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 stops ("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 each 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 balance in a cell. Enzyme inhibitors also control essential enzymes such as proteases or nucleases that, if left unchecked, may damage a cell. Many poisons produced by animals or plants are enzyme inhibitors that block the activity of crucial enzymes in prey or predators.

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) and the protease inhibitors used to treat HIV/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.

Enzyme inhibitors are a chemically diverse set of substances that range in size from organic small molecules to macromolecular proteins.

Small molecule inhibitors include essential primary metabolites that inhibit upstream enzymes that produce those metabolites. This provides a negative feedback loop that prevents over production of metabolites and thus maintains cellular homeostasis (steady internal conditions). Small molecule enzyme inhibitors also include secondary metabolites, which are not essential to the organism that produces them, but provide the organism with an evolutionary advantage, in that they can be used to repel predators or competing organisms or immobilize prey. In addition, many drugs are small molecule enzyme inhibitors that target either disease-modifying enzymes in the patient or enzymes in pathogens which are required for the growth and reproduction of the pathogen.

In addition to small molecules, some proteins act as enzyme inhibitors. The most prominent example are serpins (serine protease inhibitors) which are produced by animals to protect against inappropriate enzyme activation and by plants to prevent predation. Another class of inhibitor proteins is the ribonuclease inhibitors, which bind to ribonucleases in one of the tightest known protein–protein interactions. A special case of protein enzyme inhibitors are zymogens that contain an autoinhibitory N-terminal peptide that binds to the active site of enzyme that intramolecularly blocks its activity as a protective mechanism against uncontrolled catalysis. The N‑terminal peptide is cleaved (split) from the zymogen enzyme precursor by another enzyme to release an active enzyme.

The binding site of inhibitors on enzymes is most commonly the same site that binds the substrate of the enzyme. These active site inhibitors are known as orthosteric ("regular" orientation) inhibitors. The mechanism of orthosteric inhibition is simply to prevent substrate binding to the enzyme through direct competition which in turn prevents the enzyme from catalysing the conversion of substrates into products. Alternatively, the inhibitor can bind to a site remote from the enzyme active site. These are known as allosteric ("alternative" orientation) inhibitors. The mechanisms of allosteric inhibition are varied and include changing the conformation (shape) of the enzyme such that it can no longer bind substrate (kinetically indistinguishable from competitive orthosteric inhibition) or alternatively stabilise binding of substrate to the enzyme but lock the enzyme in a conformation which is no longer catalytically active.

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 enzyme 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 is covalent reversible inhibitors that form a chemical bond with the enzyme, but the bond can be cleaved so the inhibition is fully reversible.

Reversible inhibitors are generally categorized into four types, as introduced by Cleland in 1963. They are classified according to the effect of the inhibitor on the V_max (maximum reaction rate catalysed 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.

In competitive inhibition the substrate and inhibitor cannot bind to the enzyme at the same time. 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. 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 "methotrexate versus folate" figure in the "Drugs" section).

In uncompetitive inhibition the inhibitor binds only to the enzyme-substrate complex. 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). Uncompetitive inhibition is rare.

In non-competitive inhibition the binding of the inhibitor to the enzyme reduces its activity but does not affect the binding of substrate. This type of inhibitor binds with equal affinity to the free enzyme as to the enzyme-substrate complex. It can be thought of as having the ability of competitive and uncompetitive inhibitors, but with no preference to either type. 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.

In mixed inhibition the inhibitor may bind to the enzyme whether or not the substrate has already bound. Hence mixed inhibition is a combination of competitive and noncompetitive inhibition. Furthermore, the affinity of the inhibitor for the free enzyme and the enzyme-substrate complex may differ. By increasing concentrations of substrate [S], this type of inhibition can be reduced (due to the competitive contribution), but not entirely overcome (due to the noncompetitive component). 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 (that is, the tertiary structure or three-dimensional shape) of the enzyme so that the affinity of the substrate for the active site is reduced.

These four types of inhibition 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].

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 (shown in the "inhibition mechanism schematic" diagram), 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 K_i or K_i', respectively.

• 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).
• 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).
• 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).
• 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).

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.

Traditionally reversible enzyme inhibitors have been classified as competitive, uncompetitive, or non-competitive, according to their effects on K_m and V_max. These three types of inhibition result respectively from the inhibitor binding only to the enzyme E in the absence of substrate S, to the enzyme–substrate complex ES, or to both. 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. It is further assumed that binding of the inhibitor to the enzyme results in 100% inhibition and fails to consider the possibility of partial inhibition. 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:

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

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

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

fraction of the enzyme population bound by inhibitor

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

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 no inhibition. To account for this the equation can be easily modified to allow for different degrees of inhibition by including a delta V_max term.

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

or

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

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".

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

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 delta V_max term proposed above to modulate V_max should be appropriate in most situations:

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

An enzyme inhibitor is characterised by its dissociation constant K_i, the concentration at which the inhibitor half occupies the enzyme. In non-competitive inhibition the inhibitor can also bind to the enzyme-substrate complex, and the presence of bound substrate can change the affinity of the inhibitor for the enzyme, resulting in a second dissociation constant K_i'. Hence K_i and K_i' are the dissociation constants of the inhibitor for the enzyme and to the enzyme-substrate complex, respectively. 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. 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 to a modified Michaelis–Menten equation.

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

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

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

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

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 the inhibition becomes effectively irreversible, hence it is more practical to treat such tight-binding inhibitors as irreversible (see below).

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 Lineweaver–Burk diagrams figure. 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, it is advisable to estimate these constants using more reliable nonlinear regression methods.

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.

Substrate or product inhibition is where either an enzymes substrate or product also act as an inhibitor. This inhibition may follow the competitive, uncompetitive or mixed patterns. In substrate inhibition there is a progressive decrease in activity at high substrate concentrations, potentially from an enzyme having two competing substrate-binding sites. 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 (either the enzyme's own product, or a product to an enzyme downstream in its metabolic pathway) 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 conformational isomerism (a change in shape) 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. 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 with kinetics similar to irreversible inhibition.

Irreversible inhibitors covalently bind to an enzyme, and this type of inhibition can therefore not be readily reversed. Irreversible inhibitors often contain reactive functional groups such as nitrogen mustards, aldehydes, haloalkanes, alkenes, Michael acceptors, phenyl sulfonates, or fluorophosphonates. These electrophilic groups react with amino acid side chains to form covalent adducts. The residues modified are those with side chains containing nucleophiles such as hydroxyl or sulfhydryl groups; these include the amino acids serine (that reacts with DFP, see the "DFP reaction" diagram), and also cysteine, threonine, or tyrosine.

Irreversible inhibition is different from irreversible enzyme inactivation. Irreversible inhibitors are generally specific for one class of enzyme and do not inactivate all proteins; they do not function by destroying protein structure but by specifically altering the active site of their target. For example, extremes of pH or temperature usually cause denaturation of all protein structure, but this is a non-specific effect. Similarly, some non-specific chemical treatments destroy protein structure: for example, heating in concentrated hydrochloric acid will hydrolyse the peptide bonds holding proteins together, releasing free amino acids.

Irreversible inhibitors display time-dependent inhibition and their potency therefore cannot be characterised by an IC₅₀ value. This is because the amount of active enzyme at a given concentration of irreversible inhibitor will be different depending on how long the inhibitor is pre-incubated with the enzyme. Instead, k_obs/[I] values are used, where k_obs is the observed pseudo-first order rate of inactivation (obtained by plotting the log of % activity versus time) and [I] is the concentration of inhibitor. The k_obs/[I] parameter is valid as long as the inhibitor does not saturate binding with the enzyme (in which case k_obs = k_inact) where k_inact is the rate of inactivation.