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Basic Principles
of Enzyme
Activity
Presented by group 19 (181 – 190)
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are Master title style
Enzymes?
• Enzymes are biological catalysts that speed up biochemical
reactions without undergoing any permanent change themselves.
• They are mostly globular proteins, though some RNA molecules
(ribozymes) also act as enzymes.
• Each enzyme is highly specific for a particular substrate or type of
chemical reaction.
• Enzymes function under mild conditions of temperature and pH,
unlike chemical catalysts that require harsh environments.
• After catalysis, enzymes remain unchanged and can be reused.
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General Master titleof
Characteristics style
Enzymes
• Catalytic efficiency: Enzymes increase reaction rates by factors of
up to 10⁶–10¹⁷.
• Specificity: Each enzyme acts on specific substrates due to the
shape and chemical nature of its active site.
• Sensitivity: Enzyme activity is affected by temperature, pH, and
other conditions.
• Regulation: Enzyme activity can be increased or decreased
depending on cellular requirements.
• Reversibility: Many enzymatic reactions are reversible depending
on concentration and conditions.
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Chemical Master
Nature title style
of Enzymes
• Most enzymes are proteins, composed of amino acids
folded into specific 3D structures.
• The folding creates an active site, a small region where
catalysis occurs.
• Non-protein components such as metal ions or coenzymes
may be essential for activity.
• The structure of an enzyme determines its function;
denaturation (loss of shape) results in loss of activity.
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Principle title style
of Enzyme Action
• Chemical reactions require activation energy to reach the transition
state before forming products.
• Enzymes lower this activation energy, enabling the reaction to occur
more quickly and efficiently.
• The free energy change (ΔG) of the reaction remains the same—
enzymes affect rate, not equilibrium.
• They achieve this by stabilizing the transition state and bringing
substrates together in the correct orientation.
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Concept Master title
of Activation style
Energy
• Activation energy (Ea) is the minimum
amount of energy required for a chemical
reaction to begin.
• It represents the energy barrier that reactant
molecules must overcome to be converted
into products.
• Enzymes act by lowering the activation
energy barrier, making it easier for the
reaction to occur.
• They provide an alternative reaction
pathway where the transition state is
stabilized.
• Importantly, enzymes do not change:
• The energy of reactants or products.
• The overall free energy change (ΔG). 7 7
• The reaction equilibrium position.
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Enzymeto edit MasterComplex
Substrate title style
Formation
• The enzyme first binds to its substrate to
form a temporary enzyme–substrate (ES)
complex.
• The ES complex lowers activation energy
and brings about bond rearrangements.
• After the reaction, the product is released
and the enzyme is free to act again.
• This stepwise mechanism ensures precision
and prevents unwanted side reactions.
• Step 1: Enzyme (E) binds with substrate (S)
to form ES complex.
• Step 2: Reaction occurs, forming product (P)
and releasing enzyme.
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Mechanism Master title of
Lowering style
Activation Energy
• Enzymes lower activation energy by providing
an alternative reaction pathway with a
stabilized transition state.
• The active site binds substrates precisely,
bringing reactive groups close and orienting
them correctly.
• They stabilize the transition state through non-
covalent interactions (hydrogen bonds, ionic
bonds, hydrophobic interactions).
• Some enzymes strain substrate bonds,
making them easier to break.
• Others form temporary enzyme–substrate
intermediates, reducing the energy needed to
reach products. 9 9
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Modelsto of
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Enzyme title style
– Lock & Key Model
• Proposed by German chemist Emil Fischer in 1894.
• Suggests that the enzyme’s active site has a fixed, rigid shape complementary to the substrate’s
shape, just like a lock and its key.
• Only a specific substrate (key) can fit into the enzyme’s active site (lock).
• The binding forms an enzyme–substrate complex, leading to product formation without altering the
enzyme’s structure.
• Explains the specificity of enzymes: each enzyme acts on a particular substrate.
• The enzyme structure is rigid and unchangeable.
• Works well for simple reactions with highly specific substrates.
• Limitations:
• Fails to explain how enzymes can adapt to slightly different substrates.
• Does not account for enzyme flexibility observed experimentally.
• Cannot explain how enzymes stabilize the transition state or enhance catalysis.
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Induced Master title style
Fit Model
• Proposed by Daniel E. Koshland Jr. in 1958.
