Module 5: Enzymes

ENZYMES
• Enzymes are biological catalysts.
• A Catalyst is defined as "a substance that increases
the rate of a chemical reaction without being itself
changed in the process.”
• All enzymes are proteins with exception of some small
group of catalytic RNA molecules called ribozymes.
• Like proteins, the molecular weight of enzymes ranges
from about 2000 to more than one million Dalton.
• There are many enzymes which require cofactors for
their catalytic activity or activation. The cofactor may
be a complex organic molecule called coenzyme or
it may be a metal ion such as Fe2+, Mn2+, Zn2+,
Mg2+ .
• An enzyme plus its cofactor is called holoenzyme.
In such cases, the protein component in cofactor
requiring enzyme is called apoenzyme.
Cofactor Vs. Coenzyme

Cofactors Coenzymes
Cu2+ (Cytochrome Biocytin
oxidase)
Mg2+ (Glucose-6- Coenzyme B12
phosphate)
Mn2+ (Arginase) Coenzyme A
Ni2+ (Urease) Nicotinamide
adenine dinucleotide
(NAD)
 Properties of enzymes

• Catalytic efficiency: High efficiency, 103 to 1017

faster than the corresponding uncatalyzed reactions
• Specificity: High specific specificity, interacting
with one or a few specific substrates and catalyzing
only one type of chemical reaction.
• Mild reaction conditions: 37℃, physiological pH,
ambient atmospheric pressure.

Example:

1. The enzyme that catalyzes hydrolysis of maltose into

glucose is named as maltase.

2. Oxidoreductase enzymes are the enzymes which

can catalyze the oxidation of one substrate and
simultaneously reduce another substrate.
Classification of enzymes
 Active site of enzymes

• The catalytic reaction performed by enzymes occurs

at a particular site on the enzyme. This site is
called active site, and represents only small part of
the total size of the enzyme.
• The active site is a clearly defined pocket or cleft in
the enzyme molecule where the whole or a portion
of substrate can fit.
• Active site has a three-dimensional structure since it
consists of portions of a polypeptide chain. Various
non covalent bonds involved in enzyme substrate
binding are electrostatic interactions, hydrogen
bonds, Van Der Waals forces and hydrophobic
interactions.
• The active site often comprises non polar
environment which facilitates the binding of
substrate and the catalysis. However, some polar
residues may be present.
 Fischer’s Lock and Key Model

• The introduction of Lock and Key Model for the substrate

and enzyme interaction was proposed by Emil Fischer.
• According to this model, complementary structural features
are present between enzyme and substrate, and the active
site is pre-shaped to fit the substrate.
• The substrate can fit into its complementary site on the
enzyme as a key fits into a lock. This results in the
formation of an enzyme-substrate complex.

Koshland’s Induced Fit Model

• Daniel Koshland in 1958 proposed Induced Fit Hypothesis.

• He suggested that the structure of a substrate may be
complementary to that of the active site in the enzyme
substrate complex but not in the free enzyme.
• The interaction between the substrate and the enzyme
induces conformational changes in the enzyme which
aligns the amino acid residues or other groups for
substrate binding.
 Mechanism of enzyme action
1.Substrate Binding
1. The substrate (reactant) approaches the enzyme.
2. It binds to the enzyme's active site, forming an enzyme-substrate
complex.
3. This binding follows either the lock-and-key model (exact fit) or
induced fit model (slight shape change for better fit).
2.Catalysis (Reaction Occurs)
1. The enzyme lowers the activation energy required for the reaction.
2. It stabilizes the transition state, making it easier for the substrate to
transform into the product.
3.Product Formation
1. The chemical reaction converts the substrate into one or more products.
2. The enzyme helps break or form chemical bonds in the substrate.
4.Product Release
1. The newly formed product no longer fits well in the active site and is
released.
2. The enzyme returns to its original shape, ready to catalyze another
reaction.
5.Enzyme Reusability
1. Since the enzyme remains unchanged, it can catalyze multiple
reactions with new substrate molecules.
2. The process repeats as long as substrates and optimal conditions
are available.
 Transition state
• The transition state is the high-energy, unstable
intermediate stage that occurs during a chemical
reaction.
• It represents the point at which bonds in the reactants
are maximally strained and ready to be broken or
rearranged to form the product.
• This state is short-lived and exists at the peak of the
energy barrier that separates reactants from products.

