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Why is competitive inhibition reversible?

Why is competitive inhibition reversible?


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My Biochemistry book mentions that 'competitive inhibition' is a reversible form of inhibition.
But given that the inhibitor is blocking the active site and prevents an enzyme-substrate complex to be formed, how can it be reversible?


A competitive inhibitor typically competes for the active site with the substrate. In this textbook case, binding of a competitive inhibitor is reversible, because it binds to the active site of the enzyme, but is also released, making way for the substrate to bind. The affinity of the substrate, as well as its concentration determine the amount of inhibition (Berg et al., 2002).

An irreversible inhibitor may covalently bind to the active site, permanently disabling the enzyme (McDonald et al., 2012).

References
- McDonald et al., Enzymes: Irreversible Inhibition. In: eLS. John Wiley & Sons Ltd, Chichester (2012)
- Berg et al., Biochemistry. 5th ed. New York: W H Freeman; (2002)


Enzymes and Enzyme Regulation

Reversible Inhibition by Reaction Products

Competitive inhibition can occur in freely reversible reactions owing to accumulation of products. Even in reactions that are not readily reversible, a product can function as an inhibitor when an irreversible step precedes the dissociation of the products from the enzyme. In the alkaline phosphatase reaction, in which hydrolysis of a wide variety of organic monophosphate esters into the corresponding alcohols (or phenols) and inorganic phosphates occurs, the inorganic phosphate acts as a competitive inhibitor. Both the inhibitor and the substrate have similar enzyme-binding affinities (i.e., K m and Ki are of the same order of magnitude).


Enzymes and Enzyme Regulation

Reversible Inhibition by Reaction Products

Competitive inhibition can occur in freely reversible reactions owing to accumulation of products. Even in reactions that are not readily reversible, a product can function as an inhibitor when an irreversible step precedes the dissociation of the products from the enzyme. In the alkaline phosphatase reaction, in which hydrolysis of a wide variety of organic monophosphate esters into the corresponding alcohols (or phenols) and inorganic phosphates occurs, the inorganic phosphate acts as a competitive inhibitor. Both the inhibitor and the substrate have similar enzyme-binding affinities (i.e., K m and Ki are of the same order of magnitude).


Reversible Enzyme Inhibition | Microbiology

The main feature of competitive inhibition is that it can be reversed by increasing the substrate concentration in a reaction mixture which contains both the substrate and the inhibitor. The degree of inhibition depends on the relative concentrations of the substrate and the inhibitor. If the substrate concentration is increased, keeping the inhibitor concentration fixed, the degree of inhibition of the enzyme activity decreases.

An opposite effect is observed, if the inhibitor concentration is increased, keeping the substrate concentration fixed. This happens because the substrate and the inhibitor both bind to the same catalytic site of the enzyme by virtue of a similarity in structure of the substrate and the inhibitor. Thus, the substrate and the inhibitor compete with each other for occupying the same active site or sites of an enzyme molecule.

Because of structural similarity, the enzyme molecule cannot distinguish between the correct substrate and the false one which is the inhibitor. An often cited example of competitive inhibition is the inhibition of succinic acid dehydrogenase by malonic acid. In the TCA cycle succinic acid is dehydrogenated by succinic dehydrogenase to fumaric acid, FAD acting as H-acceptor. In presence of malonic acid, the enzyme can combine with the inhibitor, but fails to dehydrogenate it.

The structures of succinic and malonic acids are shown:

Another well-known example of competitive inhibition having clinical importance is that of sulfanilamide and p-amino benzoic acid. Sulfanilamide forms the nucleus of all sulfa-drugs which are used as chemotherapeutic agents against a variety of infections caused by bacteria. Par-amino-benzoic acid (p-ABA) is an essential vitamin required by many bacteria for synthesis of folic acid which acts as a coenzyme.

The enzyme which acts on p-ABA to convert it to the next intermediate in the biosynthesis of folic acid is competitively inhibited by sulfanilamide, because p-ABA has structural similarity with the inhibitor. The bacteria are deprived of folic acid and are unable to grow.

The structures of the two compounds are shown below:

Non-Competitive Inhibition:

A non-competitive inhibition cannot be reversed by increasing substrate concentration, because the inhibitor does not bind to the enzyme protein at the same active site as the normal substrate, but at a different site. Hence there is no competition between the substrate and the inhibitor.

