Reversible inhibitors bind to enzymes with non-covalent interactions such as hydrogen bonds, hydrophobic interactions and ionic bonds. Multiple weak bonds between the inhibitor and the active site combine to produce strong and specific binding. In contrast to substrates and irreversible inhibitors, reversible inhibitors generally do not undergo chemical reactions when bound to the enzyme and can be easily removed by dilution or dialysis.
There are three kinds of reversible enzyme inhibitors. They are classified according to the effect of varying the concentration of the enzyme's substrate on the inhibitor.
- In competitive inhibition, the substrate and inhibitor cannot bind to the enzyme at the same time, as shown in the figure on the left. This usually results from the inhibitor having an affinity for the active site of an enzyme where the substrate also binds; the substrate and inhibitor compete for access to the enzyme's active site. This type of inhibition can be overcome by sufficiently high concentrations of substrate, i.e., by out-competing the inhibitor. Competitive inhibitors are often similar in structure to the real substrate (see examples below).- In mixed inhibition, the inhibitor can bind to the enzyme at the same time as the enzyme's substrate. However, the binding of the inhibitor affects the binding of the substrate, and vice versa. This type of inhibition can be reduced, but not overcome by increasing concentrations of substrate. Although it is possible for mixed-type inhibitors to bind in the active site, this type of inhibition generally results from an allosteric effect where the inhibitor binds to a different site on an enzyme. Inhibitor binding to this allosteric site changes the conformation (i.e., tertiary structure or three-dimensional shape) of the enzyme so that the affinity of the substrate for the active site is reduced.
- Non-competitive inhibition is a form of mixed inhibition where the binding of the inhibitor to the enzyme reduces its activity but does not affect the binding of substrate. As a result, the extent of inhibition depends only on the concentration of the inhibitor.
Quantitative description of reversible inhibition
Reversible inhibition can be described quantitatively in terms of the inhibitor's binding to the enzyme and to the enzyme–substrate complex, and its effects on the kinetic constants of the enzyme. In the classic Michaelis–Menten scheme below, an enzyme (E) binds to its substrate (S) to form the enzyme–substrate complex ES. Upon catalysis, this complex breaks down to release product P and free enzyme. The inhibitor (I) can bind to either E or ES with the dissociation constants Ki or Ki', respectively.
- Competitive inhibitors can bind to E, but not to ES. Competitive inhibition increases Km (i.e., the inhibitor interferes with substrate binding), but does not affect Vmax (the inhibitor does not hamper catalysis in ES because it cannot bind to ES).- Non-competitive inhibitors have identical affinities for E and ES (Ki = Ki'). Non-competitive inhibition does not change Km (i.e., it does not affect substrate binding) but decreases Vmax (i.e., inhibitor binding hampers catalysis).
- Mixed-type inhibitors bind to both E and ES, but their affinities for these two forms of the enzyme are different (Ki ≠ Ki'). Thus, mixed-type inhibitors interfere with substrate binding (increase Km) and hamper catalysis in the ES complex (decrease Vmax).
When an enzyme has multiple substrates, inhibitors can show different types of inhibition depending on which substrate is considered. This results from the active site containing two different binding sites within the active site, one for each substrate. For example, an inhibitor might compete with substrate A for the first binding site, but be a non-competitive inhibitor with respect to substrate B in the second binding site.
Measuring the dissociation constants of a reversible inhibitor
As noted above, an enzyme inhibitor is characterized by its two dissociation constants, Ki and Ki', to the enzyme and to the enzyme-substrate complex, respectively. The enzyme-inhibitor constant Ki can be measured directly by various methods; one extremely accurate method is isothermal titration calorimetry, in which the inhibitor is titrated into a solution of enzyme and the heat released or absorbed is measured. However, the other dissociation consta
nt Ki' is difficult to measure directly, since the enzyme-substrate complex is short-lived and undergoing a chemical reaction to form the product. Hence, Ki' is usually measured indirectly, by observing the enzyme activity under various substrate and inhibitor concentrations, and fitting the data to a modified Michaelis–Menten equation
where the modifying factors α and α' are defined by the inhibitor concentration and its two dissociation constants
Thus, in the presence of the inhibitor, the enzyme's effective Km and Vmax become (α/α')Km and (1/α')Vmax, respectively. However, the modified Michaelis-Menten equation assumes that binding of the inhibitor to the enzyme has reached equilibrium, which may be a very slow process for inhibitors with sub-nanomolar dissociation constants. In these cases, it is usually more practical to treat the tight-binding inhibitor as an irreversible inhibitor (see below); however, it can still be possible to estimate Ki' kinetically if Ki is measured independently.
