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LAB #:

Title: Enzymes
Aim: To investigate the effects of limiting factors on enzyme-catalyzed reactions

Factors affecting Enzyme Activity

The activity of an Enzyme is affected by its environmental conditions. Changing these


alter the rate of reaction caused by the enzyme. In nature, organisms adjust the
conditions of their enzymes to produce an Optimum rate of reaction, where necessary,
or they may have enzymes which are adapted to function well in extreme conditions
where they live.

Temperature

Increasing temperature increases the Kinetic Energy that molecules possess. In a


fluid, this means that there are more random collisions between molecules per unit time.

Since enzymes catalyse reactions by randomly colliding with Substrate molecules,


increasing temperature increases the rate of reaction, forming more product.

However, increasing temperature also increases the Vibrational Energy that


molecules have, specifically in this case enzyme molecules, which puts strain on the
bonds that hold them together.

As temperature increases, more bonds, especially the weaker Hydrogen and Ionic
bonds, will break as a result of this strain. Breaking bonds within the enzyme will cause
the Active Site to change shape.

This change in shape means that the Active Site is less Complementary to the shape of
the Substrate, so that it is less likely to catalyse the reaction. Eventually, the enzyme
will become Denatured and will no longer function.

As temperature increases, more enzymes' molecules' Active Sites' shapes will be less
Complementary to the shape of their Substrate, and more enzymes will be Denatured.
This will decrease the rate of reaction.

In summary, as temperature increases, initially the rate of reaction will increase,


because of increased Kinetic Energy. However, the effect of bond breaking will
become greater and greater, and the rate of reaction will begin to decrease.

The temperature at which the maximum rate of reaction occurs is called the enzyme's
Optimum Temperature. This is different for different enzymes. Most enzymes in the
human body have an Optimum Temperature of around 37.0 C.

pH - Acidity and Basicity

pH measures the Acidity and Basicity of a solution. It is a measure of the Hydrogen Ion
(H+) concentration, and therefore a good indicator of the Hydroxide Ion (OH-)
concentration. It ranges from pH1 to pH14. Lower pH values mean higher H+
concentrations and lower OH- concentrations.

Acid solutions have pH values below 7, and Basic solutions (alkalis are bases) have pH
values above 7. Deionised water is pH7, which is termed 'neutral'.

H+ and OH- Ions are charged and therefore interfere with Hydrogen and Ionic bonds
that hold together an enzyme, since they will be attracted or repelled by the charges
created by the bonds. This interference causes a change in shape of the enzyme, and
importantly, its Active Site.

Different enzymes have different Optimum pH values. This is the pH value at which
the bonds within them are influenced by H+ and OH- Ions in such a way that the shape of
their Active Site is the most Complementary to the shape of their Substrate. At the
Optimum pH, the rate of reaction is at an optimum.

Any change in pH above or below the Optimum will quickly cause a decrease in the
rate of reaction, since more of the enzyme molecules will have Active Sites whose
shapes are not (or at least are less) Complementary to the shape of their Substrate.

Small changes in pH above or below the Optimum do not cause a permanent change
to the enzyme, since the bonds can be reformed. However, extreme changes in pH can
cause enzymes to Denature and permanently loose their function.

Enzymes in different locations have different Optimum pH values since their


environmental conditions may be different. For example, the enzyme Pepsin functions
best at around pH2 and is found in the stomach, which contains Hydrochloric Acid
(pH2).

Concentration

Changing the Enzyme and Substrate concentrations affect the rate of reaction of an
enzyme-catalysed reaction. Controlling these factors in a cell is one way that an
organism regulates its enzyme activity and so its Metabolism.

Changing the concentration of a substance only affects the rate of reaction if it is the
limiting factor: that is, it the factor that is stopping a reaction from preceding at a
higher rate.