• Suggests that the enzyme’s active site is flexible, not rigid.
• When the substrate approaches, the enzyme adjusts its shape slightly so that the
active site fits more snugly around the substrate.
• This conformational change helps to stabilize the transition state and enhances
catalytic efficiency.
• Explains how enzymes can act on structurally related substrates.
• The enzyme changes shape to fit the substrate — an example of dynamic
molecular recognition.
• After the reaction, the enzyme returns to its original shape.
• Advantages:
• Better explains enzyme specificity and catalytic power.
• Accounts for experimental observations such as conformational flexibility. 1212
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Comparison Master title
thestyle
Two Models
Feature Lock and Key Model Induced Fit Model
Year 1894 1958
Scientist Emil Fischer Daniel Koshland
Active Site Rigid and pre-shaped Flexible and adaptable
Enzyme molds itself around
Substrate Fit Perfect fit like a key in a lock
substrate
Explains Enzyme specificity Specificity and catalytic efficiency
More accurate and accepted
Limitation Ignores enzyme flexibility
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ActivetoSite
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• Active site is a small pocket on the enzyme where the substrate binds and a reaction
occurs.
• Contains binding and catalytic sites.
• Made up of special amino acids with unique properties—can be acidic, basic,
hydrophobic, or hydrophilic.
• Its three-dimensional shape fits only specific substrates, like a lock and key.
• The active site does two main jobs: holds the substrate in place (binding site) and
speeds up the reaction (catalytic site).
• Specificity types:
• Absolute specificity: Enzyme works on only one substrate (like urease on urea).
• Group specificity: Works on a group of similar molecules (like pepsin works on
proteins).
• Stereochemical specificity: Recognizes a specific spatial form or isomer (works
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on only one “shape” of a molecule).
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Factors edit Master title style
Enzyme Activity: 1.
Temperature
• Enzyme activity increases with rising temperature because molecules
move faster, leading to more frequent enzyme–substrate collisions.
• There is an optimum temperature (usually around 37°C in humans)
where the enzyme works at its maximum rate.
• Beyond the optimum, the enzyme’s structure begins to denature —
hydrogen bonds and weak interactions break, causing loss of the active
site shape and thus enzyme activity.
• Below optimum, reactions slow because molecular motion and collision
frequency decrease.
• Example:Human enzymes function best near 37°C; thermophilic
enzymes (from bacteria in hot springs) can remain stable up to 70–80°C.
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2. pHto edit Master title style
• Enzyme activity depends strongly on pH, which affects the ionization of amino
acid residues at the active site and substrate.
• Each enzyme has an optimum pH at which its three-dimensional structure and
charge distribution are ideal for catalysis.
• Changes in pH alter electrostatic interactions, possibly causing denaturation or
loss of substrate binding.
• Examples:
• Pepsin (stomach): optimum at pH 2 (acidic).
• Trypsin (small intestine): optimum at pH 8 (alkaline).
• Salivary amylase: optimum at pH 7 (neutral).
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3. Substrate title style
Concentration
• At low substrate concentration, the reaction rate is directly
proportional to the substrate amount — more substrate means more
frequent binding with enzyme active sites.
• As substrate concentration increases, enzymes become saturated,
and rate reaches a maximum velocity (Vmax) where all active sites
are occupied.
• Beyond this point, adding more substrate does not increase reaction
rate.
• This relationship is described by Michaelis–Menten kinetics, where
Km is the substrate concentration at half of Vmax.
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Michaelis–Menten title style
Equation
• Describes how enzyme-catalyzed reaction rate varies with substrate
concentration.
• Proposed by Leonor Michaelis and Maud Menten in 1913.
• Explains how enzymes reach a maximum velocity (Vmax) when
active sites are fully occupied.
• The model assumes:
• Formation of enzyme–substrate (ES) complex.
• ES complex either breaks down into products or dissociates back
into enzyme and substrate.
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The rateto edit
of an Master titlereaction
enzyme-catalyzed style is given by:
Where:
• V = reaction velocity
• Vmax = maximum velocity (enzyme saturated with substrate)
• [S] = substrate concentration
• Km = Michaelis constant (substrate concentration at which V = ½
Vmax)
Interpretation:
• Low [S] (< Km): rate increases nearly linearly with [S].
