Reversible Inhibitors

A substance that binds to an enzyme and reduces its activity is known as an enzyme inhibitor. An inhibitor binding can prevent a substrate from entering the active site of an enzyme and/or prevent the enzyme from catalysing its reaction. Binding of inhibitors might be reversible or irreversible. In most cases, irreversible inhibitors react with the enzyme and chemically alter it (e.g. via covalent bond formation). Key amino acid residues required for enzyme function are modified by these inhibitors. Reversible inhibitors, on the other hand, bind non-covalently and induce different types of inhibition depending on whether they bind to the enzyme, the enzyme-substrate complex, or both.

Inhibitors are divided into two categories: Reversible and Irreversible Inhibitors 

Reversible Inhibitors:

Non-covalent interactions like hydrogen bonds, hydrophobic contacts, and ionic connections are used by reversible inhibitors to attach to enzymes. The inhibitor and the active site form a series of weak connections that combine to form a strong and selective binding. Reversible inhibitors, unlike substrates and irreversible inhibitors, do not undergo chemical reactions when bound to the enzyme and can be quickly eliminated using dilution or dialysis.

Reversible enzyme inhibitors are divided into four categories. They’re categorized according to how changing the concentration of the enzyme’s substrate affects the inhibitor.

• Competitive Inhibitors 

• Uncompetitive Inhibitors 

• Non-competitive Inhibitors 

• Mixed Inhibitors

Competitive Inhibitors 

The substrate and inhibitor cannot bind to the enzyme at the same time in competitive inhibition, as indicated in the diagram to the right. This is mainly due to the inhibitor’s affinity for an enzyme’s active site, where the substrate also binds; the substrate and inhibitor compete for access to the enzyme’s active site. This form of inhibition can be circumvented by using high enough substrate concentrations (Vmax remains constant), effectively out-competing the inhibitor. However, because it requires more substrate concentration to achieve the Km point, or half the Vmax, the apparent Km will rise. Competitive inhibitors resemble the genuine substrate in structure.

Uncompetitive Inhibitors :

The inhibitor binds only to the enzyme-substrate complex in uncompetitive inhibition. This type of inhibition causes Vmax to decrease (maximum velocity decreases as activated complex is removed) and Km to decrease (better binding efficiency as a result of Le Chatelier’s principle and the effective elimination of the ES complex, resulting in a decrease in Km, indicating a higher binding affinity).

Non-competitive Inhibitors :

Non-competitive inhibition is a type of mixed inhibition in which the inhibitor binding to the enzyme affects its activity but has no effect on substrate binding. As a result, the extent of inhibition is solely determined by the inhibitor’s concentration.

Mixed Inhibitors :

In mixed inhibition, the inhibitor and the enzyme’s substrate can both bind to the enzyme. However, the inhibitor’s binding influences the substrate binding, and vice versa. Increased substrate concentrations can diminish, but not eliminate, this sort of inhibition. Although mixed-type inhibitors can bind in the active site, most inhibition occurs as a result of an allosteric action, in which the inhibitor binds to a different position on the enzyme. When an inhibitor binds to this allosteric site, the enzyme’s conformation (i.e., tertiary structure or three-dimensional shape) changes, reducing the substrate’s affinity for the active site.

Quantitative Description :

Reversible inhibition can be quantified by looking at the inhibitor binding to the enzyme and the enzyme-substrate complex, as well as its impact on the enzyme’s kinetic constants. An enzyme (E) interacts with its substrate (S) to produce the enzyme–substrate complex ES in the standard Michaelis–Menten scheme below. This complex disintegrates during catalysis, releasing product P and free enzyme. With the dissociation constants Ki and Ki’, the inhibitor (I) can bind to either E or ES.

Competitive inhibitors have the ability to bind to E but not to ES. Competitive inhibition increases Km but has no effect on Vmax (i.e., the inhibitor prevents substrate binding) (the inhibitor does not hamper catalysis in ES because it cannot bind to ES).

Mixed-type inhibitors bind to both E and ES, but their affinities for the two enzyme types differ ( Ki  Ki’). Mixed-type inhibitors obstruct catalysis in the ES complex by interfering with substrate binding (increasing Km) (decrease Vmax).

Inhibitors that aren’t competitive bind to ES. Both Km and Vmax are reduced by noncompetitive inhibition. The inhibitor impacts substrate binding and catalysis by increasing the enzyme’s affinity for the substrate (decreasing Km) (decreases Vmax)

The affinities of non-competitive inhibitors for E and ES are identical ( Ki  = Ki’). Non-competitive inhibition does not impact Km (i.e., substrate binding), but it does reduce Vmax (i.e., inhibitor binding hampers catalysis)

Special Cases :

• When an inhibitor binds solely to the enzyme–substrate complex and not to the free enzyme, uncompetitive inhibition occurs, and the EIS complex becomes catalytically inactive. This mechanism of inhibition is uncommon and results in a reduction in both Vmax and Km.

• The mechanism of partially competitive inhibition is identical to that of non-competitive inhibition, except that the EIS complex has higher catalytic activity (partially competitive activation) than the enzyme–substrate (ES) complex. This inhibition usually results in a reduced Vmax but no change in Km.

• The first enzyme–inhibitor combination EI undergoes isomerisation to a second, more tightly bound complex, EI*, resulting in slow-tight inhibition, however the whole inhibition process is reversible. This is manifested as a gradual increase in enzyme inhibition. Traditional Michaelis–Menten kinetics produce a misleading value for Ki, which is time–dependent, under these conditions. A more comprehensive examination of the on (on) and off (Koff) rate constants for inhibitor interaction can yield the true value of Ki. For more details, see the section below on irreversible inhibition.

Examples :

Because enzymes have evolved to firmly bind their substrates, and most reversible inhibitors bind in the active region of enzymes, it’s not unexpected that some of these inhibitors have structures that are strikingly similar to their targets’ substrates. DHFR inhibitors are one of the most well-known examples. Protease inhibitors, a popular family of antiretroviral medications used to treat HIV, are another example of substrate mimics. On the right, the structure of ritonavir, a protease inhibitor made up of three peptide bonds and based on a peptide. Because this medicine resembles the protein that the HIV protease uses as a substrate, it competes with it in the enzyme’s active site.

Conclusion :

Ionic connections, hydrophobic contacts, and hydrogen bonds are all examples of weak non-covalent interactions that reversible inhibitors might use to attach to enzymes. In contrast to irreversible inhibition, reversible inhibitors do not create any chemical bonds or interactions with the enzyme, therefore they are formed quickly and can be easily eliminated. As a result, the enzyme and inhibitor combination is immediately dissolved.