Catalysis: The Nature of Enzyme Activity

Allyra Liquigan

9 February 2017

BIO 115L

Introduction:

Catalysts are substances that speed up a chemical reaction, but are not permanently
altered while doing so. Enzymes are organic catalysts which execute thousands of chemical
reactions occurring within living things. They are rather large proteins made up of amino acids,
which create a concavity in the enzyme. This groove is called an active site and acts as a
placeholder for substrates, the substances to be controlled, causing an enzyme-catalyzed
reaction.

In an enzyme-substrate complex, the enzyme and substrate are held together by

hydrophobic interactions, hydrogen bonds, and ionic bonds. The enzyme takes in the substrate
and then converts it to a reaction product, a process that usually involves covalent bonds. The
product is released into the solution once it is ready, and the enzyme is then ready to take in
another substrate. The enzyme molecule goes through a recycling process, being allowed to
constantly repeat the process of forming an enzyme-substrate complex.

Both the enzyme and the active site have specific details that allow them to perform
effectively. An enzyme is specific in regards to its amino acid sequence while the specific
structure of the active site depends on what it connects to.

The following various external factors can affect the speed or efficiency of an enzymatic
reaction: temperature, pH, and other molecules. Each enzymatic reaction performs at its own
optimal speed with its own optimal conditions. However, an interference, such as a substance
blocking the active site, can halt the reaction. When the reaction is disrupted, the protein is
considered denatured, and the enzyme is no longer active.
 1. Temperature: As the temperature rises, all chemical reactions speed up. Once the
temperature of a reaction increases, so does the kinetic energy between the enzymes,
causing even greater collision. However, if the temperature increases too much, the
enzyme will then become denatured. On the other hand, if the reaction is at a low
temperature, the collisions of the enzymes will be less, resulting in a slower reaction.
2. pH: pH measures the H+ presence in a solution. A scale of 0-14 is introduced in order to
decipher the acidity of a solution. The pH of a solution is considered to be acidic if it
ranges between 0-6; the pH of a solution is considered to be neutral if it lands on 7; if the
pH of a solution ranges from 8-14, it is categorized as basic. Being both too acidic or too
basic can disrupt the enzyme. When the pH is lower, the enzyme gains H+ ions; when
the pH is higher, the enzyme loses H+ ions. Both these outcomes cause a loss in shape.
The optimal pH for enzymes is neutral, however a buffer exists in order to gain or lose
H+ so the pH does not change as often.
3. Other molecules: Just as well as a substrate, molecules called activators or inhibitors can
interact with an enzyme and can increase the rate of a reaction. They take the shape of the
active site and are used to regulate the speed of the reaction process. Inhibitors are similar
to substrates however their intention is to block the active site that is affecting the
enzyme-substrate complex. Because of their drive to block a substrate from an enzyme,
these types of inhibitors are called competitive inhibitors. There are also inhibitors
called non-competitive inhibitors which obstruct enzyme reactions but do not compete.

Objective:

The enzyme that will be used is tyrosinase. Tyrosinase is found in potatoes. The substrate
that will be used is pyrocatechol. Pyrocatechol is a clear, odorless liquid. When mixed
together, they form hydroquinone, changing into a yellowish-brown color, showing that a
reaction has occurred. A spectrophotometry device will be used in able to determine the
amount of change in color in the product. Because the absorption of color is directly related
to the concentration, the maximum absorbance of the end product is 400 nm. The device will
be set with a value of 400 nm and will be calibrated with a tube of water.

Students will work in a group of 4 in order to complete the experiment. Drive the work
among team members as such: protocol keeper, reading instructions to the team, watching
 time, and recording detail. At the end of the experiment, all students should have recorded
their data and shall examine and decipher the results.

Hypothesis:

We predict that the enzyme (tyrosinase) will only react with the substrate (pyrocatechol) and
not the sucrose. The ideal temperature for enzyme activity is 60 degrees Celsius. The ideal
enzyme concentration that will result in the highest enzyme activity will be 27 drops. The
ideal substrate concentration that will result in the highest enzyme activity will also be 27
drops. The most enzyme activity will occur at a pH of 9. At 20 drops of both inhibitors, there
will be no activity.

Methods:

