Enzymes are THE WORLD OF ENZYMES chemicals found in

living things that act to speed up

specific chemical reactions. Enzymes are catalysts for biochemical (living)
reactions. If there is any chemical reaction in any living thing there is an enzyme
that works to bring it about.

A simple definition of life would be: the transfer of energy through the breakdown
of nutrients. In other words, all living things get their energy for life by breaking
down the chemicals in other living things. Sounds simple enough but in point of
fact, the activity of getting nutrients and energy from food is maybe the most
complex group of chemical reactions in the universe. This is because chemical
reactions that occur inside living organisms can't happen without a catalyst that
would make these reactions happen and control them.

Catalysts are chemicals that while helping a reaction come about, are not
themselves changed. Catalysts, in reactions other than biochemical, generally
are common inorganic substances which have uses outside of being catalyst for
a reaction. For example, platinum is used as a catalyst the reaction that
breaks down nitrogen oxides in car exhaust, yet platinum has many other
uses. Most biological catalysts, or enzymes, on the other hand are very specific.
They exist and are created with only one purpose, to act as a catalyst for one
specific reaction biochemical reaction.

Enzymes are proteins which are used as catalysts for a specific reaction. The
exact nature of how enzymes work is not known. If is only recently that scientist
have had any clue as to the mechanism of enzyme catalyzation. Made up of a
complex of amino-acids, enzymes are part of every chemical reaction in living
things. Examples of enzyme aided reactions include all digestion, growth and
building of cells, any breakdown of substances such as vitamins, and nutrients,
all reactions involving transformation of energy. Reactions are also controlled by
enzymes. The rate and location or site of a reaction is also controlled by enzyme
action. A good example of the involvement of enzyme action is in the building of
living material within the cell.

Inside the cell, enzymes create RNA and DNA by facilitating the reaction of
ribose with adenosine. They also specify the sites for linking to build RNA along a
DNA template. Once the RNA is formed, it is the enzymes that catalyze the
construction of proteins from amino acids. It is the catalytic action of particular
enzymes that create specific structures within living cells.

Lack of specific enzymes is the cause of many disorders. Disorders such as

albinism, diabetes, and cystic fibrosis are traceable to either a lack of a specific
enzyme or an imbalance of one.
Some examples of where enzymes are visible in abundance would be in human
saliva and in the human digestive tract. Saliva contains an enzyme that breaks
down starches into their component sugars. While the stomach combines the
enzyme pepsin with acid to speed the digestion of proteins. Enzymes are carried
to the intestines to facilitate the digestion of fats.

Another benefit of enzymes in biochemical reactions is that they control the

release of energy in living reactions. The breakdown of chemical bonds releases
energy. If you were to measure the amount of energy from a candy bar, you
would see that it might have 200 calories or more. While the body needs energy
to function, the immediate release of chemical energy from the breakdown of
food and nutrients would be disastrous. The small candy bar mentioned here
would have release enough energy to raise the body temperature of a 200 pound
man 3 1/2 degrees Fahrenheit! It is the work of enzymes that allow for the
controlled release of the energy in living chemical reactions.

Plants turn the energy of sunlight directly into food by using sunlight energy for
chemical bonds in the form of sugar. Enzymes are responsible here, too, they
control the absorption of radiant energy. Think about it, have you ever sat under
a tree during a hot summer afternoon and wondered what keeps the leaves so
cool? The sunshine will be hot enough to melt tar on the streets, but plant leaves
remain barely warm to the touch. Where does all the energy go? It is slowly
being tied up in the chemical bonds of sugar. The process is photosynthesis and
it is the basis of all life on earth, but without enzymes controls this process would
be impossible.

Enzymes are at work wherever there is life. Yeast use enzymes to leaven bread and
ferment sugar into alcohol. Bacteria use enzymes to break down cellulose fiber in
the stomachs of cows and the stomachs of termites. Plants, animals, bacteria, or
fungi, if they are alive, use enzymes to control all living chemical reactions.
Reproduction, growth, metabolism, synthesis, are all enzyme regulated reactions in
living things. Enzymes are the chemicals that make life work.

