9.4. Enzyme inhibition mechanisms

Enzymes play central roles in life processes. It holds for most enzymes that their function is needed only in certain conditions. When those conditions do not apply, the activity of a given enzyme can be futile or even harmful. Accordingly, the activity of most enzymes is under strict control. Enzymes can be regulated at multiple levels, ranging from transcriptional regulation of the expression of the enzyme-encoding gene through the direct regulation of the activity of the enzyme molecule by effector molecules to the controlled proteolytic decomposition of the enzyme. In this chapter, only those inhibitors will be reviewed that reversibly and specifically bind to enzymes through non-covalent interactions and inhibit the substrate-binding and/or catalytic apparatus of the given enzyme. These inhibitors can be classified into three mechanistic groups based on their mechanism of action: competitive, uncompetitive and mixed inhibitors. The type of inhibition can be determined through enzyme kinetic measurements.

In the most frequent procedure, the kinetic parameters Vmax and Km are determined as already described in Section 9.3—however, in this case, in the presence of several pre-set inhibitor concentrations. When the experimental data are examined in the form of double reciprocal Lineweaver-Burk plots, the deviation of the obtained line from that of the uninhibited case will be diagnostic of the type of inhibition.

9.4.1. Competitive inhibition

Competitive inhibitors compete for the substrate-binding site of the enzyme with the substrate, because the substrate and the inhibitor bind to identical or overlapping sites. Due to the overlapping nature of the binding sites, a ternary complex—in which the substrate and the inhibitor would simultaneously bind to the enzyme—cannot form. Accordingly, in the enzyme-inhibitor complex, the enzyme is completely inactive.

By popular—but quite misleading—terminology, these inhibitors are said to “displace” the substrate from the enzyme. While this term is aimed to be expressive, it is totally inadequate to explain the mechanism of this type of inhibition. The popular term suggests that the inhibitor would bind to the ES complex and would thus somehow actively force the substrate to dissociate. As already mentioned, no ternary complex is formed—not even temporarily. This inhibitory mechanism simply obeys a thermodynamic principle: two equilibria exist in parallel, one between the enzyme and the inhibitor and another between the enzyme and the substrate. More precisely, the latter one is a quasi-equilibrium because, during the measurement, the enzyme and the substrate are in a steady-state (as shown in Figure 9.4). The equilibrium concentrations of the free compounds and those of the complexes are dictated by the total concentrations of the individual compounds and the affinity of their interactions.

The equilibrium between the enzyme and the inhibitor is described by Equation 9.53 in which the KI term represents a dissociation constant:


The two equilibria are not independent as the complexes, ES and EI, equilibrate with the same free enzyme pool, E. Increasing EI concentration by increasing the inhibitor concentration can be achieved only through a decrease in ES concentration, and vice versa: an elevated substrate concentration can increase the concentration of the ES complex only at the expense of the EI complex.

This mechanism is illustrated in Figure 9.6.

The scheme of competitive inhibition

Figure 9.6. The scheme of competitive inhibition

When solving the Michaelis-Menten equation, we made use of the simple fact that the total enzyme concentration can be expressed as follows: [E]T = [E]+[ES]. On the other hand, in the presence of a competitive inhibitor, [E]T = [E]+[ES]+[EI]. Solving the Michaelis-Menten equation such that this difference is taken into consideration leads to Equation 9.54. (For brevity, the intermediate steps that yield this equation are not shown.)


The meaning of the term α in Equation 9.54 is explained in Equation 9.55:


It is readily apparent that, in the absence of inhibitor, the value of α is one and, as expected, we get the original equation. In the presence of inhibitor, the value of α exceeds one. The higher the concentration of the inhibitor compared to the value of the KI dissociation constant, the higher the value of α. Equation 9.54 clearly indicates that the measured Vmax will be invariant, regardless of the presence of the inhibitor. On the other hand, in the presence of a competitive inhibitor, the measured KM will be higher than in the absence of the inhibitor. As in the case of this inhibitor type the substrate competes with the inhibitor, it is intuitively comprehensible that, at “infinitely” high substrate concentrations, the presence of the inhibitor should not affect the measurements, i.e. the maximal rate of the reaction should be unchanged. However, as in the presence of a competitive inhibitor higher than normal substrate concentration is needed to achieve a (half-) maximal rate, the value of KM must be higher than in the uninhibited case. That is exactly what Equation 9.54 expresses.

