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SKN NEW STB BIOLOGY NOTES

ENZYMES
Introduction
1. Role of Enzymes in Metabolism:
o Metabolic activities in cells rely on enzymes, which are catalytic molecules.
o Without enzymes, the steady state of the cell would cease, halting life processes.
2. Definition of Life's Biochemical Nature:
o Life involves a highly coordinated chemical relationship for reactions that either
synthesize or break down large molecules.
o These reactions require enzymes for efficiency as they occur at low temperatures
and normal pressure.
3. Specialized Substances:
o Biocatalysts, synthesized within living systems, increase reaction rates.
o The term enzyme was coined by Friedrich Wilhelm Kuhn in 1878.
o Enzymes are defined as organic substances capable of catalyzing specific
biochemical reactions in living systems.
Discovery of Enzymes
1. Enzyme Composition:
o Historically, all enzymes were believed to be proteins.
o In the 1980s, Thomas Cech and Sidney Altman discovered that certain nucleic
acids (RNA molecules) can also function as enzymes, referred to as Ribozymes.
2. Function of Ribozymes:
o Catalyze reactions involving genetic information processing.
o Commonly act as protein enzymes but exist as an exception.
Characteristics of Enzymes
1. General Features:
o Biocatalysts:
▪ Produced in the protoplasm and synthesized in the cell.
o Proteinaceous Nature:
▪ Most enzymes are protein-based macromolecules with high molecular
weight.
o Types:
▪ Some enzymes are pure proteins (e.g., amylase).
▪ Some enzymes include a non-protein part, e.g., Acetyl CoA.
2. Location and Production:
o Enzymes are created within cells and can be classified as:
▪ Endo-enzymes: Function inside the cell.
▪ Exo-enzymes: Function outside the cell.
o These enzymes differ in nature and function.
3. Enzyme-Substrate Relationship:
o Substrate: A molecule that interacts with the enzyme.

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 o Enzymes are specific to their substrates, with active sites tailored for the
substrate's size and shape.
Factors Influencing Enzymes
1. Catalytic Acceleration:
o Enzyme activity can be increased by ions or salts such as Ca²⁺, K⁺, Mg²⁺, Cl⁻.
2. Enzyme Inhibition:
o Certain factors, called inhibitors (e.g., substrate concentration, pH, or
temperature), can block enzyme activity.
3. Reusability:
o Enzymes remain chemically unchanged after completing reactions.
Key Terms Defined
1. Active Site:
o A region on the enzyme where the substrate binds to initiate a reaction.
2. Substrate:
o The specific molecule acted upon by the enzyme during the reaction.
2.1 Structure of Enzymes
1. Overview:
o Enzymes are three-dimensional globular proteins.
o They specifically react with their designated substrates.
Active Site
1. Definition:
o Each enzyme has a special charged region called the active site.
o This region complements the shape of the substrate for precise binding.
2. Formation:
o Formed by specific amino acids from the polypeptide chain, allowing it to fold
into a unique shape for the substrate.
3. Components:
o Binding Site:
▪ Responsible for attaching to the substrate.
o Catalytic Site:
▪ Contains amino acids that facilitate the chemical reaction.
Structural Parts of Enzymes
1. Protein and Non-Protein Components:
o Some enzymes are purely proteins.
o Others include non-protein parts, called co-factors.
2. Co-Factors:
o Facilitate the enzyme’s attachment to the substrate and assist in catalytic activity.
o Can be:
▪ Organic Co-Factors (e.g., vitamins).
▪ Inorganic Co-Factors (e.g., metallic ions like Fe, Mg).
3. Activator Co-Factors:
o Attach during the substrate binding process to enhance enzyme functionality.

