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COURSE OUTLINE

 Cell Metabolism

 Enzymes and Catalysis

Module-III BIOCHEMISTRY  Cell Communication and Signalling

CELL METABOLISM:
CELLULAR RESPIRATION
CATABOLISM AND ANABOLISM
 Cell Metabolism is sum of all biochemical  Living organisms store their energy in the form of
reactions that occur in a cell. These reactions
can be spontaneous or non-spontaneous. biochemical-bonds in all organic molecules such as
carbohydrates, proteins, lipids and nucleic acid.
 The spontaneous reactions release energy
whereas the non-spontaneous reactions  Cellular Respiration is a set of metabolic processes which
require energy in order to proceed. catabolize these energetic organic molecules and harness the
 The metabolism can be catabolic or anabolic: required energy for all cellular activities.

 Catabolism ( Catabolic Reactions)  It breaks down the large biomolecules into smaller ones,
 Reactions which break down molecules and releasing energy in the process, as weak (so-called "high-
release energy energy“) bonds are replaced by stronger bonds in the
 Example: Respiration products.
 Glucose molecules break down and releases
energy  In simple words, the energy in glucose is converted into ATP
(energy molecule).
 Anabolism (Anabolic Reactions)
 Reactions which synthesize molecules and use  The pathways of cellular respiration include:
stored energy
1. Glycolysis
 Example: Photosynthesis
 Plants synthesize glucose molecules by using light 2. Kreb’s cycle
energy from sun. 3. Electron transport chain.
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CELLULAR RESPIRATION & PHOTOSYNTHESIS: Energy Transformation in Living Systems

CONNECTION
PHOTOSYNTHESIS endothermic redox process
 Photosynthesis and cellular respiration are connected through an important
relationship. This relationship enables life to survive on earth.
Photosynthesis: 6CO2 + 6H2O → C6H12O6+ 6O2
Cellular Respiration: C6H12O6 + 6O2 → 6CO2 + 6H2O

 The products of one process are the

reactants of the other.
 Notice that the equation for cellular RESPIRATION
respiration is the direct opposite of
photosynthesis:
 Photosynthesis makes the glucose that is
used in cellular respiration to make ATP.
 The glucose is then turned back into
carbon dioxide, which is used in
photosynthesis.
A series of redox reactions ….

ATP AND ENERGY ATP AND ENERGY

 Cellular energy currency. ATP is the energy  Most of the ATP made in cellular respiration
carrying molecule that biological cells use for comes from the stepwise release of energy, of a
producing energy. series of oxidation-reduction reactions between
molecules embedded in mitochondria
 Regardless of whether the original form of energy (eukaryotes) or the plasma membrane
is sunlight or food, it must ultimately be (prokaryotes).
converted to ATP.
 Energy released when 3rd phosphate of ATP
 For most organisms, this conversion is molecule is broken off through hydrolysis. Now
accomplished though cellular respiration in which the left over ADP again combines with inorganic
glucose is broken down and the energy extracted phosphate to recreate the ATP molecule. Its like
is converted to ATP. rechargeable battery.
 The energy in ATP is stored in its phosphate  ATP --> ADP + P + Energy
bonds(yellow circle).  ADP + P + Energy ---> ATP
 The covalent bond holding the third phosphate  The covalent bond holding the third phosphate
group carries about 7,300 calories of energy. group carries about 7,300 calories of energy.
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BIOLOGICAL ENERGY TRANSDUCTIONS BIOLOGICAL ENERGY TRANSDUCTIONS

OBEY THE LAWS OF THERMODYNAMİCS OBEY THE LAWS OF THERMODYNAMİCS

 First Law:  Second Law:

 Law of conservation of energy: According to the  “The entropy of the universe is always
law “Energy can not be created or destroyed but it increasing”
can be changed from one form to another”. The  The sum of the entropies of a system and its
total amount of energy will remain the same surroundings must always increase. In other
before and after each transfer. words, the entropy or the disorder of the
 Photosynthesis: Light energy  Chemical energy
universe is always increasing. Within a system
there is a tendency to go towards higher
 Muscle: Chemical energy  Mechanical energy entropy.
 There seems to be a preferred direction in which  Classic Analogy example : Our bedrooms
energy flows from one form to another form.  Increase in entropy of a system is With the time it becomes
 But why it flows into preferred directions? thermodynamically favorable. messy. But it won’t
 Its answer lies in 2nd law of thermodynamics  Third Law: suddenly become neat.
 A perfectly crystalline solid at absolute zero has
an entropy of zero. As this is the most ordered
state that a substance can be in.

THERMODYNAMICS SYSTEMS : LAWS OF THERMODYNAMICS

ISOLATED, CLOSED AND OPEN APPLY TO LIVING ORGANISMS
 A system is the portion of the universe with which we’re  Biochemical reactions are the source of energy for living systems. These
reactions either release energy or absorb energy from the surrounding.
concerned (e.g., an organism or a rock or an ecosystem)
 Energy transformation in living organisms is governed by the laws of
 If it doesn’t exchange energy or matter with the outside, it’s thermodynamics.
isolated.  First Law conditions: Energy Conservation
 Living organisms cannot create or destroy energy.
 If it exchanges energy but not matter, it’s closed  Living organism may transform energy from one form to another.

 If it exchanges energy & matter, it’s open  The living systems are capable of extracting the useable energy from
biochemical reactions and release useless energy (heat) back to their
surroundings.
 Living organisms are open systems. They exchange both
material and energy with their surroundings.  Second Law conditions: Increase in Entropy
 In the process of energy transformation, the living system increases the entropy
of the system (or universe).
 Living systems are never at equilibrium with their surroundings.
 Biochemical Reactions are favorable when the free energy of products is
much lower than the free energy of reactants.
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THERMODYNAMICS AND
BIOCHEMICAL REACTIONS
G
GIBBS FREE ENERGY:
Reaction Coordinate

 Thermodynamics characterizes the energy associated with equilibrium conditions in reactions  Gibbs free energy (G) expresses the amount of energy capable of doing work
 Kinetics describes the rate at which a reaction moves toward equilibrium during a reaction at constant temperature and pressure.
 Free energy is directly related to the equilibrium of a reaction
 Means, the portion of a system's energy that can perform work
 It doesn’t tell us how fast the system will come to equilibrium
 It can be calculated as:
 Kinetics, and the way that enzymes influence kinetics, tell us about rates
 Equilibrium constant is a measure of the ratio of product concentrations to reactant 𝜟𝐆 = 𝜟H - T 𝜟S
concentrations at equilibrium.  ΔG = Change in Gibbs free energy of the reacting system (Gproducts– Greactants) (joules/mole or calories/mole )
 At equilibrium, the rate of forward reaction is equal to rate of reverse reaction. Both reactions  ΔH = Change in enthalpy of the reacting system (Hproducts – Hreactants) (Kilo Joules)
continue to occur, but the overall concentrations of products and reactants no longer change.
Each reaction has its own unique, characteristic ratio of products to reactants at equilibrium.  T = Absolute temperature (Kelvin)
 Free energy is a measure of the available energy in the products and reactants  ΔS = Change in entropy of the reacting system(Sproducts – Sreactants) (Joules/Kelvin)
 They’re related by Go = -RT ln Keq  Enthalpy(H): It is a measure of total energy of a system.
 Entropy(S): Entropy is a measure of how energy is distributed within a system.
Higher the disordered system, more is the entropy.

ENERGY REACTIONS: ENDOTHERMIC AND

ENTHALPY AND ENTROPY EXOTHERMIC

 Endothermic Process or Reaction:

 Enthalpy (H): H is the heat content of the reacting system. H reflects  Require energy or heat to begin the Endothermic Energy input
the number and kinds of chemical bonds in the reactants and products. process
Process

 When a chemical reaction releases heat, it is said to be exothermic, the heat  ΔH is positive. ΔG is positive
content of the products is less than that of the reactants and ΔH has a  Need some amount of energy to
Photons
negative value start the process or reaction.
6CO2 + 6H2O + Energy  C6H12O6 + 6O2
 Reacting systems that take up heat from their surroundings are  Absorbs free energy and stores it (glucose)
endothermic and have positive values of ΔH  Example: Photosynthesis
 The unit of ΔH is joules/mole or calories/mole
 Entropy (S): S is a quantitative expression for the randomness or a  Exothermic process or Reaction: (glucose)
disorder in a system  Chemical reactions that releases C6H12O6 + 6O2  6CO2 + 6H2O + Energy
 The unit of ΔS is joules/mole. Kelvin energy or heat
 ΔH is negative. ΔG is negative Exothermic
Process ATP Release
 Released energy is available to do Energy
work, means energetically favorable.
 Example: Cellular Respiration
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GIBBS FREE ENERGY: CELLULAR RESPIRATION

 It is used to determine whether a process is  Cell respiration is the process by which

spontaneous or not. Simply means, it will happen
on its own or not? living cells make energy.
 Cases:  Mitochondria, also called as “Power
 ΔG is negative : Spontaneous House” of the cell, produce energy in the
 ΔG is positive : Nonspontaneous form of high-energy molecules known as
 ΔG is zero : Equilibrium
ATP ( Adenosine triphosphate).