Enzymes are biological catalysts: this means that they speed up the chemical
reactions in living things. Without enzymes, our guts would take weeks and
weeks to digest our food, our muscles, nerves and bones would not work
properly and so on - we would not be living!

A catalyst is any substance which makes a chemical reaction go faster, without

itself being changed. A catalyst can be used over and over again in a chemical
reaction: it does not get used up. Enzymes are very much the same except that
they are easily denatured (destroyed: but do NOT use this word since the protein
molecule is not broken down into amino-acids, it just loses it shape and will not
work any more) by heat. Our enzymes work best at body temperature. Our
enzymes also have to have the correct pH.
All enzymes are made of protein; that is why they are sensitive to heat, pH and
heavy metal ions. Unlike ordinary catalysts, they are specific to one chemical
reaction. An ordinary catalyst may be used for several different chemical
reactions, but an enzyme only works for one specific reaction.

Human saliva contains an enzyme called amylase. This enzyme helps to turn
starch into a sugar called maltose. When you swallow a mouthful of food, the
amylase stops working because it is much too acid in the stomach pH 2.
Amyalse works best in neutral or slightly alkaline conditions, i.e. at about pH 7.
When your food gets into the small intestine, more amylase is made by the
pancreas and this turns the remaining starch into maltose. Another enzyme
(maltase) turns all this maltose into glucose. Glucose is then absorbed into the
blood.

Enzymes in the human alimentary canal and what they digest:

Enzyme Substrate
Amylase Starch
Maltase Maltose
Sucrase Sucrose
Lipase Fats
Pepsin Proteins

All animals, green plants, fungi and bacteria produce enzymes: so enzymes are
not just about digesting food. The enzymes which we use to digest our food are
extra-cellular, that means they are found outside cells. We also have enzymes
inside our cells; these are intra-cellular enzymes. Enzymes are used in ALL
chemical reactions in living things; this includes respiration, photosynthesis,
movement growth, getting rid of toxic chemicals in the liver and so on.

Viruses are rather different, but you do not need to know much about them for
GCSE, so just make sure that you don't catch any!

Enzymes must have the correct shape to do their job. They are made of proteins,
and proteins are very easily affected by heat, pH and heavy metal ions. Some
people say that enzymes work like a key in a lock. If the key has been twisted by
heat, or dissolved in acid or stuck up with chewing gum it will not work. Enzymes
change their shape if the temperature or pH changes, so they have to have the
right conditions. Copper ions are poisonous: if you get copper ions in your blood
they will block up some of the important enzymes in red and white blood cells.

Enzymes increase rates of

reactions by decreasing
the activation energy.
Note that enzymes do not change
the final products, or the
amount of product relative to
the amount of reactants when
equilibrium is reached!
Enzymes!
Enzymes increase rates of
reactions by decreasing
the activation energy.
Note that enzymes do not change
the final products, or the
amount of product relative to
the amount of reactants when (if)
equilibrium is reached!
How do enzymes work?
The ‘action’ of enzymes happens at
a specific site called the
ACTIVE SITE
The shape of each active site is specific
for a particular substrate or set of
reactants.
How do enzymes work?
Enzymes bind a substrate or
reactants and facilitate a chemical
change in it / them.
1) orient reactants so that they may
bind/interact
2) put physical stress on substrate
3) add charge to substrate

How do enzymes work?

Some proteins require other, non-protein
molecules in order to work:
1) cofactors
2) coenzymes
3) prosthetic groups

How do Enzymes Work?

An enzyme is a protein molecule that speeds up chemical reactions. Each enzyme

has a unique shape that determines its function. One enzyme works on one type
of substrate.

The enzyme, Amylase has a shape that allows its to wrap around starch
(substrate) and cut it up into individual glucose units.

Picture an enzyme as a house painter

Imagine a house decorator A about begin a task. Their task is to paint a large room
and have it ready in 12 hours. But all they have is a tiny brush and the paint is slow
to dry. Now imagine a second decorator B, given the same task but they have a
larger brush and are also provided with large fans to help the paint dry. The overall
task has not changed and at the end of the task each decorator is left with the same
set of tools they started with. But B will do the job far more efficiently than A.