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 4. Co-Enzymes:
o Organic derivatives, often derived from vitamins (e.g., NAD, FAD).
o Bind loosely and are detachable.
5. Prosthetic Groups:
o Permanently attached to the enzyme.
o Example: Mg-containing chlorophyll.
Types of Enzymes
1. Holoenzyme:
o A complete enzyme with its co-factor or prosthetic group.
2. Apoenzyme:
o An enzyme without its co-factor, which is inactive.
Example: Pepsin
1. Inactive Form:
o Pepsinogen is the inactive form of pepsin.
o Activation occurs when specific polypeptides are removed, allowing the active
site to form.
2.2 Mechanism of Enzyme Action
1. Overview:
o Enzymes catalyze metabolic reactions by binding to substrates at their active
sites.
o A temporary enzyme-substrate complex (ES) forms, converting the substrate
into the product.
o The enzyme-product complex (EP) releases the product, leaving the enzyme
unchanged for further reactions.
2. Conservation:
o The quantity and shape of the enzyme remain constant before and after the
reaction.
Models Explaining Enzyme Action
1. Lock and Key Model (Emil Fischer, 1898):
o Concept:
▪ Enzymes act like locks, and substrates are the keys.
▪ The substrate’s shape perfectly matches the enzyme's active site.
o Key Features:
▪ The enzyme's active site is rigid and specific to the substrate.
▪ Explains how enzymes demonstrate absolute specificity, catalyzing only
one specific substrate.
2. Induced Fit Model (Koshland, 1959):
o Concept:
▪ Substrates induce a conformational change in the enzyme structure upon
binding.
▪ This enhances the fit between the enzyme and the substrate.
o Key Features:
▪ Active sites are flexible and adjust to accommodate the substrate.

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 ▪ The model accounts for enzymes being able to catalyze structurally
similar molecules.
Reaction Stages:
1. E + S → ES:
o Enzyme binds to the substrate, forming the enzyme-substrate complex.
2. ES → EP:
o Substrate undergoes transformation into the product.
3. EP → E + P:
o Product is released, leaving the enzyme unchanged.
1. Flexible Active Site:
o The active site of an enzyme is flexible and adapts slightly to the shape of the
substrate.
o This adaptability allows the substrate to bind more effectively.
o This change facilitates the conversion of the substrate into the product during
the catalytic reaction.
2. Illustration Summary (Fig. 2.2 Induced Model):
o Substrate binds to enzyme: The active site adjusts its shape to fit the substrate.
o Enzyme-substrate complex: The substrate undergoes a chemical reaction.
o Product release: The enzyme returns to its original state, ready for another
reaction.
2.2.1 Energy of Activation
1. Overview:
o Enzymes enable thermodynamically favorable reactions to proceed at a faster
rate.
o Without enzymes, these reactions would occur too slowly due to high energy
barriers.
2. Chemical Transformation:
o Reactants require sufficient energy to overcome activation energy and break
chemical bonds.
o Activation Energy:
▪ The minimum energy needed for molecules to undergo transformation into
products.
Energy of Activation with and Without Enzymes
1. Without Enzymes:
o Reactants require high activation energy to proceed.
o In non-living systems, heat is often used to provide the necessary energy,
increasing molecular collisions.
2. With Enzymes:
o Enzymes lower the activation energy significantly, allowing reactions to occur
under physiological conditions (e.g., normal body temperature).
o They enhance reaction rates without being consumed in the process.

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Importance of Enzyme Action
1. Formation of Enzyme-Substrate Complex:
o Enzymes form a complex with the substrate, stabilizing the transition state and
facilitating bond breaking and formation.
o This complex reduces the energy needed for the reaction.
2. Breakdown of the Complex:
o Once the reaction is complete, the product is released, and the enzyme remains
unaltered for reuse.
3. Equilibrium:
o Enzymes accelerate reactions but do not alter the equilibrium state (ratio of
reactants to products).

