Reversible Inhibitors

Reversible inhibitors are commonly used in drug discovery research because they help study enzyme activity without permanently altering the enzymes. Read on to learn about the various types of reversible inhibitors and their kinetics including the common types: competitive, uncompetitive, and non-competitive.

Read more about

• What Are Reversible Inhibitors?
• Competitive Inhibitors
• Uncompetitive Inhibitors
• Non-Competitive Inhibitors
• Mixed Type Inhibitors
• Allosteric Inhibitors
• End Product Inhibitors

What are Reversible Inhibitors?

Reversible inhibitors are inhibitors that have no permanent effect on enzymes, meaning that once they are removed the enzyme can work normally again (i.e., inhibition is easily reversed). They reversibly bind to enzymes with noncovalent interactions, such as hydrogen bonds, ionic bonds, and hydrophobic interactions (Figure 1). Alternatively, irreversible inhibitors can react with the enzyme covalently and induce chemical changes to modify key amino acids that are required for enzymatic activity.

Figure 1. A general scheme of reversible enzyme inhibition.

Also, it is important to note that when enzymes can act on different substrates, inhibitors can display different types of inhibition depending on which substrate is being considered. This occurs because the active site may have different binding sites, one for each substrate. Hence, an inhibitor can compete with substrate A at the first binding site and act on a second binding site in a non-competitive manner with respect to substrate B.

There are three main types of reversible inhibitors: competitive, uncompetitive, and non-competitive.

Competitive Inhibitors

In competitive inhibition, the inhibitor usually has structural similarity with the natural substrate and competes with the substrate for access to the active site (Figure 2). The inhibitor has an affinity for the active site, and if it binds more tightly than the substrate, then it is an effective competitive inhibitor. Conversely, if it binds less strongly, it is considered a poor inhibitor.

Examples of competitive inhibitors include:

• Methotrexate, an anticancer agent
  • Inhibits the activity of dihydrofolate reductase, the enzyme that participates in tetrahydrofolate synthesis
• Sildenafil citrate, a smooth muscle relaxant
  • Competitively inhibits the activity of phosphodiesterase V because it is structurally similar to cyclic-GMP (cGMP) and results in the accumulation of cGMP

In competitive inhibition, the competitive inhibitor can only bind to the free enzyme and not with the enzyme-substrate complex. Hence, inhibition can be overcome by increasing the concentration of substrate in the reaction mixture.  By outcompeting with the substrate, V_max can still be achieved. However, in the presence of an inhibitor, substrate concentration has to be increased to achieve V_max. This will increase the K_m value.

Figure 2. A simplified model of competitive inhibition.

For competitive inhibition, one can determine K_i, the inhibition constant, which is the dissociation constant for the enzyme-inhibitor complex. Lower K_i values mean lower amounts of inhibitor required to reduce the rate of reaction. This relationship can be simplified as:

A general tip when studying the effect of competitive inhibitors is to add an appropriate amount of substrate to the incubation medium. An excessive amount of substrate will outcompete the inhibitor. You should always run a control reaction without an inhibitor.

Kinetics of Competitive Inhibition

The result of the kinetics of competitive inhibition can be presented in a Lineweaver-Burk plot (Figure 3). Since K_m increases as a result of competitive inhibition, the X-intercept moves closer to the origin in the presence of an inhibitor. As we increase inhibitor concentration, K_m will increase further, and the X-intercept will move even closer to the origin. Note that all lines go through the same Y-intercept because a competitive inhibitor does not affect V_max.

Figure 3. Kinetics of competitive inhibition.

Sometimes an enzyme may follow the kinetics of partial competitive inhibition. This process is similar to competitive inhibition, but the enzyme-substrate-inhibitor complex (ESI) may exhibit partial activity. This type of inhibition displays lower V_max, but K_m is not affected. With complete competitive inhibition, the velocity of reaction tends to be zero when inhibitor concentration is increased. However, with partial inhibition, the enzyme is converted into a modified, but still somewhat functional ESI complex.

Uncompetitive Inhibitors

Uncompetitive inhibitors only bind to the enzyme-substrate (ES) complex. Uncompetitive inhibition should not be confused with non-competitive inhibition. The uncompetitive inhibitor does not bind to the active site of the enzyme and it does not have to resemble the substrate. In uncompetitive inhibition, V_max is reduced because of the removal of the activated ES complex. The amount of ESI complex depends on the concentration of the inhibitor. The elimination of the ES complex also results in reduced K_m. This relationship can be represented as:

Kinetics of Uncompetitive Inhibition

In uncompetitive inhibition, both K_m and V_max decrease at the same time and the same rate. In other words, V_max/K_m is unaltered. Figure 4 shows that with an uncompetitive inhibitor, 1/V_max is increased. Hence, the Y-intercept moves up. Inhibition also increases 1/K_m to a degree that maintains the ratio of K_m/V_max, which is the slope of the curve. For this reason, Lineweaver-Burk plots for uncompetitive inhibition are parallel, with and without inhibitor.

Figure 4. Kinetics of uncompetitive inhibition.

Uncompetitive inhibition is relatively rare but may occur in multimeric enzyme systems. Evolutionarily, this is important because uncompetitive inhibition in a metabolic pathway can have larger effects on the concentrations of metabolic intermediates than competitive inhibition and may even increase toxicity. An example of an uncompetitive reversible inhibitor is oxalate, which inhibits lactate dehydrogenase.

