COLLEGE NATURAL AND APPLIED SCIENCES

DEPARTMENT OF BIOTECHNOLOGY

BIOLOGY PRACTICAL MANUAL

FOR STEM STUDENTS

EXPERIMENT 6: BIOLOGICAL CATALYSTS (Enzymes )

Objective: - at the end of this laboratory activity students will be able to

 Describe biological catalysts.

 Explain the basic properties of an enzyme as a catalyst.
 Explain the importance of enzymes in biology.
 Discuss the effect of temperature, pH, substrate concentration, and enzyme concentration on the
rate of an enzymatic reaction.

INTRODUCTION

Herein the universe, there are so many types of chemical reactions. These chemical reactions take place in
industry, laboratory, factory and living system. These chemical reactions take place spontaneously or else at
high rate. Chemical reactions that are undergoing spontaneously have low speed to give products while
chemical reactions that are taking place non-spontaneously with the addition of some substances will have
remarkably high rate of reaction and give more products with a short period of time.

These chemical substances increase the chemical reaction are known as CATALYSTS. There are two types
of catalysts namely INORGANIC AND ORGANIC catalysts. Inorganic catalysts are chemical substances
which speeding up the chemical reactions in industries, factories and laboratories. Example conc.H2SO4, Pt.

Organic catalysts are chemical substances which speeding up the chemical reactions takes place in living
system. These chemical substances are known as BIOCATALYSTS / BIOLOGICAL
CATALYSTS/ENZYMES. What you mind is that does not mean all organic catalysts are only working in
the living system, but there are some organic catalysts speeding up the chemical reactions outside the living
system. For instance carbon tetra chloride in aqueous solution (CCl4aq) acts as organic catalyst.

In fact in any chemical reaction, reactants require the minimum amount of energy to start the chemical
reaction is known as ACTIVATION ENERGY. So enzymes decrease this activation energy to speeding up
the chemical reaction.

EMZYMES

In 1878 K¨uhne introduced the term ‘enzyme’ from the Greek ‘’enzumos’’, which refers to the leavening of
bread by yeast. The study of enzyme is known as ENZYMOLOGY while the student is known as
ENZYMOLOGIST. AS the matter of fact cells function largely because of the action of enzymes. This means
that all chemical reactions in life are dynamic and are controlled by enzymes.

Enzyme kinetics

Enzyme kinetics is a discipline which studies about reaction mechanism or it is the study of the chemical
reactions that are catalysed by enzymes. The mechanism of an enzyme-catalyzed reaction is studied by:

– Determining the rate of the reaction.

– Understanding how it changes in response to changes in experimental parameters.

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. 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
isomerase, aim to measure the affinity with which the enzyme binds this substrate and the turnover rate.
Some other examples of enzymes are phosphofructokinase and hexokinase, both of which are important for
cellular respiration (glycolysis).

When enzymes bind multiple substrates, such as dihydrofolate reductase, enzyme kinetics can also show the
sequence in which these substrates bind and the sequence in which products are released. Examples of
enzymes that bind a single substrate and release multiple products are proteases, which cleave one protein
substrate into two polypeptide products. Others join two substrates together, such as DNA
polymerase linking a nucleotide to DNA. Although these mechanisms are often a complex series of steps,
there is typically one rate-determining step that determines the overall kinetics. This rate-determining
step may be a chemical reaction or a conformational change of the enzyme or substrates, such as those
involved in the release of product(s) from the enzyme. Knowledge of the enzyme's structure is helpful in
interpreting kinetic data. For example, the structure can suggest how substrates and products bind during
catalysis; what changes occur during the reaction; and even the role of particular amino acid residues in the
mechanism. Some enzymes change shape significantly during the mechanism; in such cases, it is helpful to
determine the enzyme structure with and without bound substrate analogues that do not undergo the
enzymatic reaction. Not all biological catalysts are protein enzymes; RNA-based catalysts such
as ribozymes and ribosomes are essential to many cellular functions, such as RNA splicing and translation.

