DIFFERENT FACTORS AFFECTING

ENZYME INHIBITION
ASSIGNMENT:03

JANUARY 16, 2025

SUBMITTED TO: MA’AM DR.SUMERA ZAIB
SUBMITTED BY: HUMAIRA KHAN
Topic:
Different factors affecting Enzyme Inhibition
Table of Contents:
1. Introduction

2. Types of inhibition

3. Competitive Inhibition

4. Non-Competitive Inhibition

5. Uncompetitive Inhibition

6. Concentration of Inhibitor

7. pH and Temperature

8. Effect of Temperature on Enzyme Inhibition

9. Binding Affinity (Ki)

10. Presence of Cofactors or Metal Ions

11. Reversibility of Inhibition

12. Irreversibility of Inhibition

13. Reference:
Introduction

Enzyme inhibitors are molecules that reduce or prevent the activity of enzymes by binding to
them. They play a crucial role in biological systems by regulating metabolic pathways and are
extensively used in pharmaceuticals to treat various diseases. Understanding the mechanisms
and factors that influence enzyme inhibitors is vital for designing effective drugs and
controlling enzymatic reactions in industrial processes. Several factors affect enzyme
inhibition, including the type of inhibition, concentration of the inhibitor and substrate, pH,
temperature, binding affinity, and reversibility. Each factor modifies how an enzyme interacts
with its inhibitor, ultimately impacting the rate of biochemical reactions. This assignment
explores these factors in detail, with the help of diagrams to illustrate their effects on enzyme
kinetics and functionality.

Types of Inhibition

Enzyme inhibitors can be classified into different types based on how they interact with the
enzyme and affect its activity. The three main types of inhibition are competitive, non-
competitive, and uncompetitive. Each type has distinct effects on the enzyme kinetics,
particularly on parameters like the maximum reaction velocity (Vmax) and the Michaelis
constant (Km).

1. Competitive Inhibition

In competitive inhibition, the inhibitor resembles the substrate and competes for binding at the
enzyme's active site. This type of inhibition can be overcome by increasing the concentration
of the substrate.

Effect on Kinetics:

• Increases Km (reducing the enzyme's apparent substrate affinity).

• Vmax remains unchanged.
 Figure 1:A Michaelis-Menten plot showing a shift in Km with a constant Vmax in the presence of a competitive inhibitor

2.Non-Competitive Inhibition

Non-competitive inhibitors bind to an allosteric site rather than the active site, causing a
conformational change that reduces the enzyme's activity. This binding can occur whether or
not the substrate is already bound.

Effect on Kinetics:

• Vmax decreases.
• Km remains unchanged.

Figure 2:A plot showing a decrease in Vmax without a change in Km

3.Uncompetitive Inhibition

In uncompetitive inhibition, the inhibitor binds only to the enzyme-substrate complex,

preventing the reaction from completing.
 Effect on Kinetics:

• Both Km and Vmax decrease proportionally.

Figure 3:A Lineweaver-Burk plot showing parallel lines that indicate simultaneous reductions in Km and Vmax

Concentration of Inhibitor

he concentration of an enzyme inhibitor directly affects its ability to reduce enzyme activity.
The relationship between inhibitor concentration and enzyme activity depends on the type of
inhibition involved.

1. Competitive Inhibition

In competitive inhibition, increasing the inhibitor concentration increases competition with the
substrate for the enzyme's active site, reducing the enzyme’s activity. However, this effect can
be overcome by significantly increasing the substrate concentration.

Effect:

• Higher inhibitor concentration increases Km (lower substrate affinity), but Vmax

remains unchanged.
2.Non-Competitive Inhibition

In non-competitive inhibition, the inhibitor binds to an allosteric site, not competing with the
substrate. Increasing the inhibitor concentration decreases enzyme activity by reducing the
number of active enzyme molecules.

Effect:

• Higher inhibitor concentration reduces Vmax without affecting Km.

Substrate Concentration

he concentration of the substrate significantly influences enzyme activity in the presence of

inhibitors. The type of inhibition determines how substrate concentration affects enzyme
kinetics.

1. Competitive Inhibition

In competitive inhibition, the substrate and inhibitor compete for the same active site on the
enzyme. As the substrate concentration increases, it can outcompete the inhibitor, restoring
enzyme activity.

Effect:

• At low substrate concentrations, the inhibitor significantly reduces enzyme activity.

• At high substrate concentrations, the effect of the inhibitor diminishes, and Vmax
remains the same, while Km increases.

2. Non-Competitive Inhibition

In non-competitive inhibition, the inhibitor binds to an allosteric site rather than the active site.
This binding affects enzyme activity regardless of the substrate concentration.

Effect:

• Increasing substrate concentration does not restore enzyme activity.

• Vmax decreases, while Km remains unchanged.
3.Uncompetitive Inhibition

In uncompetitive inhibition, the inhibitor binds only to the enzyme-substrate complex, locking
the substrate in place and preventing product formation.

