Effects of Enzyme Catalysis of H2O2 by Catalase in Multiple Conditions

Performed by Kalvin Foo

Lab Partners: Taylor Enoch, Jisoo Han, and Joey Dong

Submitted to Mrs. Fitzgerald

AP Biology, Period 6

October 3, 2008
Problem: To qualitatively measure the effect of the enzyme catalase on hydrogen peroxide

decomposition, then to measure the effect of the catalysis when temperature is modified.

Background: This experiment is based off of the idea that an enzyme acts to lessen a reaction’s

reaction time by lowering the energy of activation through substrate conversion via the active

site. As the reaction progresses, there is less enzyme activity due to a lowered substrate

concentration, thus slowing down the process. In the case of the reaction used for the experiment,

H2O2→ H2O+ O2, it is thermodynamically favorable (ΛH= -98.2 kJ mol-1, ΛG= -119.2 kJ mol-1,

and ΛS= 70.5 J mol-1 K-1), but has an extremely slow decomposition rate if not catalyzed.

However, it bonds directly with the active site of the enzyme catalase, thus allowing a much

quicker rate of reaction, though with the same thermodynamic net results. As the catalyzed

reaction is performed, formation of gas bubbles will be visible, and the reaction can be

effectively stopped through a denaturalization of the enzyme, most likely through injection of an

acid, H2SO4 in the case of this lab. The change in pH alters the enzymes quaternary structure and

alters the active site so that it cannot accept H2O2 as a substrate and ends catalysis. Similarly, an

increase in temperature produces the same effect as a decrease in pH, and also denaturalizes the

enzyme.

Data and Observations

(Titrations performed with KMnO4, end results when solution became reddish/brownish/pinkish)

Hydrogen Peroxide Decomposition

Baseline with Catalase Uncatalyzed Decomposition

Initial Volume 5 mL 5 mL
Final Volume 1.5 mL 1 mL
Λ Volume 3.5 mL 4 mL
Qualitative Catalyzed Decomposition
10 Sec 30 Sec 60 Sec 120 Sec 180 Sec
Initial Volume 5 5 5 5 5
Final Volume 2 2.1 2.3 2.6 3.2
Λ Volume 3 2.9 2.7 2.4 1.8

H2O2 Decomposed .5 .6 .8 1.1 1.7

Catalyzed Decomposition

1.8
1.7
Amount Decomposed (mL)

1.6
1.4
1.2
1.1
1
0.8 0.8
0.6 0.6
0.5
0.4
0.2
0
0 50 100 150 200
Tim e (s)

Rate Calculations
(.5-0)/(10-0) .005 mL/s
(.6-.5)/(30-10) .005 mL/s
(.8-.6)/(60-30) .0067 mL/s
(1.1-.8)/(120-60) .005 mL/s
(1.7-1.1)/(180-160) .005 mL/s
Avg rate: .005 mL/s

Results and Discussion

First of all, the graphed data shows an almost constant rate of catalyzed reaction up until the

endpoint. However, what should have been shown was the “leveling off” of the graph due to the

decreasing concentration of substrate available. Another anomaly was the greater amount of

decomposed hydrogen peroxide in the uncatalyzed reaction, because it should not have surpassed

the baseline catalyzation of hydrogen peroxide because it can be assumed the baseline reaction
acted to completion, showing the maximum amount of decomposition possible. Regardless, the

data obtained provides the average rate of catalyzation to be .005 mL of H2O2 per second,

calculated through the change in volume/change in time, or dV/dt. Though the curve is not

shown, another test for 360 seconds would have visibly portrayed it because the last data entry

was about at the point in which the lowered concentration would greatly decrease the rate of

reaction, approaching the zero mark as the concentration dwindles.

Analysis

In adding catalase to H2O2, a chemical change is observed in the production of a clear gas

(O2) in a fizzing form, which symbolizes that the enzyme catalyst is speeding up the

decomposition of the H2O2.

This reaction can be described as 2 H2O2 + enzyme → 2 H2O + O2.

During the reaction, the enzyme is the catalase, while the substrate is the H2O2. During the

reaction, a Catalase -H2O2 (enzyme-substrate) intermediate is formed, finally resulting in the

products, H2O and O2.

To discuss the effect of boiling on enzyme activity, the boiling subjected the enzyme to a

high temperature and a non-ideal condition for its activity, thus denaturing the quaternary and

tertiary structures. This warps the active site so that the reaction is not catalyzed because of the

inability to form the enzyme-substrate complex.

Three other factors that could affect the activity of the catalase are a change in pH,

concentration, or the addition of an inhibitor. Changing the [H+] alters hydrogen bond

interactions and affects secondary and tertiary structures and causes denaturalization similar to

an altered temperature. Next, the concentrations of both the enzyme and the substrate matter,
because the enzyme concentration determines the baseline, and as the reaction progresses, the

concentration decreases and alters the rate because of the scarcity of substrate, so changing

substrate concentration alters the rate as well. Finally, an inhibitor, whether competitive or

noncompetitive, alters the enzymes structure and also changes the enzymes active site, thus

rendering it incapable of catalysis.

Living tissue, especially from body parts such the liver, contains catalase because the body

naturally produces hydrogen peroxide as a byproduct of metabolism, and it is detoxified with

such enzymes. This is proven as H2O2 is added to a piece of potato, and the formation of bubbles

validates the decomposition of H2O2.

The baseline that was determined is necessary because it shows the extent of the reaction and

also the amount of H2O2 that is actually in solution after a catalyzed reaction.

H2SO4 was used to stop the reaction because its introduction into the system lowers the pH of

the system, so that the enzyme is denatured and the reaction is brought to a halt because the slow

uncatalyzed reaction rate is resumed.

The line on the graph represents the constant rate (amount of H2O2 decomposed is linear, rate

is the derivative) until the point in which the concentration becomes scarce and the reaction rate

decreases.

The graph’s linear portion shows that the rate of reaction over time is constant in the

concentrations tested in this lab, and the plateau was not shown.

Because there is a constant rate at first, the initial rate should correlate to the average rate

measured in the linear portion, which is .005 mL/s, which was calculated through dV/dt
Works Cited

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2008 <http://www.worthington-biochem.com/CTL/default.html>.

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<http://www.catalase.com/cataext.htm>.

"FMC Chemicals- Hydrogen Peroxide." FMC Chemicals. 01 October 2008. FMC Chemicals. 2 Oct 2008

<http://www.fmcchemicals.com/tabid/1463/Default.aspx>.

"hydrogen peroxide." Encyclopædia Britannica. 2008. Encyclopædia Britannica Online. 02 Oct. 2008

<http://www.britannica.com/EBchecked/topic/278760/hydrogen-peroxide>.