Chapter-3
Enzymes
Definition: Enzymes are biological catalysts that accelerate biochemical reactions without being
consumed during the reaction.
Each enzyme is assigned an Enzyme Commission (EC) number.
Systematic Nomenclature of Enzymes
The International Union of Biochemistry and Molecular Biology (IUBMB) assigns each enzyme
a systematic name and an Enzyme Commission (EC) number based on the reaction it catalyzes.
The EC number has the general form:
EC x.x.x.x
where:
• First number: Main enzyme class
• Second number: Subclass
• Third number: Sub-subclass
• Fourth number: Specific enzyme
Example: Alcohol dehydrogenase
EC [Link]
Interpretation:
• 1 → Oxidoreductase
• 1 → Acts on the CH–OH group of donors
• 1 → Uses NAD⁺ or NADP⁺ as the electron acceptor
• 1 → Specific enzyme (Alcohol dehydrogenase)
Six Major Classes of Enzymes
Class Function Example
EC 1 Oxidoreductases Oxidation-reduction reactions Lactate dehydrogenase
EC 2 Transferases Transfer of functional groups Hexokinase
EC 3 Hydrolases Hydrolysis reactions Trypsin
EC 4 Lyases Addition/removal without hydrolysis Fumarase
EC 5 Isomerases Rearrangement within molecules Phosphoglucose isomerase
EC 6 Ligases Formation of bonds using ATP DNA ligase
Examples:
1. Oxidoreductase
Reaction: Lactate + NAD⁺ ⇌ Pyruvate + NADH
Enzyme: Lactate dehydrogenase
2. Transferase
Reaction: Glucose + ATP → Glucose-6-phosphate + ADP
Enzyme: Hexokinase
3. Hydrolase
Reaction: Protein + H₂O → Peptides
Enzyme: Trypsin
4. Lyase
Reaction: Fumarate ⇌ Malate
Enzyme: Fumarase
5. Isomerase
Reaction: Glucose-6-phosphate ⇌ Fructose-6-phosphate
Enzyme: Phosphoglucose isomerase
6. Ligase
Reaction: DNA fragments + ATP → Joined DNA
Enzyme: DNA ligase
Cofactors
Definition
A cofactor is a non-protein chemical component required for an enzyme to become biologically
active.
Many enzymes cannot function without cofactors.
Types of Cofactors
1. Metal Ion Cofactors: These are inorganic ions.
Examples
Metal ion Enzyme
Mg²⁺ DNA polymerase
Zn²⁺ Carbonic anhydrase
Fe²⁺ Catalase
Cu²⁺ Cytochrome oxidase
Mn²⁺ Arginase
Coenzymes
Organic molecules that bind temporarily to enzymes. Mostly derived from vitamins.
Examples
Coenzyme Derived from
NAD⁺ Vitamin B3
NADP⁺ Vitamin B3
FAD Vitamin B2
Coenzyme A Vitamin B5
TPP Vitamin B1
Biotin Vitamin B7
Prosthetic Groups
Organic cofactors tightly bound to enzymes.
Examples
• FAD
• Heme group
• Biotin
Apoenzyme, Cofactor and Holoenzyme
Apoenzyme
Protein part only (inactive)
Add Cofactor
Holoenzyme
Complete active enzyme
Relationship: Apoenzyme + Cofactor = Holoenzyme
Difference Between Coenzyme and Prosthetic Group
Coenzyme Prosthetic Group
Loosely bound Tightly bound
Easily dissociates Permanently attached
Acts as carrier Integral part of enzyme
Functions of Cofactors
• Stabilize enzyme structure
• Participate in catalysis
• Carry electrons
• Transfer functional groups
• Increase catalytic efficiency
Principle of Enzyme Catalysis
Definition: Enzyme catalysis is the process by which enzymes increase the rate of biochemical
reactions by lowering the activation energy.
Activation Energy
Without enzyme: Reactants → High activation energy → Products
With enzyme: Reactants → Lower activation energy → Products
Important: Enzymes do not change:
• ΔG (Gibbs free energy)
• Equilibrium constant (K_eq)
• Reaction products
They only increase the reaction rate.
Steps in Enzyme Catalysis
Step 1: Substrate binds to enzyme.
E + S ⇌ ES
Step 2: Enzyme-substrate complex forms.
The enzyme stabilizes the transition state.
Step 3: Product forms.
ES → EP
Step 4: Product leaves.
EP → E + P
The enzyme is regenerated.
