3.4.2 Non-Competitive Inhibitors

Introduction

Non-competitive inhibition is a type of enzyme inhibition where the inhibitor binds to the enzyme at a site other than the active site. This binding changes the shape of the enzyme and prevents it from catalyzing the reaction. Unlike competitive inhibition, non-competitive inhibition cannot be overcome by increasing the substrate concentration.

Definition

Non-Competitive Inhibition: In non-competitive inhibition, the inhibitor molecule binds to an enzyme at a site other than the active site. The other binding site of the enzyme is called the allosteric site. The non-competitive inhibitors inactivate the enzyme temporarily (reversible inhibition) or they denature the enzyme permanently (irreversible inhibition).

In simple words: Non-competitive inhibition is when an inhibitor binds to a site on the enzyme other than the active site, changing the enzyme's shape and preventing it from working.

Core Concept

• In non-competitive inhibition, the inhibitor molecule binds to an enzyme other than the active site.
• The other binding site of the enzyme is called the allosteric site.
• The non-competitive inhibitors:
• Inactivate the enzyme temporarily (reversible inhibition)
• Denature the enzyme permanently (irreversible inhibition)
• Reversible non-competitive inhibitors work not by preventing the formation of enzyme-substrate complexes, but by preventing the formation of enzyme-product complexes.
• So they prevent the substrate to be converted into product.
• Feedback inhibition is an example of reversible non-competitive enzyme inhibition.
• Irreversible non-competitive inhibitors destroy the enzyme by altering its shape so that the substrate cannot bind to the active site.
• Examples of irreversible non-competitive inhibitors include cyanides and salts of heavy metals.

Detailed Explanation

Mechanism of Non-Competitive Inhibition

• The inhibitor binds to the enzyme at the allosteric site.
• The allosteric site is a site other than the active site.
• The binding is through non-covalent interactions (reversible) or covalent bonds (irreversible).

• The binding of the inhibitor causes a conformational change in the enzyme.
• This changes the shape of the active site.
• The substrate can no longer bind to the active site.
• The enzyme is inactivated.

• In reversible non-competitive inhibition:
• The substrate can still bind to the enzyme.
• But the enzyme cannot convert the substrate to product.
• The enzyme-product complex cannot form.
• In irreversible non-competitive inhibition:
• The substrate cannot bind to the active site at all.
• The enzyme is permanently destroyed.

Types of Non-Competitive Inhibition

There are two types of non-competitive inhibition:

• Reversible Non-Competitive Inhibition
• Irreversible Non-Competitive Inhibition

1. Reversible Non-Competitive Inhibition

Definition: In reversible non-competitive inhibition, the inhibitor binds reversibly to the allosteric site. The enzyme is inactivated temporarily.

• The inhibitor binds to the allosteric site.
• The enzyme changes shape.
• The substrate can still bind, but the product cannot form.
• The enzyme-product complex cannot form.
• The inhibition can be reversed if the inhibitor is removed.
• Feedback inhibition is an example of reversible non-competitive inhibition.

Example: Feedback Inhibition

• Feedback inhibition is a type of reversible non-competitive inhibition.
• The activity of almost every enzyme in a cell can be regulated by its product.
• When the activity of an enzyme is inhibited by its own product, it is called feedback inhibition.
• This phenomenon is a part of the normal regulatory mechanism.
• It usually happens during the regulation of metabolic pathways.

Example of Feedback Inhibition:

• The amino acid aspartate becomes the amino acid threonine by a sequence of five enzymatic reactions.
• When threonine (the end product of this pathway) is present in excess, it binds to an allosteric site on enzyme 1 on this pathway.
• Then the active site is no longer able to bind aspartate.
• When all the threonine is consumed in cellular events, the threonine molecule which is attached to the allosteric site is also removed.
• The pathway resumes its activity once again.

Equation: Aspartate Enzyme 1→ Intermediate 1 Enzyme 2→ Intermediate 2 Enzyme 3→ Intermediate 3 Enzyme 4→ Intermediate 4 Enzyme 5→ Threonine

• Threonine (end product) inhibits Enzyme 1 (first enzyme).
• This is an example of negative feedback.
• It prevents the overproduction of threonine.

2. Irreversible Non-Competitive Inhibition

Definition: In irreversible non-competitive inhibition, the inhibitor permanently destroys the enzyme by altering its shape so that the substrate cannot bind to the active site.

• The inhibitor binds covalently to the enzyme.
• The enzyme is permanently denatured.
• The substrate cannot bind to the active site.
• The inhibition cannot be reversed.
• The enzyme is permanently inactivated.

