NEW ERA SENIOR SECONDARY SCHOOL

AUROVILLE

DIFFERENT PROPERTIES OF ENZYMES

NAME: R.PRAVEEN
STD: 12TH STD
SUBJECT: BIOLOGY
UNDER THE GUIDANCE: MRS.TAMIZHARASI

CONTENTS
 1. INTRODUCTION
2. AIM
3. MATERIALS REQUIRED
4. THEORY
5. PROCEDURE
6. OBSERVATION
7. RESULT
8. CONCLUSION
9. PRECAUTIONS

INTRODUCTION
Enzyme, a substance that acts as a catalyst in living organisms, regulating the rate at which
chemical reactions proceed without itself being altered in the process. The biological
processes that occur within all living organisms are chemical reactions, and most are
regulated by enzymes. Without enzymes, many of these reactions would not take place at a
perceptible rate. Enzymes catalyze all aspects of cell metabolism. This includes the digestion
of food, in which large nutrient molecules (such as proteins, carbohydrates, and fats) are
broken down into smaller molecules; the conservation and transformation of chemical
energy and the construction of cellular macromolecules from smaller precursors. Many
inherited human diseases, such as albinism and phenylketonuria, result from a deficiency of
a particular enzyme. Enzymes are built of proteins folded into complicated shapes, they are
present throughout the body.The chemical reactions that keep us alive - our metabolism on
the work that enzymes carry out.Enzymes speed up (catalyze) chemical reactions; in some
cases, enzymes can make a chemical reaction millions of times faster than it would have
been without it .A substrate binds to the active site of an enzyme and is converted into
products. Once the products leave the active site, the enzyme is ready to attach to a new
substrate and repeat the process.
Enzymes also have valuable industrial and medical applications. The fermenting of wine,
leavening of bread, curdling of cheese, and brewing of beer have been practiced from earliest
times, these reactions understood to be the result of the catalytic activity of enzymes. Since
then, enzymes have assumed an increasing importance in industrial processes that involve
organic chemical reactions. The uses of enzymes in medicine include killing disease-causing
microorganisms, promoting wound healing, and diagnosing certain diseases.
Early Enzyme Discoveries
The existence of enzymes has been known for well over a century. Some of the earliest
studies were performed in 1835 by the Swedish chemist Jon Jakobe Berzelius who termed
their chemical action catalytic. It was not until 1926, however, that the first enzyme was
obtained in pure form, a feat accomplished by James B. Sumner of Cornell University.
Sumner was able to isolate and crystallize the enzyme urease from the jack bean. His work
was to earn him the 1947 Nobel Prize.
John H. Northrop and Wendell M. Stanley of the Rockefeller Institute for Medical Research
shared the 1947 Nobel Prize with Sumner. They discovered a complex procedure for
isolating pepsin. This precipitation technique devised by Northrop and Stanley has been used
to crystallize several enzymes.
Chemical Nature
All enzymes were once thought to be proteins, but since the 1980s the catalytic ability of
certain nucleic acids, called ribozymes (or catalytic RNAs), has been demonstrated. A large
protein enzyme molecule is composed of one or more amino acid chains called polypeptide
chains. The amino acid sequence determines the characteristic folding patterns of the
protein’s structure, which is essential to enzyme specificity. If the enzyme is subjected to
changes, such as fluctuations in temperature or P H, the protein structure may lose its integrity
(denature) and its enzymatic ability.
Bound to some enzymes is an additional chemical component called a cofactor, which is a
direct participant in the catalytic event and thus is required for enzymatic activity. A cofactor
may be either a coenzyme an organic molecule, such as a vitamin or an inorganic metal ion;
some enzymes require both. A cofactor may be either tightly or loosely bound to the
enzyme. If tightly connected, the cofactor is referred to as a prosthetic group.
Enzymes mechanism of action
 The digestive system - enzymes help the body break down larger complex molecules
into smaller molecules, such as glucose, so that the body can use them as fuel.

 DNA replication - each cell in your body contains DNA. Each time a cell divides, that
DNA needs to be copied. Enzymes help in this process by unwinding the DNA coils
and copying the information.

 Liver enzymes - the liver breaks down toxins in the body. To do this, it uses a range of
enzymes.

 Enzymes can only work in certain conditions. Most enzymes in the human body work
best at around 37°C - body temperature. At lower temperatures, they will still work but
much more slowly.

 Similarly, enzymes can only function in a certain PH range (acidic/alkaline). Their

preference depends on where they are found in the body. For instance, enzymes in the
intestines work best at 7.5 pH, whereas enzymes in the stomach work best at pH 2
because the stomach is much more acidic.