 Spontaneous process are both enthalpically or  The food we eat is broken down by various
entropically favorable. enzymes in order to produce the energy
source (ATP) of the cell.

Relationships among K'eq, ΔG'˚, and the Direction

 The pathway by which ATP is produced
depends on the availability of oxygen in
of Chemical Reactions cells.
Starting with all
When K'eq is . . . ΔG'˚ is . . . components at 1 M, the
 Based upon the requirement of oxygen,
cellular respiration can be categorized into
reaction . . .
two types:
>1.0 Negative proceeds forward  Aerobic Respiration
1.0 zero is at equilibrium  Anaerobic Respiration

<1.0 positive proceeds in reverse

AEROBIC AND ANAEROBIC RESPIRATION AEROBIC AND ANAEROBIC RESPIRATION

 If enough oxygen is present in cell, Aerobic Respiration takes

place in mitochondria. It produces more amount of ATP  Anaerobic respiration is common in those organisms that do not need
molecules as compared to anaerobic respiration. oxygen in order to grow. The chief source of energy in such organisms
 The energy metabolism process begins with the glycolysis
which occurs in cytoplasm. The glucose is broken down
rely on fermentation or anaerobic respiration. The main example of such
through series of reaction from 6 carbon compound to 3 organism is bacteria.
carbon compound, called Pyruvate (or Pyruvic acid).
 The Pyruvate is the key molecule responsible of aerobic or  In humans while exercising, when there is not enough oxygen is
anaerobic respiration. available for a moment in skeletal muscles, the cells obtain the
 If sufficient oxygen is available in cells, pyruvate gets
transported to the mitochondria for aerobic respiration.
adequate energy by fermentation process only.
 Rest of the pathways of aerobic respiration occurs in  In the muscle, the by- products of fermentation include molecules
mitochondria which includes:
 Pyruvate oxidation, called lactate (also known as lactic acid). The deposition of lactic acid in
 Krebs cycle, muscles is responsible for a few diseases. It also hurts while we exercise.
 Oxidative phosphorylation (or electron transport chain).
 Fermentation produces lower amounts of energy and releases various
 Oxidation of glucose under aerobic conditions results in 32
mol of ATP per mol of glucose. by-products.
 If insufficient of oxygen is available to cells, energy is
produced through anaerobic respiration. The anaerobic  In yeast the by-product of fermentation is alcohol.
pathways are also called as fermentation.
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ANAEROBIC RESPIRATION
(FERMENTATION)

Lactic Acid Fermentation Alcohol Fermentation

THREE STAGES OF AEROBIC RESPIRATION TOTAL ATP FROM 1 GLUCOSE

 Stage 1. Glycolysis (cytoplasm): break  Glycolysis(anaerobic respiration)

glucose in half and charge 2 ATP; no makes 2 ATP
oxygen needed
 With O2, Aerobic Respiration (all
• Stage 2. Krebs Cycle (mitochondria): three stages) makes 36-38 ATP
finish the job to turn glucose into CO2
and release energy; requires oxygen;  Some energy will be lost as heat and
• Stage 3. Electron Transport Chain used to maintain body temperature
(mitochondria): use the energy from
stage 1 and 2 to charge 32-34 ATP;  ATP TOTALS to Memorize!
requires oxygen  (aerobic) = around 36 ATP
 (anaerobic) = only 2 ATP
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GLYCOLYTIC
GLYCOLYSIS PATHWAY STEPS
 The glycolytic pathway completes in 10 steps.
 Glycolysis. (Greek: glyk-”sugar” + lysis “breakdown.”). Means breakdown of
sugar molecules.  The first 4 steps are often referred as Priming
Phase or preparatory phase or energy investment
phase because 2 ATPs are utilized by the pathway.
 Also called as Embden-Meyerhof pathway
 The last 6 steps are referred to as energy payoff
 Glycolysis is the first step of energy metabolism. It occurs in the cytoplasm of phase because it produces 4 ATP molecules and 2
NADH (energy carrier).
the cell.
 2NADH produced here will make 4ATP in
 Glycolysis is a series of linked chemical reactions that convert glucose (6C) Electron transport chain.
into pyruvic acid or pyruvate (3C).  While each NADH can theoretically yield 3 ATP
molecules in the ETC, the exact number can vary
depending on the shuttle mechanism used to
 At the end of the 10 steps pathway, one molecule of glucose converts into transport NADH from the cytoplasm (where
glycolysis occurs) into the mitochondria.
two molecules of pyruvate and the pathway  Shuttle Mechanisms: Malate-Aspartate Shuttle,
 Produces 4 ATP molecules but consumes 2 ATP so net gain of 2 ATP molecules Glycerol-3-Phosphate Shuttle ( These shuttles
require energy to transport NADH to
 2 NADH molecules ( electron carrier) mitochondria).

25

GLYCOLYSIS: INPUTS AND OUTPUTS

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PYRUVATE IS A FLEXIBLE GLUCONEOGENESIS

INTERMEDIATE
 Pyruvate is a flexible intermediate. For energy production, it  When levels of pyruvate are high and energy
normally diffuses into the mitochondrion where it will be oxidized
further. demands are low, pyruvate can be converted back
into glucose by a series of reactions called
 However, mitochondrial oxidation requires oxygen. If oxygen is “gluconeogenesis.”
lacking in the tissue cells of animals (hypoxic condition), then
pyruvate is converted into lactic acid.  Glycolysis occurs primarily in the muscles, while
 The reduction of pyruvate’s ketone functional group into an alcohol
gluconeogenesis occurs in the liver.
requires a reducing agent. NADH provides the electrons and enough
reduction potential to do the job.  Lactate formed during anaerobic glycolysis is
usually transported to the liver where it is converted
 In fact, consuming NADH is the main goal of this reaction. Cellular all the way back to glucose via gluconeogenesis.
levels of NAD+/NADH are limited, and oxidation of NADH back to
NAD+, provides an ongoing supply of this reactant for continued  This process is often called the “Cori” cycle, named
oxidation of GAP and continued production of ATP. for the husband and wife team who first described
 Lactate is a “dead end” in this provisional shunt, accumulating in it.
muscle cells during strenuous activity.
 Gluconeogenesis shares some of the same
 Eventually, it must be oxidized back to pyruvate (a task normally (reversible) reactions as the glycolysis pathway,
performed by the liver and the pathway called Gluconeogenesis). however three of the reactions are very different
due to their irreversible nature.

PYRUVATE AND ETHANOL FERMENTATION BIOENERGETICS PATHWAYS

 The Anaerobic conversion of glucose into ethanol is called fermentation,

one of the most studied and applied biochemical pathways of all time.
 In yeast and other microorganisms, hypoxic conditions result in a different
product to maintain redox equivalence (NAD+ supply). Formation of Acetyl CoA

 These organisms first decarboxylate pyruvate, forming acetaldeyde and

then reduce it to ethanol.
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KREB'S CYCLE FORMATION OF ACETYL COA

( CITRIC ACID CYCLE OR TCA CYCLE)

 The Kreb's Cycle is an aerobic process consisting of eight

definite steps. It occurs in the matrix of mitochondria in the  Junction between glycolysis and Krebs cycle
presence of oxygen.
 Oxidation of pyruvate to acetyl CoA
 Cyclical series of oxidation reactions that give off CO2 and
produce one ATP per cycle.  Pyruvate molecules are translocated from the cytosol into the mitochondrion by a
 Glycolysis generates two pyruvate molecules, so it runs twice carrier protein in the mitochondrial membrane.
for each glucose molecule and produces two ATP molecule.
 A CO2 is removed from pyruvate – making a 2C compound.
 In order for pyruvate from glycolysis to enter the Kreb's Cycle it
must first be converted into Acetyl-CoA by the pyruvate  Coenzyme A is attached to the acetyl group.
dehydrogenase complex which is an oxidative process wherein
NADH and CO2 are formed. Another source of acetyl-CoA is
beta oxidation of fatty acids.
 The reason oxygen is required is because the NADH and
[FADH2] produced in the Kreb's Cycle are able to be oxidized in
the electron transport chain (ETC) thus replenishing the supply
of NAD+ and [FAD].
 The Krebs cycle transfers much of this energy to electron
carriers NAD+ and FAD.
 During ETC, 1 NAD gives 3 ATPs and 1 FAD gives 2 ATP.