The main difference between ribozymes and enzymes is that RNA catalysts are composed of nucleotides,
whereas enzymes are composed of amino acids. Ribozymes also perform a more limited set of reactions,
although their reaction mechanisms and kinetics can be analyzed and classified by the same methods. All
chemical reactions are theoretically reversible, i.e., they proceed in both the directions, although at different
rates:

A+B C + D. The rates of forward and reverse reactions depend on:

– The concentrations of reactants (e.g., A and B).

– The concentration of products (e.g., C and D).
– The "rate constants" for the forward and backward reactions.
The reaction rate increases with substrate concentration in first-order kinetics. The rate stabilizes when there
is an excess of substrate – zero-order kinetics. What Michaelis and Menten discovered was a simple means
of solving the equations for two variables. If multiple plots of enzyme activity vs. substrate concentration
are made with increasing enzyme concentration, the value of V continues to increase, but the substrate
concentration which corresponds to 1/2 V remains constant. This concentration is the Michaelis
Constant for an enzyme. As mentioned, it is designated as K and is operationally the concentration of
substrate which will give exactly 1/2 V when reacted with an enzyme with maximum pH, temperature
and cofactors. Therefore, catalytic action of an enzyme on a given substrate can be described by TWO
parameters:
– Vmax – the maximal velocity of the reaction at saturating substrate concentrations.
– Km – the Michaelis constant, a measure of the affinity of an enzyme for its substrate.
– The Km is defined as the substrate concentration that yields a half-maximal reaction rate (i.e. Vmax).

According to the Michaelis-Menten equation:

V (S)
V = -----------
K + (S)
This equation is derived from the formula for a hyperbola (c=xy) where K = (S)(V /v-1)

When v=V /2, K = (S)(V /(V /2)-1) = (S) confirming that the units of this constant are those of
concentration. The Michaelis-Menten curve describes constant Km, the substrate concentration that
corresponds to ½ V max – the maximum reaction velocity or rate.

The smaller the value of Km, the more strongly an enzyme can bind substrate from a dilute solution
and the smaller the substrate concentration needed to reach half-maximal velocity.
Smaller Km = more efficient enzyme; larger km = less efficient enzyme

In 1934, two individuals, Lineweaver and Burke made a simple mathematical alteration in the process by
plotting a double inverse of substrate concentration and reaction rate (velocity).

The Lineweaver/Burke equation is:

This equation fits the general form of a straight line, y= mx+b, where m is the slope of the line and b is the
intercept. Thus, the Lineweaver/Burke Plot for an enzyme is more useful than Michaelis-Menten, since as
velocity reaches infinity, 1/V approaches 0. Moreover, since the plot results in a straight line, the slope
is equal to K /V , the y intercept equals 1/V (1/S=0). Hence, Lineweaver-Burk transformation gives
inverse: 1/Vmax and 1/Km Projection of the line back through the x axis yields the value -1/K (when
1/V=0). These values can easily be determined by using a linear regression plot and calculating the
corresponding values for x=0 and y=0. The inverse of the intercept values will then yield V and K .
.

• X intercept = -1/K
M

• Y intercept = 1/Vmax

• Slope = K /Vmax
M

PROPERTIES OF ENZYMES

Enzymes do have several types of characteristics or properties. Thus includes

i) They are protein. All enzymes are protein but the vice versa is not true. Due to the fact that they
have the three dimensional structure of protein they act as catalysts.
ii) They speed up the chemical reaction. How? This is because they do have an ability to reduce
activation energy.