• High [S] (> Km): rate approaches Vmax (enzyme saturated). 22
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Significance titleVmax
of Km and style
• Km indicates enzyme affinity for the substrate:
• Low Km → high affinity (enzyme binds substrate tightly).
• High Km → low affinity.
• Vmax represents the maximum rate achieved when all enzyme
molecules are bound to substrate.
• Both parameters help in comparing enzyme efficiency and
understanding drug inhibition and metabolic regulation.
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4. Enzyme Master title style
Concentration
• When substrate is present in
excess, increasing enzyme
concentration leads to a
proportional increase in reaction
rate since more enzymes mean
more active sites for catalysis.
• Eventually, if substrate becomes
limiting, further increase in
enzyme concentration does not
affect rate.
• The reaction rate thus depends
on the availability of both enzyme
and substrate.
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5. Inhibitors title style
and Activators
• Inhibitors are molecules that reduce or block enzyme activity:
• Competitive inhibitors compete with substrate for the active site (e.g.,
malonate inhibits succinate dehydrogenase).
• Non-competitive inhibitors bind elsewhere, altering enzyme conformation
(e.g., heavy metals).
• Activators enhance enzyme activity by stabilizing active conformation or
participating in catalysis (e.g., metal ions like Mg²⁺ for kinases).
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Inhibition
• Enzyme inhibition occurs when a molecule (inhibitor) reduces or
prevents enzyme activity.
• Inhibitors are vital in regulating metabolic pathways, designing
drugs, and understanding enzyme mechanisms.
• There are three major types of reversible inhibition:
• Competitive
• Non-competitive
• Uncompetitive
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Competitive title style
Inhibition
• The inhibitor closely resembles the substrate and competes for the
enzyme’s active site.
• Binding is reversible and can be overcome by increasing substrate
concentration.
• As a result: Km increases (more substrate needed to reach half Vmax).
Vmax remains unchanged (at high substrate, enzyme fully active).
• Example: Malonate inhibits succinate dehydrogenase (in the Krebs
cycle).
• Mechanism Summary:
E + S ⇌ ES → E + P
E + I ⇌ EI (inactive complex) 27
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Non-Competitive title style
Inhibition
• Inhibitor binds to a site other than
the active site (allosteric site).
• Binding causes a conformational
change that reduces catalytic
efficiency.
• Substrate can still bind, but the
enzyme cannot convert it effectively.
• Effect: Vmax decreases (less active
enzyme available). Km remains
constant (affinity unaffected).
• Example: Cyanide inhibits
cytochrome oxidase, blocking
cellular respiration. 29
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Uncompetitive title style
Inhibition
• Inhibitor binds only to the enzyme–
substrate complex, not to free
enzyme.
• This stabilizes the ES complex and
prevents product formation.
• Effect: Both Km and Vmax
decrease.
• Rare type, but important in multi-
substrate reactions.
• Example: Lithium inhibition of
inositol monophosphatase (used in
bipolar disorder treatment). 30
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Co-factors
• Enzymes often require non-protein components for their activity. These non-
protein components are collectively called co-factors.
• Co-factors are non-protein substances that assist enzymes in catalyzing
biochemical reactions.
• They may help by:
• Stabilizing enzyme structure
• Participating in the reaction (acting as carriers of electrons, atoms, or
functional groups)
• Types of Cofactors:
• Metal ions
• Co-enzymes
• Prosthetic groups
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Co-factors
1. Metal Ions:
• Certain enzymes require metal ions (inorganic cofactors) for activity.
• These ions can help in substrate binding, stabilizing enzyme-substrate
complex and participating directly in catalysis
2. Co-enzymes
• Coenzymes are organic, non-protein molecules that act as transient
carriers of specific atoms or functional groups during enzymatic
reactions. They are often derived from vitamins, especially B-complex.
3. Prosthetic Group
• A prosthetic group is a cofactor that is tightly or covalently bound to the
enzyme throughout the catalytic cycle. Examples: FAD in succinate
dehydrogenase, Heme in cytochromes and catalase, Biotin in pyruvate
carboxylase 32
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Examples of Master title style
Metal Ions
Metal ion Example enzyme Role
Catalytic role in hydration of
Zn²⁺ Carbonic anhydrase
CO₂
Hexokinase, DNA Stabilizes ATP and
Mg²⁺
polymerase substrates
Fe²⁺ / Fe³⁺ Catalase, cytochromes Electron transfer
Cu²⁺ Cytochrome oxidase Electron transport
Arginase, superoxide
Mn²⁺ Catalytic role 33
dismutase 33
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Examples of Master title style
Co-enzymes
Coenzyme Vitamin Source Function
NAD (Nicotinamide Adenine Acts as an electron carrier in oxidation–reduction reactions (e.g.,
Niacin (Vitamin B₃)
Dinucleotide) glycolysis, TCA cycle).