STEPS IN KREB’S CYCLE KREB’S CYCLE SUMMARY

Pyruvate
CO2 NAD+
NADH  Krebs cycle enzymes are located in the
•All compounds are tricarboxylic Acetyl CoA
mitochondrial matrix
acids
 Each turn of the Krebs Cycle also
•Carbons from glucose are shown
produces 3NADH, 1FADH2, and 2CO2
in red
 Therefore, For each Glucose molecule,
•Carbons from glucose are lost as
the Krebs Cycle produces 6NADH,
CO2 (decarboxylation)
2FADH2, 4CO2, and 2ATP
•Several NADH + H+ are
generated via oxidation of
intermediates
•One high energy phosphate
compound (GTP)is produced
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ELECTRON TRANSPORT CHAIN

OXIDATIVE PHOSPHORYLATION

 The electron transport chain is a series of protein

complexes that are found in the inner membrane of
mitochondria and is a vital component of oxidative
phosphorylation.
 The protein complexes are named Complex I, II, III, and IV.
 As electrons are transported from one complex to another,
protons (H+) are pumped out into the space between the
inner and outer membrane of the mitochondria.
 As protons are pumped into the space between the two
membranes, a proton gradient forms – more protons are
present in the space between the two membranes.
 The proton gradient is essential in ATP production.  Complex I : ubiquinone (UQ), or Coenzyme Q (CoQ).
 The protons that accumulate between the two membranes  Complex II : Succinate-Coenzyme Q Reductase
are then transported through a molecule called ATP
synthase. It uses energy of the ion-gradient to power ATP  Complex III : Coenzyme Q-Cytochrome c Reductase
synthesis.
 Complex IV : Cytochrome c oxidase
 ATP synthase then produces ATP molecules that the cell
uses as its source of energy. For every H+ ion that flows  Complex V : ATP Synthase
through ATP synthase, one ATP can be formed from
ADP.

ELECTRON TRANSPORT CHAIN ELECTRON CARRIERS FROM THE KREBS CYCLE

ARE USED TO POWER AN ELECTRON
TRANSPORT CHAIN AND PROTON PUMP.
 These four protein complexes, labeled I-IV, move the
electrons from reduced electron carriers such as NADH and
FADH2 to the another protein complex V (ATP Synthase),
where oxidative phosphorylation take place, which finally Oxygen is required
generates ATP molecules. to accept energy- Flow of H+ down
depleted electrons. concentration gradient
powers ATP synthesis.
 The electron transport chain is initiated by the redox High-energy electron carriers
from acetyl CoA formation, Krebs
reactions in citric acid cycle where NAD and FAD is cycle, and glycolysis feed into the ETC.
reduced to NADH and FADH2
NADH FADH2 1/2O2 2e– 2H+ H2O matrix
 This is an oxidation reaction where 2 hydrogen atoms (or 2 ADP
+
ATP
FAD
hydrogen ions and 2 electrons) are removed from the organic Pi
NAD
metabolite.

 The reaction can be represented simply where M = any

inner
metabolite. membrane
MH2 + NAD+ -----> NADH + H+ + M: + energy

 Single hydrogen is removed with 2 electrons as a hydride ion

(H-) while the other is removed as the positive ion (H+). intermembrane
space

Energy from high-energy

electrons powers active High H+ concentration is H+ channel is coupled to
transport of H+ by ETC. generated by active transport. ATP synthesizing enzyme.
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CELLULAR RESPIRATION: ATP

ETC: SUMMARY SUMMARY
1. Occurs in the inner membrane of the mitochondria and 5 protein complexes are involved.  Glycolysis
 2 ATP
2. Electron transport proteins in the inner mitochondrial membrane  2 NADH  4-6 ATP (Depends on
how this NADH molecule gets to
3. Oxygen pulls the electrons from NADH and FADH2 down the electron transport chain to a the ETC. To make things simple we
lower energy state will say that these two NADH’s
make 4 ATP )
4. Process produces 34 ATP or 90% of the ATP in the body.
 Formation of Acetyl CoA
5. Requires oxygen, the final electron acceptor. Produce H2O in the end.  2 NADH  6 ATP

6. For every FADH2 molecule – 2 ATP’s are produced.  Krebs Cycle and ETC
 2 ATP
7. For every NADH molecule – 3 ATP’s are produced.
 6 NADH  18 ATP
8. Chemiosmosis – the production of ATP using the energy of H+ gradients across membranes to  2 FADH2  4 ATP
phosphorylate ADP.
 Grand Total = 36 ATP

COURSE OUTLINE WHAT ARE ENZYMES ?

 Enzymes are Biocatalyst , which increases the speed of biological

 Cell Metabolism reactions, without itself undergoing any change in the process.
 Mostly enzymes are protein molecules with a few exceptions of Riboyzmes
 Enzymes and Catalysis ( which is RNA) and are specialized to catalyse biological reactions.
 They are vital to living organisms as life depends on biochemical reactions.
 Cell Communication and Signalling Millions of biochemical reactions and pathways are running continuously
to maintain the organisms to be alive.
 Any change in its structure may lead to disfunction of enzyme activity and
can cause a disease or lethal effects.
 They are very specific to substrate(s), a chemical substance on which the
enzyme act and convert it to specific product.

Maltase
Maltose Glucose
(Substrate) (Product)
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ENZYMES DISCOVERY PROPERTIES OF ENZYMES

 The word ‘enzyme’ was first used by the German physiologist Wilhelm Kühne in 1. Colloidal nature
1878, while describing the ability of yeast to produce alcohol from sugars, and it is  Their molecular weights usually range from 12000 to over a million Daltons, Hence, they are very large
derived from the Greek words en (meaning ‘within’) and zume (meaning ‘yeast’). compared to their substrates or the functional group they act upon . Due to their giant size, the enzymes
exhibit many colloidal properties such as:
 In early twentieth century (1920s) the enzymes were crystallized, revealing that  Diffusion rates are very slow and forms colloidal systems in water
catalytic activity is associated with protein molecules.
 May produce considerable high light scattering. They form turbidity in solution known as Tyndall effect
 For the next 60 years, it was believed that all enzymes are proteins only, but in 2. Catalytic nature
1980s, Ribozymes (RNA molecules as enzymes) were discovered which were also
 A universal feature of all enzymatic reactions is the virtual absence of any side product. An enzyme is
found to exert catalytic effect. Ribozymes were found to play a role in gene precisely adapted to catalyze a particular reaction
expression.
3. Turnover number
 During 80s, scientists developed the technology to generate antibodies that possess  The catalytic power of an enzyme is measured by the turnover number or the molecular activity
catalytic properties, called as Abzymes. These abzymes have significant potential to  It is defined as the number of substrate molecules converted into the product in a given unit of time by a
be used as industrial catalysts and also used in therapeutics. single enzyme molecule, when the enzyme is fully saturated with the substrate
 The value of the turnover number varies with different enzymes and it depends upon the conditions in
 Over 2000 enzymes are known and ~ 200 have been crystallized. which the reaction is taking place