Activation energy
• All chemical reactions have an energy barrier, called the
activation energy, separating the reactants and the
products.
• Activation energy: amount of energy needed to disrupt
stable molecule so that reaction can take place.
• Activation Energy is the minimum energy needed for
a reaction to start.
• It is like a small energy "push" that reactants need to
reach the transition state before forming products.
• Enzymes lower activation energy, making reactions
happen faster and easier.
 Factors affecting enzyme activity
Temperature
1. The rate of an enzyme catalyzed reaction increases with the increase in
temperature up to a maximum and then falls.
2. Optimum Temperature: The temperature at which enzyme activity is highest,
typically between 40°C-45°C for most enzymes.
3. Human Enzymes: Most human enzymes have an optimum temperature of
37°C (98.6°F) and denature at extreme temperatures, while some enzymes (e.g.,
Taq polymerase) remain active at high temperatures (up to 100°C).

Hydrogen ion concentration (pH)

1. Enzyme activity is also affected by pH.
2. Optimum pH: Each enzyme has a specific optimum pH where its activity is
highest, typically between pH 6-8 for many enzymes. When a graph is plotted between temperature/pH versus enzyme
activity, a bell-shaped curve is obtained
3. Exceptions: Some enzymes, like pepsin (pH 1-2) and alkaline phosphatases
(pH 10-11), have unique pH ranges.

Substrate concentration
1. The substrate concentration also influences enzyme activity.
2. As the substrate concentration increases the rate of reaction also increases.
This is because the more substrate molecules will interact with enzyme
molecules, the more products will be formed.
3. However, after a certain concentration, further increase in substrate
concentration will have no effect on the rate of reaction, since the substrate
concentration will no longer be the limiting factor
4. At this stage, enzyme molecules become saturated and work at their maximum When a graph is plotted between substrate concentration versus
possible rate enzyme activity, a hyperbolic curve is obtained
 Amylase (Digestion of Starch)

Enzyme: Amylase

Function: Breaks down starch (a complex

carbohydrate) into simple sugars (maltose and
glucose).

Where it Works: Found in saliva and the

pancreas.

How it Works:
• You eat starchy food (e.g., bread or rice).
• Amylase in saliva binds to starch molecules.
• It breaks the starch into smaller sugar
molecules.
• These sugars are further broken down and
absorbed in the intestines for energy.

Without amylase, starch digestion would

be much slower!
 Catalase (Breakdown of Hydrogen Peroxide)

Enzyme: Catalase

Function: Breaks down hydrogen peroxide (H₂O₂) into

water (H₂O) and oxygen (O₂).

Where it Works: Found in almost all living cells,

especially in the liver.

How it Works:
• Cells produce hydrogen peroxide as a byproduct of
metabolism.
• Hydrogen peroxide is harmful and can damage cells.
• Catalase quickly converts H₂O₂ into harmless water
and oxygen.
• Oxygen bubbles can sometimes be seen (e.g., when
applying hydrogen peroxide to a wound).

Without catalase, toxic hydrogen peroxide would

build up and harm cells!
 Kinetics of enzyme-catalyzed reaction

• During catalysis, substrate S binds at the

active site of the enzyme E and results in
formation of enzyme substrate complex
ES, which is finally converted into product
P.
• The reaction can be represented as:
E+s → Es→ E+P
• Where, E forms weakly bonded complex
ES with the substrate S.
• The ES complex decomposes to yield
product P and the free enzyme E.
• The kinetics of enzyme catalyzed
reactions was explained by Leonor
Michaelis and Maud Menten in 1913. The
equation is called Michaelis-Menten
equation.
 Enzyme Kinetics and Kinetic Parameters

Enzyme kinetics is the study of the rates of enzyme-catalyzed reactions and how factors like substrate
concentration, temperature, and pH influence these rates. Understanding enzyme kinetics is crucial for
designing drugs, understanding metabolic pathways, and optimizing industrial processes.