The inhibition may be caused due to a change in the shape of the substrate-site due to binding of the inhibitor to the same enzyme molecule though at a different site. This type of non-competitive inhibition is also known as allosteric inhibition and has been dealt with separately.

The more common type of non-competitive inhibition is that exerted by the heavy metal ions that bind reversibly with the sulfhydryl group (-SH) of cysteine residues of enzyme proteins. For many enzymes, the free -SH groups are essential for catalytic activity, because they are often involved in maintaining the correct three-dimensional configuration of the enzyme protein required for its catalytic function. Heavy metal ions, like Hg ++ and Ag ++ bind to the -SH groups of enzyme proteins reversibly, causing non-competitive inhibition.

Non-competitive inhibition may also be due to some agents which bind inorganic co-factors required by certain apo-enzymes to form functional holoenzymes. These inorganic co-factors are usually divalent metal ions, like Mg ++ , Ca ++ , Fe ++ etc. The inhibitors which bind such metal ions include cyanide which binds Fe ++ or Fe +++ , fluoride which binds Ca ++ or Mg ++ , ethylene diamino tetra acetic acid (EDTA) which binds Mg ++ and other divalent metal ions, etc.

Whether an inhibior acts as a competitive or a non-competitive one can be recognized from their kinetics. Lineweaver-Burk plots using varying concentrations of the inhibitor and a fixed concentration of the substrate reveal the difference between a competitive and a non-competitive inhibitor. In case of competitive inhibition, plots of 1/v against 1/[S] produce straight lines of different slopes, intersecting the 1/v axis at a common point.

The curves indicate that Vmax is not altered by the presence of the inhibitor, but the Km is. In presence of the inhibitor Km has a higher value (Fig. 8.39 A). In case of non­competitive inhibition, on the other hand, the straight lines also show different slopes with varying concentrations of the inhibitor, but they do not intercept the 1/v axis at the same point as observed in case of competitive inhibition.

Rather the lines meet at a common point on the -1/[S] axis. This indicates that increasing concentration of inhibitor causes decrease in Vmax which is not restored by increasing substrate concentration. The Km, however, remains unchanged, because the substrate and the inhibitor do not bind to the same site of the enzyme (Fig. 8.39B).


Reversible inhibitor

A reversible inhibitor is one that, once removed, allows the enzyme it was inhibiting to begin working again. It has no permanent effects on the enzyme - it does not change the shape of the active site, for example. Reversible Inhibition may be Competitive, Non-Competitive or Uncompetitive. 

Competitive

A competitive inhibitor directly competes with the substrate in a reaction. It will be a very similar molecule to the substrate - similar enough that it fits into the active site - but once inside the active site it will not be affected in any way by the enzyme. The inhibitor will probably have a higher affinity for the enzyme than the substrate does - however, a competitive inhibitor can be overcome simply by adding more substrate. With enough substrate, the chance of a collision between the substrate and an enzyme is much higher than the chance of a collision between an enzyme and the inhibitor, causing the effect of the inhibitor molecule to be overcome. 

Non-Competitive

The inhibitory molecule in this case does not bind to the active site, but to another site known as the Allosteric site. The substrate may still bind to the active site of the enzyme, but the enzyme will be unable to affect the substrate, and thus no product is produced. As this form of inhibition cannot be overcome by increasing concentration, cells must have other methods to overcome this, by removing the inhbior and allowing the enzyme to resume function. 

Uncompetitive

Only takes place in multi-substrate reactions.  It cannot be overcome by increasing the substrate concentration. It binds only to the enzyme-substrate complex [1] .


What is Reversible enzyme inhibitors?

Reversible inhibitors form non-covalent bonds with the enzyme. They are characterized by a rapid dissociation from their target.

Reversible inhibitors can be classified into two main categories, competitive and non-competitive inhibitors.

A reversible inhibitor is competitive when the enzyme can bind with its active site, either to the inhibitor forming an enzyme-inhibitor complex (EI), or to the substrate forming an enzyme-substrate (ES) complex.

In this case of competitive inhibition, the binding of the enzyme to the substrate or to the inhibitor are mutually exclusive: the enzyme can never bind to the inhibitor and to the substrate at the same time.

The reduction of the catalytic activity of the enzyme is achieved by the reduction of the proportion of the enzyme-substrate complex.