Effect:

• Increasing substrate concentration enhances inhibitor binding, further reducing

enzyme activity.
• Both Km and Vmax decrease proportionally.

pH and Temperature

Both pH and temperature are crucial environmental factors that affect enzyme activity and
the efficiency of enzyme inhibition. Changes in these parameters can alter the structure of the
enzyme or the inhibitor, impacting how they interact.

1. Effect of pH on Enzyme Inhibition

Enzymes have an optimal pH range in which they are most active. Deviations from this range
can:

• Change the ionization state of amino acids in the active site.

• Affect the binding affinity of inhibitors.

Competitive Inhibition

• A change in pH may alter the structure of the inhibitor or the active site, affecting the
ability of the inhibitor to compete with the substrate.
• Effect: At extreme pH levels, competitive inhibition may weaken due to reduced
substrate and inhibitor binding efficiency.

Non-Competitive and Uncompetitive Inhibition

• In these types of inhibition, changes in pH may disrupt the enzyme's overall structure
or the allosteric site, reducing enzyme-inhibitor interactions.
 Effect: Reduced enzyme activity regardless of substrate concentration.

2. Effect of Temperature on Enzyme Inhibition

Temperature affects both enzyme activity and inhibitor binding:

• Increasing temperature generally speeds up reaction rates until the enzyme denatures at
high temperatures.
• Inhibitors may lose or gain binding affinity at different temperatures.

1. Optimal Temperature
Enzymes have an optimal temperature range for maximum activity. Inhibitors may also
have temperature-dependent binding efficiency.
2. High Temperature
Effect: High temperatures can denature the enzyme, reducing both substrate and
inhibitor binding.
Irreversible inhibitors may permanently inactivate the enzyme faster at elevated
temperatures.
3. Low Temperature
Effect: At low temperatures, enzyme and inhibitor molecules move slower, reducing
the frequency of binding interactions.

Binding Affinity (Ki)

The binding affinity of an enzyme inhibitor is quantified by the inhibition constant (Ki),
which represents the concentration of an inhibitor required to reduce the enzyme’s activity by
half. A lower Ki value indicates a stronger affinity between the inhibitor and the enzyme,
meaning the inhibitor is more effective at lower concentrations.

1. Competitive Inhibition and Ki

In competitive inhibition, the Ki value affects how much substrate is needed to
overcome the inhibitor’s effect.

Effect of Low Ki:

 • Strong binding of the inhibitor to the active site.
• Higher substrate concentration required to achieve maximum reaction velocity
(Vmax).

2. Non-Competitive Inhibition and Ki

For non-competitive inhibition, Ki reflects the binding affinity of the inhibitor to the enzyme’s
allosteric site, independent of substrate concentration.

Effect of Low Ki:

• Reduced Vmax at lower inhibitor concentrations.

• Km remains unchanged.

Uncompetitive Inhibition and Ki

In uncompetitive inhibition, Ki describes how effectively the inhibitor binds to the enzyme-
substrate complex. A lower Ki means stronger binding, leading to more significant reductions
in both Km and Vmax.

Ki and Inhibitor Potency

Ki is a critical parameter in pharmacology and drug design. A lower Ki indicates a potent

inhibitor that effectively reduces enzyme activity at lower concentrations, making it preferable
in therapeutic contexts.

Presence of Cofactors or Metal Ions

Cofactors and metal ions are non-protein components that are essential for the catalytic activity
of many enzymes. The presence or absence of these molecules can significantly influence
enzyme inhibition.

1. Role of Cofactors

Cofactors can either enhance or reduce enzyme activity and influence the effectiveness of
inhibitors. Some enzymes require specific cofactors to maintain their active site configuration.
 • Activator Cofactors: Enhance enzyme activity by stabilizing the active site.
• Inhibitory Cofactors: Alter the enzyme structure to favor inhibitor binding.

Example:

• Zinc (Zn²⁺) is a cofactor for carbonic anhydrase, and its removal disrupts enzyme
activity.

Figure 4: showing an enzyme with and without a cofactor

2. Role of Metal Ions

Some inhibitors act by chelating metal ions essential for enzyme function, reducing or
eliminating catalytic activity. Conversely, the presence of metal ions can sometimes facilitate
inhibitor binding.

• Example: EDTA binds metal ions and inhibits metalloproteinases.

Reversibility of Inhibition

Inhibition can be classified as either reversible or irreversible based on how the inhibitor
interacts with the enzyme.

1. Reversible Inhibition
Reversible inhibitors bind non-covalently to the enzyme. The effects can be reversed by
removing the inhibitor or increasing the substrate concentration (depending on inhibition type).

• Types: Competitive, non-competitive, and uncompetitive inhibition.

• Bond Type: Hydrogen bonds, ionic interactions, or hydrophobic forces.

Example:

• Ibuprofen reversibly inhibits cyclooxygenase (COX) enzymes.

2. Irreversible Inhibition

Irreversible inhibitors form covalent bonds with the enzyme, permanently inactivating it.

• Effect: Complete loss of enzyme function, even after removing the inhibitor.
• Example: Penicillin irreversibly inhibits bacterial transpeptidase.
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