In a way enzymes are like this. They are the tools that the cells employ in order to
metabolise nutrients and synthesise the molecules that they require to survive and
reproduce. The above analogy is crude but does serve to illustrate how enzymes
work. Instead of a "task" think of instead of a biochemical reaction and imagine a
tool that has taken millions of years to evolve into a precise protein structure. Like
the tools above, at the end of the reaction the enzyme returns to it’s original state.
Enzymes therefore also fit the definition of a catalyst although to distinguish them
from other (mainly metallic) catalysts they are usually referred to as biological or
biochemical catalysts.

Most (bio)chemical reactions have an energy barrier to be overcome beforethe

reaction can happen.This is known as activation energy or Ea. On a biochemical level
enzymes work by lowering this activation energy of a reaction.

Imagine two drivers that have to travel a short journey on 1 litre of petrol. Driver A
has a road map and knows that by using a suitable bridge that the journey will be
about 6Km. Driver A therefore manages to complete the journey. Driver B does not
know about the shortcut and runs out of petrol before completing the task. Both
drivers set out with the same chance of completing the same task but only one
succeeded. In this analogy the enzyme could be considered to be the bridge that
allows the shorter path to be taken and the road map / knowledge of the driver as
the genetic knowledge stored within a cell.

Enzymes lower or take a shortcut around the activation energy required for a
reaction to occur in many ways.
EXPERIMENTS WITH ENZYME ACTIVITY

Expt 1: Enzymes in Saliva

OVERVIEW:

Enzymes are proteins that speed up or slow down a chemical reaction and are not
consumed by the reaction. They play an important role in the living process. The most
easily understood use of enzymes is in the digestive process. Digestion can include
nutritious molecules or potentially harmful molecules like alcohol. Students will use
active enzymes in their saliva to demonstrate how enzymes work on common food
molecules. They are also asked to apply this concept to alcoholism.

PURPOSE:

The activity will demonstrate enzyme activity and how changes in the physical
conditions affects their performance. Students will work with active enzymes and explore
the boundaries of their activity.

OBJECTIVES: (The student will be able to:)

1. define enzyme
2. observe enzyme activity
3. explain how changing the physical conditions affects enzyme activity
4. discuss the relationship between drug use and enzyme activity
5. explain the enzyme deficiencies of an alcoholic

RESOURCES/MATERIALS:

Starch solution, 6 test tubes, Benedict's solution, Iodine solution, hot water bath

ACTIVITIES AND PROCEDURES:

1. Demonstration - add hydrogen peroxide to separate beakers containing potato

cubes and liver extract. Have students list their observations and formulate an
explanation. As a group discuss enzymes and how they react.
2. Perform the lab activity and answer the lab questions

TYING IT ALL TOGETHER: Using a minimum of 50 words, answer the following

questions.

1. What are some of the important properties and characteristics of enzymes?

2. Explain the action of the saliva on the starch. How does this affect digestion in
your body?
3. What happens to enzyme activity when the physical conditions are changed?
4. Alcoholics do not produce the enzyme needed for the digestion of alcohol. How
will this affect their ability to metabolize alcohol?
Discuss the opening demonstration allow students to changetheir explanation of enzyme
activity. Discuss the objectives and the lab activity.

ADDITIONAL ACTIVITIES: SPIT

Before you start you will need to set up a chart that lists testtubes #1 - #6. Leave room
for a description of the test tube contents and your observation of results.