Mechanisms of Enzyme Catalysis
1. Proximity Effect: Brings substrates close together.
2. Acid–Base Catalysis: Enzyme donates or accepts H⁺.
Example: Ribonuclease A
3. Covalent Catalysis: Temporary covalent bond forms between enzyme and substrate.
Example: Serine proteases
4. Metal Ion Catalysis: Metal ions stabilize charges or participate in electron transfer.
Example: Carbonic anhydrase (Zn²⁺)
5. Transition State Stabilization: The enzyme binds the transition state more tightly than the
substrate, lowering the activation energy.
Factors Affecting Enzyme Catalysis
• Temperature
• pH
• Enzyme concentration
• Substrate concentration
• Inhibitors
• Activators
• Availability of cofactors
Main Characteristics of Catalysis
The main characteristics of catalysis are:
1. Increases the reaction rate: A catalyst accelerates a chemical reaction by lowering the
activation energy.
2. Remains unchanged: The catalyst is not consumed during the reaction and can be reused.
3. Does not alter the equilibrium: A catalyst speeds up both the forward and reverse
reactions equally, so it does not change the equilibrium constant (Keq) or the equilibrium
position.
4. Effective in small amounts: Even a small quantity of catalyst can catalyze a large amount
of reactant.
5. Highly specific (especially enzymes): Enzymes exhibit high substrate and reaction
specificity, catalyzing only particular reactions.
6. Provides an alternative reaction pathway: Catalysts lower the activation energy by
offering a different reaction mechanism.
7. Does not change the Gibbs free energy (ΔG): Catalysts do not affect the thermodynamics
of the reaction; they only influence the reaction rate.
8. Activity depends on reaction conditions: The effectiveness of catalysts, particularly
enzymes, depends on factors such as temperature, pH, and the presence of inhibitors or
activators.
Enzyme-Catalyzed Reaction Show First-Order and Zero-Order Kinetics
The reaction order depends on the substrate concentration ([S]) relative to the enzyme
concentration.
(a) First-Order Kinetics
An enzyme-catalyzed reaction follows first-order kinetics when the substrate concentration is
much lower than the Michaelis constant (Km).
Condition:
[S]≪Km
Characteristics:
• Most enzyme active sites are unoccupied.
• The reaction rate is directly proportional to the substrate concentration.
• Doubling the substrate concentration approximately doubles the reaction rate.
(b) Zero-Order Kinetics
An enzyme-catalyzed reaction follows zero-order kinetics when the substrate concentration is
much higher than the Michaelis constant (Km).
Condition:
[S]≫Km
Characteristics:
• All enzyme active sites are saturated with substrate.
• The reaction rate reaches its maximum value (Vmax).
• Increasing substrate concentration further does not increase the reaction rate.
Enzyme-Catalyzed Reactions Having One Substrate
Introduction: Many enzymes catalyze reactions involving only one substrate. These reactions are
known as single-substrate (uni-substrate) reactions and provide the basis for understanding
Michaelis–Menten enzyme kinetics.
The general reaction scheme is: E + S ⇌ ES → E + P
Where:
• E = Enzyme
• S = Substrate
• ES = Enzyme–substrate complex
• P = Product
Mechanism of a Single-Substrate Reaction
Step 1: Substrate Binding: The substrate binds reversibly to the enzyme's active site to form the
enzyme–substrate complex.
E + S ⇌ ES
Step 2: Catalysis: The enzyme converts the substrate into the product while the substrate is bound
to the active site.
ES → EP
where EP is the enzyme–product complex.
Step 3: Product Release
The product is released, and the enzyme is regenerated unchanged.
EP → E + P
The regenerated enzyme is then free to catalyze another reaction.
Michaelis–Menten Equation
The initial reaction velocity (v) is related to the substrate concentration ([S]) by the Michaelis–
Menten equation:
Where:
• v = Initial reaction velocity
• Vmax = Maximum reaction velocity
• [S] = Substrate concentration
• Km = Michaelis constant
Significance of Km
Interpretation:
• Low Km → High affinity between enzyme and substrate.
• High Km → Low affinity between enzyme and substrate.
Effect of Substrate Concentration on Reaction Rate
Characteristics:
• All enzyme active sites are saturated with substrate.
• The reaction reaches its maximum velocity (Vmax).
• Further increases in substrate concentration do not increase the reaction rate.
• The reaction follows zero-order kinetics.
Rate equation: v ≈ Vmax
Velocity–Substrate Concentration Curve
A graph of reaction velocity (v) versus substrate concentration ([S]) gives a rectangular hyperbola.