Non-Competitive Inhibitors

In non-competitive inhibition, the binding of the inhibitor reduces enzyme activity but does not affect the binding of substrate (Figure 5). Hence, the degree of inhibition is only dependent on the concentration of the inhibitor. An example of a non-competitive reversible inhibitor is digitalis, which blocks the activity of Na⁺-K⁺ ATPase and is used for the treatment of cardiac arrhythmia. These non-competitive inhibitors bind noncovalently to sites other than the substrate-binding site. Inhibitor binding does not influence the availability of the binding site for the substrate. Therefore, the binding of the substrate and the inhibitor are independent of each other and inhibition cannot be overcome by increasing substrate concentration. 

Figure 5. A simplified model of non-competitive inhibition.

Kinetics of Non-Competitive Inhibition

Non-competitive inhibitors have identical affinities for enzymes and ES complexes; therefore, K_i = K’_I (ES complex dissociation constant = K’_i). Hence, V_max is reduced, but K_m is unaffected. V_max cannot be attained in the presence of a non-competitive inhibitor. Equilibria for non-competitive inhibition can be simplified as follows:

The effect of a non-competitive inhibitor is graphically presented in Figure 6. Since the Y-intercept is 1/V_max, as V_max decreases, 1/V_max increases. However, K_m remains the same for any concentration of the non-competitive inhibitor. Hence, all lines go through the same X-intercept. 

Figure 6. Kinetics of non-competitive inhibition.

Mixed Type Inhibitors

In certain cases, an inhibitor can bind to both the free enzyme (with a dissociation constant K_i) as well as the ES complex (with dissociation constant K’_i). However, their affinities are different, hence, K_i ≠ K’_i. Here, the inhibitor binding can be reduced by adding more substrate, but the inhibition cannot be totally overcome as in competitive inhibition. An example of mixed-type inhibition is that of xanthine oxidase by palladium ions.

Kinetics of Mized Type Inhibition

Mixed type inhibitors interfere with binding and reduce the effectiveness of turnover. This type of inhibition is mostly allosteric in nature, where the inhibitor binds to a site other than the active site to cause a conformational change in the enzyme structure, reducing the affinity of substrate for the active site. Hence, K_m is increased while V_max is reduced (Figure 7). 

Figure 7. Kinetics of mixed type inhibition.

Allosteric Inhibitors

Allosteric enzymes belong to a group of enzymes that do not obey Michaelis-Menten kinetics. They generally have a regulatory role in the cell. They function through reversible, noncovalent binding of effector molecules (activators and inhibitors) to their regulatory site. The binding of an activator promotes active shape and enzyme activity, whereas binding of an inhibitor to the regulatory site causes the allosteric enzyme to adopt the inactive shape and causes a reduction in activity (Figure 8).

Figure 8. A model of allosteric inhibition.

Allosteric inhibition occurs when the binding of one ligand decreases the affinity for the substrate at other active sites. A classic example of allosteric inhibition is that of phosphofructokinase (PFK). PFK catalyzes the phosphorylation of fructose-6-phosphate to form fructose-1-6-bisphosphate. When levels of ATP increase, the activity of PFK is allosterically inhibited. ATP binds to an allosteric site on PFK, causing a change in the enzyme’s shape. This reduces its affinity for fructose-6- phosphate and ATP at the active site, reducing the rate of glycolysis.

The active and inactive forms of the allosteric enzyme exist in an equilibrium that is dependent on the relative concentrations of substrate and inhibitor. The binding of an allosteric inhibitor causes the enzyme to adopt the inactive conformation and can promote the cooperative binding of a second inhibitor.

Kinetics of Allosteric Inhibition

The kinetics of allosteric inhibitors generates a sigmoidal curve instead of a hyperbolic curve. This is because these enzymes possess multiple binding sites and can bind to more than one substrate molecule. They exhibit saturation with the substrate when [S] is sufficiently high. In this sigmoidal reaction curve, the substrate concentration at which reaction velocity is half-maximal cannot be designated as K_m, because the Michaelis-Menten model does not apply. Instead, the symbol [S]_0.5 or K_0.5 is used to represent the substrate concentration at which half-maximum velocity is observed.

End Product Inhibitors

Many enzyme-catalyzed reactions occur sequentially, in a biochemical pathway, in which the product of one reaction becomes the substrate for the next reaction. If the end product of the pathway accumulates in quantities more than needed, then this end product can inhibit the activity of the first enzyme. A pathway is shut down when the end product of the pathway is bound to an allosteric site on the first enzyme of the pathway (Figure 9). 

Figure 9. In end product inhibition, the end product of a pathway (D) can directly bind to (and inhibit) the enzyme that converts A to B, thus shutting down the pathway.

Upon this binding, the enzyme undergoes a conformational change and cannot react with the first substrate. This is a form of biological control that prevents an excessive buildup of the product. An example is the formation of L-isoleucine from L-threonine, catalyzed by L-threonine dehydratase, which is strongly inhibited by L-isoleucine.

Explore the other main category of enzyme inhibitors in our article on irreversible inhibitors.