Procedure:

1. Spit into a test tube, filling it one-fourth full of saliva. All lab group members may
contribute to this effort. It may help to think about mouth-watering food while
contributing (lemons). Sometimes something inedible, such as a rubber band,
helps. Do not put any food or drink other than water into you mouth for at least
two hours preceding this laboratory. Otherwise you will contaminate your sample.
2. Add an equal amount of water to the spit in the test tube, and gently mix it all
together.
3. Number the remaining test tubes #3 - #6.
4. Add 5 ml. starch solution to each of the test tubes #3 - #6.
5. Add 1 ml. saliva solution to test tubes #3 and #4. Allow both test tubes to set for
five minutes.
6. While these test tubes are setting, place remaining saliva solution in the water
bath.
7. Add 3 drops of iodine solution to test tubes #1 & #3. Record your observations.
8. Add 10 drops of Benedict's solution to test tubes #2 . Heat these solutions in the
hot water bath for five minutes. Record your observations.
9. Add 1 ml. boiled saliva solution to test tubes #5 and #6. Allow both test tubes to
set for five minutes.
10. Add 3 drops of iodine solution to test tube #5. Record your observations.
11. Add 10 drops of Benedict's solution to test tube #6. Heat this solution in the hot
water bath for five minutes. Record your observations.

Answer the following questions, summarizing the lab.

1. What conclusions can you reach about the action of salivary enzymes on the
digestion of starch?
2. What varied in the treatment of test tubes #1 and #3? Why is this difference
important in your conclusion?
3. How did heating the saliva solution affect the action of the saliva?
Expt 2: ENZYME SALAD LAB

Have you ever noticed the warning on a JELL-O package not to use fresh or
frozen pineapple? Today we are going to put the fruit into gelatin and see what
happens. What are some possible results?

BACKGROUND: The enzymes in some tropical fruits can break down

protein. For example meat tenderizers have the enzyme, papain,
that breaks down some of the fiber. We use it to soften up a
tough piece of meat. The papain comes from the papaya fruit.
Pineapple has a digestive enzyme called bromelin.

MATERIALS: Four Test Tubes Fresh Pineapple

Hot Gelatin Canned Pineapple
Ice Water Bath Frozen Pineapple
Glass Marker Single-edged Razor Blade
Test Tube Rack

PROCEDURE:
l. With the razor blade carefully cut equal sized pieces of fresh canned, and
frozen pineapple.
The pieces should be just small enough to slide into your tubes.

2. Label the test tubes l-4.

3. Pour 3 ml of liquid gelatin into each of the four tubes.

4. Place the different pieces of pineapple into the first three tubes.
See the table below to match up the contents. Notice that test tube #4 contains
only gelatin.

5. Mix the contents of the tubes by rolling them upright between the palms of
your hands.

6. Place all four test tubes into the ice water bath.

7. Every few minutes check to see if the gelatin is setting in the tubes. When test
tube#4 has gelled firmly you can remove all the tubes and compare the
consistencies

8. Record you observations in the table below.

TUBE CONTENTS RESULTS

1. Gelatin and Fresh Pineapple

2. Gelatin and Canned Pineapple

3. Gelatin and Frozen

4. Gelatin

DISCUSSION QUESTIONS:

1. Why did test tube #4 contain only gelatin?

2. What could account for the different results in the tubes?

3. How could the canning process change bromelin?

4. What could you do to fresh pineapple that would allow the salad to gel?

5. Design an experiment to find a temperature at which a meat tenderizer works the best?

NOTES TO THE TEACHER:

We usually do this lab when we are discussing digestive enzymes and temperature dependence
of enzymes. The students like the application of biochemistry to something they eat.

Here are some suggestions that have helped make the lab more smoothly.
Preparing the gelatin: Stir two envelopes of KNOX unflavored gelatin into 800 ml of hot water.
This should be plenty for three classes. Store it in a warm water bath when the students are using
it so it will pour easily.

Remind the students to rinse of the razor blade between types of pineapple. Clean up is easier if
they cut the fruit on paper plates.

Canned Pineapple: Use the chunky style.

Fresh Pineapple: You will probably have to buy a whole pineapple and most of it will be left after
the labs. You and the students might enjoy eating it.

Ice baths made in beakers or cans will eliminate too much movement through the room.

The name of the digestive enzyme you were working with is bromelin. It is named after the group
of plants to which the pineapple belongs - the Bromeliads.
 BjBarton.com, - Lesson Plans With Fun Labs & Activities for High School
Science.

