NEW ERA SENIOR SECONDARY SCHOOL

AUROVILLE

AFFILIATION NO: 1980010

BIOLOGY INVESTIGATORY PROJECT

ON

DIFFERENT PROPERTIES OF ENZYMES

BY

S.Akash

XII-SCIENCE

2019-2020
 CONTENT

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 PH, 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 PH 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 PH and temperature. Most
mammalian enzymes operate maximally at around physiological PH 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 PH 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 pH 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 O2 as there is no
reaction whereas second test tube shows froth thus indicating release of O2
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 PH. 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.