normally to form the enzyme-substrate complex. However, notice that upon the formation of the enzyme-substrate complex, that induces the binding site for the uncompetitive inhibitor to form. Now the uncompetitive inhibitor can bind to the enzyme substrate complex to form the ESI complex. Whenever the inhibitor is again bound to the enzyme, that's going to prevent the reaction from taking place. However, what's really important here again is this idea that with uncompetitive inhibitors there's absolutely no competition between the uncompetitive inhibitor and the substrate. It's important to emphasize here that the uncompetitive inhibitor does not compete with the substrate and the reason is that the substrate is actually uncompetitive inhibitors will only bind to the enzyme, when, the enzyme substrate complex forms. Uncompetitive inhibitors will only bind to the enzyme substrate complex which is again very very different from competitive inhibitors. In our next lesson video, we'll be able to revisit our analogy but change it up a little bit for uncompetitive inhibitors and we'll also give you guys a little memory tool to help you guys memorize the effects that uncompetitive inhibitors have on enzymes. I'll see you guys in those videos.

Uncompetitive Inhibitor Effects

In this video, we're going to talk about the effects that uncompetitive enzyme inhibitors have on enzymes. And so recall from our previous lesson videos, we mentioned the fact that uncompetitive enzyme inhibitors will actually make the apparent kilometers of an enzyme appear to get better. However, in the process, it makes the apparent V_max of the enzyme worse. And so even though the apparent kilometers seem to get better in the presence of an uncompetitive inhibitor, uncompetitive inhibitors are still going to inhibit the enzyme-catalyzed reaction because the better apparent kilometers is accompanied by V_max getting worse. And so recall that a better apparent kilometers just means that the enzyme is going to appear to have a stronger affinity for the substrate. But of course, a stronger affinity corresponds with a decreased k_m. And, of course, a worse apparent V_max is going to correspond with a decreased V_max as well. And so essentially what we're saying here is that uncompetitive enzyme inhibitors will proportionally decrease both the apparent kilometers as well as the apparent V_max of an enzyme. Now also recall from our previous lesson videos that uncompetitive enzyme inhibitors do not compete. There's no competition between the substrate and the uncompetitive inhibitor. Now, the real question is exactly how and why is it that uncompetitive enzyme inhibitors have these particular effects on an enzyme. In order to understand that, let's take a look down below at our image. Notice that we're using the same exact image from our previous competitive inhibition lesson where this image represents an analogy for our enzyme-catalyzed reaction. We have this adorable cute little puppy, whose name is Zyme, to represent our free enzyme. Zyme is binding to our substrate so Zyme can eat the bone, catalyze the reaction unaltered while creating a poop on the ground. Notice that we do not have the competitive inhibitor, a soccer ball, in this position, as we did in our previous lesson videos. Instead, we're using the owner of our adorable little puppy to represent the uncompetitive inhibitor. Notice that the owner is one of the most uncompetitive people you would ever meet. Notice, he's wearing a shirt that says "We are all winners" because he has no interest in competing with anyone or anything, including the substrate. This reminds us that uncompetitive enzyme inhibitors do not compete with the substrate for a binding position on the enzyme's active site, which again, the active site of the enzyme is represented by the dog's mouth. What this means is that our uncompetitive inhibitor owner is not going to be binding to the dog's mouth, which makes sense. And so notice that our owner, our uncompetitive inhibitor, is actually in a pretty upset mood. You can tell by just looking at his facial expressions. The reason he's in such a bad mood is that Zyme, when he has a bone in his mouth, keeps creating poops on the ground inside the house. Notice that our owner, when he's in this upset mood, only cares about Zyme when he has the bone in his mouth. He's saying, "Drop the bone, Zyme." This means that our uncompetitive enzyme inhibitor will only bind to the enzyme-substrate complex or the dog with the bone in his mouth. When our owner is in this bad mood, he's not going to care much about the free enzyme, and he's not going to give much attention to Zyme when he doesn't have the bone in his mouth. What you'll notice is that we have the symbol α', which you might recall, is the degree of inhibition on the enzyme-substrate complex. This is a reminder to us that uncompetitive enzyme inhibitors will only bind to the enzyme-substrate complex. Whenever the owner catches Zyme with a bone in his mouth, he will quickly grab his leash and take Zyme outside to form the enzyme-substrate-inhibitor complex. When this enzyme-substrate-inhibitor complex is formed, it will prevent Zyme from creating a poop on the ground, and so notice that our uncompetitive inhibitor, the owner, is saying "No pooping." When they are forming this complex, it is preventing the reaction from proceeding and is thus preventing poops from being created. Now that we understand our image a bit better, let's go back up to our text to revisit exactly how and why uncompetitive enzyme inhibitors have these particular effects on an enzyme. We're going to start off with how uncompetitive inhibitors decrease the apparent kilometers. The reason that uncompetitive inhibitors decrease the apparent kilometers is that uncompetitive enzyme inhibitors also lower or decrease the concentration of the enzyme-substrate complex. If we take a look at our image down below, notice that the uncompetitive inhibitor here is going to bind to the enzyme-substrate complex to form the enzyme-substrate-inhibitor complex, but in