PROPERTIES OF ENZYMES PROPERTIES OF ENZYMES

4. Specificity of enzyme action 6. Solubility

 Enzymes are highly specific in their action when compared with a chemical  The solubility of enzymes is markedly influenced by the pH. Solubility is lower at
catalytic reaction; i.e., a particular enzyme attacks only a particular substrate. pI and increases with increasing acidity or alkalinity
 Enzymes shows optical specificity, reaction specificity, substrate specificity, 7. Optical activity
Geometrical specificity
 All proteins rotate the plane polarized light to the left; i.e., they are laevorotatory
5. Amphoteric nature
8. Denaturation of enzymes
 Enzymes are amphoteric. They act as both acids and bases.
 Denaturation is a result of change in conformation or unfolding of the enzyme
 Each enzyme has a fixed value of isoelectric point (pI) at which it will move in an molecule, i.e., 2 or 3 structure of the enzyme is completely lost in the process
electric field. Isoelectric field or isoelectric point is the pH value at which the without any break in the 1 structure. Denaturation is defined as the total loss or
number of cations equals the number of anions. Thus, at pI, the net electric charge randomization of the 3-D structure
on an enzyme is always 0.
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PROPERTIES OF ENZYMES NOMENCLATURE

9. Reversibility of a Reaction  Typically add “-ase” to name of substrate

 Example
 Enzymes are capable of bringing about reversion in a chemical reaction. The  Lactase ( Substrate Lactose -> Glucose and galactose)
digestive enzymes catalyze the hydrolytic reactions which are reversible.  Maltase ( Substrate Maltose -> Glucose )
 Sucrase ( Substrate Sucrose -> Fructose and Glucose)
 For example, lipase, which catalyzes the synthesis of fat from glycerol and fatty
acids, can also hydrolyze them into their component units  Combination of Substrate and Catalytic Reaction Type
 Example
 Lactate dehydrogenase
 Pyruvate decarboxylase
 Some names are historical - no direct relationship to substrate or reaction
type
 Example
 Trypsin
 Rennin
 Pepsin

INTRACELLULAR AND
EXTRACELLULAR ENZYMES CLASSIFICATION OF ENZYMES

 Intracellular enzymes (Endo enzymes)

 Enzyme Commission (EC) – according to International
 They can work inside cells .
 Example: DNA replication enzymes such as helicase and
Union of Biochemistry & Molecular Biology (IUBMB)
DNA polymerase.
 Each enzyme was given 4 digit numbes [1.2.3.4]
 Extracellular enzymes ( Exo enzymes)  1st one of the 6 major classes of enzyme activity
 They can work outside cells  2nd the subclass (type of substrate or bond cleaved)
 Example: digestive enzymes such as lipase and amylase
 3rd the sub-subclass (group acted upon, cofactor required, etc...)
 4th a serial number… (order in which enzyme was added to list)
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SIX CLASSES OF ENZYMES OXIDOREDUCTASE

Classes of enzymes based on organic chemical reaction catalyzed:  Catalyzing oxidation reduction reaction where one substrate is oxidized and other is reduced
 Oxidases-
 Catalyzing oxidation of the substrate and atomic oxygen acts as recipient of hydrogen e.g. Ascorbic acid
1. Oxidoreductase – oxidase, Cytochrome oxidase, Tyrosinase
catalyze redox reactions: Eg, dehydrogenases, oxidases, peroxidases  Ascorbic acid Oxidase [ Ascorbic acid  Dehydro ascorbic acid ]
2. Transferases –  Aerobic Dehydrogenases
catalyze group transfer reactions; often require coenzymes  Catalyzing oxidation of the substrate and molecular oxygen acts as recipients of hydrogen e.g. Glucose
oxidase, L amino acid dehydrogenase, Xanthene dehydrogenase
3. Hydrolases –  Glucose Oxidase [Glucose  Gluconolactone]
catalyze hydrolysis reactions
4. Lyases –  Anaerobic Dehydrogenases
 Catalyzing oxidation of the substrate and coenzymes act as recipients of hydrogen e.g. Lactate Dehydrogenase
lysis of substrate; produce contains double bond with NAD and Glucose 6 phosphate dehydrogenase with NADP
5. Isomerases –  Lactate dehydrogenase [Lactic acid + NAD  Pyruvic acid + NADH – H+ ]
catalyze structural changes; isomerization  Oxygenases
6. Ligases –  Catalyzing oxidation of the substrate and oxygen is added to the substrate eg are Homogentisate oxygenase, L
Tryptophan dioxygenase.
ligation or joining of two substrates with input of energy, usually from ATP hydrolysis;
often called synthetases or synthases  Phenylalanine Hydroxylase [Phenylalanine + NADPH – H+ + O2  Tyrosine + NADP + H2O ]

TRANSFERASES HYDROLASES
 Transaminases.  Catalyzing hydrolytic breakdown of different bonds. Most of the GIT enzymes belong to this class
 Catalyzing transfer of amino group between an amino acid and a ketoacid e.g. Aspartate transaminase (AST), Alanine transaminase (ALT)
 Aspartate transaminase (AST)  Enzymes hydrolyzing carbohydrates
 Polysaccharidases: Amylase [Starch  Maltose, maltotrios, dextrins ]
 Glutamic acid + Oxalo acetic acid   ketoglutaric acid + Aspartic acid
 Oligosaccharidases Dextrinase [Dextrins  glucose]
 Alanine transaminase (ALT)
 Disacharidases [Maltose, Lactose, Sucrose  monosaccharides]
 Glutamic acid + Pyruvic acid   ketoglutaric acid + Alanine

 Transmethylases  Enzymes Hydrolysing Lipids

 Lipase [ Triacyl glycerol  monoacyl glycerol + 2 F.F.A ]
 Catalyzing transfer of methyl group between to substrates e.g. Catechol O methyltransferase (COMT)
 Cholesterol esterase [Cholesterol ester  free cholesterol + FFA]
 COMT [ Noradrenalin + CH3  Adrenaline ]

 Enzymes Acting on Peptide Bonds

 Transpeptidases
 Exopeptidases Carboxypeptidase, Aminopeptidase [ Protein  amino acids]
 Catalyzing transfer of amino acids to substrates e.g. Benzyl-SCoA transpeptidase
 Endopeptidase Pepsin [ Proteins -> smaller peptides]
 Benzyl-SCoA transpeptidase [Benzyl - SCoA + Glycine  Hippuric acid ]
 Dipeptidase [ Dipeptide  AA ]
 Phosphotransferases
 Phosphatases
 Catalyzing transfer of phosphate group to substrates e.g. Hexokinase, glucokinase
 Phosphomonoesterases
 Hexokinase [ATP + Glucose Hexokinase  ADP + D-Glucose -6-Phosphate]
 Phosphatase [Glucose – 6.P. + H2O  G 6. Phosphate + Glucose + Pi ]
 Acetyltransferase  Phosphodiesterases
 Catalyzing transfer of acetyl group to substrates e.g. choline acetyltransferase  Removal of phosphate Group of diesters breakdown of 3’-5’ p linkages in cyclic AMP
 Choline acetyltransferase [Acetyl-CoA+ Choline  CoA + Acetyl- Choline ]
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LYASES ISOMERASES

 Catalysing reactions in which groups are removed without hydrolysis  Involved in inter conversion of pair of isomeric compounds
leaving a double bond or add groups to already existing double bonds
 Phosphogluco Mutase
 Pyruvate Decarboxylase  Glucose 6. P  glucose 1.P
 CH3. CO. COOH (Pyruvate)  CH3. CHO+ CO2 (Acetaldehyde)
 Phosphohexose Isomerase
 Fumerase [Fumaric acid  Malic Acid]  Glucose 6.P  Fructose 6.P
 COOH.CH = CH. COOH   COOH-CHOH. CH2-COOH

LIGASES

 Catalyze reactions in which linking together of two molecules

occur coupled with the breakdown of a high energy phosphate
bonds like ATP, GTP STRUCTURE AND MECHANISM
 Acetyl CoA Synthetase OF ENZYME ACTION
 Acetate + CoA +ATP  Acetyl CoA+AMP+PP

 Succinyl CoA Synthetase

 Succinate + CoA + ATP  Succinyl CoA + ADP+ Pi

 Pyruvate Carboxylase
 Pyruvate + CO2 + ATP  Oxaloacetate + ADP + Pi
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ENZYME STRUCTURE COFACTOR AND COENZYME

 Chemically, enzymes may be divided into two categories: simple and complex.
 Enzymes are globular proteins and each enzyme has
its own tertiary structure with specific 3D  Simple protein enzymes
 These contain simple proteins only. E.g., urease, amylase etc.
conformation which is very essential for its catalytic
activity.  Complex or Conjugated protein enzymes
 These complex enzymes require a non-protein substance for their catalytic activity.
 In such enzymes this non-protein part is called as Prosthetic Group and the protein part is called
 The catalytic activity of enzymes is performed at a as Apoenzyme. The two parts together called as Holoenzyme. E.g., catalase.
specific site, called Active site.
 Prosthetic Group
 The substrate molecules tend to bind with the R-groups  COFACTOR: If the prosthetic group is an inorganic substance, it is called as Cofactor
 COENZYME: If the prosthetic group is an organic substance, it is called as Coenzyme
of amino acids forming the active site through a weak  The coenzyme or the cofactor may be an integral part of the enzyme or Its presence may be
non-covalent interactions. required during the reaction

 Enzymes either catalyze Anabolic (to synthesize) or

Catabolic reactions(to break down).