Enzymes work by weakening bonds which lowers activation energy

iii) They never consumed at the end of the chemical reaction and hence they reused for the next
reaction. This simply summarized as follows
E + S ===== ES ====== E+ P
E – Enzyme S- Substrate ES- enzyme substrate complex P- Product

Q) What is substrate? It is a substance on which an enzyme is acted up on it.

iv) Do not change the equilibrium constant of the reaction. But they can change rate at which
equilibrium is established.
v) Enzymes are specific to their action. That means one enzyme only catalyses one substrate only.
E.g. salivary amylase only catalyses starch, but never protein, lipid, disaccharides, cellulose etc.
vi) Enzymes can act on only one form of isomers of the substrates. For example Lactate
dehydrogenase can recognize only the L-form but the D-form lactate.
vii) Enzymes have active site. Active site is a part of enzymes at which a substrate is fitting in to for
catalytic reactions. Substrates are specific for their enzymes due to this active site. This principle
gives what we call it LOCK AND KEY model of enzymatic action.

Fig. for key and lock model, active site, appropriate substrate

viii) Enzymes cannot work independently as a whole rather joining with other biological molecules
like called coenzymes, apoenzymes and cofactors.
COENZYMES are organic molecules which must bind to protein portion of enzyme in order to
from correct configuration for a reaction.
COFACTORS are inorganic molecules which bind to the protein portion of enzyme to form the
right reaction.
 APOENZYME is proteineous molecule fit in to an enzyme. Therefore maximum rate further
assumes with the presence of any coenzymes and or cofactors that an enzyme requires.
ix) Enzymes are sensitive to different types of parameters/factors that they influence a type of
enzymatic rate of reaction.
x) Enzymes show fast of reaction rate. Reactions with enzymes are up to 10 billion times faster than
those without enzymes. Enzymes typically react with between 1 and 10,000 molecules per second.
Fast enzymes catalyze up to 500,000 molecules per second.

Mechanism of Enzyme Action

This equation represents an enzymatic reaction:
• E+S ↔ ES → P+E E = enzyme, S = substrate, P = product

Formation of the ES complex occurs rapidly. There are two models for specific binding of substrate to
enzyme
– Lock and key specificity (Fisher’s)

– Induced Fit Mode

Key-and-Lock model
According to this principle, the enzyme’s active center is exactly complementary to the substrate. In the
active site there is NO covalent bonding, but only noncovalent ones (like H-bonding) exist. The substrate is
inserted into the active site of the enzyme to form an ES complex. This type of interaction results
CATALYSIS. The following diagram depicting a Key-and-Lock model.
Induced-Fit model

An enzyme-substrate complex forms when the enzyme’s active site binds with the substrate like a key fitting
a lock. Therefore, in induced fit model enzyme and its substrate are not exactly complementary and hence
the shape of the enzyme must match the shape of the substrate. The active site of an enzyme is not rigid.
Binding of substrate to the enzyme, induces a change in shape on the active site of enzyme molecule.
Ultimately, resulting in an optimum fit for S-E interaction. This structural change can put strain on the
substrate and this stress may help bonds to break, thus promoting the reaction

Enzymes are therefore very specific; they will only function correctly if the shape of the substrate matches
the active site. The substrate molecule normally does not fit exactly in the active site. This induces a change
in the enzymes conformation (shape) to make a closer fit. In reactions that involve breaking bonds, the inexact
fit puts stress on certain bonds of the substrate. This lowers the amount of energy needed to break them. The
enzyme does not form a chemical bond with the substrate. After the reaction, the products are released and
the enzyme returns to its normal shape. Because the enzyme does not form chemical bonds with the substrate,
it remains unchanged. As a result, the enzyme molecule can be reused. Only a small amount of enzyme is
needed because they can be used repeatedly.
Factors affecting rate of enzymatic action

The rate at which an enzyme works is influenced by several factors. Such as

A) Temperature
B) PH
C) Concentration of enzyme itself.
D) Concentration of substrate
E) Concentration of salt (salinity)
F) The presence of inhibitors like heavy metals, etc.
G) Inhibitors

TEMPERATURE

It is the degree of hotness and coldness. Temperature has three cardinal values:-

 Minimum value( below the optimum)

 Optimum value ( the correct/ right value)
 Maximum value

As the temperature rises, molecular motion and hence collisions between enzyme and substrate speed up.
But as enzymes are proteins, there is an upper limit beyond which the enzyme becomes denatured and
ineffective. Higher temperature generally causes more collisions among the molecules and therefore
increases the rate of a reaction. More collisions increase the likelihood that substrate will collide with the
active site of the enzyme, thus increasing the rate of an enzyme-catalyzed reaction. Above a certain
temperature, activity begins to decline because the enzyme begins to denature. The rate of chemical
reactions therefore increases with temperature but then decreases.