the process, it's going to decrease the concentration of the enzyme-substrate complex. What that means is that this equilibrium over here is going to respond to the decreased enzyme- substrate complex by shifting to the right. That's exactly what we're seeing up above. Notice that by Le Chatelier's principle, a lowered or decreased concentration of enzyme-substrate complex causes the k₁ reaction to shift to the right. Notice that the number 1 up above corresponds with the number 1 down below. We have Le Chatelier's principle here, and we have this pink arrow shooting to the right, reminding us that this equilibrium is going to shift to the right when uncompetitive enzyme inhibitors decrease the concentration of enzyme-substrate complex. If this equilibrium shifts to the right, the enzyme-substrate complex is going to form, and that's going to make it appear as if the enzyme has a stronger affinity for the substrate. And a strengthened enzyme-substrate affinity, we know, corresponds with a decreased k_m. That's exactly how and why uncompetitive enzyme inhibitors decrease the apparent kilometers. And again, the decreased apparent kilometers appears to make the enzyme perform better because the enzyme is going to appear to have a stronger affinity for the substrate. But just because the apparent kilometers appears to get better does not mean that uncompetitive inhibitors make the enzyme better. That's because this decreased apparent k_m that appears to make the enzyme better is also accompanied by the apparent V_max getting worse. Inhibitory effect on the enzyme-catalyzed reaction. And so now we're going to move on to the fact that uncompetitive enzyme inhibitors also decrease the apparent V_max. This has to do with the fact that uncompetitive enzyme inhibitors do not compete. And again, we already know that the owner of our puppy, which represents the uncompetitive inhibitor, is one of the most uncompetitive people that we know. Again, he's wearing a shirt that says "We are all winners", and so he has no interest in competing with anyone or anything, including the substrate. If our owner uncompetitive inhibitor is not competing with the substrate, that also means that our substrate cannot outcompete the effects of the uncompetitive inhibitor. And so this is exactly what we're saying up above in our text. We're saying that since the substrate can't outcompete or compete at all with the uncompetitive inhibitors, the effects of the uncompetitive inhibitor are not going to be reversed simply by increasing the substrate concentration to saturating levels. And so this is very different from the competitive inhibitor because we recall from our previous lesson videos with competitive inhibitors that increasing the substrate concentration to saturating levels allowed the competitive inhibitor to be outcompeted by the substrate. But again, this is not possible with uncompetitive inhibitors since there's no competition feature. And the uncompetitive inhibitor will not compete with the substrate, so the substrate cannot outcompete the uncompetitive inhibitor. What this means is that even if we had a huge pile of bones in front of our puppy, the owner, our uncompetitive inhibitor, will always be there and ready to bind to our puppy with the bone to prevent our puppy from creating a poop on the ground. Our uncompetitive inhibitor owner will ensure that the maximum amount of poops will not be created. What this means is if the maximum amount of poops is not being created, this means that the apparent V_max of the enzyme is going to be decreased by these uncompetitive inhibitors. This is exactly how and why uncompetitive inhibitors will decrease the apparent V_max and make it worse. Since the uncompetitive inhibitors decrease the apparent V_max, this also means that the catalytic constant or the k_cat or the turnover number is also going to be decreased. And recall from our previous lesson videos that the k_cat is the maximum catalytic efficiency under saturating substrate concentrations. Even if we had a huge pile of bones in front of our puppy, the owner will be able to prevent the maximum amount of poops from being created, therefore it will decrease the V_max and decrease the k_cat. And so, notice that down below, here what we're saying is that the decreased catalytic constant or k_cat or turnover number is going to be equal to the V_max also being decreased. The total enzyme concentration is not going to be affected by the uncompetitive inhibitor. The decreased V_max is going to lead to the decreased k_cat. And so this is a lot of effects to remember about uncompetitive enzyme inhibitors. How could you guys possibly go about memorizing all of these different effects that uncompetitive inhibitors have? And so that's exactly why we have this box down below. When it comes to uncompetitive enzyme inhibitors, we really want to focus on this U here, which is quite unique to uncompetitive inhibition. This u here reminds me of a U-turn, that just tells me that everything's going to be going down, including the Kilometers and the V_max are both going to be decreased as we said above. Notice that with uncompetitive inhibition, even though it says the kilometers is going to be increased, the kilometers is actually going to decrease when we consider the U-turn. And, of course, with uncompetitive inhibition, there's absolutely no competition between the uncompetitive inhibitor and the substrate, which means that the substrate can't compete. If the substrate can't compete, then it can't keep the same V_max, and that means that the V_max is going to be decreased. Again, all you need to remember with uncompetitive inhibition is that it represents a U-turn and everything is going to be going down, so the Kilometers being decreased, and so is the V_max. This here concludes our introduction to the effects of uncompetitive enzyme inhibitors. We'll be able to get some practice later in our course. But in our next lesson video, we'll talk about how uncompetitive enzyme inhibitors affect the Michaelis-Menten plot. So I'll see you guys there.