COFACTORS COENZYME
ENZYMES CO FACTORS
 They bind covalently or non-covalently to the apoenzyme. Coenzyme acts as a
 They are mainly inorganic metal co-substrate or a second substrate in the group transfer reactions.
complexes which are tightly bound Catalase
Peroxidase
Iron : Fe2+ or Fe3+
Cytochrome oxidase  The coenzyme either donates or accepts the group that is being transferred.
to the enzyme
 Coenzymes account for 1% of the entire enzyme molecule.
 They are highly required for normal Cytochrome oxidase Copper : Cu+2
 The coenzymes generally contain vitamins of B-complex family
conformational structure and  Some act as coenzymes by themselves e.g. biotin OR
Carbonic anhydrase alcohol Zinc : Zn2+
function of the enzyme dehydrogenase
 Some are converted into coenzymes e.g. thiamin, riboflavin, niacin, pantothenic acid,
pyridoxine, folic acid and vitamin B12
Hexokinase Magnesium Mg2+
 They act as donors or acceptors in Glucose-6-phosphatase
Pyruvate kinase  Coenzymes are generally required in group transfer reactions, Eg
oxidation and reduction reactions  Oxidation-reduction
 Transamination
 E.g., Mg2+, Ca2+, Cu2+, Zn2+, K+, Arginase Manganese Mn2+
 Phosphorylation
Fe2+, Mn2+, Mo2+, etc. Pyruvate kinase Potassium K+
 Coenzymes can be divided into two groups:
Urease Nickel N 2+
 Coenzymes involved in transfer of hydrogen, Eg: FAD, NAD, FMN,
 Coenzymes involved in transfer of groups other than hydrogen, Eg: ATP, Biotin, CoA,
Vit B12, Lipoic Acid, TPP
Glutathione Peroxidase Selenium : Se
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Mechanism of enzyme action Mechanism of enzyme action

MECHANISM OF ENZYME ACTION MECHANISM OF ENZYME ACTION

 At temperatures above absolute zero (– 273°C), molecules are in  The mechanism of action of enzymes can be explained by two
constant motion because of their kinetic energy. perspectives-
 A chemical reaction occurs when molecules of reactants collide with 1. Thermodynamic changes
each other in the correct orientation (kinetic theory of reaction)  All enzymes accelerate reaction rates by lowering down the activation energy of a
reaction.
 The greater the frequency of collisions between the reactant 2. Processes at the active site
molecules, the greater will be the rate of reaction.  Catalysis by proximity
 The frequency of collisions can be increased by raising the  Acid base catalysis
temperature.  Catalysis by strain
 Covalent Catalysis
 Rise in temperature would increase:
Molecular motion
Frequency of collisions
Rate of reaction

Mechanism of enzyme action Mechanism of enzyme action

ACTIVATION ENERGY ACTIVATION ENERGY

 Energy level of reactants has to be  Enzymes are natural reaction

raised to a critical level for the reaction accelerator. They increase the
to occur. reaction rate by lowering down
the energy needed for
 Energy input required to reach the activation.
critical level is known as the energy of
activation  Enzymes are required in very low
concentrations and they speed
 In a general chemical reaction, it is up reactions by increasing the
commonly achieved by heating the
velocity of chemical reaction
reaction. without themselves being
 The option of raising temperature is not consumed during the reaction.
available in living organisms.  So, they remain unchanged at Without enzyme, high amount of activation energy
 In living organisms, the enzymes provide the end of the reactions and free is required to get a chemical reaction to take place.
an alternate pathway for the reaction. for next reaction.
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Mechanism of enzyme action Mechanism of enzyme action

ENZYME-SUBSTRATE INTERACTION ENZYME-SUBSTRATE INTERACTION

 The size of the enzyme molecules are much larger than their substrates.
 Enzyme possesses substrate specific sites, called Active site ( or catalytic site). The substrate  On binding of two substrates to the enzyme, a
binds and fits into these active site and forms the Enzyme-Substrate (ES) Complex. The
formation of ES complex stabilizes the transition-state between substrate to product formation.
chemical group may be transferred from one
substrate to another
 The binding may bring two substrates in close proximity (bond-forming distance) in the correct
orientation so that a bond is formed between the two.
 The binding of a substrate to the enzyme many induce a strain in the substrate. As a result, a
bond is broken in the substrate. The substrate is split into two or more products which are
released. Enzyme gets free at the end and available for next reaction.

Mechanism of enzyme action Mechanism of enzyme action

MODELS OF ENZYME ACTION LOCK AND KEY MODEL

 Two models has been proposed  Assumed the conformation of enzymes

 Lock and Key Model very rigid.
 By Emil Fischer in 1894  Lock and key type of complementarity
 based upon rigid conformations between substrate and enzyme.
 Induced fit Model  Complementarity responsible for
 By Koshland in 1958. specificity of enzymes.
 Induced conformational change based
 Fischer’s model did not agree with
certain experimental findings obtained
later.
 Conformation of enzyme was found to
change when it combined with its
substrate
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Mechanism of enzyme action Mechanism of enzyme action

THE LOCK AND KEY INDUCED FIT MODEL

1. The enzyme has a cleft/ depression called the active site  Koshland’s model explained the conformation
changes in enzyme during cataysis
2. Active site and the specific substrate has complementary shape
 In the absence of substrate, complementarity
3. The substrate meets the enzymes by random movement between enzyme and substrate is not apparent.
4. The substrates fits into the cleft  Approach of substrate induces change in
conformation of the enzyme
5. The R group binds with the substrate
 The substrate site becomes complementary to
6. An enzyme-substrate complex is formed the substrate
7. The enzyme catalyzes the reaction – breaking it apart or joining  Change in conformation of the enzyme produces
'induced fit'
8. An enzyme-product complex is formed
 The substrate binds to the enzyme, and is
9. The product leaves the enzymes converted into the product
10. The enzymes remain unchanged – ready to go for the next  Release of the product restores the enzyme to its
substrate original conformation

Mechanism of enzyme action Mechanism of enzyme action

ALLOSTERIC ENZYMES ALLOSTERIC ENZYMES

 Some enzymes possess a site, in addition to the substrate site, known as the allosteric site.
 Binding of an allosteric molecule to allosteric site changes the conformation of the
enzyme.
 Enzymes having allosteric site are termed as allosteric enzymes
 The allosteric molecule is also known as:
 Allosteric effector
 Allosteric modifier
 Allosteric regulator

 Some allosteric molecules:

 Facilitate the conformational change required for substrate binding, They are known as
allosteric activators (positive modifiers)
 An example is N-acetylglutamate which activates carbamoyl phosphate synthetase
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Mechanism of enzyme action Enzyme specificity

SOME ALLOSTERIC REGULATORS: ENZYME SPECIFICITY

 Prevent the conformational change required for
the binding of the substrate.  One of the properties of enzymes that makes them so
 Such regulators are known as allosteric inhibitors important as diagnostic and research tools is the specificity,
(negative modifiers). they exhibit relative to the reactions they catalyse.
 An example is glucose-6-phosphate which inhibits
hexokinase  Enzyme specificity is based on the mode of accepting the
 Negative Modulation of allosteric enzyme substrate and their reaction
 Enzymes subject to allosteric inhibition are usually
present at the start of long pathways  There are four distinct types of specificities:
 The allosteric inhibitor is generally the product of the
pathway 1. Absolute or Reaction Specificity
 The allosteric enzyme regulates the rate of formation
of the product 2. Stereochemical or optical specificity
 If the product is not being utilised, it will accumulate 3. Substrate specificity
 It inhibits the allosteric enzyme, and further synthesis
of the product ceases 4. Geometrical specificity
 When the product is used up, the allosteric enzyme
becomes free and active again