FAD (Flavin Adenine Participates in redox reactions, particularly in the electron transport chain
Riboflavin (Vitamin B₂)
Dinucleotide) and fatty acid oxidation.
Pantothenic acid (Vitamin Transfers acyl groups in metabolism; essential in the formation of acetyl-
CoA (Coenzyme A)
B₅) CoA and fatty acid synthesis.
Coenzyme for decarboxylation of α-keto acids (e.g., pyruvate
TPP (Thiamine Pyrophosphate) Thiamine (Vitamin B₁)
dehydrogenase, α-ketoglutarate dehydrogenase).
Involved in transamination, deamination, and decarboxylation of amino
PLP (Pyridoxal Phosphate) Pyridoxine (Vitamin B₆)
acids.
Acts as a coenzyme for carboxylation reactions (e.g., pyruvate
Biotin Biotin (Vitamin B₇) 34
carboxylase, acetyl-CoA carboxylase). 34
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Apoenzyme Master title style
Holoenzyme
• Apoenzyme: Inactive protein
part of the enzyme.
• Cofactor: Non-protein
component essential for
catalytic activity.
• Holoenzyme: Complete, active
enzyme formed by combination
of apoenzyme + co-factor.
• Cofactor ensures correct
folding, substrate orientation,
and charge stabilization during
catalysis.
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Regulation
• Ensures reactions occur at appropriate rates for cellular balance.
• Prevents excess formation or depletion of intermediates.
• Regulation mechanisms include:
• Allosteric regulation
• Feedback inhibition
• Covalent modification
• Genetic control (enzyme synthesis rate)
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Allosteric Master title style
Regulation
• Allosteric enzymes have separate regulatory (allosteric) sites distinct
from the active site.
• Allosteric effectors (activators or inhibitors) bind to these sites,
causing conformational changes.
• Positive effectors: enhance enzyme activity.
• Negative effectors: reduce activity.
• Often found in enzymes that regulate key metabolic pathways.
• Example: Phosphofructokinase (PFK): Inhibited by ATP (high energy
state). Activated by AMP (low energy state).
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Feedback Master title style
Inhibition
• The end product of a metabolic pathway inhibits the first
committed enzyme in that pathway.
• Acts as an automatic control mechanism to prevent
overproduction.
• Maintains homeostasis and efficient resource use.
• Example: Isoleucine inhibits threonine deaminase in amino acid
synthesis.
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Covalent Master title style
Modification
• Involves reversible chemical changes in enzyme structure that
affect activity.
• Common types:
• Phosphorylation (addition of phosphate group).
• Methylation, acetylation, etc.
• Modifications are usually enzyme-controlled and can rapidly switch
enzymes on/off.
• Example: Glycogen phosphorylase activated by phosphorylation
→ enhances glycogen breakdown.
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Clinical edit Master title style
of Enzymes
• Enzyme levels serve as diagnostic markers for tissue damage or
disease.
• ALT (Alanine Aminotransferase), AST (Aspartate Aminotransferase):
Liver damage (hepatitis, cirrhosis).
• CK-MB: Myocardial infarction (heart attack).
• Amylase, Lipase: Pancreatitis.
• Therapeutic enzymes: Streptokinase: Dissolves blood clots.
• Asparaginase: Used in leukemia treatment.
• Enzyme assays help in diagnosis, prognosis, and therapy monitoring.
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Industrial Applications
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• Enzymes are widely used in industrial, medical, and environmental
applications.
• Food Industry:
• Amylases: Convert starch into sugar (brewing, baking).
• Proteases: Tenderize meat, clarify beer.
• Detergent Industry:
• Proteases & lipases: Remove protein and fat stains at low
temperatures.
• Textile Industry:
• Cellulases: Soften fabrics and enhance texture.
• Biotechnology:
• DNA polymerases, restriction enzymes: Used in genetic engineering, 43
PCR, and cloning. 43
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Thank
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