Eddy Egan

October 31, 2008

Contreras Pd. ¾
AP Biology
Relative Amount of H2O2 Decomposed per second by Catalase measured by use of a
Titrant

Enzymes are globular proteins that catalyze metabolic reactions by reducing the
activation energy needed. This occurs when the substrate binds to the enzyme at its active
site. Once the substrates, or reactants, have absorbed the activation energy needed from
their surroundings, bonds will be broken because the reactants would have become
unstable. This would therefore make them more reactive as the reach their transition state.
(CITE: 1)The reason for this occurrence is because the thermal energy absorbed by the
reactants increases their speed, resulting in many forceful collisions, which will break the
reactants bonds. The amount of energy absorbed, the activation energy, needed is lower
by the enzyme, therefore, catalyzing the reaction. Enzymes are very selective in the
reactions which they catalyze, determining the reactions occurring with the cell at any
given time. (CITE: 2) In order to begin catalysis, the substrate must bind to the enzyme,
forming and enzyme-substrate complex. While joined, the enzyme’s catalytic action
converts the substrate into the product. Additionally, enzymes can recognize their own
substrate from a group of many others, further increasing their selectivity (CITE: 2).
These enzymes can even differentiate between isomers. This is a result of their unique
shape, since they are proteins. The specific active site on the enzyme, the region where
the substrate binds, is the cause of the enzyme’s specificity. The substrate does not need
to perfectly fit, however, the chemical groups on their surface and the amino acids on the
active site can slightly change there shape to fit better (CITE:2). This is an example of the
induced fit model.
The induced fit model illustrates how the substrates bind to its enzyme to form the
enzyme-substrate complex by showing a slight change in the shape of the active site. No
other shape will be able to fit into this active site including isomers (CITE: 2). The only
exceptions are inhibitors, which prevent the normal substrate from binding. This model
differs from the lock and key model, which says that the substrate must fit exactly with its
corresponding enzyme. This model is no longer in use. While substrates are held in the
active site, though, hydrogen bonds and van der Waals forces keep the substrate in place
(CITE: 2) The R groups of several of the amino acids that compose the active site
catalyze the reaction, converting the substrate into the product. The product will then be
released after the reaction has finished. This process is completed so quickly that one
enzyme can catalyze approximately one thousand reactions per second (CITE: 2). After
the reaction has completed and the product has been released, the enzyme will return to
its original shape, allowing it to act upon other molecules. In some cases, the R groups
may be acidic, basic, or alkaline, in an otherwise neutral cellular environment. This
would affect whether the substrate would have been facilitated by H+ or OH- to form the
product. (CITE: 3)
The initial reaction rates of enzyme-catalyzed reactions are affected by changes in
temperature, pH, enzyme concentration, and substrate concentration. The rate will also be
affected when the enzyme is denatured. This would occur as a result in the drop pH or
increase in temperature. Denaturing would break the bonds of the enzyme’s quaternary
structure, including hydrogen and ionic bonds, and van der Waals interactions. This
would cause the enzyme to unravel and lose its shape, which would prevent it from
catalyzing reactions when it is renatured. (CITE: 4)
As stated before, a catalyst speeds up the process of a reaction, in our lab we used
an catalytic enzyme known as catalase. In the experiment, catalase will catalyze the
decompositions of hydrogen peroxide, producing water and oxygen gas (H2O2  H2O +
O2)
I believe that the enzyme catalase will increase the reactions’ speed (speed of
H2O2 decomposition into H2O + O2). This is because enzymes catalyze reactions by
lowering their activation energy, therefore increasing the rate of reaction. The rate will
then decrease, though, once the concentration of H2O2 begins to substantially decrease.

Procedure:
Exercise 2B: The Base Line Assay
1, 2. 3.