Uncompetitive Inhibition & Michaelis-Menten Plots

So now that we know that uncompetitive enzyme inhibitors will proportionally decrease both the apparent Kilometers and the apparent V_max of an enzyme, in this video, we're going to talk about how uncompetitive inhibitors affect Michaelis Menten plots. Recall from our previous lesson videos that all enzyme inhibitors, regardless of what type they are, including uncompetitive inhibitors, will decrease the initial reaction velocity or the V₀ of an enzyme-catalyzed reaction. Also, by looking at our image below on the left-hand side, we have a Michaelis Menten plot showing 2 different curves. This black curve represents the enzyme-catalyzed reaction in the absence of inhibitor, and this blue curve represents the enzyme-catalyzed reaction in the presence of uncompetitive inhibitor, with the initial reaction rate being decreased as we indicated above.

Recall from our previous lesson videos that uncompetitive enzyme inhibitors will only bind to the enzyme-substrate complex, which means that alpha prime is going to measure its degree of inhibition on the enzyme-substrate complex. Therefore, alpha prime, the degree of inhibition on the enzyme-substrate complex of an uncompetitive inhibitor, is going to proportionally decrease both the apparent Kilometers and the apparent V_max of an enzyme. Notice that the apparent Kilometers is defined as Kilometers over alpha prime and the apparent V_max is also defined as V_max over alpha prime.

To get the Michaelis-Menten equation in the presence of an uncompetitive inhibitor, all we need to do is substitute the V_max with the apparent V_max, and substitute the Kilometers with the apparent Kilometers, as we did below. Looking again at this Michaelis Menten plot, it is clear that in the presence of an inhibitor, the V_max is being decreased, unlike with competitive inhibition. Recall with competitive inhibition, the blue curve eventually reaches the V_max so that it is unchanged. But here, with uncompetitive inhibition, the V_max is being decreased. That's because no matter how much we increase the substrate concentration, essentially, no matter how many "bones" we add, of course, Shaggy is always there to make sure that Scooby Doo does not convert the maximum amount of bones into poop, and so Scooby is only going to be able to convert a lowered amount of bones into poop, indicated by a lowered V_max.