I. Characteristics of the Enzyme Reaction

A. The Reaction
Test tube 1 was labeled and filled full of distilled water. 10 drops of tyrosinase
were added, as well as 10 drops of pyrocatechol. The end of the tube was covered
with parafilm and flipped around to mix. The initial color was recorded. The tube
was then placed in a rack and observed for 5 minutes. At 5 minutes, the color and
absorbance at 400 nm were recorded.
B. Control
Test tube 2 was labeled and filled full of distilled water. 10 drops of
pyrocatechol were added. The end of the tube was covered with parafilm and
flipped around to mix. The initial color was recorded. The tube was then placed in
a rack and observed for 5 minutes. At 5 minutes, the color and absorbance at 400
nm were recorded.
C. Substrate specificity
Test tube 3 was labeled and filled full of distilled water. 10 drops of tyrosinase
were added, as well as 10 drops of sucrose. The end of the tube was covered with
parafilm and flipped around to mix. The initial color was recorded. The tube was
then placed in a rack and observed for 5 minutes. At 5 minutes, the color and
absorbance at 400 nm were recorded.
II. The Effect on Temperature on Enzyme Activity
5 test tubes were labeled #1-#5. Each were filled full of distilled water. Tube 1 was
placed in ice mixture. Tube 2 stayed at room temperature. Tubes 3-5 were placed in
pre-warmed warmers. Each warmer had its temperature regulated according to the
temperatures listed on Table 2. All tubes stayed for 5 minutes. After 5 minutes, 10
drops of enzyme solution and 10 drops of substrate solution were added to all tubes.
They were mixed and the initial color was recorded. The tubes were returned to their
experimental temperatures and placed for 5 minutes. After 5 minutes, tubes were
removed, color was recorded, and absorbance was measured.
III. The Effect of Enzyme Concentration
3 test tubes were labeled #1-#3. Each were filled full of distilled water. 3 drops of
enzyme were added to tube 1, 9 drops of enzyme into tube 2, and 27 drops of enzyme
into tube 3. 10 drops of substrate were added to each tube. Each tube was covered
with parafilm and mixed. The initial color was recorded. The tubes were observed for
3 minutes. At 3 minutes, the color was recorded and the absorbance was measured.
IV. The Effect of Varying the Substrate Concentration
3 test tubes were labeled #1-#3. Each were filled full of distilled water. 3 drops of
substrate were added to tube 1, 9 drops of substrate into tube 2, and 27 drops of
substrate into tube 3. 10 drops of enzyme were added to each tube. Each tube was
covered with parafilm and mixed. The initial color was recorded. The tubes were
observed for 5 minutes. At 5 minutes, the color was recorded and the absorbance was
measured.
V. The Effect of pH
5 test tubes were labeled #1-#5. Each were filled full with buffer solutions of pH
levels shown on Table 5. Tubes were placed in a rack. 10 drops of enzyme and 10
drops of substrate were added to each tube. The tubes were covered with parafilm and
mixed. The initial color was recorded. The tubes were observed for 5 minutes. At 5
minutes, the color was recorded and the absorbance was measured.
VI. The Effect of Inhibitors
6 test tubes were labeled #1-#6. Each were filled full of distilled water. 1 drop of
phenylthiourea was added to tube 1, 10 drops of phenylthiourea into tube 2, and 20
 drops of phenylthiourea into tube 3. 1 drop of tyrosine was added to tube 4, 10 drops
of tyrosine into tube 5, and 20 drops of tyrosine into tube 6. 10 drops of enzyme and
10 drops of substrate solutions were added to each tube. Each tube was covered with
parafilm and mixed. The initial color was recorded. The tubes were observed for 5
minutes. At 5 minutes, the color was recorded and the absorbance was measured.

Results:

Table 1: Characteristics of the Enzyme Reactions

Tube Contents of Tube Initial Color End Color (at 5 min) A400 (at 5 min)
1 Reaction (10 drops enzyme, 10 Greenish- Light yellow 0.259
drops substrate) yellow
2 Control (10 drops substrate) Clear Clear 0.013
3 Substrate specificity (10 drops Clear Clear 0.015
enzyme, 10 drops sucrose)
Summary: In this table, the reaction, control, and substrate specificity were tested. Each
addition of drops was placed into test tubes for experimenting. Results are shown above.

Table 2: Effects of Temperature on Enzyme Activity

Tube Temperature (C) Initial Color End Color (at 5 min) A400 (at 5 min)
1 0 Slightly yellow Light yellow 0.308
2 20 Slightly yellow Light yellow 0.320
3 40 Slightly yellow Light yellow 0.140
4 60 Slightly yellow Slightly yellow 0.056
5 100 Clear Pinkish clear 0.058
Summary: In this table, the temperature of each enzyme reaction was altered to show a
change in reaction speed. Each tube was placed in various temperature settings ranging
from 0-100 degrees Celsius. Results are shown above.
Table 3: Effects of Enzyme Concentration on Enzyme Activity

Tube Contents of Tube Initial Color End Color (at 3 min) A400 (at 3 min)
1 3 drops enzyme, 10 drops Clear Slightly yellow 0.105
substrate
2 9 drops enzyme, 10 drops Clear Light yellow 0.309
substrate
3 27 drops enzyme, 10 drops Slightly yellow Light yellow 0.592
substrate
Summary: In this table, the enzyme concentration was tested by adding different amounts
of the enzyme and 10 drops of the substrate to each tube. Results are shown above.