Increasing the substrate concentration can relieve the inhibition of the enzyme.

In the reversible non-competitive inhibition, the substrate and the inhibitor bind simultaneously to different sites of the enzyme, rendering it inactive. This inhibition cannot be overcome by increasing the substrate concentration.


Noncompetitive inhibition

The inhibitor prevents the enzyme from catalyzing the reaction by binding to an allosteric site whether or not the substrate is in the active site.

The binding of the inhibitor changes the shape of the active site such that the reaction can no longer be catalyzed.

This is different from competitive inhibition because the inhibitor can bind to the enzyme and stop the reaction, even if the substrate is already bound to the active site. As a result, it no longer matters “who got there first.” This means that you cannot overcome noncompetitive inhibition by simply increasing the amount of substrate present.

How does this impact &?

Noncompetitive inhibition decreases /> because no matter how much substrate is present, some of the enzymes will always be prevented from catalyzing the reaction by the inhibitor. The means that the enzyme can never reach its original />.

Noncompetitive inhibition does not change because affinity for the enzyme is unchanged, there are just fewer functioning enzymes.


C4: Noncompetitive and Mixed Inhibition

  • Contributed by Henry Jakubowski
  • Professor (Chemistry) at College of St. Benedict/St. John's University

Reversible noncompetitive inhibition occurs when I binds to both E and ES. We will look at only the special case in which the dissociation constants of (I) for (E) and (ES) are the same. This is called noncompetiive inhibition. It is quite rare as it would be difficult to imagine a large inhibitor which inhibits the turnover of bound substrate having no effect on binding of (S) to (E). However covalent interaction of protons with both E and ES can lead to noncompetitive inhibition. In the more general case, the (K_d)'s are different, and the inhibition is called mixed. Since inhibition occurs, we will hypothesize that ESI can not form product. It is a dead end complex which has only one fate, to return to (ES) or (EI). This is illustrated in the chemical equations and in the molecular cartoon below.

Let us assume for ease of equation derivation that I binds reversibly to E with a dissociation constant of Kis (as we denoted for competitive inhibition) and to ES with a dissociation constant (K_) (as we noted for uncompetitive inhibition). Assume for noncompetitive inhibition that Kis = Kii. A look at the top mechanism shows that in the presence of I, as S increases to infinity, not all of (E) is converted to (ES). That is, there is a finite amount of ESI, even at infinite S. Now remember that

if and only if all (E) is in the form (ES). Under these conditions, the apparent (V_m), (V_) is less than the real (V_m) without inhibitor. In contrast, the apparent Km, (K_), will not change since I binds to both (E) and (ES) with the same affinity, and hence will not perturb that equilibrium, as deduced from Le Chatelier's principle. The double reciprocal plot (Lineweaver Burk plot) offers a great way to visualize the inhibition. In the presence of I, just Vm will decrease. Therefore, -1/Km, the x-intercept will stay the same, and (1/V_m) will get more positive. Therefore the plots will consists of a series of lines intersecting on the x axis, which is the hallmark of noncompetitive inhibition. You should be able to figure out how the plots would appear if (K_) is different from (K_) (mixed inhibition).

An equation, shown in the diagram above can be derived which shows the effect of the noncompetitive inhibitor on the velocity of the reaction. In the denominator, Km is multiplied by (1+I/K_), and (S) by (1+I/K_). We would like to rearrange this equation to show how Km and Vm are affected by the inhibitor, not S, which obviously isn't. Rearranging the equation as shown above shows that

This shows that the (K_m) is unchanged and (V_m) decreases as we predicted. The plot shows a series of lines intersecting on the x axis. Both the slope and the y intercept are changed, which are reflected in the names of the two dissociation constants, Kis and Kii. Note that if (I) is zero, (K_ = K_m) and (V_ = V_m). Sometimes the (K_) and (K_) inhibition dissociations constants are referred to as (K_c) and (K_u) (competitive and uncompetitive inhibition dissociation constants.

Mixed (and non-)competitive inhibition (as shown by mechanism above) differ from competitive and uncompetiive inhibition in that the inhibitor binding is not simply a dead end reaction in which the inhibitor can only dissociate in a single reverse step. In the above equilibrium, (S) can dissociate from (ESI) to form (EI) so the system may not be at equilibrium. With dead end steps, no flux of reactants occurs through the dead end complex so the equilibrium for the dead end step is not perturbed.