 If the temperature is too high or if the environment is too acidic or alkaline, the enzyme
changes shape; this alters the shape of the active site so that substrates cannot bind to it
- the enzyme has become denatured.
Structure of enzymes
Enzymes always act as catalysts and small quantities compared to their substrate are required
to considerably increase the rate of chemical reactions, wherein the enzymes themselves
experience no overall change .An enzyme does not alter the ultimate equilibrium position of
a reaction, which is thermodynamically determined, thus merely the rate of completion of
equilibrium of a feasible reaction is augmented. In addition to catalytic properties, enzymes
exhibit the physico-chemical behaviour of proteins: their solubility, electrophoretic
properties, electrolytic behaviour and catalytic action of enzymes is determined by the linear
chain of amino acid residues linked via peptide bonds, which constitute a protein molecule.
Localized folding of the primary structure is called a secondary structure, whereas the
complete folding of the molecule is known as a tertiary structure. In contrast to these
structural configurations, a quaternary structure is the agglomeration of several folded
chains. The structural features of enzymes. In contrast to traditional chemical catalysts, e.g.
hydrogen ions, heavy metals or metal oxides, which are most effective in organic solvents, at
very high temperatures or at extreme pH values, enzymes operate most efficiently under very
mild conditions. When using enzymes, there are certain issues that require attention, such as
deviation from homogeneous aqueous solutions, physiological P H and temperature, which
can rapidly destroy enzyme activity. Under normal conditions the increase in reaction rate is
rarely matched by their non-protein counterparts.

STRUCTURE OF ENZYME
Nomenclature
An enzyme will interact with only one type of substance or group of substances, called the
substrate, to catalyze a certain kind of reaction. Because of this specificity, enzymes often
have been named by adding the suffix “-ase” to the substrate’s name (as in urease, which
catalyzes the breakdown of ureurea. A classification system has been developed based on the
type of reaction the enzyme catalyzes. There are six principal categories and their reactions:
(1) Oxidoreductases, which are involved in electron transfer
(2) Transferases, which transfer a chemical group from one substance to another
(3) Hydrolases, which cleave the substrate by uptake of a water molecule (hydrolysis)
(4) Lyases, which form double bonds by adding or removing a chemical group
(5) Isomerases, which transfer a group within a molecule to form an isomer
(6) Ligases, or synthetises, which couple the formation of various chemical bonds to the
breakdown of a pyrophosphate bond in adenosine triphosphate or a similar nucleotide.

Factors Affecting Enzyme Activity

Because enzymes are not consumed in the reactions they catalyze and can be used over and
over again, only a very small quantity of an enzyme is needed to catalyze a reaction. A
typical enzyme molecule can convert 1,000 substrate molecules per second. The rate of an
enzymatic reaction increases with increased substrate concentration, reaching maximum
velocity when all active sites of the enzyme molecules are engaged. The enzyme is then said
to be saturated, the rate of the reaction being determined by the speed at which the active
sites can convert substrate to product.

i. Enzyme affected by temperature

Like most chemical reactions, the rate of an enzyme-catalysed reaction

increases as the temperature is raised. A ten degree Centigrade rise in temperature will
increase the activity of most enzymes by 50 to 100%.Over a period of
time, enzymes will be deactivated at even moderate temperatures.

ii. Enzyme affected by PH

Changes in pH may not only affect the shape of an enzyme but it may also change the
shape or charge properties of the substrate so that either the substrate cannot bind to
the active site or it cannot undergo catalysis.
Enzymes have optimum PH which is not same for each enzymes.
iii. Enzyme affected by low temperature

This flexibility is essential to how enzymes bind to other molecules and cause
chemical reactions to happen on those molecules. Lowering the temperature slows the
motion of molecules and atoms, meaning this flexibility is reduced or lost. As
the temperature decreases, so does enzyme activity

Enzyme Inhibition mechanisms

Enzyme activity can be inhibited in various ways.
i. Competitive inhibition occurs when molecules very similar to the substrate molecules
bind to the active site and prevent binding of the actual substrate. Penicillin, for
example, is a competitive inhibitor that blocks the active site of an enzyme that many
bacteria use to construct their cell walls.

ii. Non-competitive inhibition occurs when an inhibitor binds to the enzyme at a location
other than the active site. In some cases of non-competitive inhibition, the inhibitor is
thought to bind to the enzyme in such a way as to physically block the normal active
site. In other instances, the binding of the inhibitor is believed to change the shape of
the enzyme molecule, thereby deforming its active site and preventing it from reacting
with its substrate. This latter type of non-competitive inhibition is called allosteric
inhibition; the place where the inhibitor binds to the enzyme is called the allosteric
site. Frequently, an end-product of a metabolic pathway serves as an allosteric
inhibitor on an earlier enzyme of the pathway. This inhibition of an enzyme by a
product of its pathway is a form of negative feedback.