Illustration Summary (Fig. 2.3 Energy of Activation):

1. Activation Energy Without Enzymes:
o The red curve shows the high activation energy required for a reaction to proceed
naturally.
2. Activation Energy With Enzymes:
o The green curve illustrates how enzymes significantly reduce the activation
energy.
3. Energy of Reactants and Products:
o The overall energy difference between reactants and products (ΔG) remains
unchanged.
2.3 Factors Affecting Enzyme Activity
1. Definition of Enzyme Activity:
o The rate of enzyme activity is defined as the quantity of substrate transformed
into product within a unit time (e.g., 1 second).
o The amount of product formed per second is directly proportional to the
enzyme activity.
o Enzyme activity can vary based on several factors such as:
▪ Enzyme concentration.
▪ Substrate concentration.
▪ Environmental conditions like temperature and pH.

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 2. Allosteric Regulation:
o Inhibitors:
▪ Inhibitors bind to the enzyme at sites away from the active site, known as
allosteric sites.
▪ Binding causes the enzyme's shape to change, making its active site
unsuitable for substrate binding.
▪ As a result, the enzyme's activity decreases.
o Activators:
▪ Certain molecules bind at the allosteric site and enhance the enzyme’s
ability to bind the substrate, increasing enzyme activity.
o Characteristics of Allosteric Regulation:
▪ Allosteric inhibitors and activators do not bind covalently to enzymes.
▪ Their interactions are reversible.
o The activity of enzymes is influenced by:
▪ Thermal factors (temperature).
▪ Concentration changes in substrates and inhibitors.
2.3.1 Temperature
1. Temperature and Reaction Rate:
o The rate of chemical reactions depends on molecular motion, which increases
with higher temperatures.
o A 10°C increase in temperature can double the rate of reaction because of:
▪ Higher collision frequency between molecules.
▪ Faster formation of enzyme-substrate complexes.
2. Optimum Temperature:
o Enzymes exhibit maximum activity at an optimum temperature.
o This temperature range is generally around 25°C to 42°C for enzymes in human
systems.
o At this point:
▪ Enzymes achieve their highest rate of catalysis.
3. Effects of Exceeding Optimum Temperature:
o Above Optimum Temperature:
▪ Enzyme activity decreases rapidly due to structural denaturation.
▪ Denaturation causes the enzyme's active site to lose its functional shape,
making it incapable of binding substrates.
o At 100°C or Higher:
▪ Most enzymes are completely inactive and destroyed.
o At Minimal Temperatures:
▪ Enzymes remain undamaged but show reduced activity because of slowed
molecular motion.
4. Thermophiles:
o Certain bacteria, called thermophiles, thrive at extremely high temperatures (e.g.,
70°C or higher).
o Enzymes from thermophiles are heat-stable and find applications in:

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 ▪ Biological washing detergents designed for high-temperature usage.
▪ Other industrial processes requiring high-heat conditions.

Illustration Summary: Fig. 2.4 Effect of Temperature on Enzymes

1. Graph Breakdown:
o Temperature Increase Phase:
▪ As the temperature rises, enzyme activity steadily increases due to higher
collision rates between enzymes and substrates.
o Optimum Temperature:
▪ The peak of enzyme activity where the rate of reaction is at its highest.
▪ Beyond this temperature, activity begins to decline.
o Denaturation Phase:
▪ A sharp drop in enzyme activity occurs as enzymes lose their structural
integrity.
o Inactive Zone:
▪ Enzymes are fully denatured and no longer functional beyond this point.
2. Enzymatic Stability:
o Enzymes show maximum efficiency within their optimum temperature range.
o Stability diminishes at extreme temperatures.
2.3.2 Effect of pH
1. Definition:
o Enzyme activity is highly sensitive to pH, with each enzyme having an optimum
pH at which it works most efficiently.
o A narrow pH range is generally effective for enzymes.
2. Effect of pH on Enzyme Structure:
o Changes in pH affect the ionic and hydrogen bonds in amino acid side chains.
o These changes can alter the tertiary and quaternary structure of proteins,
impacting the active site of the enzyme.
3. Examples of Enzymes and Their Optimum pH:
o Pepsin:
▪ Found in the stomach, works best in highly acidic conditions (optimum
pH: 1.4).
▪ Functional pH range: 1.5 to 2.5.