Examples:

• Cyanides are potent poisons of living organisms.
• They can kill an organism by inhibiting cytochrome oxidase, essential for cellular respiration.
• They block the action of these enzymes by combining with iron which may be present in the prosthetic group.
• This prevents electron transport and ATP production.
• The organism dies due to lack of energy.

• Ions of heavy metals such as mercury (Hg), silver (Ag), and copper (Cu) combine with thiol (-SH) groups in the enzyme.
• This breaks the disulphide bridges.
• Disulphide bridges are important in maintaining tertiary structure.
• When these bridges are broken, the enzyme becomes denatured and inactive.

Comparison: Competitive vs Non-Competitive Inhibition

Comparison: Reversible vs Irreversible Non-Competitive Inhibition

Effect of Non-Competitive Inhibition on Enzyme Kinetics

Michaelis-Menten Equation: V₀ = V_max [S]K_m + [S]

• Vmax decreases.
• Km remains unchanged.
• This means the enzyme has the same affinity for the substrate, but the maximum rate is reduced.
• Increasing substrate concentration cannot overcome the inhibition.

Lineweaver-Burk Plot:

Feedback Inhibition in Detail

Definition: When the activity of an enzyme is inhibited by its own product, it is called feedback inhibition.

• It is a type of reversible non-competitive inhibition.
• It is a part of normal regulatory mechanisms.
• It usually happens during the regulation of metabolic pathways.
• The end product of a pathway inhibits the first enzyme of the pathway.
• This prevents the overproduction of the end product.
• It is also called end-product inhibition or negative feedback.

• Aspartate → Threonine pathway:
• Aspartate is converted to threonine by five enzymatic reactions.
• Threonine (end product) inhibits Enzyme 1 (first enzyme).
• This is an example of feedback inhibition.

• Prevents overproduction of products.
• Saves energy and resources.
• Maintains metabolic balance.
• Regulates metabolic pathways.

Clinical Significance

Intermediate Level

Mechanism in Detail

Types of Non-Competitive Inhibition

Comparison: Reversible vs Irreversible

Feedback Inhibition in Detail

Effect on Enzyme Kinetics

Important Points

• 1. In non-competitive inhibition, the inhibitor molecule binds to an enzyme other than active site.
• 2. The other binding site of enzyme is called allosteric site.
• 3. The noncompetitive inhibitors inactivate the enzyme temporarily (reversible inhibition) or they denature the enzyme permanently (irreversible inhibition).
• 4. Reversible non-competitive enzyme inhibitors work not by preventing the formation of enzyme-substrate complexes, but by preventing the formation of enzyme-product complexes.
• 5. So they prevent the substrate to be converted into product.
• 6. Feedback inhibition is an example of reversible non-competitive enzyme inhibition.
• 7. On the other hand, an irreversible non-competitive enzyme inhibitor destroys enzyme by altering its shape so that the substrate cannot bind to the active site.
• 8. The examples of irreversible non-competitive inhibitors include cyanides and salts of heavy metals.
• 9. Cyanides are potent poisons of living organism because they can kill an organism by inhibiting cytochrome oxidase essential for cellular respiration.
• 10. They block the action of these enzymes by combining with iron which may be present in the prosthetic group.
• 11. Ions of heavy metals such as mercury, silver and copper (Hg, Ag, and Cu) combine with thiol (-SH) groups in the enzyme breaking the disulphide bridges.
• 12. These bridges are important in maintaining tertiary structure.
• 13. When these bridges are broken, the enzyme becomes denatured and inactive.
• 14. Feedback Inhibition:
• - The activity of almost every enzyme in a cell can be regulated by its product.
• - When the activity of an enzyme is inhibited by its own product, it is called feedback inhibition.
• - This is a type of reversible non-competitive inhibition.
• - This phenomenon is a part of normal regulatory mechanism and usually happens during the regulation of metabolic pathways.
• - For example, the amino acid aspartate becomes the amino acid threonine by a sequence of five enzymatic reactions.
• - When threonine, the end product of this pathway, is present in excess, it binds to an allosteric site on enzyme 1 on this pathway and then the active site is no longer able to bind aspartate.
• - When all the threonine is consumed in cellular events, the threonine molecule which is attached to the allosteric site is also removed; the pathway resumes its activity once again.