If it is the limiting factor, increasing concentration will increase the rate of reaction
up to a point, after which any increase will not affect the rate of reaction. This is
because it will no longer be the limiting factor and another factor will be limiting the
maximum rate of reaction.

As a reaction proceeds, the rate of reaction will decrease, since the Substrate will get
used up. The highest rate of reaction, known as the Initial Reaction Rate is the
maximum reaction rate for an enzyme in an experimental situation.

Substrate Concentration

Increasing Substrate Concentration increases the rate of reaction. This is because


more substrate molecules will be colliding with enzyme molecules, so more product
will be formed.

However, after a certain concentration, any increase will have no effect on the rate of
reaction, since Substrate Concentration will no longer be the limiting factor. The
enzymes will effectively become saturated, and will be working at their maximum
possible rate.

Enzyme Concentration

Increasing Enzyme Concentration will increase the rate of reaction, as more enzymes
will be colliding with substrate molecules.

However, this too will only have an effect up to a certain concentration, where the
Enzyme Concentration is no longer the limiting factor.

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Cues:

Questions:

ENZYMES note
Enzymes - are proteins made of amino acids
- are catalysts- they speed up chemical reactions &
lower the activation energy
- are reusable- they remain unchanged after the
reaction
- are specific- there is a perfect enzyme for a certain
substrate
- have a 3D shape which can be destroyed/denatured
w/extreme changes in pH & temperature
- have active sites = places where substrate (food &
waste) molecules attach
Draw the enzyme with an active site + a substrate:

Eg., ) Catalase breaks down hydrogen peroxide (waste produced in


cells) into
2H2O2 ----> 2H2O + O2
Lactase breaks down lactose in milk
Protease breaks down proteins
Lipase breaks down lipids
How Enzymes Work?
Active sites on enzymes = places to which a specific
substrate binds
Enzyme-substrate complexes form (when substrates
attach to active sites on the enzymes) to break
apart or put together substances at a fast rate
There are two models of enzyme action
1. lock & key model substrate & the enzyme fit
together perfectly
2. induced-fit model enzymes change shape slightly to
accommodate the substrate
Factors affecting enzyme action
1. Temperature enzymes work best at certain
temperatures, 37oC is best for human enzymes in
the body
2. pH enzymes work best at certain pH; basic,
neutral, and/or acidic environments
eg.) Amylase in saliva at pH 7, Pepsin in the stomach
at pH 2-3, & Trypsin in the intestines at pH 9
3. Substrate & enzyme concentrations how fast
reactions take place depends on how much of the
substrate & enzyme is available.
4. Coenzymes helpers such as vitamins & minerals
Summary

ENZYMES book questions


Answer the following questions in complete sentences.
1. What are enzymes?
2. Are enzymes proteins?
3. What are coenzymes?
4. What are the functions of enzymes?
5. What are the elements found in enzymes?
6. What is a catalyst?
7. What enzyme breaks-down proteins?
8. Compare and contrast the two models of enzyme action.
9. Draw figure 4-15 and copy its caption.
10. What are the factors that affect how enzymes work?
11. Classify the following substances as carbohydrate, lipid, protein, or
nucleic acid: maltose, chlorophyll, DNA, vegetable oil, fructose, RNA,
wax, glycogen, insulin, and albumin.

Observing the enzyme CATALASE


INTRODUCTION: what would happen to your cells if they made a
poisonous chemical? You might think that they would die. In fact,
your cells are always making poisonous chemicals. They do not die
because your cells use enzymes to break down these poisonous
chemicals into harmless substances. Enzymes are proteins that
speed up the rate of reactions that would otherwise happen more
slowly. The enzyme is not altered by the reaction. You have hundreds
of different enzymes in each of your cells. Each of these enzymes is
responsible for one particular reaction that occurs in the cell.
In this lab, you will study an enzyme that is found in the cells of
many living tissues. The name of the enzyme is catalase (KAT-uhLAYSS); it speeds up a reaction which breaks down hydrogen
peroxide, a toxic chemical, into 2 harmless substances--water and
oxygen. The reaction is as follows:
2H2O2 ----> 2H2O + O2
This reaction is important to cells because hydrogen peroxide (H2O2)
is produced as a byproduct of many normal cellular reactions. If the
cells did not break down the hydrogen peroxide, they would be
poisoned and die.