4. Remove a 5 mL sample and place in another beaker to assay for the amount of
H2O2 by doing the following
5. Put the beaker with the sample over a white piece of paper and use a syringe
to add KMnO4 ONE DROP at a time. Gently swirl the beaker after each drop
is put in. Do this until the solution turns a persistent pink or brown color.
6. Record data in Table BL1

Exercise 2C: The uncatalyzed Rate of H2O2 Decomposition

1. Pour 10 mL of 1.5% H2O2 in a beaker and store it uncovered at room
temperature for about 24 hours.
2. Repeat steps 2-5 from exercise 2B to determine the amount of H2O2 still
present.
3. Record in Table UD

Exercise 2D: An Enzyme-Catalyzed Rate of H2O2 Decomposition

1. 10 seconds:
a. Pour 10 mL of 1.5% H2O2 in a 50 mL beaker and then add 1 mL
catalase extract
i. Catalase will be on ice and may settle to the bottom of the
test tube. To overcome this follow these steps:
1. Tightly close lid on test tube.
2. Invert the test tube and place in a vortex until
cloudy
b. Swirl gently for 10 seconds
c. When time is up, add 10 mL 1.0 M H2SO4 (Added in order to stop
any more reactions from occurring)
 2. 30, 60, 90, 120, 180, 360 seconds
a. Repeat steps a-c in step 1, but swirl the length of time until the
timer has reaches 30, 60, 90, 120, 180, and 360 seconds.
3. Each time, obtain a 5 mL sample of the solution and assay for the
amount of H2O2. To find this, repeat step 5 of Exercise 2B and record
data in Table KT
4. Graph the data from the enzyme-catalyzed reaction.

Analysis:
The rate is highest in the first interval with a rate of 0.20 mL H2O2/sec.
This is because the largest amounts of H2O2 able to be catalyzed were in the first
interval since the reactions were first starting. They decreased over time as more
and more H2O2 was decomposed and less became available. The reaction rate can
be the highest on any point in a graph as long as it contains the most amount of
substrate that is available to react with enzymes.
The rate is lowest at the last interval with a rate of .0005 mL H2O2/sec.
This is a result of the very little H2O2 that has not been decomposed in the
solution. All reactions similar to this will slow down when concentration of the
substrate decreases.
The initial rate of reaction of enzymes is determined by the characteristics
it possesses. This initial reaction rate remains constant for any enzyme and its
substrate at a specific pH and temperature, as long as the substrate is in excess. It
is represented on a graph by the linear portion of the curve. This rate is
determined by finding the slope after choosing two points.
Free energy is the surrounding energy that can be readily absorbed by
molecules and other particles. Part of this energy is absorbed as activation energy
for a reaction. In order to prevent the denaturing of proteins, killing the cell, and
speeding up all reactions, an enzyme is used to lower the activation energy (CITE:
2) It does this by stretching out the substrate so that it is more unstable, making it
easier to reach the transition state (CITE: 3) To reach it, free energy is absorbed,
which breaks bonds and forms new ones. By the enzyme lowering the activation
energy barrier, less free energy is needed to be absorbed as activation energy. This
will not affect the free-energy change of a reaction whether an is used or not.
(CITE: 2)
Sulfuric acid was used to stop decomposition in the experiment because of
its ability to denature catalase. Catalase and other enzymes will denature as a
result of a drop in pH of their surroundings. Since catalase is not in its optimal
pH, hydrogen bonds, ionic bonds, and other weak forces are broken, changing the
enzymes three-dimensional structure. This prevents further catalyzation of the
decomposition of the H2O2 with this enzyme, inhibiting the function of catalase.
(TALK MORE ABOUT THIS!!) (Tertiary structure too!)
By lowering the temperature of an enzyme’s environment, the rate of
reaction will also decrease. This is because enzymes have an optimum
temperature to function and if it is significantly dropped or raised, the rate of
reaction will slow down. However, icrease in temperature generally almost
always increases reaction rate until a certain point. Since the particles would be
moving so slowly because of the cold, few collisions would occur. (TALK MORE
ABOUT THIS!!)
Hypothesis proved or disproved
The graph shows the amount of H2O2 that has reacted as a function of
time. After the third measured point, the graph shows a decrease in the reaction
rate representing the decrease of available H2O2 for decomposition. The dotted
line extending from the third point shows how the amount of H2O2 decomposed
would continue to increase at a constant rate if H2O2 supplies did not diminish.
Sources of Error:
Misreading values of reactants used
Wrong amounts of solutions added
Further Study: