2.3.2: Overview of Enzyme Inhibition

Regulation of enzyme activity

Apart from their ability to greatly speed the rates of chemical reactions in cells, enzymes have another property that makes them valuable. This property is that their activity can be regulated, allowing them to be activated and inactivated, as necessary. This is tremendously important in maintaining homeostasis, permitting cells to respond in controlled ways to changes in both internal and external conditions.

Inhibition of specific enzymes by drugs can also be medically useful. Understanding the mechanisms that control enzyme activity is, therefore, of considerable importance.

Inhibition

We will first discuss four types of enzyme inhibition – competitive, non-competitive, uncompetitive, and suicide inhibition. Of these, the first three types are reversible. The last one, suicide inhibition, is not.

Competitive inhibition

Probably the easiest type of enzyme inhibition to understand is competitive inhibition and it is the one most commonly exploited pharmaceutically. Molecules that are competitive inhibitors of enzymes resemble one of the normal substrates of an enzyme. An example is methotrexate, which resembles the folate substrate of the enzyme dihydrofolate reductase (DHFR). This enzyme normally catalyzes the reduction of folate, an important reaction in the metabolism of nucleotides.

Figure 4.33 - Competitive inhibitors resemble the normal substrate and compete for binding at the active site. Image by Aleia Kim

Inhibitor binding

When the drug methotrexate is present, some of the DHFR enzyme binds to it, instead of to folate, and during the time methotrexate is bound, the enzyme is inactive and unable to bind folate. Thus, the enzyme is inhibited. Notably, the binding site on DHFR for methotrexate is the active site, the same place that folate would normally bind. As a result, methotrexate ‘competes’ with folate for binding to the enzyme. The more methotrexate there is, the more effectively it competes with folate for the enzyme’s active site. Conversely, the more folate there is, the less of an effect methotrexate has on the enzyme because folate outcompetes it.

Figure 4.34 - Methotrexate and dihydrofolate. Image by Ben Carson

No effect on Vmax

How do we study competitive inhibition? It is typically done as follows. First, one performs a set of V0 vs. [S] reactions without inhibitor (20 or so tubes, with buffer and constant amounts of enzyme, varying amounts of substrate, equal reaction times). V0 vs. [S] is plotted (Figure 4.35 red line), as well as 1/V0 vs. 1/[S] (Figure 4.36 green line). Next, a second set of reactions is performed in the same manner as before, except that a fixed amount of the methotrexate inhibitor is added to each tube. At low concentrations of substrate, the methotrexate competes for the enzyme effectively, but at high concentrations of substrate, the inhibitor will have a much reduced effect, since the substrate outcompetes it, due to its higher concentration (remember that the inhibitor is at fixed concentration).

Figure 4.35 V0 vs [S] Plots for uninhibited reactions (red) and competitively inhibited reactions (blue). Both ultimately have same Vmax

Graphically, the results of these inhibitor experiments are shown in Figure 4.35 (blue line) and Figure 4.36 (orange line). Notice that at high substrate concentrations, the competitive inhibitor has essentially no effect, causing the \(V_{max}\) for the enzyme to remain unchanged. To reiterate, this is due to the fact that at high substrate concentrations, the inhibitor doesn’t compete well. However, at lower substrate concentrations, it does.

Figure 4.36 - Lineweaver-Burk plots - uninhibited reactions (green). Competitively inhibited reactions (orange). Lines cross on Y-axis at 1/Vmax Since Vmax is the same for both reactions. Image by Aleia Kim

Increased \(K_m\)

In competitively inhibited reactions, the apparent Km of the enzyme for the substrate increases (\(-1/K_m\) gets closer to zero - red line in Figure 4.36) when the inhibitor is present compared to when the inhibitor is absent, thus illustrating the better competition of the inhibitor at lower substrate concentrations. It may not be obvious why we call the changed Km the apparent Km of the enzyme. The reason is that the inhibitor doesn’t actually change the enzyme’s affinity for the folate substrate. It only appears to do so. This is because of the way that competitive inhibition works. When the competitive inhibitor binds the enzyme, it is effectively ‘taken out of action.’ Inactive enzymes have NO affinity for substrate and no activity either. We can’t measure Km for an inactive enzyme.

The enzyme molecules that are not bound by methotrexate can, in fact, bind folate and are active. Methotrexate has no effect on them and their Km values are unchanged. Why then, does Km appear higher in the presence of a competitive inhibitor? The reason is that the competitive inhibitor is having a greater effect of reducing the amount of active enzyme at lower concentrations of substrate than it does at higher concentrations of substrate. When the amount of enzyme is reduced, one must have more substrate to supply the reduced amount of enzyme sufficiently to get to Vmax/2.