Table 4: Effect of Varying the Substrate Concentration on Enzyme Activity

Tube Contents of Tube Initial Color End Color (at 5 min) A400 (at 5 min)
1 3 drops substrate, 10 drops Clear with Clear with light 0.128
enzyme green tint green tint
2 9 drops substrate, 10 drops Clear with Light yellow 0.227
enzyme green tint
3 27 drops substrate, 10 drops Clear with Darker light yellow 0.294
enzyme green tint than above
Summary: In this table, the substrate concentration was tested by adding different
amounts of the substrate and 10 drops of the enzyme to each tube. Results are shown
above.
Table 5: Effect of Ph On Enzyme Activity

Tube pH of Tube Initial Color End Color (at 5 min) A400 (at 5 min)
1 3 Clear Clear 0.027
2 5 Clear Clear (little yellow) 0.170
3 7 Yellow Yellow 0.348
4 9 Yellow (little clear) Clear (little yellow) 0.116
5 11 Clear (little yellow) Clear (little yellow) 0.204
Summary: In this table, the pH levels were tested using the buffer solution in each tube. 5
test tubes were tested, each with a specific level of buffer solution, along with 10 drops of
both the enzyme and substrate solutions. Results are shown above.

Table 6: Effect of Inhibitors on Enzyme Activity

Tube Contents of Tube Initial Color End Color (at 5 min) A400 (at 5 min)

1 1 drop phenylthiourea, 10 drops Clear Clear/foggy yellow 0.029

enzyme and 10 drops substrate
2 10 drops phenylthiourea, 10 drops Clear Clear 0.025
enzyme and 10 drops substrate
3 20 drops phenylthiourea, 10 drops Clear Clear 0.018
enzyme and 10 drops substrate
4 1 drop tyrosine, 10 drops enzyme and Very light yellow Light yellow 0.082
10 drops substrate
5 10 drops tyrosine, 10 drops enzyme Very light yellow Light yellow 0.024
and 10 drops substrate
6 20 drops tyrosine, 10 drops enzyme Very light Foggy yellow 0.022
and 10 drops substrate yellow/blue
Summary: In this table, 7 test tubes test the inhibitors reaction on the enzyme process.
Specific amounts of phenylthiourea and tyrosine were used. Results are shown above.
Discussion:

A. Characteristics of the Enzyme Reaction

Test tube 1 had an absorbance of color that was detected by the Spectronic 20. The
addition of pyrocatechol to the tyrosinase caused a color change from greenish-yellow to
light yellow. Although the control tube contained pyrocatechol, it was lacking because it
was a control experiment and only contained pyrocatechol, thus it had nothing to react
with. It is important to include controls in the experiment to help determine if an error
was made and to calculate the amount of change. A control with just tyrosinase could
have been used as well. This enzyme demonstrates substrate specificity because when
added to the sucrose, there was no reaction/color change, whereas when added with the
pyrocatechol, there was a reaction/color change.
B. The Effect of Temperature on Enzyme Activity
All of the test tubes had some sort of change in most temperatures, with an exceptionally
microscopic change at 60 and 100 degrees Celsius. The enzyme reaction occurs more
slowly at temperatures below the optimum because it reduces the speed of kinetic energy
between the molecules. The enzyme at temperatures higher than the optimum will have a
faster reaction because of the increased kinetic energy between the molecules. However,
if the temperature is either too high or too low, it can cause a disruption between the
molecules, making the enzyme denatured.
C. The Effect of Enzyme Concentration
Changing the concentration of enzyme affects the rate of the reaction because more
enzymes causes an increase in the amount of active sites that are available. With a small
amount of substrate and a large amount of active sites, the substrates can then be
accounted for and handled in the reaction. If you continued to add increasing amounts of
enzyme but kept the substrate concentration constant, the enzymatic activity would
greatly overflow.
Enzyme Activity

Enzyme Concentration
D. The Effect of Varying the Substrate Concentration
Increasing the concentration of substrate affects the rate of the reaction because there will
not be enough active sites to promote enzyme activity. Because the enzyme is typically
reusable, it would take longer for each substrate to be changed in an enzymatic reaction.
E. The Effect of pH
The pH level at which the enzyme is most effective is 7 (neutral) because the enzyme is
most effective in a neutral state rather than when it is too acidic or too basic. The enzyme
reaction fails to occur at very low and very high pH because these pH levels can disrupt
the enzyme and cause it to become denatured.
Enzyme Activity

pH

F. The Effect of Inhibitors

The tyrosinase reaction proved the inhibitor was non-competitive due to the change in
color of the solution. The two mechanisms by which inhibitors interfere with the enzyme
action are competitive and non-competitive inhibitors. Competitive inhibitors are driven
to block a substrate from the enzyme. Non-competitive inhibitors obstruct enzyme
reactions; however, they do not compete. Increasing the inhibitor concentration affected
the enzyme reaction because it resulted in a slower reaction process due to the blocking
of active sites from the substrate to the enzyme.
Conclusion:

Based on the results of this experiment, the optimum temperature is 20 degrees Celsius (room
temperature), the pH at 7 (neutral), and relatively the same amount of enzyme to substrate
concentrations caused the most effective reactions. My hypotheses were somewhat supported by
this evidence, however I was proven wrong in terms of the temperature, pH, and inhibitors. It is
important to learn/note that these conditions are not necessarily the same for every enzyme
because most enzymes are substrate specific, meaning they can only connect with certain
substrates. This can cause a limitation on organisms.
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