In biology both extremes are BAD. This implies that too much minimum and too much maximum are not
good, but the best is optimum. As it is mentioned before, enzymes are proteins and hence they will be
disrupted by different temperature value either below or above the optimum value. When we try to sketch
enzymatic reaction rate in relation to temperature, it shows as follows.
Fig. Enzymatic reaction rate in relation to temperature value

As you understand from the above graph, as the temperature increases enzymatic rate of reaction increases
until it becomes optimum and when temperature rises above the optimum enzymatic reaction rate becomes
cease off. Therefore from this you can understand that at lower temperature value enzymes become
INACTIVE and at higher temperature enzymes become DENATURE.

PH

It refers to the point of concentration of hydronium ion or simply the negative logarithm of concentration of
hydronium ion. In the form of equation it can be represented as follows.

PH = -log [H+].

The conformation of a protein is influenced by pH and as enzyme activity is crucially dependent on its
conformation, its activity is likewise affected. A change in p H can alter the ionization of the R groups of the
amino acids. When the charges on the amino acids change, hydrogen bonding within the protein molecule
change and the molecule changes shape. The new shape may not be effective.

The diagram below shows that pepsin functions best in an acid environment. This makes sense because
pepsin is an enzyme that is normally found in the stomach where the pH is low due to the presence of
hydrochloric acid. Trypsin is found in the duodenum, and therefore, its optimum pH is in the neutral range
to match the pH of the duodenum. Enzymes do have different requirement of PH value for their specific
catalyzing reaction. The pH has the following types of values
 i) Acidic value, when pH is less than 7
ii) Neutral value, when pH is equal to 7
iii) Basic or alkaline value, when pH is greater than 7

So that enzymes work best at acidic condition will be affected in basic condition. For example, salivary
amylase works best at pH nearly equal to 7 and pepsin works best at pH of 4. These enzymes can’t function
properly at pH values other than the aforementioned ones. When we try to sketch enzymatic reaction rate in
relation to pH, it shows as follows.

Fig. Enzymatic reaction rate in relation to PH value.

As you observe from the above graph nearly all enzymes work best at neutral condition since as pH value
goes to pH 7 reaction rate of enzymes increase. In general speaking as PH value increases to the optimum
the reaction rate of enzymes so does, but as PH value increases beyond the optimum reaction rate of enzymes
become decreases.

ENZYME CONCENTRATION [E]

If there is insufficient enzyme present, the reaction will not proceed as fast as it otherwise would because all
of the active sites are occupied with the reaction. Additional active sites could speed up the reaction. As the
amount of enzyme is increased, the rate of reaction increases. It means that when enzyme concentration
increases, the collision with substrate increases and hence the enzymatic reaction rate increases until all active
sites are occupied. If there are more enzyme molecules than are needed, adding additional enzyme will not
increase the rate. Reaction rate therefore increases as enzyme concentration increases but then it levels off.
Addition of more concentration of enzyme no further increases the reaction rate instead it goes in constant.
This can be summarized in the following type of graph.

Fig. Enzymatic reaction rate in relation to enzyme concentration itself

As you can see from the above graph the enzymatic reaction rate will only increases as the concentration of
enzyme increases until the occupation of all active site by any more substrate. Therefore any Addition of
more concentration of enzyme no further increases the reaction rate instead it goes in constant.