Allosteric control can involve stimulation of enzyme action as well as inhibition. An

activator molecule can be bound to an allosteric site and induce a reaction at the active
site by changing its shape to fit a substrate that could not induce the change by itself.
Common activators include hormones and the products of earlier enzymatic reactions.
Allosteric stimulation and inhibition allow production of energy and materials by the
cell when they are needed and inhibit production when the supply is adequate.
Enzyme inhibition decreases the activity of an enzyme without significantly disrupting
its three-dimensional macromolecular structure. Inhibition is therefore distinct from
 denaturation and is the result of a specific action by a reagent directed or transmitted

to the active site region. When low molecular weight compounds interfere with the
activity of enzymes by partially reducing or completely inhibiting the enzyme activity
either reversibly or irreversibly, it is known as enzyme inhibition. The compounds
responsible for such inhibition are called enzyme inhibitors. To protect the enzyme
catalytic site from any change, a ligand binds with a critical side chain in the enzyme.
Chemical modification can be performed to test the inhibitor for any drug value.
Studies of enzymes can yield much information about the following:

Cofactors
Some enzymes cannot function unless they have a specific non-protein molecule attached to
them. These are called cofactors. For instance, carbonic anhydrase, an enzyme that helps
maintain the pH of the body, cannot function unless it is attached to a zinc ion.
Advantages of using enzymes
• A number of drugs useful in medicine, which seem to function because they can inhibit
certain enzymes in malfunctioning cells.
• The convenience of elucidating metabolic pathways in cells.
• The mechanism of the catalytic activity.
• The nature of the functional group at the active site.
The substrate specificity of the enzyme.
The pharmacological action of drugs is mainly based on enzyme inhibition, e.g.
sulphonamides and other antibiotics. In the majority of cases the enzyme inhibited is known.
The development of nerve gases, insecticides and herbicides is based on enzyme inhibition
studies. There are two major types of enzyme inhibition: reversible and irreversible.

Reversible inhibitors efficiently bind to enzymes by forming weak non-covalent interactions

Example’s: ionic bonds, hydrophobic interactions and hydrogen bonds. Reversible inhibitors
do not form any strong chemical bonds or reactions with the enzyme; they are formed
quickly and can easily be removed, in contrast to irreversible inhibitors. Reversible
inhibition includes competitive inhibition, uncompetitive inhibition and non-competitive
inhibition. Irreversible inhibition includes group specific inhibition (reacts only to a certain
chemical group), reactive substrate analogs (affinity label) and inhibitors that are structurally
similar to the substrate and will bind to the active site, and mechanism-based inhibitors
(enzymes transform the inhibitor into a reactive form within the active site).
 Competitive inhibitors - a molecule blocks the active site so that the substrate has to
compete with the inhibitor to attach to the enzyme.
 Non-competitive inhibitors - a molecule binds to an enzyme somewhere other than the
active site and reduces how effectively it works.
 Uncompetitive inhibitors - the inhibitor binds to the enzyme and substrate after they
have bound to each other. The products leave the active site less easily, and the
reaction is slowed down.

Enzyme Kinetics
Enzyme kinetics is the study of the chemical reactions that are catalysed by enzymes. In
enzyme kinetics, the reaction rate is measured and the effects of varying the conditions of the
reaction are investigated. Studying an enzyme's kinetics in this way can reveal the catalytic
mechanism of this enzyme, its role in metabolism, how its activity is controlled, and how
a drug or an agonist might inhibit the enzyme.
Enzymes are usually protein molecules that manipulate other molecules—the
enzymes' substrates. These target molecules bind to an enzyme's active site and are
transformed into products through a series of steps known as the enzymatic mechanism
E + S ⇄ ES ⇄ ES* ⇄ EP ⇄ E + P

These mechanisms can be divided into single-substrate and multiple-substrate mechanisms.

Kinetic studies on enzymes that only bind one substrate, such as triose phosphate isomers,
aims to measure the affinity with which the enzyme binds this substrate and the turnover rate
can be studied.
The rate of an enzyme-catalyze reaction varies with the substrate concentration. At very low
substrate concentrations, the rate will be directly proportional to the concentration of the
substrate and will exhibit first-order kinetics. At very high substrate levels, all of the active
sites are occupied.
Erepsin: converts peptones and polypeptides into amino acids.
Maltase: converts maltose into glucose.
Lactase: This is a significant enzyme that converts lactose into glucose and galactose.
Sucrase: converts sucrose into glucose and fructose.
Other disaccharides.