Along with the V_max being decreased, the Kilometers is also decreased. The apparent Kilometers is also being decreased, with respect to the Kilometers in the absence of inhibitor. Over here on the right, this Michaelis Menten plot is just trying to show us what happens if we increase the concentration of uncompetitive inhibitor even further. Notice we have these 3 different curves: the black curve that represents the enzyme-catalyzed reaction in the absence of any inhibitor, the purple curve that represents the enzyme-catalyzed reaction in the presence of just 1 Molar concentration of uncompetitive inhibitor, and the green curve that represents the enzyme-catalyzed reaction in the presence of plus 2 Molar concentration of uncompetitive inhibitor. Increasing the concentration of uncompetitive inhibitor even further will decrease the V_max even further and it will also decrease the Kilometers even further. Thus, the Kilometers is still going to be decreased here along with the decreases in the V_max.

This lesson on how uncompetitive inhibition affects Michaelis Menten plots is concluded. In our next lesson video, we'll talk about how uncompetitive inhibition affects the Lineweaver Burk plot. I'll see you guys in that video.

Uncompetitive Inhibition & Lineweaver-Burk Plots

So, in this video, we're going to talk about how uncompetitive inhibitors affect Lineweaver Burk plots. Recall that uncompetitive inhibitors proportionally decrease both the apparent Kilometers as well as the apparent Vmax of an enzyme. Even though both the apparent Kilometers and the apparent Vmax are decreased, it turns out that the slope of the line on a Lineweaver Burk plot, which is the ratio of the Kilometers over the Vmax, is actually not changed. Here, we can say that the slope is not going to change even in the presence of the slope of the line not changing. Since a Lineweaver Burk plot is also known as a double reciprocal plot since it plots the reciprocal of the initial reaction velocity on the y-axis and the reciprocal of the substrate concentration on the x-axis. What this means is that both the y intercept, which is the reciprocal of the Vmax, as well as the absolute value of the x intercept, which is the negative reciprocal of the Km, are going to be proportionally increased. This has to do with the nature of the fact that the reciprocals are plotted on these axes.

If we take a look at our image below on the left-hand side, you can see that in the presence of an uncompetitive inhibitor, this is how the Lineweaver Burk equation changes. Notice that the slope of our line, which is again the ratio of the Km over the Vmax, is actually not being affected by the degree of inhibition factor. There's no alpha prime affecting this slope here. This means that the slope is not being changed and this has to do with the fact that both the apparent Kilometers and the apparent Vmax are proportionally being decreased.

If we take a look at the Lineweaver Burk equation over here on the right, notice we have two different lines. We have this black line here that represents the enzyme catalyzed reaction in the absence of inhibitor indicated by a negative concentration of I. And then we have this purple line here that represents the enzyme catalyzed reaction in the presence of an uncompetitive inhibitor, 1 molar concentration. Notice that even though the y-intercept is being increased and the x-intercept, the absolute value of the x-intercept is also being increased. These values here are getting further and further away from our 0 markers, which we know acts as the infinity marker for the Vmax and the Km themselves. You can see that the Vmax here is getting further away from the infinity marker, meaning it's being decreased. And, the Km here is also getting further away from the infinity marker, which means it's being decreased as well. They're being decreased proportionally, which is what allows the slope here of our line to stay exactly the same.

Notice that the example here is asking us if the concentration of uncompetitive inhibitor was doubled. Here we have plus 1 concentration of I. So if we were to double this, it would be plus 2 concentration of I. In the presence of even more uncompetitive inhibitor, the Vmax is going to decrease even further. Here, we can say that the y-intercept is going to get further away from the 0 infinity marker and the same goes for the Km, it's going to get further away and it's going to be proportionally decreased so that the slope stays exactly the same. Notice that all of these slopes here are exactly the same and that is a unique identifying feature that uncompetitive inhibitors are present. When you think again about uncompetitive inhibitors, think about the unique U, think about the U-turn. Both the Km and the Vmax are going to be decreased because of this U-turn. With this U-turn, it can also help you remember that there are going to be parallel lines as well. Here's our line for, in the absence of inhibitor, and notice that it does a U-turn here, and it pretty much keeps the same line exactly parallel. Hopefully, these little, hints, that help me remember uncompetitive inhibitor effects on Lineweaver Burk Plots will also help you guys remember it a bit as well. This concludes our lesson and we'll be able to get some practice as we move forward in our course. So, I'll see you guys in our next video.