SUBSTRATE CONCENTRATION [S]

The concentration of substrate molecules (the more of them available, the quicker the enzyme molecules
collide and bind with them). The concentration of substrate is designated [S] and is expressed in units
of molarity. At lower concentrations, the active sites on most of the enzyme molecules are not filled because
there is not much substrate. Higher concentrations cause more collisions between the molecules. With more
molecules and collisions, enzymes are more likely to encounter molecules of reactant.

Reaction rate increases proportionately with an increase in substrate concentration[S] it is then defined as
first-order kinetics. Km is a constant specific for each enzyme: the [S] that corresponds to ½ maximum
velocity. [S] increases until available enzyme is saturated and reaction velocity flattens out or plateaus and
then rate does not change with added substrate such mechanism is defined as zero-order kinetics. The
maximum velocity of a reaction is reached when the active sites are almost continuously filled. Increased
substrate concentration after this point will not increase the rate. Reaction rate therefore increases as substrate
concentration is increased but it levels off. As the substrate concentration increases, the collision with
enzymes increases so that the enzymatic reaction rate increases as all active sites of enzymes are fitted /
occupied. Therefore, any Additional supplement of more concentration of substrates no further increases
the reaction rate instead it goes in constant. This is simply representing as the following type of graph of
enzymatic reaction rate in relation to substrate concentration.

Fig. Enzymatic reaction rate in relation to substrate concentration itself

SALINITY

It refers to the amount or concentration of salt. As the concentration of salt increases, the rate of enzymatic
reaction be affected or cease off. This is because those enzymes are affected by high concentration of salt as
the proteins are also affected.

THE PRESENCE OF INHIBITORS

competitive inhibitors are molecules that bind to the same site as the substrate — preventing the substrate
from binding as they do so — but are not changed by the enzyme.

Types of inhibitors

 Competitive inhibition
 Non-competitive inhibition
Competitive inhibition

In competitive inhibition, a similar-shaped molecule competes with the substrate for active sites. Certain
molecules compete with the substrate for the active site of the enzyme and it prevent formation of product
thus the mechanism is known to have a higher Km than the preferred substrate. The competitive inhibitions
can be overcome by addition of more substrate.

Example; Lactate and -dehydroxybutyrate for LD

Noncompetitive Inhibition

Another form of inhibition involves an inhibitor that binds to an allosteric site of an enzyme. An allosteric
site is a different location than the active site. Noncompetitive inhibitors are molecules that bind to some
other site on the enzyme reducing its catalytic power.

Non-competitive inhibitors bind on allosteric site but not the active sites of enzyme and it prevent formation
of product despite the enzyme-substrate complex. The non-competitive inhibition cannot be overcome by
addition of more substrate. The binding of an inhibitor to the allosteric site alters the shape of the enzyme,
resulting in a distorted active site that does not function properly. The binding of an inhibitor to an allosteric
site is usually temporary. Poisons are inhibitors that bind irreversibly. For example, penicillin inhibits an
enzyme needed by bacteria to build the cell wall.

Feedback Inhibition

Negative feedback inhibition is like a thermostat. When it is cold, the thermostat turns on a heater which
produces heat. Heat causes the thermostat to turn off the heater. Heat has a negative effect on the thermostat;
it feeds back to an earlier stage in the control sequence as diagrammed below.

Many enzymatic pathways are regulated by feedback inhibition. As an enzyme's product accumulates, it
turns off the enzyme just as heat causes a thermostat to turn off the production of heat. The end product of
the pathway binds to an allosteric site on the first enzyme in the pathway and shuts down the entire sequence.
Feedback inhibition occurs in most cells.
THE PRACTICAL ASPECT

This practical aspect focuses on testing

i) Enzyme specificity
ii) Effect of enzyme concentrations
iii) Effect of substrate concentration
iv) Effect of temperature
v) Effect of PH