Characteristics:
• At low substrate concentrations, the reaction rate increases rapidly.
• As substrate concentration increases, the rate rises more slowly.
• At very high substrate concentrations, the curve approaches Vmax asymptotically.
Explanation
The velocity–substrate concentration curve for a Michaelis–Menten enzyme is a rectangular
hyperbola.
• At low substrate concentration ([S]), the reaction velocity increases almost linearly with
increasing substrate concentration (first-order kinetics).
• As [S] increases, more enzyme active sites become occupied, and the rate increases more
slowly.
• At high substrate concentration, all enzyme active sites are saturated, and the reaction
reaches the maximum velocity (Vmax). Further increases in substrate concentration do
not increase the reaction rate (zero-order kinetics).
• The substrate concentration at which the reaction velocity is half of Vmax is called the
Michaelis constant (Km). It is a measure of the enzyme's affinity for its substrate.
Effect of pH and Temperature on Enzyme Catalysis
Effect of pH on Enzyme Catalysis
The catalytic activity of an enzyme is strongly influenced by the pH of its environment. Each
enzyme has an optimum pH at which it exhibits maximum catalytic activity. Changes in pH alter
the ionization state of amino acid residues at the enzyme's active site and may also affect the
substrate. As a result, enzyme activity decreases when the pH deviates from the optimum value.
At very low or very high pH, the enzyme may become denatured, leading to a loss of its three-
dimensional structure and catalytic function.
Characteristics
• Every enzyme has a characteristic optimum pH.
• Enzyme activity is highest at the optimum pH.
• Activity decreases above or below the optimum pH.
• Extreme pH values can denature the enzyme.
Examples
Enzyme Optimum pH
Pepsin 1.5–2.0
Salivary amylase 6.7–7.0
Trypsin 7.5–8.5
Alkaline phosphatase 9–10
Short Explanation:
The graph is bell-shaped because enzyme activity increases as the pH approaches the optimum
value, reaches a maximum at the optimum pH, and then decreases as the pH moves away from the
optimum.
Effect of Temperature on Enzyme Catalysis
Temperature also has a significant effect on enzyme activity. As the temperature increases, the
kinetic energy of enzyme and substrate molecules increases, leading to more frequent collisions
and a faster reaction rate.
Each enzyme has an optimum temperature at which its catalytic activity is maximum. For most
human enzymes, this temperature is approximately 37°C.
Above the optimum temperature, the enzyme begins to denature, causing a rapid decline in
catalytic activity. At low temperatures, enzyme activity is reduced because molecular motion is
slower, but the enzyme usually remains structurally intact.
Characteristics
• Enzyme activity increases with temperature up to the optimum.
• Maximum activity occurs at the optimum temperature.
• Above the optimum temperature, activity decreases rapidly due to denaturation.
• Low temperatures reduce reaction rate but generally do not denature the enzyme.
Temperature–Activity Curve
Short Explanation:
The graph shows that enzyme activity increases with temperature until the optimum temperature
is reached. Beyond this point, the enzyme denatures, and its catalytic activity decreases sharply.
Define Enzymes and Enzyme Inhibitors. Compare the Molecular Mechanisms of
Competitive and Uncompetitive Inhibition, Highlighting Their Effects on Enzyme–Substrate
Binding Equilibria and Kinetic Parameters (Km and Vmax).
Definition of Enzymes
Enzymes are biological catalysts, mainly proteins (some are RNA molecules called ribozymes),
that accelerate biochemical reactions by lowering the activation energy without being consumed
during the reaction. They are highly specific for their substrates and are essential for regulating
cellular metabolism.
Definition of Enzyme Inhibitors
An enzyme inhibitor is a molecule that decreases or prevents the catalytic activity of an enzyme
by binding either to the enzyme itself or to the enzyme–substrate (ES) complex. Inhibitors regulate
metabolic pathways and are widely used as therapeutic drugs and research tools.
Comparison of Competitive and Uncompetitive Inhibition
1. Competitive Inhibition
Molecular Mechanism
In competitive inhibition, the inhibitor (I) closely resembles the substrate and competes for binding
at the active site of the enzyme. The inhibitor binds only to the free enzyme (E), preventing
substrate binding.
Reaction scheme:
E + S ⇌ ES → E+P
E + I ⇌ EI
Since the inhibitor and substrate compete for the same binding site, increasing the substrate
concentration can overcome the inhibition.
Effect on Enzyme–Substrate Binding Equilibrium
The inhibitor shifts the equilibrium
E+S⇌ES
toward the left by decreasing ES complex formation.