PRELAB REVIEW:
Before you begin this lab, review pH. Recall that pH is the measure of
the acidity or alkalinity of a solution. An acidic solution has many hydrogen
ions (H+) and a pH below 7. An alkaline, or basic, solution has very few
hydrogen ions and a pH above 7. A neutral solution has a pH of 7.
Recall that the substrate is the molecule that the enzyme acts on,
and the products are the molecules produced by the reaction. Review why
enzymes are reusable. Under certain conditions enzymes are denatured.
An enzyme is denatured when the protein molecule loses its proper shape
and cannot function. Some things that can denature an enzyme are high
temperatures, extremes of pH, heavy metals, and alcohol.

PRE-LAB PREP:
Mix 1 molar concentration solutions of hydrochloric acid and sodium
hydroxide. For the HCl, mix 2.2 ml of concentrated acid with enough
distilled water to make a total volume of 25 ml. (REMEMBER: NEVER
ADD WATER TO ACID, ALWAYS ADD ACID TO WATER). For the sodium
hydroxide, add 1.0 g of NaOH to enough distilled water to make a total
volume of 25 ml. 3% Hydrogen peroxide is what you buy in the grocery
store.

MATERIALS:
6 Test tubes and
rack
Test tube holder
Thermometer
Stirring rod
pH paper

10-ml Graduated cylinder


Straight-edged razor
blade
3 beakers for water baths
Scissors and Forceps
(tweezers)

1molar HCl solution (in dropper


bottle)
1molar NaOH solution (in dropper
bottle)
40 ml 3% Hydrogen peroxide
solution
Fresh liver, chicken meat, Apple,
and Potato

PROCEDURES AND ANALYSIS QUESTIONS


Part I PROCEDURE -- Normal Catalase Activity
NOTE: Be sure to clean your stirring rod (and test tubes) between steps.
1. Place 2 ml of the 3% hydrogen peroxide solution into a clean test tube
2. Add a small piece of liver to one test tube. Observe the bubbles; what gas is being
released?
Throughout this investigation you will estimate the rate of the reaction (how rapidly the
solution bubbles) on a scale of 0-5 (0=no reaction, 1=slow,...., 5= very fast). Assume that
the reaction in step 2 proceeded at a rate of "4" and record the speed in DATA TABLE 1,
and DATA TABLE 2 as the rate at room temperature.
3. Recall that a reaction that absorbs heat is endothermic; a reaction that gives off heat is
exothermic. Now, feel the temperature of the test tube with your hand.
Has it gotten warmer or colder? Is the reaction endothermic or exothermic?
Is Catalase Reusable?
4. Pour off the liquid into a second clean test tube. Assuming the reaction is complete.
What is this liquid composed of? What do you think would happen if you added more
liver to this liquid? Why?
5. Add another 2 ml of hydrogen peroxide to the liver remaining in the first test tube.
Can you observe a reaction? What do you think would happen if you poured off this
liquid and added more hydrogen peroxide to the remaining liver?
Are enzymes reusable?
Occurrence of Catalase
Catalase is present in many kinds of living tissues. You will now test for the presence of
catalase in tissues other than liver.
6. Place 2 ml of hydrogen peroxide in each of 3 clean test tubes. To the first tube, add a
small piece of potato. To the second tube, add a small piece of chicken. To the last tube,
add a small piece of apple. As you add each test substance, record the reaction rate (0-5)
for each tube in TABLE 1.
Which tissues contained catalase?