This practical session is applying on the action of catalase on hydrogen peroxide. Hydrogen peroxide is toxic,
poisonous substances to cells. It is produced as lipid metabolism as of synthesis. Hydrogen Peroxide (H2O2)
is a powerful oxidizing agent produced as a toxic byproduct of aerobic metabolism. Without rapid enzymatic
catalysis, H2O2 would quickly destroy essential biomolecules in a living cell, resulting in cell damage and
death. You will have two different solutions of H2O2 available for your experiments. This substance therefore
must be degraded or broken down to nontoxic, useful substances called water and oxygen the action of
biocatalyst/enzyme called CATALASE. This enzyme is secreted or produced in some types of tissues like
muscle, kidney, and liver from animals and potatoes tuber from plants. This can be summarized as follows:
H2O2 -------------------catalase---------------H2O + O2

MATERIALS REQUIRED

APPARATUS CHEMICALS

 Test tubes with its rack hydrogen peroxide (3%H2O)

 Cork borer distilled water (dH2O)
 Ruler sodium chloride (10M NaCl)
 Pipette sodium hydroxide (6N NaOH)
 Tit hydrochloric acid (6N HCl)
 Pyrex beaker
 Stove/Bunsen burner
 Spatula/forceps
 Scalpel/knife
 Petridish/plate
 Potato tuber
PROCEDURES OR PROTOCOL

I) Enzyme specificity
1) Cut potato cylinders in to equal cubes by measuring its length
2) Prepare two test tubes.
3) Add 4ml of H2O2 in one of the test tube and 4ml of dH2O in to another test tube.
4) Add equal amount of potato cylinder in both test tubes.
5) Observe the result

Questions

A) What do you observe?

B) What are the substrate and the enzyme in the reactions?
C) From which test tube gas is evolved? Why?
D) What is the gas evolved as the result of the reaction?
E) What is the purpose of this experiment?
F) What is the conclusion be drawn?
II) Enzyme concentration
1) Prepare two test tubes
2) Add equal amount of hydrogen peroxide in both test tubes (4ml).
3) Add three potato cylinders in one of the two test tubes and add one potato cylinder in to
another test tube.
4) Look the result

Questions

A) In which test tube do you observe the reaction? Why?

B) In which test tube do you observe more gas evolution? Why?
C) What is the conclusion from the above reaction?
III) Substrate concentration
1) Prepare two test tubes.
2) Add 4ml H2O2 in test tube number one and 2ml H2O2 and 2ml H2O in test tube number
two.
 3) Add equal potato cylinder.
4) Look the result.

Questions

a) In which test tube do you observe reaction takes place? Why?

b) In which test tube do you observe least gas evolution? Why?
c) What is your conclusion from the above reaction?
d) What will happen if you add more hydrogen peroxide in one test tube?
IV) Effect of temperature
1) Prepare two test tubes.
2) Add 4ml H2O2 in both test tubes.
3) Add fresh potato cylinder in one test tube and boiled potato cylinder in on other test
tube (equal amount of potato).
4) Look the result.

Questions

a) In which test tube do you observe reaction takes place? Why?

b) In which test tube do you observe gas evolution? Why?
c) What is your conclusion from the above reaction?
vi) Effect of PH
1) Prepare six test tubes.
2) Add two or three fresh cube potato in test tubes containing of 5 ml of conc. HCl, NaOH and
water respectively from 10-20 minutes.
3) Pick out each potato cylinder and add in to another three test tubes containing of 2 ml
hydrogen per oxide
4) Look the result.

Questions

a) In which test tube do you observe reaction takes place? Why?

b) In which test tube do you observe gas evolution? Why?
c) From which test tube no reaction? Why?
d) What is your conclusion from the above reaction?
Summary questions

1) Explain how enzymes can be so specific toward the substrate they bind?
2) Explain dependence of the rate of an enzyme catalyzed reaction on temperature?
3) How is it possible to have a reaction characterized by large ΔG and small EA? Large EA and small ΔG ?
4) List the mechanisms by which enzymes accelerate rate of chemical reactions?
5) Enzymes have no effect on the thermodynamics of the reaction. Explain it briefly what it infers?
6) Enzymes accelerate bond breaking and bond forming process. A) True B) false reason out for your
selective answer!