The effects of different types of reversible enzyme inhibitors on enzymatic activity can be visualized using graphical representations of the Michaelis–Menten equation, such as Lineweaver–Burk and Eadie-Hofstee plots. For example, in the Lineweaver-Burk plots at the right, the competitive inhibition lines intersect on the y-axis, illustrating that such inhibitors do not affect Vmax. Similarly, the non-competitive inhibition lines intersect on the x-axis, showing these inhibitors do not affect Km. However, it can be difficult to estimate Ki and Ki' accurately from such plots, so it is advisable to estimate these constants using more reliable nonlinear regression methods, as described above.
Special cases
The mechanism of partially competitive inhibition is similar to that of non-competitive, except that the EIS complex has catalytic activity, which may be lower or even higher (partially competitive activation) than that of the enzyme–substrate (ES) complex. This inhibition typically displays a lower Vmax, but an unaffected Km value.
Uncompetitive inhibition occurs when the inhibitor binds only to the enzyme–substrate complex, not to the free enzyme; the EIS complex is catalytically inactive. This mode of inhibition is rare and causes a decrease in both Vmax and the Km value.
Substrate and product inhibition is where either the substrate or product of an enzyme reaction inhibit the enzyme's activity. This inhibition may follow the competitive, uncompetitive or mixed patterns. In substrate inhibition there is a progressive decrease in activity at high substrate concentrations. This may indicate the existence of two substrate-binding sites in the enzyme. At low substrate, the high-affinity site is occupied and normal kinetics are followed. However, at higher concentrations, the second inhibitory site becomes occupied, inhibiting the enzyme.Product inhibition is often a regulatory feature in metabolism and can be a form of negative feedback.
Slow-tight inhibition occurs when the initial enzyme–inhibitor complex EI undergoes isomerisation to a second more tightly held complex, EI*, but the overall inhibition process is reversible. This manifests itself as slowly increasing enzyme inhibition. Under these conditions, traditional Michaelis–Menten kinetics give a false value for Ki, which is time–dependent. The true value of Ki can be obtained through more complex analysis of the on (kon) and off (koff) rate constants for inhibitor association. See irreversible inhibition below for more information.
Examples of reversible inhibitors
As enzymes have evolved to bind their substrates tightly, and most reversible inhibitors bind in the active site of enzymes, it is unsurprising that some of these inhibitors are strikingly similar in structure to the substrates of their targets. An example of these substrate mimics are the protease inhibitors, a very successful class of antiretroviral drugs used to treat HIV.The structure of ritonavir, a protease inhibitor based on a peptide and containing three peptide bonds, is shown on the right. As this drug resembles the protein that is the substrate of the HIV protease, it competes with this substrate in the enzyme's active site.
Enzyme inhibitors are often designed to mimic the transition state or intermediate of an enzyme-catalysed reaction. This ensures that the inhibitor exploits the transition state stabilising effect of the enzyme, resulting in a better binding affinity (lower Ki) than substrate-based designs. An example of such a transition state inhibitor is the antiviral drug oseltamivir; this drug mimics the planar nature of the ring oxonium ion in the reaction of the viral enzyme neuraminidase.
However, not all inhibitors are based on the structures of substrates. For example, the structure of another HIV protease inhibitor tipranavir is shown on the left. This molecule is not based on a peptide and has no obvious structural similarity to a protein substrate. These non-peptide inhibitors can be more stable than inhibitors containing peptide bonds, because they will not be substrates for peptidases and are less likely to be degraded.