Effect on Kinetic Parameters
• Km increases (apparent affinity decreases).
• Vmax remains unchanged, because sufficiently high substrate concentration can
outcompete the inhibitor.
2. Uncompetitive Inhibition
Molecular Mechanism
In uncompetitive inhibition, the inhibitor binds only to the enzyme–substrate (ES) complex, not
to the free enzyme. The binding forms an inactive enzyme–substrate–inhibitor (ESI) complex that
cannot produce product.
Reaction scheme:
E+S⇌ES
ES+I⇌ESI
Because the inhibitor binds only after substrate binding, increasing substrate concentration does
not reverse the inhibition.
Effect on Enzyme–Substrate Binding Equilibrium
The inhibitor removes ES by converting it into the inactive ESI complex. According to Le
Châtelier's principle, this shifts the equilibrium
E+S⇌ES
toward the right, promoting additional ES complex formation.
Effect on Kinetic Parameters
• Km decreases (apparent affinity increases).
• Vmax decreases, because a fraction of the enzyme remains trapped as inactive ESI
complexes.
Comparison Table
Feature Competitive Inhibition Uncompetitive Inhibition
Binding site Active site Enzyme–substrate (ES) complex only
Inhibitor binds free
Yes No
enzyme
Inhibitor binds ES
No Yes
complex
Competition with
Yes No
substrate
Effect on ES Shifts equilibrium toward free Shifts equilibrium toward ES formation
equilibrium enzyme (less ES formed) (ES converted to ESI)
Effect of increasing
Overcomes inhibition Does not overcome inhibition
substrate
Km Increases Decreases
Vmax Unchanged Decreases
Malonate inhibiting succinate Lithium inhibiting inositol
Example
dehydrogenase monophosphatase
Catalyst Influence the Activation Energy of a Reaction as Explained by the Arrhenius
Equation
Effect of a Catalyst
A catalyst increases the reaction rate by providing an alternative reaction pathway with a lower
activation energy (Ea). According to the Arrhenius equation, the rate constant k depends
exponentially on the activation energy.
Important Points
• A catalyst lowers the activation energy (Ea).
• It increases the rate constant (k) according to the Arrhenius equation.
• It provides an alternative reaction pathway.
• It does not change the Gibbs free energy (ΔG), the equilibrium constant (Keq), or the
overall enthalpy change (ΔH) of the reaction.
• The catalyst is not consumed during the reaction and can be reused.
Classify Proteins in Different Ways and Briefly Explain Each Classification. (Contain in
Chapter-2)
How the Structure of Proteins Determines Enzymatic Activity
Enzymes are globular proteins whose catalytic activity depends on their specific three-dimensional
(3D) structure. The unique folding of the protein creates an active site, where the substrate binds
and is converted into the product. Therefore, the structure of a protein is directly related to its
enzymatic activity.
The primary structure, which is the sequence of amino acids linked by peptide bonds, determines
how the protein folds into its functional shape. Even a single change in the amino acid sequence
can alter the enzyme's structure and reduce or abolish its activity.
The secondary structure, consisting of α-helices and β-pleated sheets, provides stability to the
protein through hydrogen bonding. These structural elements help maintain the proper
arrangement of the enzyme.
The tertiary structure is the overall three-dimensional folding of a single polypeptide chain. This
level of structure is the most important for enzyme activity because it forms the active site. The
active site has a specific shape and chemical properties that allow only particular substrates to
bind, ensuring high substrate specificity. The active site also contains amino acid residues that
participate directly in catalysis by lowering the activation energy of the reaction.
Some enzymes contain more than one polypeptide chain. Their quaternary structure is formed by
the association of two or more subunits. The proper arrangement of these subunits is essential for
the full catalytic activity and regulation of many enzymes.
The enzymatic activity of proteins depends on maintaining this precise three-dimensional
structure. Factors such as high temperature, extreme pH, heavy metals, organic solvents, or genetic
mutations can disrupt the secondary, tertiary, or quaternary structure of the protein. This process,
known as denaturation, changes the shape of the active site, preventing substrate binding and
causing a partial or complete loss of enzyme activity.
In conclusion, the structure of a protein determines its enzymatic activity by forming a specific
active site that binds the substrate and catalyzes the reaction. Any alteration in the protein's three-
dimensional structure can impair substrate binding, reduce catalytic efficiency, or completely
inactivate the enzyme. Thus, the relationship between protein structure and enzyme function is
fundamental to all biological processes.