True or false: Increasing [S] in the presence of an uncompetitive inhibitor will lower the inhibition constant (K_I).

• A. True.
• B. False.

Answer: (B)

In the presence of an uncompetitive inhibitor that binds __________ the substrate, the apparent V_max ____________ and the apparent K_m ____________ with respect to the V_max and K_m of the uninhibited enzyme.

• A. Before, Decreases, Increases.
• B. After, Decreases, Decreases.
• C. After, Increases, Increases.
• D. Before, Stays the same, Decreases.
• E. After, Decreases, Stays the same.

Answer: (B)

What is the effect of an uncompetitive inhibitor on the equilibrium between free enzyme & the ES-complex?

• A. A shift to the right due to decreased [ES].
• B. A shift to the left due to decreased [ES].
• C. A shift to the right due to increased [ES].
• D. A shift to the left due to increased [ES].

Answer: (A)

Here’s what students ask on this topic:

What is uncompetitive inhibition in enzyme kinetics?

Uncompetitive inhibition occurs when an inhibitor binds exclusively to the enzyme-substrate complex (ES), forming an enzyme-substrate-inhibitor (ESI) complex. This type of inhibition decreases both the apparent Michaelis constant (Km) and the maximum reaction velocity (Vmax) of the enzyme. Unlike competitive inhibitors, uncompetitive inhibitors do not bind to the free enzyme. Instead, they only bind after the substrate has bound to the enzyme, preventing the conversion of the substrate into the product. This results in a stronger apparent affinity for the substrate (lower Km) but a reduced Vmax.

How does uncompetitive inhibition affect the Michaelis-Menten plot?

In a Michaelis-Menten plot, uncompetitive inhibition causes the reaction curves to shift downwards. This is because both the apparent Km and Vmax are proportionally decreased. The black curve represents the enzyme-catalyzed reaction without the inhibitor, while the blue curve shows the reaction in the presence of the uncompetitive inhibitor. The Vmax is lowered, and the Km is also decreased, indicating a stronger apparent affinity for the substrate. This shift reflects the inhibitor's effect on the enzyme-substrate complex, reducing the overall reaction rate.

What is the effect of uncompetitive inhibition on Lineweaver-Burk plots?

In Lineweaver-Burk plots, uncompetitive inhibition results in parallel lines. This is because both the apparent Km and Vmax are proportionally decreased, leaving the slope (Km/Vmax) unchanged. The y-intercept (1/Vmax) and the absolute value of the x-intercept (1/Km) both increase, moving further from the origin. This parallel shift is a key indicator of uncompetitive inhibition, as it shows that the inhibitor affects the enzyme-substrate complex without altering the ratio of Km to Vmax.

Why does uncompetitive inhibition decrease both Km and Vmax?

Uncompetitive inhibition decreases both Km and Vmax because the inhibitor binds only to the enzyme-substrate complex, reducing the concentration of this complex. According to Le Chatelier's principle, this shift causes the equilibrium to favor the formation of more enzyme-substrate complex, which appears as a stronger affinity for the substrate (lower Km). However, since the inhibitor prevents the conversion of the substrate to the product, the maximum reaction rate (Vmax) is also reduced. This dual effect is unique to uncompetitive inhibition.

How can you identify uncompetitive inhibition in enzyme kinetics experiments?

Uncompetitive inhibition can be identified by analyzing both Michaelis-Menten and Lineweaver-Burk plots. In Michaelis-Menten plots, the presence of an uncompetitive inhibitor causes the reaction curves to shift downwards, indicating a decrease in both Km and Vmax. In Lineweaver-Burk plots, the lines remain parallel, as the slope (Km/Vmax) is unchanged despite the proportional decrease in both parameters. These characteristic shifts help distinguish uncompetitive inhibition from other types of enzyme inhibition.