Enzyme factors
This represents saturation, and the reaction rate is at its maximum and is designated Vmax.
Other factors that influence enzyme activity include P H and temperature. Most mammalian
enzymes operate maximally at around physiological P H and body temperature. The stomach
digestive enzyme pepsin works best at around PH 2.0, the approximate PH of the stomach.
Bacteria found in hot springs have enzymes that operate at or near the boiling point of water.
In most cases, however, extremes of P H or temperature will destroy enzymes in an enzyme-
unfolding process known as denaturation.

Symptoms of inadequate enzymes:

 Feeling tired easily
 Muscle pain
 Eat a lot, but still look skinny
 Indigestion
 No appetite
 Aged earlier than expected
 Low blood sugar
 Imbalance fat secretion
AIM
To determine the important properties of enzymes

MATERIALS REQUIRED
Test tube standard, test tubes, tape, breakers, pipette, water bath, thermometer, spirit
lamp,biuret reagent, phenolphthalein, Iodine, heavy metal ions (like AgNO3,HgCl),
H2O2,urea and starch

THEORY
Enzymes are biocatalysts that influence biochemical reaction rates without altering the
equilibrium of reaction. Enzymes work upon lock and key mechanism.
Properties of enzymes:
1. They are proteinaceous, biocatalysts.
2. Enzymes are specific in nature.
3. They exhibit inhibition by heavy metals
4. Their activity depends on temperature
5. Their activity depends on P

PROCEDURE
1. For demonstrating proteinaceous nature:
Biuret reagent gives test with peptide bonds and proteins have peptide bonds.
2ml Enzymes +2ml biuret reagent = violet
Bluish colour indicates proteinaceous nature .Higher is the concentration of proteins
darker is the colour

2. For demonstrating specific nature in their activity:

In two test tubes take 2 ml of amylase. To one test tube add 2 ML starch and add 2 ml
of Urea in another test tube. Put a drop of phenolphthalein in the second test tube and
drop of iodine in the first test tube. Observe the change of colours into test tubes.
 3. For demonstrating inhibition by heavy metals:
Heavy metals bind to sulphidal group of enzyme and make it ineffective and now
enzyme cannot bind to the substrate. In two test tubes take 2ml of catalase. In one tube
add 2 ml of inhibit or HgCl or AgNO3. Now in both tubes add 2 ml of substrate, H.
Now observe the appearance of froth.

4. For demonstrating effect of temperature:

In one test tube take 2 ml of unboiled enzyme at room temperature and in another test
tube take 2 ml of boiled catalase enzyme. In both the tubes add 2 ml H2O and study
the appearance of froth.

5. For demonstrating effect of PH:

In 2 tubes add 2 ml of enzymes catalase. In one tube add p H tablet for ph3. Now add
H2O2 to both the tubes and observe the appearance of froth.

OBSERVATION
1. Proteinaceous nature:
Violet are bluish colour indicates protein nature of enzymes

2. Specific nature of enzymes:

In first test tube no blue colour appears showing starch has been broken down by
amylase. In second test tube there is no formation of pink colour showing urea has not
been acted upon by amylase.

3. Inhibition by heavy metals:

In first test tube no froth appears showing no release of O 2 as there is no reaction
whereas second test tube shows froth thus indicating release of O 2 due to breakdown of
H2O2 to H2O and or O2.

4. Effect of Temperature:
Froth appears in first test tube with unboiled enzyme and no froth is seen in second
test tube because enzyme gets denatured at high temperature.

5. Effect of pH:
First test tube with normal pH shows appearance of froth but second test tube does not
shows froth because of change in PH the enzymes has been inactivated.
RESULT
The various tests demonstrate the above stated properties of enzymes.

CONCLUSION
The rate of a chemical reaction increases as the substrate concentration increases. Enzymes
can greatly speed up the rate of a reaction. However, enzymes become saturated when the
substrate concentration is high. The reaction rate depends on properties of the enzymes, the
enzyme concentration (E) .Enzymes work best at optimal P H. Just like PH, enzymes perform
at their best in a small range of temperature. These biological catalyst increase the rate of
reaction when exposed to the optimum temperature and would underperform when
temperature is lower or higher than the optimal temperature are introduced. Enzymes remain
active at optimal PH and temperature. Enzymes are used in various fields which benefit
humans in day to day life.

PRECAUTIONS
1. Equal amounts of enzymes and substrate should be taken.
2. Boiled enzymes should be cooled to room temperature before adding substrate.