Enzyme specificity Enzyme specificity

ENZYME SPECIFICITY ENZYME SPECIFICITY

1. Absolute or Reaction specificity 2. Stereochemical or optical specificity

 Enzymes are specific towards any particular reaction, which means one enzyme will
catalyzes only one reaction that substrate can undergo.  Stereoisomers are those compounds which have the same molecular formula but
 For Example : differ in their structural configuration. The enzymes act on only one isomer and
 Oxaloacetate is an important metabolic intermediate which can undergo several thus exhibit specificity towards that particular stereoisomer.
reactions namely, reduction to give maleic acid; decarboxylation to give pyruvate; or
accept an amino group to give Asp and so on.  For example
 But because of absolute specificity, each reaction of Oxaloacetate (OAA) is catalyzed  l-amino acid oxidase and d-amino acid oxidase; hexokinase acts only on d-
by separate enzyme, which catalyzes only that reaction and none other. hexoses, arginase acts only on l-Arg and not on d-Arg, amylase acts only on
α-1,4-glycosidic linkages and not on beta- glycosidic linkages, and so on.
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Enzyme specificity Enzyme specificity

ENZYME SPECIFICITY ENZYME SPECIFICITY

3. Substrate specificity  3 A) Group dependent substrate specificity

 Absolute group specificity or relative group specificity or broad specificity
 The extent of substrate specificity varies from one enzyme to the  Some enzymes catalyze the reaction of a structurally related group of
other compounds
 For example, hexokinase adds a phosphate group to all hexoses like glucose,
 Substrate specificity may be either absolute or relative fructose, mannose, etc.
 Absolute specificity:  Group specificity is exhibited by proteolytic enzymes with respect to the
peptide bond between 2 specific amino acids
 one to one specificity - It is seen in some enzymes which are capable  Examples:
of acting on only 1 substrate. For example, urease acts only upon  Trypsin splits peptide bonds in which the carboxylic group is contributed by either
urea Lys or Arg
 Chymotrypsin preferentially splits peptide bonds in which the carboxyl group is from
 Relative specificity: an aromatic amino acid
 Thrombin splits peptide bonds in which the side chain on the carboxyl site of the
 It can be further classified into two types: susceptible peptide bond must be Arg, while the one on the amino group must be
Gly
 Group dependent substrate specificity  These enzymes help in the elucidation of the arrangement of the amino acid
 Bond dependent substrate specificity residues in a protein.

Enzyme specificity Factors affecting enzyme activity

ENZYME SPECIFICITY FACTORS AFFECTING ENZYME ACTIVITY

3 B)Bond dependent substrate specificity  The activity of enzyme can be effected by

 Bond dependent class of enzymes, are very specific in catalyzing bond-dependent
reactions Temperature
 Examples: pH value
 Proteases  Peptide bonds of proteins
 lycosidases  Glycosidic bonds of carbohydrates
Enzyme concentration
 Esterases  Ester linkages in lipids Substrate concentration
 Exonucleases and endonucleases  Phosphodiester linkages in nucleic acids

4. Geometrical specificity
 Some enzymes exhibit specificity towards the cis/trans forms (geometric isomers)
 For example, fumarase catalyzes the interconversion of fumarate (trans form) and malate (cis
form)
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Factors affecting enzyme activity Factors affecting enzyme activity

EFFECT OF TEMPERATURE EFFECT OF pH

 To see the effect of temperature, velocity of a reaction is measured at  The rate of almost all enzyme-catalyzed reactions exhibits a significant
different temperatures . A curve is plotted between velocity and dependence on pH or hydrogen ion concentration. Change of pH can
temperature. A bell-shaped curve is obtained. disturb the ionic bond which is important to the tertiary structure of a
 When the temperature rises, the velocity initially increases. This is due to proteins.
increase in the kinetic energy of the reactants.
 If velocity is measured at different pH levels, and is plotted against
 A further rise in temperature leads to progressive denaturation of the pH, a bell-shaped curve is obtained.
enzyme. The velocity begins to decrease as the enzyme gets denatured.
The reaction practically stops when the enzyme is completely  A change in pH alters electrical charges on the enzyme molecules, and
denatured. often on substrate molecules as well. This may affect binding of the
 The temperature at which the velocity is maximum is known as the substrate to the enzyme or the catalytic activity of the enzyme or
optimum temperature of the enzyme. For all human enzymes, the both.
optimum temperature is 37°C
 At an optimum pH, the velocity of the reaction is the highest because:
 The temperature coefficient (Q10) of an enzyme is the factor by which  The electrical charges on the enzyme and the substrate are the most suitable
the rate of a biologic process increases when temperature rises by 10 °C. for both enzyme-substrate binding and catalysis
 For most of the enzymes, the temperature coefficient is two. This
means that the velocity is doubled when the temperatures rises by 10°C  As we move away from the optimum pH, the velocity of the reaction
decreases. At extremely low or high pH, the enzyme may be
 Most animal enzymes rapidly become denatured at temperatures above denatured. The optimum pH is different for different enzymes
40oC. The optimal temperatures of the enzymes in higher organisms
rarely exceed 50 °C .  Most intracellular enzymes exhibit optimal activity at pH values
between 5 and 9.

Factors affecting enzyme activity Factors affecting enzyme activity

EFFECT OF ENZYME CONCENTRATION

EFFECT OF SUBSTRATE
CONCENTRATION
 As the amount of enzyme is increased, the rate  At lower concentrations, the active sites on
of reaction increases. most of the enzyme molecules are not filled
because there is not much substrate. Higher
 If there are more enzyme molecules than are concentrations cause more collisions
needed, adding additional enzyme will not between the molecules. The rate of reaction
increase the rate. Reaction rate therefore increases(First order reaction).
increases then it levels off.
 The maximum velocity of a reaction is
 Rate of the first reaction leading to formation of reached when the active sites are almost
ES is proportional to the product of molar E+S ES E+P continuously filled. Increased substrate
concentrations of E and S concentration after this point will not
 Rate of formation of ES Complex ∝ [E] [S] increase the rate. Reaction rate therefore
increases as substrate concentration is
 Therefore, the rate of the overall reaction is increased but it levels off (Zero order
proportional to the enzyme concentration. But reaction)
this is true only if enough substrate is available
to combine with the enzyme.
The shape of the curve that relates activity to
substrate concentration is hyperbolic.
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Factors affecting enzyme activity Factors affecting enzyme activity

MICHAELIS-MENTEN EQUATION MICHAELIS-MENTEN EQUATION Vmax . [S]

V=
Effect of substrate concentration Km + [S]
 Rate of the reaction should be proportional to substrate  The relationship between the velocity of the reaction and
concentration also like enzyme concentration. But this is the substrate concentration can be expressed by Michaelis-
possible only if enough enzyme is available to bind the Menten equation.
substrate.
 When the substrate concentration is very low, the sum of
 However, the availability of enzymes in the cells is limited , Km and [S] is nearly equal to Km as [S] is negligible.
When the substrate concentration rises, initially the
velocity of the reaction rises proportionately.
Vmax . [S]
 But later the rise in velocity becomes slower until a  Hence, the equation may be rewritten as: v =
maximum velocity (Vmax) is reached. Km
 At Vmax, all the enzyme molecules are saturated with  Since both Vmax and Km are constant, v ∝ [S]
substrate, and velocity cannot increase further if the
Vmax . [S]
substrate concentration is raised.  When the substrate concentration is very high, the sum of Km and [S] is nearly equal to
[S] as Km is relatively negligible
 The substrate concentration at which the velocity is half of v = Vmax .[s]
Vmax is known as the Michaelis constant (Km) of the
enzyme.
Km + [S] Hence, the equation may be rewritten as: v =
[s]
 The relationship between the velocity of the reaction and [S] and [S] are cancelled; the equation may be rewritten as: v = Vmax
the substrate concentration can be expressed by Michaelis- Michaelis-Menten equation
Menten equation.