It is worth noting that in competitive inhibition, the percentage of inactive enzyme changes drastically over the range of [S] values used. To start, at low [S] values, the greatest percentage of the enzyme is inhibited. At high [S], no significant percentage of enzyme is inhibited. This is not always the case, as we shall see in non-competitive inhibition.

Non-competitive inhibition

A second type of inhibition employs inhibitors that do not resemble the substrate and bind not to the active site, but rather to a separate site on the enzyme (Figure 4.37). The effect of binding a non-competitive inhibitor is significantly different from binding a competitive inhibitor because there is no competition. In the case of competitive inhibition, the effect of the inhibitor could be reduced and eventually overwhelmed with increasing amounts of substrate. This was because increasing substrate made increasing percentages of the enzyme active. With non-competitive inhibition, increasing the amount of substrate has no effect on the percentage of enzyme that is active. Indeed, in non-competitive inhibition, the percentage of enzyme inhibited remains the same through all ranges of [S].

Figure 4.37 - Non-competitive inhibition - inhibitor does not resemble the substrate and binds to a site other than the active site. Image by Aleia Kim

This means, then, that non-competitive inhibition effectively reduces the amount of enzyme by the same fixed amount in a typical experiment at every substrate concentration used The effect of this inhibition is shown in Figure 4.38 & 4.39. As you can see, \(V_{max}\) is reduced in non-competitive inhibition compared to uninhibited reactions.

Figure 4.38 - V0 vs [S] plots of uninhibited reactions (red) and non-competitively inhibited reactions (blue). Vmax is reduced, but Km values are unchanged in non-competitively inhibited reactions

This makes sense if we remember that Vmax is dependent on the amount of enzyme present. Reducing the amount of enzyme present reduces \(V_{max}\). In competitive inhibition, this doesn’t occur detectably, because at high substrate concentrations, there is essentially 100% of the enzyme active and the \(V_{max}\) appears not to change. Additionally, Km for non-competitively inhibited reactions does not change from that of uninhibited reactions. This is because, as noted previously, one can only measure the \(K_m\) of active enzymes and \(K_m\) is a constant for a given enzyme.

Figure 4.39 - Lineweaver Burk plots of uninhibited reactions (green) and non-competitively inhibited reactions (purple). Image by Aleia Kim

Uncompetitive inhibition

A third type of enzymatic inhibition is that of uncompetitive inhibition, which has the odd property of a reduced Vmax as well as a reduced Km. The explanation for these seemingly odd results is rooted in the fact that the uncompetitive inhibitor binds only to the enzyme-substrate (ES) complex (Figure 4.40). The inhibitor-bound complex forms mostly under concentrations of high substrate and the ES-I complex cannot release product while the inhibitor is bound, thus explaining the reduced \(V_{max}\).

Figure 4.40 - Uncompetitive inhibition. Image by Aleia Kim

The reduced Km is a bit harder to conceptualize. The reason is that the inhibitor-bound complex effectively reduces the concentration of the ES complex. By Le Chatelier’s Principle, a shift occurs to form additional ES complex, resulting in less free enzyme and more enzyme in the forms ES and ESI (ES with inhibitor). Decreases in free enzyme correspond to an enzyme with greater affinity for its substrate. Thus, paradoxically, uncompetitive inhibition both decreases \(V_{max}\) and increases an enzyme’s affinity for its substrate (\(K_m\) - Figures 4.41 & 4.42).

Figure 4.41 - V0 vs [S] plot for uncompetitive inhibition (blue) and uninhibited reactions (red)

Figure 4.42 - Uncompetitive inhibition (purple) and uninhibited reactions (green). Image by Aleia Kim

Suicide inhibition

In contrast to the first three types of inhibition, which involve reversible binding of the inhibitor to the enzyme, suicide inhibition is irreversible, because the inhibitor becomes covalently bound to the enzyme during the inhibition. Suicide inhibition rather closely resembles competitive inhibition because the inhibitor generally resembles the substrate and binds to the active site of the enzyme. The primary difference is that the suicide inhibitor is chemically reactive in the active site and makes a bond with it that precludes its removal. Such a mechanism is that employed by penicillin (Figure 4.43), which covalently links to the bacterial enzyme, DD transpeptidase and stops it from functioning. Since the normal function of the enzyme is to make a bond necessary for the peptidoglycan complex of the bacterial cell wall, the cell wall cannot properly form and bacteria cannot reproduce.

Figure 4.43 - Action of penicillin. DD-transpeptidase builds peptidoglycan layer of bacterial cell wall (1-3). Binding of penicillin by DD-transpeptidase stops peptidoglycan synthesis (4-5). Wikipedia