Enzyme Inhibition

Diagrams showing how different types of inhibition affect reaction rate

Core Concepts

In this article, you will learn about competitive, noncompetitive, and uncompetitive enzyme inhibition, and the effect of each type of inhibitor on Km and Vmax.

Topics Covered in Other Articles

Introduction to Enzyme Inhibition

Enzymes are necessary to catalyze many important biological reactions. Many reactions can be regulated through enzyme inhibition. Some drugs also utilize principles of enzyme inhibition to prevent harmful reactions from proceeding. There are three main types of enzyme inhibition: competitive, non-competitive, and uncompetitive. We will look at each of these in turn.

Competitive Enzyme Inhibition

Competitive inhibitors compete with the substrate to bind an enzyme. When the inhibitor binds, it prevents the substrate from binding by creating an inactive enzyme-inhibitor (EI) complex instead of an active enzyme-substrate (ES) complex. The enzyme-inhibitor complex will not yield product, so the reaction does not proceed.

There are two main types of competitive inhibition. In the first, the inhibitor and substrate are competing for the active site. The inhibitor is structurally similar to the substrate, and so is able to bind to the active site and block the substrate. The other type of competitive inhibition is allosteric inhibition. This is when the inhibitor binds at an allosteric, or non-active, site on the enzyme. This allosteric binding changes the enzyme’s conformation such that the substrate is no longer able to bind the active site. In both types of competitive inhibition, the enzyme-substrate complex is not formed.

Competitive inhibitors are more effective at lower substrate concentrations because they are better able to outcompete the substrate for enzyme binding. Because of this, the Km of the reaction increases in the presence of competitive inhibitors. Effectively, this means that it takes a higher concentration of substrate than usual to reach one half of Vmax. If you need a reminder about enzyme kinetics, check out our article here!

While competitive inhibitors are effective at low substrate concentrations, they are not as effective at high substrate concentrations. This is because the substrate outcompetes the inhibitors. Because of this, the presence of competitive inhibitors does not change the Vmax (the maximum velocity of the reaction).

This Lineweaver-Burk plot showcases how competitive inhibitors increase Km (the x-intercept represents -1/K m). On this plot, a= substrate concentration.

Noncompetitive Enzyme Inhibition

In noncompetitive inhibition, the inhibitor and substrate do not compete to bind to the same site on the enzyme, and the binding of one does not affect the ability of the other to bind. The inhibitor can bind to the enzyme, creating an enzyme-inhibitor (EI) complex. It can also bind to the enzyme-substrate (ES) complex, creating an enzyme-substrate-inhibitor (ESI) complex. Both the EI and ESI complexes are inactive. This means that once the noncompetitive inhibitor has bound to an enzyme or to the enzyme-substrate complex, the reaction will not proceed.

Because noncompetitive inhibitors do not affect the substrate’s affinity for the enzyme, reaction Km is unchanged in their presence.

Since these inhibitors can bind the enzyme-substrate complex and prevent it from creating a product, a higher concentration of substrate does not overcome inhibition. In the presence of noncompetitive inhibitors, fewer enzymes are able to catalyze the reaction, which decreases Vmax.

This Lineweaver-Burk plot shows that noncompetitive inhibitors do not affect Km, but do decrease Vmax.

Uncompetitive Enzyme Inhibition

Uncompetitive inhibition is another type of inhibition in which the inhibitor and substrate do not compete to bind the enzyme. It is distinct from noncompetitive inhibition in that the inhibitor only binds the enzyme-substrate complex, and does not bind unbound enzymes. The resulting enzyme-substrate-inhibitor (ESI) complex is inactive and does not produce any product.

Uncompetitive inhibition does not affect how much substrate is able to bind to enzymes. Additionally, when an uncompetitive inhibitor binds an ES complex, it prevents substrate from dissociating. This actually increases the apparent affinity of substrate for enzymes, resulting in an apparent decreased Km. This means that it takes a lower substrate concentration than usual to reach one half of Vmax.

Interestingly, though Km is decreased in the presence of uncompetitive inhibitors, Vmax is decreased as well. This is because the inhibitor binds ES complexes before they can make product, and ESI complexes prevent the progress of the reaction. 

This Lineweaver-Burk plot shows that in the presence of uncompetitive inhibitor, Vmax and Km are both decreased.

Real-World Examples

Now that we understand the types of enzyme inhibition and the effects of different inhibitors on Km and Vmax, let’s look at some real world examples.

Competitive Inhibition: Methotrexate

Methotrexate is a chemotherapy drug that inhibits an enzyme called dihydrofolate reductase, or DHFR. DHFR catalyzes the synthesis of tetrahydrofolate, which is necessary for the synthesis of thymine. DHFR is often upregulated in cancer cells, allowing for increased nucleotide production, which contributes to uncontrolled cell division.

Methotrexate is structurally similar to folate, one of DHFR’s substrates, but with an even higher affinity for DHFR. When Methotrexate binds DHFR, it blocks folate from binding and stops the synthesis of tetrahydrofolate.

Here, we can see the structural similarity between folic acid (top) and methotrexate (bottom).

Noncompetitive Inhibition: Cyanide

Cyanide (HCN) is a deadly poison that prevents the production of ATP, which is essential to life. It inhibits an enzyme called cytochrome oxidase, the last enzyme in the electron transport chain. Cyanide binds to cytochrome oxidase and prevents it from transporting electrons across the mitochondrial membrane, which is a key step in cellular respiration. It does not bind to the active site, or prevent electrons from binding to the enzyme, so it is non-competitive. It creates a complex with either cytochrome oxidase (an EI or enzyme-inhibitor complex), or with both cytochrome oxidase and the electron substrate (an ESI or enzyme-substrate-inhibitor complex). In either case, the inhibitor prevents the final step of electron transport. This causes cellular asphyxiation, and is lethal.

Uncompetitive Inhibition: L-Phenylalanine inhibition of alkaline phosphatase

Alkaline phosphatase is an enzyme that catalyzes dephosphorylation of phosphate compounds at basic pH levels. L-phenylalanine specifically inhibits intestinal forms of alkaline phosphatase, preventing dephosphorylation. L-phenylalanine binds the enzyme-substrate complex, forming an ESI complex that is inactive. This causes a decrease of both Km and Vmax, as described in this study.