Remember

• - Non-competitive inhibitor = binds to allosteric site
• - Allosteric site = site other than active site
• - Changes the shape of the enzyme
• - Cannot be overcome by increasing substrate concentration
• - **Vmax** = decreased
• - **Km** = unchanged
• - Reversible = temporary; Irreversible = permanent
• - Examples: Cyanide, heavy metals
• - Feedback inhibition = reversible non-competitive inhibition
• - Cyanide = inhibits cytochrome oxidase
• - Heavy metals = combine with thiol groups; break disulphide bridges

Common Mistakes

• 1. Confusing competitive and non-competitive inhibition – Competitive = binds active site; Non-competitive = binds allosteric site.
• 2. Thinking non-competitive inhibition can be overcome – Non-competitive inhibition cannot be overcome by increasing substrate concentration.
• 3. Thinking Vmax increases in non-competitive inhibition – Vmax decreases; Km remains unchanged.
• 4. Thinking the inhibitor is similar to the substrate – Non-competitive inhibitors are not similar to the substrate.
• 5. Forgetting that non-competitive inhibition can be reversible or irreversible – It can be either.
• 6. Confusing the examples – Cyanide and heavy metals are non-competitive inhibitors.
• 7. Thinking feedback inhibition is competitive – Feedback inhibition is reversible non-competitive inhibition.
• 8. Forgetting the role of cyanide – Cyanide inhibits cytochrome oxidase by combining with iron.
• 9. Forgetting the role of heavy metals – Heavy metals combine with thiol groups and break disulphide bridges.
• 10. Thinking the enzyme is permanently destroyed in reversible inhibition – In reversible inhibition, the enzyme can recover.
• 11. Confusing Km and Vmax – Vmax decreases; Km remains unchanged.
• 12. Thinking substrate concentration has no effect – Substrate concentration does not overcome non-competitive inhibition.

Quick Revision

• In non-competitive inhibition the inhibitor molecule binds to an enzyme other than active site.
• The other binding site of enzyme is called allosteric site.
• The noncompetitive inhibitors inactivate the enzyme temporarily (reversible inhibition) or they denature the enzyme permanently (irreversible inhibition).
• Reversible non-competitive enzyme inhibitors work not by preventing the formation of enzyme-substrate complexes, but by preventing the formation of enzyme-product complexes.
• So they prevent the substrate to be converted into product.
• Feedback inhibition is an example of reversible non-competitive enzyme inhibition.
• On the other hand, an irreversible non-competitive enzyme inhibitor destroys enzyme by altering its shape so that the substrate cannot bind to the active site.
• The examples of irreversible non-competitive inhibitors include cyanides and salts of heavy metals.
• Cyanides are potent poisons of living organism because they can kill an organism by inhibiting cytochrome oxidase essential for cellular respiration.
• They block the action of these enzymes by combining with iron which may be present in the prosthetic group.
• Ions of heavy metals such as mercury, silver and copper (Hg, Ag, and Cu) combine with thiol (-SH) groups in the enzyme breaking the disulphide bridges.
• These bridges are important in maintaining tertiary structure.
• When these bridges are broken, the enzyme becomes denatured and inactive.
• Feedback Inhibition:
• The activity of almost every enzyme in a cell can be regulated by its product.
• When the activity of an enzyme is inhibited by its own product, it is called feedback inhibition.
• This is a type of reversible non-competitive inhibition.
• This phenomenon is a part of normal regulatory mechanism and usually happens during the regulation of metabolic pathways.
• For example, the amino acid aspartate becomes the amino acid threonine by a sequence of five enzymatic reactions.
• When threonine, the end product of this pathway, is present in excess, it binds to an allosteric site on enzyme 1 on this pathway and then the active site is no longer able to bind aspartate.
• When all the threonine is consumed in cellular events, the threonine molecule which is attached to the allosteric site is also removed; the pathway resumes its activity once again.

60-Second Revision

• Non-competitive inhibitor = binds to allosteric site
• Allosteric site = site other than active site
• Changes the shape of the enzyme
• Cannot be overcome by increasing substrate concentration
• **Vmax** = decreased
• **Km** = unchanged
• Reversible = temporary; Irreversible = permanent
• Examples: Cyanide, heavy metals
• Feedback inhibition = reversible non-competitive inhibition
• Cyanide = inhibits cytochrome oxidase
• Heavy metals = combine with thiol groups; break disulphide bridges
• Warning: Non-competitive inhibition cannot be overcome by increasing substrate concentration. Vmax decreases; Km remains unchanged. The inhibitor binds to the allosteric site, not the active site. Non-competitive inhibition can be reversible or irreversible. Cyanide and heavy metals are irreversible non-competitive inhibitors. Feedback inhibition is reversible non-competitive inhibition.