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 o Salivary Amylase:
▪ Found in saliva, works best in neutral pH (around 7.0).
o Alkaline Phosphatase:
▪ Found in the pancreas, works best at a highly alkaline pH (optimum pH:
8.5).
4. Illustration Summary (Fig. 2.5 Effect of pH):
o Graph shows the relative activity of enzymes like pepsin, salivary amylase, and
alkaline phosphatase at varying pH levels, with distinct peaks corresponding to
their optimum pH.
2.3.3 Enzyme Concentration
1. Definition:
o Enzyme activity is influenced by the availability of enzymes and substrate
molecules.
2. Impact of Enzyme Concentration:
o At low substrate concentrations:
▪ Enzyme activity is directly proportional to enzyme concentration.
o At high substrate concentrations:
▪ Once substrate availability becomes constant, increasing enzyme
concentration will initially increase activity.
▪ After a certain point, enzyme activity remains constant as all substrates
are already bound to enzymes.
3. Illustration Summary (Fig. 2.6 Enzyme Concentration Graph):
o Graph shows that enzyme activity increases with enzyme concentration until
reaching a plateau where further increases have no effect.
2.3.4 Substrate Concentration
1. Definition:
o Enzyme activity increases with substrate concentration up to a certain point.
2. Substrate Saturation:
o At low substrate concentrations:
▪ Enzyme activity increases as more substrate molecules are available for
binding.
o At high substrate concentrations:
▪ A point of saturation is reached where all enzyme active sites are
occupied, and further increases in substrate concentration do not affect
reaction rate.
o Beyond saturation:
▪ Excess substrate may exert a retarding effect on enzyme action.
3. Illustration Summary (Fig. 2.7 Substrate Concentration Graph):
o The graph depicts enzyme activity rising sharply with substrate concentration
before leveling off at the saturation point.

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2.4 Enzyme Inhibition
1. Definition:
o Enzyme inhibition refers to the reduction or halting of enzyme activity due to
certain conditions or molecules.
o It occurs when:
▪ The system no longer requires the products.
▪ Enzymatic activity needs to be regulated or stopped.
2. Causes of Enzyme Inhibition:
o Excess of substrate relative to enzyme.
o Accumulation of the end product, which may feedback to inhibit the enzyme.
3. Effect on Reaction Rate:
o Initially, enzyme activity increases proportionally with substrate concentration.
o Once the enzyme reaches its maximum velocity (Vmax), further increases in
substrate concentration have no effect on the reaction rate.
2.4.1 Types of Enzyme Inhibition
1. Definition of Inhibitors:
o Inhibitors are substances that decrease enzyme activity by:
▪ Binding to enzymes (either at the active site or other regions).
▪ Blocking interaction between the enzyme and activators.
2. Three Major Types of Inhibition:
o Competitive Inhibition:
▪ Mechanism:
▪ Molecules resembling the substrate in structure compete with the
actual substrate for binding to the enzyme's active site.
▪ These inhibitors block the active site, preventing substrate binding.
▪ Example:
▪ Penicillin acts as a competitive inhibitor by blocking bacterial
enzymes responsible for cell wall synthesis.
▪ Reversibility:
▪ This inhibition can be reversed by increasing substrate
concentration. A higher concentration of the substrate can
outcompete the inhibitor and restore enzyme activity.
o Non-Competitive Inhibition:
▪ Mechanism:
▪ Inhibitors bind to an enzyme at a site other than the active site,
known as the allosteric site.
▪ This binding causes a change in the enzyme's shape, reducing or
completely blocking its activity.
▪ Effect on Function:
▪ Even if the substrate binds to the enzyme, the enzyme cannot
catalyze the reaction effectively.