Key Kinetic Parameters:

1. Reaction Velocity (V): The rate at which a reaction occurs, typically expressed as the amount of product
formed per unit time (e.g., μmol/min). It depends on substrate concentration, enzyme concentration,
temperature, and other factors.

2. Vmax (Maximum Velocity): The highest rate of reaction achieved when the enzyme is fully saturated with
substrate. It is the maximum velocity at which an enzyme can convert substrate into product.

3. Km (Michaelis Constant): The substrate concentration at which the reaction rate is half of Vmax.

4. Turnover Number (kcat): The number of substrate molecules converted to product by one enzyme
molecule per unit of time when the enzyme is fully saturated with substrate.

Formula: 𝑘cat=𝑉max/[𝐸] , where [E] is the enzyme concentration.

• A graph of V0 against [S] results in rectangular hyperbola.
• Vmax is the maximum velocity at particular enzyme
concentration.
• Vmax and Km can be determined from the graph as shown in
Fig.
• In the graph, we can see that at very low substrate
concentration (when [S]<< Km),

i.e., the reaction rate (V0) is directly proportional to

substrate concentration [S] .

• At high substrate concentration (when [S]>>Km), V0=Vmax ,

i.e., reaction rate is maximum and independent of
substrate concentration.
• When [S]=Km , then V0= Vmax/2. Thus, Km is substrate
concentration at which half of the maximum reaction
rate is obtained.
 Enzyme inhibition

• Substances which decrease the rate of an

enzyme catalyzed reaction are called as enzyme
inhibitors and the process is known as enzyme
inhibition.
• Enzyme inhibition can be classified as reversible
inhibition and irreversible inhibition.
• In irreversible inhibition, the inhibitor binds
very tightly to the enzyme and does not
dissociate from it. For example, the antibiotic
penicillin acts as inhibitor and binds with the
enzyme transpeptidase, which is responsible for
synthesis of bacterial cell wall. Hence, binding
of this drug to the enzyme prevents cell wall
synthesis, thus killing the bacteria.
• In reversible inhibition, the inhibitor rapidly
dissociates from the enzyme-inhibitor complex.
• There are three types of reversible inhibitions:
competitive, non-competitive and
uncompetitive inhibition.
The Lineweaver–Burk plot, also called the double
reciprocal plot, is a graphical method used in enzyme
kinetics to analyse the Michaelis-Menten equation. It
helps determine key enzyme parameters such as Km​
(Michaelis constant) and Vmax (maximum reaction
velocity) and is particularly useful for identifying different
types of enzyme inhibition.

The Michaelis-Menten equation describes how enzyme

activity depends on substrate concentration:

Taking the reciprocal of both sides gives the

Lineweaver–Burk equation, which has a linear form:

This equation is similar to the straight-line equation:

Competitive inhibition

• In competitive inhibition, there is close resemblance in the

structure of inhibitor I and substrate S, therefore, they both
compete for the same active site on the enzyme.
• The enzyme can form enzyme-substrate ES complex or it can
form enzyme-inhibitor EI complex but not both ESI.
• Competitive inhibitors decreases the rate of reaction by
reducing the amount of active enzyme molecules bound to a
substrate.
• 𝐾𝑚 (Michaelis-Menten constant): Increases
• The inhibitor reduces the enzyme’s ability to bind to the
substrate by occupying the active site. As a result, a higher
substrate concentration is required to reach half of the
maximum velocity (𝑉𝑚𝑎𝑥), increasing the apparent 𝐾𝑚
• 𝑉𝑚𝑎𝑥 (Maximum reaction velocity): Unchanged
• Since the inhibition is reversible, adding a sufficiently high
concentration of substrate can outcompete the inhibitor,
allowing the enzyme to achieve the same 𝑉𝑚𝑎𝑥 .Thus, in
competitive inhibition, the 𝐾𝑚 increases, but 𝑉𝑚𝑎𝑥 remains
unchanged.
Non-Competitive inhibition

