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An enzyme inhibitor is a molecule that binds to an enzyme and blocks its activity. Enzymes are proteins that speed up chemical reactions necessary for life, in which substrate molecules are converted into products. An enzyme facilitates a specific chemical reaction by binding the substrate to its active site, a specialized area on the enzyme that accelerates the most difficult step of the reaction.

An enzyme inhibitor hinders ("inhibits") this process, either by binding to the enzyme's active site (thus preventing the substrate itself from binding) or by binding to another site on the enzyme such that the enzyme's catalysis of the reaction is blocked. Enzyme inhibitors may bind reversibly or irreversibly. Irreversible inhibitors form a chemical bond with the enzyme such that the enzyme is inhibited until the chemical bond is broken. By contrast, reversible inhibitors bind non-covalently and may spontaneously leave the enzyme, allowing the enzyme to resume its function. Reversible inhibitors produce different types of inhibition depending on whether they bind to the enzyme, the enzyme-substrate complex, or both.

Enzyme inhibitors play an important role in all cells, since they are generally specific to one enzyme and serve to control that enzyme's activity. For example, enzymes in a metabolic pathway may be inhibited by molecules produced later in the pathway, thus curtailing the production of molecules that are no longer needed. This type of negative feedback is an important way to maintain homeostasis in a cell. Enzyme inhibitors also control essential enzymes that, if unchecked, may damage a cell, such as proteases or nucleases. A well-characterised example of this is the ribonuclease inhibitor, which binds to ribonucleases in one of the tightest known protein–protein interactions.^[1] Many poisons found in animals are enzyme inhibitors that block the activity of a crucial enzyme in the victim.

Many drug molecules are enzyme inhibitors that inhibit an aberrant human enzyme or an enzyme critical for the survival of a pathogen such as a virus, bacterium or parasite. Examples include methotrexate (used in chemotherapy and in treating rheumatic arthritis) as well as the protease inhibitors used to treat AIDS. Since anti-pathogen inhibitors generally target only one enzyme, such drugs are highly specific and generally produce few side effects in humans, provided that no analogous enzyme is found in humans. (This is often the case, since such pathogens and humans are genetically distant.) Medicinal enzyme inhibitors often have low dissociation constants, meaning that only a minute amount of the inhibitor is required to inhibit the enzyme. A low concentration of the enzyme inhibitor reduces the risk for liver and kidney damage and other adverse drug reactions in humans. Hence the discovery and refinement of enzyme inhibitors is an active area of research in biochemistry and pharmacology.^[2]

Reversible inhibitors

Types

Reversible inhibitors attach 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 irreversible inhibitors, reversible inhibitors generally do not undergo chemical reactions when bound to the enzyme and can be easily removed by dilution or dialysis. A special case are covalent reversible inhibitors that covalently bind to the enzyme, but the chemical reaction that forms the bond is fully reversible.^[3]

There are four kinds of reversible enzyme inhibitors. They are classified according to the effect of the inhibitor on the V_max (maximum reaction rate catalyzed by the enzyme) and K_m (the concentration of substrate resulting in half maximal enzyme activity) as the concentration of the enzyme's substrate is varied.^[4]^[5]^[6]

Competitive

In competitive inhibition, the substrate and inhibitor cannot bind to the enzyme at the same time,^[8]^: 134 as shown in the figure on the right. 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 (V_max remains constant), i.e., by out-competing the inhibitor.^[8]^{: 134–135} However, the apparent K_m will increase as it takes a higher concentration of the substrate to reach the K_m point, or half the V_max. Competitive inhibitors are often similar in structure to the real substrate (see for example the folic acid methotrexate comparison below).^[8]^: 134

Uncompetitive

In uncompetitive inhibition, the inhibitor binds only to the enzyme-substrate complex.^[8]^: 139 This type of inhibition causes V_max to decrease (maximum velocity decreases as a result of removing activated complex) and K_m to decrease (due to better binding efficiency as a result of Le Chatelier's principle and the effective elimination of the ES complex thus decreasing the K_m which indicates a higher binding affinity).^[9]

Non-competitive

In non-competitive inhibition, 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. V_max will decrease due to the inability for the reaction to proceed as efficiently, but K_m will remain the same as the actual binding of the substrate, by definition, will still function properly.^[10]

Mixed

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.^[8]^{: 136–138} 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.