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 ▪ Reversibility:
▪ This inhibition is reversible when the inhibitor detaches from the
enzyme, restoring normal enzyme activity.
o Uncompetitive Inhibition:
▪ Mechanism:
▪ Inhibitors bind only to the enzyme-substrate complex (ES-
complex), not to the free enzyme.
▪ This prevents the release of the product, halting the reaction.
▪ Effect:
▪ The inhibition specifically affects reactions involving two or more
substrates or when the complex is essential for catalysis.
▪ Outcome:
▪ The inhibitor permanently reduces the efficiency of the reaction
until it is removed.
Key Highlights:
• Competitive Inhibition:
o Inhibitor binds to the active site.
o Can be overcome by increasing substrate concentration.
o Example: Penicillin.
• Non-Competitive Inhibition:
o Inhibitor binds to the allosteric site (not the active site).
o Alters enzyme structure and function.
o Substrate binding may still occur, but no reaction takes place.
• Uncompetitive Inhibition:
o Inhibitor binds only to the enzyme-substrate complex.
o Prevents product formation and release.
o Common in multi-substrate reactions.
2.4.2 Significance of Enzyme Inhibition
1. Role in Biological Systems:
o Enzyme inhibition is a critical control mechanism in biological systems.
o It is essential for regulating metabolic pathways and maintaining homeostasis.
2. Applications:
o Drug Discovery:
▪ Used to identify molecules that can inhibit enzymes related to diseases.
o Metabolic Pathway Analysis:
▪ Helps understand substrates and conditions essential for catalysis.
o Disease Screening:
▪ Enzyme inhibitors are employed to detect diseases driven by overactivity
of enzymes.
2.4.3 Feedback Inhibition
1. Definition:
o A biological control mechanism where the end product of a metabolic pathway
regulates the activity of an enzyme earlier in the pathway.

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 2. Mechanism:
o Usually occurs through non-competitive inhibition.
o When the product of a pathway accumulates to a high concentration, it binds to an
enzyme at an allosteric site, inhibiting further production.
o This ensures the pathway is regulated and avoids overproduction of unnecessary
compounds.
3. Examples in Enzymatic Pathways:
o Feedback inhibition ensures that metabolic pathways shut down when sufficient
product is present, saving energy and resources.
2.5 Classification of Enzymes
1. Basis of Classification:
o Enzymes are classified based on:
▪ The type of reaction they catalyze.
▪ Their substrate-specific activity.
2. Classification Based on Reaction Types:
o 1. Oxido-Reductase:
▪ Catalyze oxidation and reduction reactions.
▪ Example: Removal of hydrogen or addition of oxygen, as in Ferredoxin
reducing substance.
o 2. Transferase:
▪ Transfer functional groups from one molecule to another.
▪ Example: Phosphates are transferred from ATP to hexose.
o 3. Hydrolase:
▪ Responsible for breaking down substances by adding water (hydrolysis).
▪ Example: Digestive enzymes.
o 4. Lyase:
▪ Catalyze the breakdown of bonds without water.
▪ Example: Removal of a specific group.
o 5. Isomerase:
▪ Facilitate the rearrangement of atoms within a molecule.
▪ Example: Conversion of glucose-6-phosphate to fructose-6-phosphate
by enzyme phosphohexose.
6. Ligase (Synthetase):
o Function:
▪ Catalyzes the condensation reactions such as the formation of DNA and
RNA.
▪ Example: DNA polymerase and RNA polymerase.
2.5.1 Classification Based on Substrate:
1. Protease:
o Acts on proteins to break peptide bonds.
o Examples:
▪ Trypsin, pepsin, etc.

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2. Lipase:
o Catalyzes the hydrolysis of fats into glycerol and fatty acids.
3. Glycosidase:
o Acts on carbohydrate molecules.
o Examples:
▪ Amylase:
▪ Converts starch into maltose.
▪ Cellulase:
▪ Acts on cellulose.
▪ Maltase:
▪ Acts on maltose.
▪ Lactase:
▪ Acts on lactose.
▪ Sucrase:
▪ Acts on sucrose.
4. Nuclease:
o Acts on nucleic acids (DNA and RNA).
o Examples:
▪ RNAase, DNAase, ATPase, etc.

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