Catalase Lab
A Bio ENZYME ACTIVITY LABORATORY

INTRODUCTION

Hydrogen peroxide (H2O2) is a poisonous byproduct of metabolism that can damage

cells if it is not removed. Catalase is an enzyme that speeds up the breakdown of
hydrogen peroxide into water (H2O) and oxygen gas (O2).

2H2O2--------catalase--------------> 2H2O + O2

REMEMBER: A CATALYST is a substance that lowers the activation energy required for
a chemical reaction, and therefore increases the rate of the reaction without being used
up in the process. CATALASE is an enzyme, a biological (organic) catalyst. Hydrogen
peroxide is the substrate for catalase.
****YOU WILL BE WORKING WITH HOT WATER, ACIDS AND BASES IN THIS
LABORATORY. USE EXTREME CAUTION AND WEAR GOGGLES AT ALL TIMES!!!

The general procedure for the lab is outlined below, and specific details for each variable
follow.

GENERAL DIRECTIONS:

The assay system used in this lab consists of a filter paper disc which is coated with the
enzyme and then dropped into a vial of substrate (hydrogen peroxide). As the catalyst
breaks down the hydrogen peroxide into water and oxygen gas, the bubbles of oxygen
collect underneath the filter and make it rise to the surface of the hydrogen peroxide.
The time it takes for the filter to rise is an indication of the rate of enzyme activity.

RATE:

Rate of enzyme activity = distance (depth of hydrogen peroxide in mm)/time (in sec). We
will assume that each filter is coated with the same amount of catalase (except in the
investigation of the effect of enzyme concentration on enzyme activity).
The enzyme has been prepared for you as follows: 50g of peeled potato was mixed with
50 ml cold distilled water and crushed ice and homogenized in a blender for 30 seconds.
This extract was filtered through cheesecloth and cold distilled water was added to a
total volume of 100 ml. Extract concentration is arbitrarily set at 100 units/ml. ENZYME
SHOULD BE KEPT ON ICE AT ALL TIMES!!

MATERIALS:
catalase, hydrogen peroxide 3% and 1.0%, forceps, filter paper discs, water, ice, water
baths, vials, marking pencils, stopwatch or timer

PLAN:

Each group will investigate and report on two of the following questions. Suggestions for
each question are given below. EVERY STUDENT IS RESPONSIBLE FOR
RECORDING THE RESULTS OF ALL FOUR EXPERIMENTS in his/her lab report.

1. What is the effect of enzyme concentration on enzyme activity?

2. What is the effect of substrate concentration on enzyme activity?
3. What is the effect of pH on enzyme activity?
4. What is the effect of temperature on enzyme activity?

1. What is the effect of enzyme concentration on enzyme activity?

• Set up five vials containing 40 ml of 3% hydrogen peroxide each. Measure and

record the depth of the hydrogen peroxide in the vials.
• Dilute the enzyme as follows. Make each dilution in a separate cup.
100 units/ml = 20 ml 100 units/ml
80 units/ml = 12 ml 100 units/ml + 3 ml cold dH2O
50 units/ml = 10 ml 100 units/ml + 10 ml cold dH2O
20 units/ml = 3 ml 100 units/ml + 12 ml cold dH2O
0 units/ml = 20 ml cold dH2O

Using forceps, dip a filter into the enzyme solution at 100 units/ml, then remove it and
drain it on a paper towel. Drop the disc into the vial of hydrogen peroxide labeled 100
units/ml and time how long it takes the filter to rise to the surface. Repeat this procedure
for each of the other enzyme dilutions, and be sure to use a FRESH vial of substrate for
each filter (WHY?) Record your results in the appropriate data chart.

2. What is the effect of substrate concentration on enzyme activity?

• Obtain 1 vial of catalase at 100 units/ml

• Dilute the substrate (hydrogen peroxide) as described below. Each dilution
should be made in a separate labeled vial. Measure and record the depth of the
hydrogen peroxide.