Part II PROCEDURE -- Effect of Temperature on Catalase Activity


7. Put a piece of liver into the bottom of a clean test tube and cover it with a small amount
of distilled water. Place this test tube in a boiling water bath for 5 minutes. What will
boiling do to an enzyme?
8. Remove the test tube from the hot water bath, allow it to air cool, then pour out the water.
Add 2 ml of hydrogen peroxide. CAUTION: Use a test-tube holder when handling the
hot test tubes. What is happening in the test tube? Record the reaction rate (0-5) in
DATA TABLE 2.
9. Put equal quantities of liver into 2 clean test tubes and 1 ml H2O2 into 2 other test tubes.
Put one test tube of liver and one of H2O2 into each of the following water baths: Ice
bath (0 deg.C) and Warm water bath (37 deg.C)
10. After 3 minutes, pour each tube of H2O2 into the corresponding tube of liver and
observe the reaction. Record the reaction rates (0-5) in DATA TABLE 2. You recorded
the reaction rate for room temperature earlier.
What is the "optimum" temperature for catalase? (This is the temperature at which the
reaction proceeds fastest.)
Why did the reaction proceed slowly at 0 deg.C?
Why did the reaction not proceed at all at 100 deg.C?
Part III PROCEDURE -- Effect of pH on Catalase Activity
12. Add 2 ml hydrogen peroxide to each of 3 clean test tubes. Treat each tube as follows:
Tube 1--add a drop of 1molar HCl (acid) at a time until pH 3.
Tube 2--add a drop of 1molar NaOH (base) at a time until pH 10.
Tube 3--adjust the pH to 7 by adding single drops of either 1molar HCl or 1molar NaOH
as needed.
CAUTION: Do not let acids or bases contact your skin or clothing. Swirl each test tube
after adding each drop and measure the pH of each solution with pH paper. To do this,
remove a drop or two of solution from a test tube using a clean glass stirring rod. Rinse
your stirring rod and wipe dry before you dip it into each test tube. Place the drop on pH
paper. Record the pH of each solution in DATA TABLE 3.
13. Next, add a small piece of liver to each test tube. Estimate the reaction rates (0-5) and
record in DATA TABLE 3.
14. Does there appear to be a pH "optimum"? At what pH?

What is the effect of low or high pH on enzyme activity?

TABLE 1: Occurrence of Catalase


Sample

Rate of Enzyme Activity

Liver
Potato
Chicken
Apple

TABLE 2: Temperature effect on Liver Catalase Activity


Temperature
___ oC Freezing Temp.
___ oC Room Temp.
___ oC Body Temp.
___ oC Boiling Temp.

Rate of Enzyme Activity

TABLE 3: pH effect on Catalase Liver Activity


pH
3
7
10

Rate of Enzyme Activity

Understanding the enzyme CATALASE Lab #

PRELAB REVIEW:
Before you begin this lab, review pH. Recall that pH is the measure of the
acidity or alkalinity of a solution. An acidic solution has many hydrogen ions (H+)
and a pH below 7. An alkaline, or basic, solution has very few hydrogen ions and a
pH above 7. A neutral solution has a pH of 7.
Recall that the substrate is the molecule that the enzyme acts on, and the
products are the molecules produced by the reaction. Review why enzymes are
reusable. Under certain conditions enzymes are denatured. An enzyme is
denatured when the protein molecule loses its proper shape and cannot function.
Some things that can denature an enzyme are high temperatures, extremes of pH,
heavy metals, and alcohol.

Objectives:
1. identify tissues that have the enzyme catalase
2. study the enzyme catalase activity
3. explore factors that affect enzyme action (pH, Temperature, etc.)
4. understand that enzymes are specific and reusable

Pre-Lab Questions

1. The reaction is

2. Why is this reaction necessary in the body?

3. The enzyme is ________________ & the substrate is ___________________ (H2O2).

4. The reactants are _______________________ & the products are ________________

5. Protein Denaturation is ____________________________________________.

Enzyme lab