Mirielsa A.

Palor
AP BIO 1/30/14
Pd. 7
th
and 8
th


pH Levels Effects on Enzyme Inhibition
Background
pH at extremely low or high levels similar to temperature denatures enzymes acting as an
enzyme inhibitor changing its structure which affects the activity of the enzyme. pH affects the enzyme
inhibiting it changing its structure so that the substrate can no longer fit or be catalyzed at all. Enzymes
that have optimum pH in the neutral pH range in most cases are irreversibly denatured at extremely
high or low pH. In living systems there are buffers that equilibrate and will gain or lose H+ ions at a
minimum. As for salinity of enzymes concerning concentration if the concentration is very lo r zero,
the charged amino acidside chains will stick together and the enzyme will denture and form an inactive
substrate. If the salt concentration is high which in our experiment was high because we added salt into
our catalase this caused the normal interactions of charged groups to be blocked causing a new
interaction to occur, this also causes the enzyme to precipitate.

Introduction
In this experiment we tested the affects of pH level changing from neutral to acidic and basic as well as
salinity (salt concentration a second inhibitor variable we tested which has no pH) to see which will
inhibit the potato catalase the most from hydrolysis of hydrogen peroxide. Our testable question was
How does possible enzyme inhibitors affect the catalysis of the potato catalase (enzyme) which
will accelerate the hydrolysis of hydrogen peroxide (substrate)? We simulated the hydrolysis of
the potatoe catalase of hydrogen peroxide by submerging filter papers in enzymes which we have
added an enzyme inhibitor into and then submerging into the hydrogen peroxide to time the amount of
time they take to completely rise to the top. We had a positive control which was just the regular potato
catalase with the pH of 5 submerged in hydrogen peroxide and a negative control which was just
regular potato catalase with the pH of also 5 submerged into water. We used these two to compare our
data of the experimental groups of potato catalase containing pH levels of basic and acidic. Our basic
enzyme with the pH of 6 is the potato catalase + 70% isopropyl alcohol and our acidic enzyme with the
pH of 2 is the potato catalase + lemon concentrate which are both non- competitive inhibitors and are
irreversible. Another enzyme inhibitor that we tested was salinity hence salt concentration in this lab
we added salt to the potato catalase increasing the salinity of the enzyme disrupted the normal
interactions causing a new interaction to occur between the enzyme and the substrate. Our hypothesis
for variable 1 (pH level) was If the pH level decreases, then it will inhibit the most due to its
increased acidity because when the pH level of an enzyme decreases the carboxyl (-COOH) and the
amino group (-NH2) readily gain H+ ions and continue until the side chains are affected disrupting the
shape or in other words denaturing the enzyme affecting its ability to catalyze properly. Our second
variable which was If the salt concentration increases, then the enzyme will be non-competitively
inhibited creating a new interaction to occur because when the salt concentration of an enzyme
increases the normal interactions of the charged groups will be blocked which creates new interactions
meaning the inhibitor bind with the enzyme on another site which changes the shape of the enzyme
affecting its ability to properly function in catalyzing. This showed in the data because compared to the
positive control which was the catalase + hydrogen peroxide it rose 1.16 seconds later and compared to
the negative control which was catalase + water it rose 2.17 seconds later so (this leads me to the
conclusion that) the enzyme activity slowed.




Method and Materials
We used a graduated cylinder to measure out 40 mL of 1% hydrogen peroxide and poured it into 12
beakers. And 40 mL of water into 3 beakers. In total we had 13 beakers in total to conduct 3 trials. We
then punched out filter papers and separated them for each catalase with the different pH level and
salinity. We then divided the potato catalase into 5 beakers and individually added salt, lemon
concentrate, and 70% isopropyl alcohol to three of the catalase filled beakers. We then used pH papers
to read the pH of each potato catalase. Then we used forceps to take each individual filter papers and
submerged them into the medicated catalase for 5 seconds and then let it sit for 5 seconds on a paper
towel for another 5 seconds, then after 5 seconds have passed we submerged the filter paper with
catalase into hydrogen peroxide and timed how many seconds it took for it to rise to the top completely.

Observations
The filter paper in the basic enzyme took longer to rise to the top compare ot the negative and positive
controls while the acidic enzyme did not rise at all.