These types can also be distinguished by the effect of increasing the substrate concentration [S] on the degree of inhibition caused by a given amount of inhibitor. For competitive inhibition the degree of inhibition is reduced by increasing [S], for noncompetitive inhibition the degree of inhibition is unchanged, and for uncompetitive (also called anticompetitive) inhibition the degree of inhibition increases with [S].^[11]

Quantitative description

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.^[12]^: 6 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.^[12]^: 55 The inhibitor (I) can bind to either E or ES with the dissociation constants K_i or K_i', respectively.^[12]^: 87

Competitive inhibitors can bind to E, but not to ES. Competitive inhibition increases K_m (i.e., the inhibitor interferes with substrate binding), but does not affect V_max (the inhibitor does not hamper catalysis in ES because it cannot bind to ES).^[12]^: 102
Uncompetitive inhibitors bind to ES. Uncompetitive inhibition decreases both K_m and V_max. The inhibitor affects substrate binding by increasing the enzyme's affinity for the substrate (decreasing K_m) as well as hampering catalysis (decreases V_max).^[12]^: 106
Non-competitive inhibitors have identical affinities for E and ES (K_i = K_i'). Non-competitive inhibition does not change K_m (i.e., it does not affect substrate binding) but decreases V_max (i.e., inhibitor binding hampers catalysis).^[12]^: 97
Mixed-type inhibitors bind to both E and ES, but their affinities for these two forms of the enzyme are different (K_i ≠ K_i'). Thus, mixed-type inhibitors affect substrate binding (increase or decrease K_m) and hamper catalysis in the ES complex (decrease V_max).^[13]^: 63–64

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.^[14]

Traditionally reversible enzyme inhibitors have been classified as competitive, uncompetitive, or non-competitive, according to their effects on K_m and V_max.^[7] These different effects result from the inhibitor binding to the enzyme E, to the enzyme–substrate complex ES, or to both, respectively. The division of these classes arises from a problem in their derivation and results in the need to use two different binding constants for one binding event.^[15] The binding of an inhibitor and its effect on the enzymatic activity are two distinctly different things, another problem the traditional equations fail to acknowledge. In noncompetitive inhibition the binding of the inhibitor results in 100% inhibition of the enzyme only, and fails to consider the possibility of anything in between.^[15] The common form of the inhibitory term also obscures the relationship between the inhibitor binding to the enzyme and its relationship to any other binding term be it the Michaelis–Menten equation or a dose response curve associated with ligand receptor binding. To demonstrate the relationship the following rearrangement can be made:^[16]

{\begin{aligned}{\cfrac {V_{\max }}{1+{\cfrac {\ce {[I]}}{K_{i}}}}}&={V_{\max }}\left({\cfrac {K_{i}}{K_{i}+[{\ce {I}}]}}\right)&&{\text{multiply by }}{\cfrac {K_{i}}{K_{i}}}=1\\&={V_{\max }}\left({\cfrac {K_{i}+[{\ce {I}}]-[{\ce {I}}]}{K_{i}+[{\ce {I}}]}}\right)&&{\text{add }}[{\ce {I}}]-[{\ce {I}}]=0{\text{ to numerator}}\\&={V_{\max }}\left(1-{\cfrac {[{\ce {I}}]}{K_{i}+[{\ce {I}}]}}\right)&&{\text{simplify }}{\cfrac {K_{i}+[{\ce {I}}]}{K_{i}+[{\ce {I}}]}}=1\\&=V_{\max }-V_{\max }{\cfrac {\ce {[I]}}{K_{i}+[{\ce {I}}]}}&&{\text{multiply out by }}V_{\max }\end{aligned}}

This rearrangement demonstrates that similar to the Michaelis–Menten equation, the maximal rate of reaction depends on the proportion of the enzyme population interacting with its substrate.

fraction of the enzyme population bound by substrate

{\cfrac {\ce {[S]}}{[{\ce {S}}]+K_{m}}}

fraction of the enzyme population bound by inhibitor

{\cfrac {\ce {[I]}}{[{\ce {I}}]+K_{i}}}

the effect of the inhibitor is a result of the percent of the enzyme population interacting with inhibitor. The only problem with this equation in its present form is that it assumes absolute inhibition of the enzyme with inhibitor binding, when in fact there can be a wide range of effects anywhere from 100% inhibition of substrate turn over to just >0%. To account for this the equation can be easily modified to allow for different degrees of inhibition by including a delta V_max term.

V_{\max }-\Delta V_{\max }{\cfrac {\ce {[I]}}{[{\ce {I}}]+K_{i}}}

or

V_{\max 1}-(V_{\max 1}-V_{\max 2}){\cfrac {\ce {[I]}}{[{\ce {I}}]+K_{i}}}

This term can then define the residual enzymatic activity present when the inhibitor is interacting with individual enzymes in the population. However the inclusion of this term has the added value of allowing for the possibility of activation if the secondary V_max term turns out to be higher than the initial term. To account for the possibly of activation as well the notation can then be rewritten replacing the inhibitor "I" with a modifier term (stimulator or inhibitor) denoted here as "X".^[16]^{: eq 13}

V_{\max 1}-(V_{\max 1}-V_{\max 2}){\cfrac {\ce {[X]}}{[{\ce {X}}]+K_{x}}}

While this terminology results in a simplified way of dealing with kinetic effects relating to the maximum velocity of the Michaelis–Menten equation, it highlights potential problems with the term used to describe effects relating to the K_m. The K_m relating to the affinity of the enzyme for the substrate should in most cases relate to potential changes in the binding site of the enzyme which would directly result from enzyme inhibitor interactions. As such a term similar to the one proposed above to modulate V_max should be appropriate in most situations:^[16]^{: eq 14}