Factors affecting enzyme activity Factors affecting enzyme activity

MICHAELIS-MENTEN EQUATION Vmax . [S] DETERMINATION OF Km VALUE

V=
Km + [S]
• Every enzyme has got a characteristic Km.
 When the substrate concentration is exactly
• Determination of Km is important in:
equal to Km, the sum of Km and [S] may be • Study of enzyme kinetics
taken as 2 [S] • Assay of enzyme activity
• Evaluation of enzyme inhibitors
The equation may be rewritten as:
• Plotting v versus [S] is a lengthy process, The velocity has to be
measured at a number of substrate concentrations.
Vmax .[s] Vmax
v= = • The substrate concentration has to be raised until Vmax is reached
2[s] 2
• Lineweaver and Burk devised a simple method for determination of Km
• Velocity is measured at a small number (5-6) of substrate concentrations
Thus, when the substrate concentration is
• A graph is plotted between the reciprocal of v and the reciprocal of [S]
equal to Km, the velocity is half of Vmax
Lineweaver-Burk plot
The 1/v versus 1/[S] plot is known as: OR
Double reciprocal plot
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Factors affecting enzyme activity Factors affecting enzyme activity

MICHAELIS-MENTEN EQUATION IS INVERTED

THE DOUBLE RECIPROCAL PLOT
Determination of Km VALUE: Double Reciprocal Plot
At the x-intercept (where the line meets the x-axis), the
 In reality, the enzyme continues to work, so the rate
value of y = 0 increases little by little and would only flatten out at
Therefore, at the x-intercept: infinity
 Because infinity is not on the graph – we can’t accurately
ax + b = 0 read off the Vmax - we can guess at best
ax = – b  Solution: Since 1/infinity = 0 (if n tends to infinity but if an
infinity value is fixed, then it’s 1)
x = – b/a  Therefore, by plotting a graph of 1/(Substrate
concentration) against a graph of 1/ (velocity)
 we receives a reciprocal graph that at whichever point that it
reaches the 0 (zero) substrate concentration
This is the equation for a straight line Thus, the x-intercept i.e. the value of 1/[S] at  the point when it touches the y axis will be equal to 1/Vmax
(because that is the infinity substrate concentration in
y (y-axis) is 1/v the x-intercept gives the value of 1/Km, and reciprocal term).
a (slope of the line) is Km/Vmax
the reciprocal of this will be the Km  -1/Km (because we can’t have negative Km) can be found
x (x-axis) is 1/[S] at the point of x-axis interception
b (y-intercept) is 1/Vmax

Enzyme inhibition Enzyme inhibition

ENZYME INHIBITION COMPETITIVE INHIBITION:

 Catalytic activity of some enzymes can be inhibited by certain  This is also known as substrate-analogue
inhibition because the inhibitor has a close
compounds. structural resemblance with the substrate. Due
to structural resemblance Inhibitor (I) binds to
 Enzyme inhibition may be of two types: the substrate site of enzyme forming enzyme-
 Competitive inhibitor (EI) complex, However, the inhibitor
 High similarity in structure cannot form the product.
 Non-competitive  Thus, in the presence of the inhibitor, the
catalytic activity of the enzyme is inhibited. The
inhibitor competes with the substrate to bind to
the enzyme. Due to competition between
substrate and inhibition, this type of inhibition is
known as competitive inhibition.
 Many competitive inhibitors are used as drugs
 Example: Amethopterin and aminopterin,
Allopurinol , Mevastatin and lovastatin
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Enzyme inhibition Uses of enzymes

 In medicine and Diagnostics
 to diagnose diseases

NON-COMPETITIVE INHIBITION
 used as drugs to treat a disease
 used as laboratory tool to measure the level of biomolecules

 In Genetic Engineering
 A set of enzymes is used in Biotech for genetic modification
of organisms
 Restriction Endonucleases, Ligases, S1 Nucleases, Taq
 The non-competitive inhibitors have no structural DNA Polymerases etc.
resemblance with the substrate. They do not compete
with the substrate for the substrate site on the  In chemical industries for household items
enzyme.  Cleaning: In detergents for removing stains of blood and
sweat.
 They bind to some other region of the enzyme and
render it inactive. USES OF  Proteases, Amylases and Lipases
 Beauty care and Surgical threads
 Non-competitive inhibition may be reversible or
irreversible. Generally it is irreversible. ENZYMES  Chitinase – cosmetics and surgical threads

 Paper and Textile Industry

 Examples are iodoacetamide, p-  Bio-bleaching
chloromercuribenzoate, heavy metals etc
 Hemicellulase – degrade plant cell wall
 In kinetics, In the presence of a non-competitive inhibitor,  Laccases – Lignolytic enzyme from plants
Lineweaver-Burk plot shows that:
 x-intercept is unchanged  Catalase – is used to remove any hydrogen peroxide
residues after bleaching
 y-intercept is higher  Deinking
 This means that non-competitive inhibitors lower the  Multiple enzymes have been adopted for the deinking process.
Some of them are esterases, lipases, hemicellulases,
Vmax but do not affect the Km ligninolytic enzymes, and cellulases.
 The pulp that has enzymatically deinked has superior quality
physical properties.

Uses of enzymes Uses of enzymes

USES OF ENZYMES In Dairy and

Meat and Leather Industry
• For Meat tenderization
Food Processing
• Bromelain – ( from Pineapple stem) Making Fructose from Starch
• Papain – ( from unripe papaya) • Amylase and Glucose isomerase

• Ficin – ( from fig fruit latex)

USES OF Cheese Making

ENZYMES
• Chymosin (Rennet) – from Calf abomasum
In agriculture and animal feed •
•
Pepsin - ( Bovine abomasum)
Lipase – for cheese flavor enhancement
• Catalase - Pasteurization and cheese flavor
• Animal feed:
Low lactose dairy milk – for lactose intolerants
• Phytate, Glucan, Raffinose etc. • Lactase or beta-galactosidase ( from fungi aspergillus oryzae)

• Agriculture: Phytases Baby milk – predigested proteins for easy digestions

which enhances the nutritional • Trypsin – Proteolytic activity converts milk protein(casein) into small peptides and free amino acids

values of the food grains Other Dairy Enzymes

• Phosphatase – Quality testing and Pasteurization
Biofuels •
•
Lysozymes - Antimicrobial ( to control bacteria)
Chitinases - Antimicrobial ( to control fungi)
• Beta- Glucanases – Antimicrobial ( to control yeast)
• Lacto-Peroxidases – used instead of preservative agents
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ISOENZYMES ZYMOGENS
 Enzymes that exist in multiple molecular forms but catalyse
the same reaction  Zymogens are inactive precursors of enzymes. Most digestive
 They may occur in different tissues of same organs, in and blood-clotting enzymes exist in the zymogen form, until
different cell types, or in sub cellular compartments. The activated.
tissue distribution of isoenzymes is highly specific.
Measurement of isoenzymes can be of great diagnostic  In the case of digestive enzymes, this is necessary to prevent
importance. Lactate dehydrogenase on electrophoresis digestion of pancreatic and gastric tissue. For blood clotting, it is
 Differ slightly in physical, chemical and immunological
gives 5 different bands and has 4 protomers
to avoid premature of blood cells
properties. The normal pattern of LD isoenzymes
in serum is LD2 >LD1 >LD3 >LD4 >LD5 ZYMOGEN ACTIVE FORM OF ENZYME
 Isoenzymes Usually differ in their Km and Vmax values
pepsinogen pepsin
 These can be separated by electrophoresis, Chromatography trypsinogen trypsin
and Immunochemical methods.
prothrombin thrombin
 Isoenzymes of diagnostic importance include:
 Lactate dehydrogenase
 Creatine kinase
 Alkaline phosphatase

Immobilization of enzymes Immobilization of enzymes

IMMOBILIZATION OF ENZYMES EXAMPLE: LACTOSE-FREE MILK

USE: Lactose intolerance patients . Lactose causes allergy due to insufficient production of lactase enzyme in body.

• Lactase obtained from commonly from yeast (bacteria is

 Enzyme immobilization is a technique to produce stable enzymes an alternative)
which can used for commercial purposes.
 Generally enzymes are natural bio-catalysts that produced inside • Lactase mixed with sodium alginate – then each droplet
the body of living organism, animals, plants and microorganisms. put into calcium chloride – which then immediately
When enzymes isolated out of the body for external use, they get
damaged easily. forms beads.
 So in order to avoid damage, Enzyme immobilization techniques is • Lactase is bound to the surface of alginate beads. The
developed which is used to isolate enzymes and store them
externally for a longer duration. Thus help in utilizing the complete enzyme converts the substrate into product as it flows
potential of enzyme and its reuse without loss of its activity. by the vessels.
 Immobilization of enzymes greatly simplifies the recovery process,
enhances process control, and reduces operational costs. • Milk is passed (repeatedly) over the beads.
 A small amount of enzyme can catalyse the formation of large • The lactose is broken down into glucose and galactose
amount of product, making their application very economical for
industry.
• The immobilized enzyme remains to be used again and
 These industrial enzymes are commercially used in a variety of does not affect the quality of the lactose free milk
industries such as pharmaceuticals, chemical
production, biofuels, food & beverage, and consumer products.
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COURSE OUTLINE WHAT IS CELL SIGNALING?