DATA TABLE

Reg Catalase:pH 5
Catalase+Alc:pH 6
Catalase+Salt:pH 4
Catalase+Lemon juice: pH
2
Time to
Float
(seconds)

Trial 1 Trial 2 Trial 3 Average
Positive control
(Reg. Catalase & H2O2)
1 sec 3.53 sec 3.98 sec 2.84 sec
Negative control
(Reg. Catalase + H2O)
2.20 sec 1.46 sec n/a 1.83 sec
(catalase + isopropyl
alcohol 70 %)
10.40 sec 7.51 sec 9.79 sec 9.23 sec
Catalase + Salt 3.00 sec 4.23 sec 4.78 sec 4 sec
Catalase + Lemon Juice n/a n/a n/a n/a

Data Table
Our collected data table shows that both the enzyme which had a significant change in pH level
disrupted the catalase by slowing or stopping the enzyme activity from occurring. We can see that the
basic catalase (catalase + isopropyl alcohol 70 %) with the pH of 6 denatured the catalase by slowing
the enzyme activity because it took the average of 9.23 seconds in three trials to hydrolyze the
hydrogen peroxide. In comparison to the positive control it took 6.39 seconds longer to rise to the top
completely as for the negative control it took about 7.4 seconds longer to rise to the top. This proves
that the increasing of pH level of enzyme by just 1 pH level (regular catalase pH:5 and catalase+ 70%
isopropyl alcohol pH:6) can easily disrupt an enzyme and its rate to hydrolyze a substrate. On the other
hand we can see that the acidic catalase with the pH of 2 which is the decrease of 3 pH levels
completely denatured the catalase causing the filter paper to not rise at all. As for the salinity in other
words salt concentration of an enzyme the data shows that it inhibited the potato catalase at a minimum
because it took an average of seconds to rise to the top. In comparison to positive control time it took
an average of about 1.16 seconds longer to rise to the top, and in comparison to the negative control it
took an average time of about 2.17 seconds longer to rise to the top. We did not create a graph for this
data table because there was no relationship or correlation of each variable.

Conclusion
Overall we can conclude that changing an enzymes pH just by 1 or more significantly alters the
enzyme by causing a disruption to the enzyme affecting its ability to catabolize. This experiment
proved our hypothesis with the first variable of pH that If the pH level decreases, then it will inhibit
the most due to its increased acidity is correct because the data collected showed that the enzyme as
completely denatured by the filter paper no rising to the top at all when submerged in the hydrogen
peroxide which meant that there was no catabolic reaction occurring to the hydrogen peroxide. The
second variable If the salt concentration increases, then the enzyme will be non-competitively
inhibited creating a new interaction to occur was supported by this experiment because accodring
to the data collected the filter paper submerged into the salt and catalase took about 1.16 seconds longer
to rise to the top compared to the positive control and 2.17 seconds longer to rise to the top compared
the the negative control. Based on our entire data we can conclude that salt concentration and pH level
changes at an extreme causes a disruption to the enzymes ability to catabolize.

Discussion Questions
How does enzyme activity vary with enzyme concentration?
Enzyme activity is the rate at which an enzyme breaks down the substrate while enzyme concentration
is the amount of enzyme. These two are positively correlated to each other because if the concentration
of an enzyme increases then the enzyme activity will increase due to the amount of enzyme. The more
enzyme the faster the rate the enzyme canalizes the substrate until it reaches a constant due to the
limiting factor of substrate being less than the enzyme.
How is the rate of enzyme activity affected by increasing the concentration of the substrate?
The enzyme activity increases as the enzyme concentration increases because there are more enzymes
to catalyze the substrate until it reaches a constant.
What do you think would happen if you increased the substrate concentration to 40.0%
hydrogen peroxide?
Since substrate concentration and enzyme activity are positively correlated meaning if the
concentration of substrate increases so will enzyme activity to a certain point and vise versa with
enzyme concentration increasing the substrate concentration to 40% will increase the enzyme activity
until it reaches the maximum causing no change in enzyme activity.
How does changing the substrate concentration compare to changing the enzyme concentration
in this experiment?
Increasing the concentration of the substrate will increase the enzyme activity because more enzymes
are being used, however it can only increase to a maximum hence adding more substrate wont affect
the activity anymore once the max is reached. Similarly increasing the enzyme concentration will
increase the enzyme activity until the constant is reached.
Explain the results when the hydroxylamine is added to the enzyme.
Not available

From the temperature data, what can you conclude about how temperature affects enzyme
activity? How would you explain the results?
Temperature increase speeds up all chemical reactions however raising the temperature above the
optimum temperature of an enzyme will denature the enzyme. Based on the data as the temperature
increased in celsius the time for the filter paper to rise and float increased.

Source of error and limiting factors
One source of error is the 70% isopropyl alcohol being expired because when we tried dipping the pH
papers the pH paper change to yellow reading the alcohols pH to be acidic rather than basic. The pH
paper could have also been a contributor to the source of error because it read the pH of the alcohol to
be acidic rather than basic.Another source of error is driving the filter paper into the glass when we
submerged it so that it stuck to the glass for a little bit altering the correct time that it should rise.