When Equation 9.54 is rearranged according to the double reciprocal transformation, we get Equation 9.56, which is analogous to Equation 9.52 introduced previously for the uninhibited case:


Equation 9.56 is graphically illustrated by the plots shown in Figure 9.7.The double reciprocal plots clearly show that, in the presence of a competitive inhibitor, the lines are steeper than in the uninhibited case; but the intercept on the y axis, which refers to the 1/Vmax value, remains the same. The plot nicely illustrates the didactical strength of double reciprocal data analysis to demonstrate the mechanism of a reversible inhibitor.

Double reciprocal Lineweaver-Burk analysis of competitive inhibition

Figure 9.7. Double reciprocal Lineweaver-Burk analysis of competitive inhibition

As competitive inhibitors compete with the substrate for overlapping binding sites on the enzyme, it is not surprising that competitive inhibitors often resemble the substrate in terms of chemical structure, shape and polarity pattern. Due to this, competitive inhibitors are often used as useful reagents to study the substrate binding mechanism of enzymes. Comparative analysis of the structure of the substrate and that of a set of different competitive inhibitors can help in identifying the functionally most important parts of the substrate—those that provide the most binding energy in the ES complex. Note that such indirect approaches are important because direct analysis of the short-lived ES complex is a demanding scientific challenge.

9.4.2. Uncompetitive inhibition

Some inhibitors bind only to the ES complex without binding to the free enzyme. This interaction scheme is illustrated in Figure 9.8.

Uncompetitive inhibition

Figure 9.8. Uncompetitive inhibition

The kinetic equation of this type of inhibition can also be expressed as a modified version of the uninhibited case, as shown in Equation 9.57:


The meaning of the KI’ term is shown in Equation 9.58:


The meaning of the α’ term in Equation 9.57 is analogous to the meaning of α introduced in Equation 9.55. However, as shown in Equation 9.59, α’ is associated with KI’ instead of KI:


Double reciprocal transformation of Equation 9.57 results in Equation 9.60:


A graphical illustration of Equation 9.60 is presented in Figure 9.9.

Double reciprocal Lineweaver-Burk analysis of uncompetitive inhibition

Figure 9.9. Double reciprocal Lineweaver-Burk analysis of uncompetitive inhibition

The plot clearly illustrates that both the KM and Vmax values are divided by the value of α’ (i.e. their reciprocal value is multiplied by the value of α’). This means that, unlike in the case of competitive inhibitors, the presence of an uncompetitive inhibitor results in a decreased Vmax value. Moreover, the KM (i.e. the substrate concentration at which the reaction rate reaches its half maximum) also decreases, and it does so to exactly the same extent as the Vmax. As both kinetic parameters decrease to the same degree, the slopes of the lines do not change. This type of inhibition is dramatically different from the competitive one. Namely, the effect of an uncompetitive inhibitor, although its binding is reversible, cannot be abolished by increasing substrate concentration.

9.4.3. Mixed inhibition

There are inhibitors that can bind both to the free enzyme as well as to the ES complex. These inhibitors represent a combination of the two already discussed types. The scheme of mixed inhibition is illustrated in Figure 9.10.

Mixed inhibition

Figure 9.10. Mixed inhibition

In most cases of mixed inhibition, the inhibitor binds to the free enzyme and to the ES complex with different affinities—however the case where KI is equal to KI` cannot be excluded (see below).

As the scheme of mixed inhibition is the combination of the schemes of the previously mentioned inhibition types, the kinetic Equation 9.61 of mixed inhibition is also a combination of the previous equations:


The double reciprocal transformation of Equation 9.61 results in Equation 9.62:


The meaning of the terms α and α’ is the same as was discussed for the competitive and uncompetitive cases, respectively. These represent the contribution of the competitive and uncompetitive components, respectively, to the observed inhibition.

A graphical illustration of the double reciprocal equation is shown in Figure 9.11.

Double reciprocal Lineweaver-Burk analysis of mixed inhibition

Figure 9.11. Double reciprocal Lineweaver-Burk analysis of mixed inhibition

In conclusion, the three different types of inhibition are accompanied by characteristically different double reciprocal plots. Therefore, these plots are of diagnostic value in the quick assessment of the type of the inhibitor.

It is worth noting that a certain (rather theoretical) variant of mixed inhibition is a type in which the inhibitor would bind to the free enzyme and the ES complex with exactly the same affinity. This would result only a single type of α term with a single value. This would alter the plot in Figure 9.11 such that each line corresponding to a given inhibitor concentration would intercept the x axis in the same point (as the factor of −1/KM would be α/α = 1), while the y axis intercepts would depend on inhibitor concentration, having different α/Vmax values.

In the case of all inhibitor types, the values of α and/or α’ can be experimentally determined and, through these parameters, the corresponding values of the inhibitory constants can also be computed.