K_{m1}-(K_{m1}-K_{m2}){\cfrac {\ce {[X]}}{[{\ce {X}}]+K_{x}}}

Dissociation constants

An enzyme inhibitor is characterised by its two dissociation constants, K_i and K_i', to the enzyme and to the enzyme-substrate complex, respectively.^[17]^: Glossary The enzyme-inhibitor constant K_i can be measured directly by various methods; one especially 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.^[18] However, the other dissociation constant K_i' is difficult to measure directly, since the enzyme-substrate complex is short-lived and undergoing a chemical reaction to form the product. Hence, K_i' is usually measured indirectly, by observing the enzyme activity under various substrate and inhibitor concentrations, and fitting the data via nonlinear regression^[19] to a modified Michaelis–Menten equation^[20]

V={\frac {V_{max}[S]}{\alpha K_{m}+\alpha ^{\prime }[S]}}={\frac {(1/\alpha ^{\prime })V_{max}[S]}{(\alpha /\alpha ^{\prime })K_{m}+[S]}}

where the modifying factors α and α' are defined by the inhibitor concentration and its two dissociation constants

\alpha =1+{\frac {[I]}{K_{i}}}

\alpha ^{\prime }=1+{\frac {[I]}{K_{i}^{\prime }}}.

Thus, in the presence of the inhibitor, the enzyme's effective K_m and V_max become (α/α')K_m and (1/α')V_max, 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 K_i' kinetically if K_i is measured independently.

The effects of different types of reversible enzyme inhibitors on enzymatic activity can be visualised using graphical representations of the Michaelis–Menten equation, such as Lineweaver–Burk, Eadie-Hofstee or Hanes-Woolf plots. An illustration is provided by the three Lineweaver–Burk plots depicted in the adjacent figure on the right. In the top diagram, the competitive inhibition lines intersect on the y-axis, illustrating that such inhibitors do not affect V_max. In the bottom diagram, the non-competitive inhibition lines intersect on the x-axis, showing these inhibitors do not affect K_m. However, since it can be difficult to estimate K_i and K_i' accurately from such plots,^[21] it is advisable to estimate these constants using more reliable nonlinear regression methods.

Special cases

Partially competitive

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 V_max, but an unaffected K_m value.^[9]

Substrate and product

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.^[2] 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.^[22] Product inhibition is often a regulatory feature in metabolism and can be a form of negative feedback.

Slow-tight

TGDDF / GDDF MAIs where blue depicts the tetrahydrofolate cofactor analog, black GAR or thioGAR and red, the connecting atoms.

Structure diagram of a TGDDF/GDDF multi-substrate adduct inhibitor

Peptide-based HIV-1 protease inhibitor ritonavir with substrate binding sites located in enzyme labeled as S2, S1, S1', and S2'

Nonpeptidic HIV-1 protease inhibitor tipranavir

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 K_i, which is time–dependent.^[2] The true value of K_i can be obtained through more complex analysis of the on (k_on) and off (k_off) rate constants for inhibitor association. See irreversible inhibition below for more information.

Bi-substrate analogues

Bi-substrate analogue inhibitors are high affinity and selectivity inhibitors that can be prepared for enzymes that catalyze bi-molecular reactions by capturing the binding energy of each substrate into one molecule.^[23]^[24] For example, in the formyl transfer reactions of purine biosynthesis, a potent Multi-substrate Adduct Inhibitor (MAI) to glycinamide ribonucleotide (GAR) TFase was prepared synthetically by linking analogues of the GAR substrate and the N-10-formyl tetrahydrofolate cofactor together to produce thioglycinamide ribonucleotide dideazafolate (TGDDF),^[25] or enzymatically from the natural GAR substrate to yield GDDF.^[26] Here the subnanomolar dissociation constant (KD) of TGDDF was greater than predicted presumably due to entropic advantages gained and/or positive interactions acquired through the atoms linking the components. MAIs have also been observed to be produced in cells by reactions of pro-drugs such as isoniazid^[27] or enzyme inhibitor ligands (e.g., PTC124) ^[28] with cellular cofactors such as nicotinamide adenine dinucleotide (NADH) and adenosine triphosphate (ATP) respectively.

Examples

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. Inhibitors of DHFR are prominent examples. Other example of these substrate mimics are the protease inhibitors, a very successful class of antiretroviral drugs used to treat HIV.^[29] 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-catalyzed reaction. This ensures that the inhibitor exploits the transition state stabilising effect of the enzyme, resulting in a better binding affinity (lower K_i) 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.^[30]

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.^[31]

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 (ATP), 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.^[32]