 Cell signaling refers to the cell-cell communications. That is, the way by which the cells
communicate with each other.
 Cell Metabolism
 Response to stimulus is the nature of Living beings. All live organisms keep receiving signals
(external cues) from external environments or surroundings through their sensory organs.
 Enzymes and Catalysis These signals transmit to the brain to take further actions.
 The cellular response to these stimulus is carried out by a series of biological events which
 Cell Communication and Signalling involve different cells to communicate and generate response according to the commands
received from brain.

WHAT IS CELL SIGNALING? THREE STAGES OF CELL SIGNALING

 In simple words one cell (signaling 1. Reception:

 A cell detects a signaling molecule from
cell) sends signals (or information) to the outside of the cell. A signal is detected
another cell (responding cell) to take when the chemical signal (also known as a
action or generate response. ligand) binds to a receptor protein on the
surface of the cell or inside the cell.
 Mostly the signal or information is 2. Transduction:
transferred in the form of chemical  When the signaling molecule binds the
message ( also called ligand receptor it changes the receptor protein in
molecule). This message is received by some way. This change initiates the
a specific group of cells having process of transduction. Signal
transduction is usually a pathway of
receptors on their surface. several steps. Each relay molecule in the
signal transduction pathway changes the
 Receptors on surface receive the next molecule in the pathway.
signals and starts the signal
transduction pathway which as a 3. Response:
 Finally, the signal triggers a specific cellular
results alters the gene expression response.
levels of responding proteins.
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SIGNAL TRANSDUCTION PATHWAY SIGNAL TRANSDUCTION PATHWAY

 Signal transduction refers to the overall process of converting extracellular signals into intracellular  Example: Protein Tyrosine kinase pathway
responses.
 In this signal transduction pathway,
 A signal transduction pathway represents the flow of information in a cell. The pathway starts when activation of the kinase function of the
a signaling molecule or ligand binds to its receptor protein. receptor by ligand binding results in
 The receptor-ligand interaction triggers a change in the receptor that is carried or transduced to the phosphorylation of an adaptor or other
next protein in the pathway. protein.
 Receptors and signal transduction systems have evolved to detect and respond to hormones, growth  If the next protein in the sequence is an
factors, neurotransmitters, pheromones, oxygen, nutrients, light, touch, heat, etc. inactive kinase, then the addition of a
phosphate group will alter its conformation,
 There are an enormous number of signal molecules and receptors in cells. In contrast, there are converting it into an active kinase.
relatively few types of intracellular signal transduction systems.
 The resulting phosphorylation cascade will
 Key players in signal transduction are signaling molecules, receptors, signal transduction proteins and eventually result in a change in the cell’s
second messengers, and effector proteins. behavior either by modifying the activity of
 Cells respond to signals by changing the activity of existing enzymes (fast) and/or the levels of proteins already present within the
expression of enzymes and cell components (slower) by gene regulation. cytoplasm or by altering gene expression.

TYPES OF CELL SIGNALING ENDOCRINE SIGNALING

 Cell-cell signaling involves the transmission of a signal
(chemical message) from a sending cell to a receiving cell. • Endocrine signaling acts over long distances within the organism (e.g.,
 There are four basic categories of chemical signaling found in insulin by endocrine gland).
multicellular organisms:
 Paracrine signaling • A cell targets a distant cell through the bloodstream.
 Autocrine signaling • A signaling molecule is released by one cell, then travels through the
 Endocrine signaling bloodstream to bind to receptors on a distant target cell elsewhere in the
 Cell-to-cell direct contact signaling body.
 The main difference between the different categories of
signaling is the distance that the signal travels through the
organism to reach the target cell.
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PARACRINE SIGNALING AUTOCRINE SIGNALING

• Paracrine signaling acts over very short distances, for example between neighboring  In autocrine signaling, a cell signals to itself, releasing a ligand that binds to receptors
cells.
on its own surface (or, depending on the type of signal, to receptors inside of the cell).
 Example: Neurotransmitters and developmental signals typically act in this manner.
• Paracrine signaling allows cells to locally coordinate activities with their neighbors.  Many growth factors act in this fashion, and cultured cells often secrete growth
• Cells that are near one another communicate through the release of chemical factors that stimulate their growth and proliferation.
messengers (ligands that can diffuse through the space between the cells).
 This may seem like an odd thing for a cell to do, but autocrine signaling plays an
• Paracrine signals are especially important during development, when they allow one
group of cells to tell a neighboring group of cells what cellular identity to take on. important role in many processes.
Example: spinal cord development  For instance, autocrine signaling is important during development, helping cells
• The conduction of an electric impulse from one nerve cell to another or from one take on and reinforce their correct identities.
nerve cell to an effector or muscle cell occurs via paracrine way.  From a medical standpoint, autocrine signaling is important in cancer and is
thought to play a key role in metastasis (the spread of cancer from its original site
to other parts.
 In many cases, a signal may have both autocrine and paracrine effects, binding to the
sending cell as well as other similar cells in the area.

Cell-to-celldirect contact signaling SIGNALING RECEPTORS AND

LIGANDS
 In this type of signaling systems cells communicates directly via cell-to-cell contact through cell
membrane.  Receptor is a molecule which receives specific chemical signal in
 It involves plasma membrane-attached proteins for communication.
cellular communication.
 Gap junctions in animals and plasmodesmata in plants are example of such cellular communication  These receptors either found on the surface of a cell (cell-
where a tiny channels directly connect neighbouring cells and transduce signal through diffusion.
surface receptors) or inside the cell in cytoplasm ( nuclear
 The transfer of signaling molecules transmits the current state of one cell to its neighbor. This allows a
group of cells to coordinate their response to a signal that only one of them may have received. receptors).
 Small molecules, such as calcium ions (Ca2+), are able to move between cells, but large molecules like
proteins and DNA cannot fit through the channels without special assistance.  A molecule which binds to a receptor is called a ligand. Ligand
may be a peptide (short protein) or other small molecules such
as neurotransmitter, a hormone, a drug or a toxin.
 The accurate binding of ligand to receptors opens or elicit a
biochemical pathway as a response.
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DIFFERENCE B/W INTRA AND EXTRA

TYPES OF RECEPTORS CELLULAR RECEPTORS

 Intracellular
 These are mostly proteins and found inside the cell, usually in  Cell surface receptors
cytoplasm or nucleus.
 The signal (or ligand) is basically very small and hydrophobic (water-  Receives hydrophilic Signals
hating) in nature.  Membrane bound
 Small size (of ligand) enable it to cross the plasma membrane and
reach their receptors  Signals released into extracellular
 Example space – local (paracrine) or long-
 Sex hormones estradiol (estrogen) and testosterone distance (endocrine)
 Extracellular
 Found on cell surface. Also called Cell-Surface Receptors.  Intracellular receptors
 They are membrane-anchored proteins that bind to ligands on the  Receives hydrophobic signals on carrier
outside surface of the cell.
 In this type of signaling, the ligand does not need to cross the plasma proteins
membrane. So, many different kinds of molecules (including large,
hydrophilic or "water-loving" ones) may act as ligands.  Pass directly through cellmembrane
 Example:  Receptors present in cytosol or nucleus
 Most common extracellular signaling molecules are hormones,
neurotransmitters, cytokines, growth factors and cell recognition molecules.
These molecules attached to the receptor and triggers the signal transduction
pathway.

TRANSDUCTION - SUMMARY
 Since signaling systems need to be responsive
to small concentrations of chemical signals and
act quickly, cells often use a multi-step
pathway that transmits the signal quickly, while
amplifying the signal to numerous molecules at
each step.
 Steps in the signal transduction pathway often
involve the addition or removal of phosphate
groups which results in the activation of
proteins.
 Enzymes that transfer phosphate groups from
ATP to a protein are called protein kinases.
 Many of the relay molecules in a signal
transduction pathway are protein kinases and
often act on other protein kinases in the
pathway. Often this creates a phosphorylation
cascade, where one enzyme phosphorylates
another, which then phosphorylates another
protein, causing a chain reaction.