• In this type of inhibition, inhibitor has no structural

similarity with substrate and binds with the enzyme at
different site other than the active site. Therefore, there
is no competition between S and I, and formation of
ES, EI and ESI takes place.
• The inhibitor I and substrate S can bind simultaneously
to the same enzyme molecule as their binding sites are
different and hence do not overlap.
• Non competitive inhibitor lowers the Vmax by
decreasing the proportion of enzyme molecules that
are bound to the S. Thus, the non-competitive inhibition
in contrast to competitive inhibition, cannot be
overcome by increasing substrate concentration.
• The substrate can still bind to the EI complex.
However, the ESI does not form product. The I
effectively lowers the concentration of active enzyme
and hence lowers the Vmax.
• There is no effect on Km as the inhibitor decrease the
amount of functional enzyme .
Uncompetitive inhibition

• In this type of inhibition, inhibitor does

not bind to free enzyme. It binds only to
enzyme substrate (ES) complex directly
or its binding is facilitated by the
conformational change that takes place
after substrate binds to enzyme.
• In both the cases, the inhibitor does not
compete with substrate for the same
binding site. Therefore, the inhibition
cannot be overcome by increasing
substrate concentration.
 Allosteric enzymes

• Enzymes that are regulated by the binding of effector

molecules (activators or inhibitors) at specific sites
called allosteric sites, distinct from the active site.
• Effector molecules:
❖ Positive effectors (activators): Increase enzyme
activity.
❖ Negative effectors (inhibitors): Decrease enzyme
activity.
• Conformational states: Exist in at least two forms:
❖ Tense (T): Less active or inactive
❖ Relaxed (R): More active
• Allosteric enzymes do not obey Michaelis Menten
kinetics.
• These enzymes in general consist of more than one
protein subunits, therefore, more than one active sites
are present. Allosteric enzymes result in sigmoidal
graph instead of rectangular hyperbola when V0 is
plotted against substrate concentration [S].
 RNA catalysis
RNA catalysis refers to the ability of RNA molecules to function as enzymes or ribozymes, catalyzing
biochemical reactions, much like protein enzymes. This discovery was groundbreaking because it
demonstrated that RNA could not only store genetic information but also participate actively in chemical
reactions, challenging the assumption that only proteins could act as biological catalysts.

What is RNA Catalysis?

RNA catalysis occurs when RNA molecules facilitate or accelerate chemical reactions without being
consumed in the process. These RNA molecules, known as ribozymes, have the ability to:
•Speed up chemical reactions (like enzymes).
•Interact with substrates to form or break bonds.
•Maintain their own structure and function as catalysts.
•They work by adopting specific structures that allow them to accelerate reactions.
 Hammerhead Ribozymes

Hammerhead Ribozymes:
•Found in some viruses and have a self-
cleaving mechanism.
•They catalyze the cleavage (breaking) of
their own RNA strands by forming a specific
structure that facilitates the chemical
reaction.
•Reaction: Hammerhead ribozymes can
catalyze the cleavage of RNA in the
presence of metal ions (e.g., Mg²⁺).
 Mechanisms of RNA Catalysis

RNA catalysis works through several Types of Reactions Catalysed by Ribozymes

mechanisms, most of which involve the RNA Ribozymes can catalyse various types of reactions,
molecule adopting a specific tertiary structure including:
that brings key functional groups into proximity •RNA Cleavage: Breaking phosphodiester bonds in
to perform the catalysis. Some of the RNA molecules. The hammerhead ribozyme catalyzes
mechanisms include: the cleavage of RNA by breaking the phosphodiester
•Acid-Base Catalysis: The RNA can act as an bond between nucleotides in the RNA molecule.
acid or base to donate or accept protons, •RNA Splicing: Removing introns (non-coding
facilitating bond formation or breaking. regions) from RNA sequences (e.g., Group I and II
•Stabilization of Transition States: RNA can introns).
stabilize high-energy intermediate states •Ligations: Joining RNA molecules
(transition states), lowering the activation •T4 RNA ligase is a viral enzyme that catalyzes the
energy of a reaction. ligation of RNA molecules
•Nucleophilic Attack: Certain RNA molecules •can catalyze the ligation of two RNA molecules by
can perform nucleophilic attacks on substrates, joining their 3' and 5' ends using a phosphodiester
which is common in self-cleaving ribozymes. bond.