Enzyme inhibitor



 

Enzyme inhibitors are enzymatic activity.

The binding of an inhibitor can stop a enzyme, the enzyme-substrate complex, or both.

Many drug molecules are enzyme inhibitors, so their discovery and improvement is an active area of research in side effects and thus low toxicity.

Enzyme inhibitors also occur naturally and are involved in the regulation of metabolism. For example, enzymes in a protein–protein interactions.[1] Natural enzyme inhibitors can also be poisons and are used as defenses against predators or as ways of killing prey.

Reversible inhibitors

Types of reversible inhibitor

Reversible inhibitors bind to enzymes with non-covalent interactions such as 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.[2]

  • 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 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 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.[3]

Measuring the dissociation constants of a reversible inhibitor

 

As noted above, an enzyme inhibitor is characterized by its two Michaelis–Menten equation


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 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,[6] 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.[7]
  • 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.[7]
  • 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 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 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 neuraminidase.[10]

 

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.[11]

In drug design it is important to consider the concentrations of substrates to which the target enzymes are exposed. For example, some 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.[12]

Irreversible inhibitors

Types of irreversible inhibition

 

Irreversible inhibitors usually tyrosine.[13]

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 peptide bonds holding proteins together, releasing free amino acids.[14]

Analysis of irreversible inhibition

 

As shown in the figure to the left, irreversible inhibitors form a reversible non-covalent complex with the enzyme (EI or ESI) and this then reacts to produce the covalently modified "dead-end complex" EI*. The rate at which EI* is formed is called the inactivation rate or kinact. Since formation of EI may compete with ES, binding of irreversible inhibitors can be prevented by competition either with substrate or with a second, reversible inhibitor. This protection effect is good evidence of a specific reaction of the irreversible inhibitor with the active site.

The binding and inactivation steps of this reaction are investigated by incubating the enzyme with inhibitor and assaying the amount of activity remaining over time. The activity will be decrease in a time-dependent manner, usually following exponential decay. Fitting these data to a rate equation gives the rate of inactivation at this concentration of inhibitor. This is done at several different concentrations of inhibitor. If a reversible EI complex is involved the inactivation rate will be saturable and fitting this curve will give kinact and Ki.[15]

Another method that is widely used in these analyses is peptides that can be analysed using a mass spectrometer. The peptide that changes in mass after reaction with the inhibitor will be the one that contains the site of modification.

Special cases

 

Not all irreversible inhibitors form covalent adducts with their enzyme targets. Some reversible inhibitors bind so tightly to their target enzyme that they are essentially irreversible. These tight-binding inhibitors may show kinetics similar to covalent irreversible inhibitors. In these cases, some of these inhibitors rapidly bind to the enzyme in a low-affinity EI complex and this then undergoes a slower rearrangement to a very tightly bound EI* complex (see figure above). This kinetic behaviour is called slow-binding.[18] This slow rearrangement after binding often involves a conformational change as the enzyme "clamps down" around the inhibitor molecule. Examples of slow-binding inhibitors include some important drugs, such acyclovir.[21]

Examples of irreversible inhibitors

 

synapses of neurons, and consequently is a potent neurotoxin, with a lethal dose of less than 100 mg.[23]

Suicide inhibition is an unusual type of irreversible inhibition where the enzyme converts the inhibitor into a reactive form in its active site. An example is the inhibitor of imine, a highly electrophilic species. This reactive form of DFMO then reacts with either a cysteine or lysine residue in the active site to irreversibly inactivate the enzyme.[17]

Since irreversible inhibition often involves the initial formation of a non-covalent EI complex, it is sometimes possible for an inhibitor to bind to an enzyme in more than one way. For example, in the figure showing nitrogen mustard group.[24]

Discovery and design of inhibitors

 

New drugs are the products of a long combinatorial chemistry approaches that quickly produce large numbers of novel compounds and high-throughput screening technology to rapidly screen these huge chemical libraries for useful inhibitors.[25]

More recently, an alternative approach has been applied: rational drug design uses the M.[27]

Uses of inhibitors

Enzyme inhibitors are found in nature and are also designed and produced as part of disinfectants such as triclosan.

Chemotherapy

 

 

 

The most common uses for enzyme inhibitors are as drugs to treat disease. Many of these inhibitors target a human enzyme and aim to correct a pathological condition. However, not all drugs are enzyme inhibitors. Some, such as membrane receptors.

An example of a medicinal enzyme inhibitor is cyclic guanosine monophosphate.[28] This signalling molecule triggers smooth muscle relaxation and allows blood flow into the corpus cavernosum, which causes an erection. Since the drug decreases the activity of the enzyme that halts the signal, it makes this signal last for a longer period of time.

Another example of the structural similarity of some inhibitors to the substrates of the enzymes they target is seen in the figure comparing the drug chemotherapy.[29]

Drugs also are used to inhibit enzymes needed for the survival of ribbon-diagram).

fatty acids.

Metabolic control

Enzyme inhibitors are also important in metabolic control. Many NADH and pyruvate. A key step for the regulation of glycolysis is an early reaction in the pathway catalysed by phosphofructokinase-1 (PFK1). When ATP levels rise, ATP binds an allosteric site in PFK1 to decrease the rate of the enzyme reaction; glycolysis is inhibited and ATP production falls. This negative feedback control helps maintain a steady concentration of ATP in the cell. However, metabolic pathways are not just regulated through inhibition since enzyme activation is equally important. With respect to PFK1, fructose 2,6-bisphosphate and ADP are examples of metabolites that are allosteric activators.[31]

Physiological enzyme inhibition can also be produced by specific protein inhibitors. This mechanism occurs in the protein phosphatases.[35]

Acetylcholinesterase inhibitors

Acetylcholinesterase (AChE) is an enzyme found in animals from insects to humans. It is essential to nerve cell function through its mechanism of breaking down the neurotransmitter chlorpyrifos irreversibly inhibit acetylcholinesterase.

 

Natural poisons

Animals and plants have evolved to synthesize a vast array of poisonous products including cytoskeleton.[37]

Many natural poisons act as muscarinic acetylcholine receptors.[39]

Although many natural toxins are secondary metabolites, these poisons also include peptides and proteins. An example of a toxic peptide is protein phosphatases.[41] This toxin can contaminate water supplies after algal blooms and is a known carcinogen that can also cause acute liver hemorrhage and death at higher doses.[42]

Proteins can also be natural poisons, such as the castor oil beans. This enzyme is a glycosidase that inactivates ribosomes. Since ricin is a catalytic irreversible inhibitor, this allows just a single molecule of ricin to kill a cell.[43]

See also

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This article is licensed under the GNU Free Documentation License. It uses material from the Wikipedia article "Enzyme_inhibitor". A list of authors is available in Wikipedia.