3.0% H2O2: 40 ml 3% H2O2

1.5% H2O2: 20 ml 3% H2O2 + 20 ml distilled water
0.75% H2O2: 10 ml 3% H2O2 + 30 ml distilled water
0.38% H2O2: 5 ml 3% H2O2 + 35 ml distilled water
0.0% H2O2 : 40 ml distilled water
• Dip a filter into the catalase, drain on a paper towel and then drop the filter into
the 3% H2O2. Time how long it takes the filter to rise to the top. Repeat this
procedure for each of the substrate dilutions. Record your results in the
appropriate data chart.

3. What is the effect of pH on enzyme activity?

 • Obtain 1 vial of 40 ml 1% H2O2. Measure and record the depth of the hydrogen
peroxide.
• Label 5 small vials as follows: pH3, pH5, pH7, pH9, pH11 and dilute catalase into
the appropriate vial as directed below:
pH 3: 5 mL catalase + 5 mL pH 3 Buffer
pH 5: 5 mL catalase + 5 mL pH 5 Buffer
pH 7: 5 mL catalase + 5 mL pH 7 Buffer
pH 9: 5 mL catalase + 5 mL pH 9 Buffer
pH 11: 5 mL catalase + 5 mL pH 11 Buffer
• Dip a filter into the catalase at pH 3, drain on a paper towel and drop it into the
1% H2O2. Time how long it takes the filter to rise to the top. Remove the filter and
repeat this procedure for each of the pH's. Record your results in the appropriate
data chart.

4. What is the effect of temperature on enzyme activity?

• Set up an ice bath (0oC), a room temp water bath, a 37 oC bath and a boiling
water bath
• Place 5 ml of catalase at 100 units/ml in each of 4 test tubes. Place 1 test tube in
each of the water baths.
• Place 40 ml 1% H2O2 in each of 4 vials. Measure and record the depth of the
H2O2. Place 1 vial in the 0oC bath and leave 3 at room temperature. This is
necessary because heat will destroy the hydrogen peroxide.
• Allow the catalase and substrate to incubate at each temperature for about 5
minutes, then test the reaction time at each temperature by dipping a filter into
enzyme at that temperature, draining it, and then dropping it into substrate at the
same temp. Use the second room temp. vial of hydrogen peroxide for the boiled
catalase. DO NOT BOIL HYDROGEN PEROXIDE!!! Time how long it takes the
filter to rise at each temperature. Record your results in the appropriate data
chart.

CATALASE LAB NAME_______________

DATA CHARTS

What is the effect of enzyme concentration on enzyme activity?

2. What is the effect of substrate concentration on enzyme activity?
What is the effect of pH on enzyme activity?

4. What is the effect of temperature on enzyme activity?

ANALYSIS:

For EACH variable, use the AVERAGE rates to construct a graph of the independent
variable vs. the dependent variable. This means you will have four graphs:

1. Enzyme concentration (x axis) vs. Rate (y axis)

2. Substrate concentration (x axis) vs. Rate (y axis)
3. pH (x axis) vs. Rate ( y axis)
4. Temperature (x axis) vs. Rate (y axis)

CONCLUSION:
Answer the following questions in complete sentences:

1. What is the effect of enzyme concentration on enzyme activity? Explain how

enzyme activity changes as enzyme concentration decreases, and discuss why
this occurs (on a molecular level).
2. What is the effect of substrate concentration on enzyme activity? How does
enzyme activity change as substrate concentration decreases? Explain your
observations by discussing this reaction on a molecular level.
3. How does temperature affect the activity of catalase? Explain your observations
by discussing the effect of temperature on protein structure. Discuss both high
and low temperature effects.
4. How does pH affect the activity of catalase? Consider both high and low pH, and
explain your observations by discussing the effect of pH on protein structure.
5. Ectothermic organisms have body temperatures that vary with the temperature of
their surroundings. Discuss the effect this variation might have on the functioning
of enzymes in these organisms. Suggest some ways ectothermic organisms
might cope with this problem.

BjBarton.com, - Lesson Plans With Fun Labs & Activities for High School Science.