In drug design it is important to consider the concentrations of substrates to which the target enzymes are exposed. For example, some protein kinase inhibitors have chemical structures that are similar to adenosine triphosphate, one of the substrates of these enzymes. However, drugs that are simple competitive inhibitors will have to compete with the high concentrations of ATP in the cell. Protein kinases can also be inhibited by competition at the binding sites where the kinases interact with their substrate proteins, and most proteins are present inside cells at concentrations much lower than the concentration of ATP. As a consequence, if two protein kinase inhibitors both bind in the active site with similar affinity, but only one has to compete with ATP, then the competitive inhibitor at the protein-binding site will inhibit the enzyme more effectively.
The mechanism of partially competitive inhibition is similar to that of non-competitive, except that the EIS complex has catalytic activity, which may be lower or even higher (partially competitive activation) than that of the enzyme–substrate (ES) complex. This inhibition typically displays a lower Vmax, but an unaffected Km value.
Uncompetitive inhibition occurs when the inhibitor binds only to the enzyme–substrate complex, not to the free enzyme; the EIS complex is catalytically inactive. This mode of inhibition is rare and causes a decrease in both Vmax and the Km value.
Substrate and product inhibition is where either the substrate or product of an enzyme reaction inhibit the enzyme's activity. This inhibition may follow the competitive, uncompetitive or mixed patterns. In substrate inhibition there is a progressive decrease in activity at high substrate concentrations. This may indicate the existence of two substrate-binding sites in the enzyme. At low substrate, the high-affinity site is occupied and normal kinetics are followed. However, at higher concentrations, the second inhibitory site becomes occupied, inhibiting the enzyme.Product inhibition is often a regulatory feature in metabolism and can be a form of negative feedback.
Slow-tight inhibition occurs when the initial enzyme–inhibitor complex EI undergoes isomerisation to a second more tightly held complex, EI*, but the overall inhibition process is reversible. This manifests itself as slowly increasing enzyme inhibition. Under these conditions, traditional Michaelis–Menten kinetics give a false value for Ki, which is time–dependent. The true value of Ki can be obtained through more complex analysis of the on (kon) and off (koff) rate constants for inhibitor association. See irreversible inhibition below for more information.
Examples of reversible inhibitors
As enzymes have evolved to bind their substrates tightly, and most reversible inhibitors bind in the active site of enzymes, it is unsurprising that some of these inhibitors are strikingly similar in structure to the substrates of their targets. An example of these substrate mimics are the protease inhibitors, a very successful class of antiretroviral drugs used to treat HIV.The structure of ritonavir, a protease inhibitor based on a peptide and containing three peptide bonds, is shown on the right. As this drug resembles the protein that is the substrate of the HIV protease, it competes with this substrate in the enzyme's active site.
Enzyme inhibitors are often designed to mimic the transition state or intermediate of an enzyme-catalysed reaction. This ensures that the inhibitor exploits the transition state stabilising effect of the enzyme, resulting in a better binding affinity (lower Ki) than substrate-based designs. An example of such a transition state inhibitor is the antiviral drug oseltamivir; this drug mimics the planar nature of the ring oxonium ion in the reaction of the viral enzyme neuraminidase.However, not all inhibitors are based on the structures of substrates. For example, the structure of another HIV protease inhibitor tipranavir is shown on the left. This molecule is not based on a peptide and has no obvious structural similarity to a protein substrate. These non-peptide inhibitors can be more stable than inhibitors containing peptide bonds, because they will not be substrates for peptidases and are less likely to be degraded.
In drug design it is important to consider the concentrations of substrates to which the target enzymes are exposed. For example, some protein kinase inhibitors have chemical structures that are similar to adenosine triphosphate, one of the substrates of these enzymes. However, drugs that are simple competitive inhibitors will have to compete with the high concentrations of ATP in the cell. Protein kinases can also be inhibited by competition at the binding sites where the kinases interact with their substrate proteins, and most proteins are present inside cells at concentrations much lower than the concentration of ATP. As a consequence, if two protein kinase inhibitors both bind in the active site with similar affinity, but only one has to compete with ATP, then the competitive inhibitor at the protein-binding site will inhibit the enzyme more effectively.
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