Other mechanisms can commonly give mixed inhibition. For example, the product released in a ping pong mechanism (discussed in the next chapter) can give mixed inhibition.

If (P), acting as a product inhibitor, can bind to two different forms of the enzyme ((E') and also (E)), it will act as an mixed inhibitor.

Java Applet: Noncompetitive Inhibition

4/26/13Wolfram Mathematica CDF Player - Mixed Inhibition v vs S curves Kis and Kii called Kc and Ku (start sliders at high values) (free plugin required)

4/26/13Wolfram Mathematica CDF Player - Mixed Inhibition v vs S curves (start sliders at high values) (free plugin required). Note where the inhibited and inhibited curves intersect at different values of Kis and Kii (in the graph termed Kc and Ku).

If you can apply Le Chatelier's principle, you should be able to draw the Lineweaver-Burk plots for any scenario of inhibition or even the opposite case, enzyme activation!


Explaining why Gleevec is a specific and potent inhibitor of Abl kinase

Tyrosine kinases present attractive drug targets for specific types of cancers. Gleevec, a well-known therapeutic agent against chronic myelogenous leukemia, is an effective inhibitor of Abl tyrosine kinase. However, Gleevec fails to inhibit closely homologous tyrosine kinases, such as c-Src. Because many structural features of the binding site are conserved, the molecular determinants responsible for binding specificity are not immediately apparent. Some have attributed the difference in binding specificity of Gleevec to subtle variations in ligand-protein interactions (binding affinity control), whereas others have proposed that it is the conformation of the DFG motif, in which ligand binding is only accessible to Abl and not to c-Src (conformational selection control). To address this issue, the absolute binding free energy was computed using all-atom molecular dynamics simulations with explicit solvent. The results of the free energy simulations are in good agreement with experiments, thereby enabling a meaningful decomposition of the binding free energy to elucidate the factors controlling Gleevec's binding specificity. The latter is shown to be controlled by a conformational selection mechanism and also by differences in key van der Waals interactions responsible for the stabilization of Gleevec in the binding pocket of Abl.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Structural comparison of Abl and…

Structural comparison of Abl and c-Src in the Gleevec-bound kinase domain.

One-dimensional PMF of DFG-flip in…

One-dimensional PMF of DFG-flip in Abl (red line) and c-Src (blue line) as…

PMFs of rmsd of Gleevec in bulk solution (black line) and in the…

( Top ) Difference in the average interaction energies of the DFG-flip ΔΔ…


Three types of reversible inhibition

Before we get into the three kinds of inhibition, we need to imagine a scenario. Imagine that you want to do some work when your body and your intent come together, the work takes place, which leads to the end product of the work getting completed. The enzyme here is you, the intent is the substrate (the chemical with which the enzyme reacts), you and your intent leads to working, and working is the equivalent of a complex formed by the reaction of the enzyme and substrate (called the enzyme substrate complex). The end product&mdashthe work getting completed&mdashis the equivalent of the end product of the reaction.

Competitive Inhibition

Taking into consideration the scenario mentioned above, imagine that you have to work, but are feeling lazy, which competes with your intent to work. It might make you lazy to the extent that you won&rsquot move and the laziness will fight your intent to do work, but if you could avoid the laziness, you would do the work. This is called competitive inhibition. The inhibitor reacts with the free enzyme in the first stage, and the enzyme then must compete to bind with either the substrate or the inhibitor.

Non-competitive Inhibition

Imagine another scenario in which you are healthy and have the intent to work, but your internet is slow. There are two ways this could play out: the slow internet affects your working and you&rsquore unable to do it, despite your intent, or you eventually get frustrated with the slow internet and no longer want to do the work. This is called non-competitive inhibition, in which the inhibitor can either bind with the free enzyme or the enzyme substrate complex.

Uncompetitive Inhibition

Finally, imagine a scenario in which you are present, with the intent to work, but due to the battery of your computer dying, your work didn&rsquot get saved. This is called uncompetitive inhibition, in which the inhibitor only binds with the enzyme substrate complex, and thus only the working is affected.

In all three cases, in the absence of the laziness, the slow internet, or the battery dying, you could complete the work smoothly. In the same way, in the absence of the inhibitor, the enzyme would work perfectly and the reaction would go forward to completion. This is just a brief understanding of how tiny enzymes help our body to function!



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