BIO107.

2 • Grp 3 • LR 1

ENZYME ASSAY: EFFECTS OF MODIFIED CONDITIONS

AND INHIBITOR B ON ENZYME 4
Alarcon AR, Arsad H, Frenila R Jr., Namion J, Pinongpong J
BIO107.2 Experiment 1 Group 3
Science Department, College of Natural Sciences and Mathematics
Mindanao State University-General Santos
Brgy. Fatima, General Santos City 9500, Philippines
15 October 2021

ABSTRACT

This laboratory activity was conducted to determine if the activity of the enzyme is
affected by some important factors and conditions – adding a competitive inhibitor,
change in pH, incubation time, amount of enzyme, incubation temperature, and
substrate concentration. Using the simulated laboratory, the students ran the
simulation using Enzyme 4 at maximum range and with no inhibitor as an initial setup.
After performing the initial setup, each condition was modified three times with the
presence of inhibitor B to observe different enzyme activity and to determine the
optimum for each condition. For the pH of incubation, the optimum observed is around
9.2. The optimum incubation time is at 12 min while the optimum incubation
temperature is at 66 ℃. Moreover, when the amount of enzyme increased, it also
increases the rate of activity of the enzyme. Similarly, increasing substrate
concentration also resulted in the increasing rate of enzyme activity. Finally, the
addition of Inhibitor B decreases the activity of the enzyme for any condition.

Introduction

In a living organism, an enzyme is a protein that accelerates the pace of a chemical process. It
is a protein that works as a catalyst in chemical processes, transforming one set of reactants (called
substrates) into another set of products. The shape of an enzyme's active site, which is determined by
the three-dimensional arrangement of amino acids, determines its specificity. Enzymes alter shape at
the active site during catalysis as a result of their flexibility. Small metabolites (inhibitors) in cells can
also interact with, prevent the substrates from binding, and change the shapes of several enzymes due
to this flexibility (Bergtrom, G., 2018). Furthermore, this research will also look at the effects of pH, time,
enzyme concentration, incubation temperature, and substrate concentration on enzyme 4 activity. In
addition, this research also aims to see how changing the parameter/condition affects enzyme activity.
Finally, this research seeks to discover the enzyme 4 optimum under each condition as well as the
influence of inhibitor B on enzyme 4 activity.

Methodology

This activity aims to systematically evaluate the effect of an enzyme in different incubation
conditions by using an online laboratory platform, the simulated virtual laboratory: Enzymes assay which
was developed by David A. Bender (1982-2005) of the Department of Biochemistry and Molecular
Biology, UCL. The group is tasked to run the simulation using Enzymes 4, inhibitor B as per the
instruction of the instructor. the experiment includes the pH of incubation (between 1-14), incubation
time (between 0-60 min), Amount of Enzymes (between 0-250µL), incubation temperature (between 0-
100 °C), and concentration of substrates (0-250mmol/L).

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Data Gathering

1. Without inhibitor
1.1 Initial set-up for pH incubation
The experiment used the incubation of enzyme 4, inhibitor B on this set-up. Minimum
and maximum pH levels from 1.0-14.0 respectively are used with the selected condition that is
specified directly by the simulation that includes: 250µL of enzymes, the temperature at 70°C,
incubation time of 12min and 250mmol/L of substrates. The optimum pH level is determined to
be the best enzymes condition.
1.2. Initial set-up for incubation time
A 0-60 minutes incubation time of enzyme 4 is used in this set-up. The optimum pH
level, the pH Buffer of 8.80 that was obtained on 1.1 is used, thus another condition is specified
default.
1.3. Initial set-up for the number of Enzymes
The amount of Enzymes ranges from 0-250µL on the incubation of enzyme 4 is used
in the experiment. In this set-up, the group used the optimum incubation time, which is 12
minutes obtained from 1.2 to determine the optimum amount of enzymes and the optimum pH
level of 8.80 as the other condition are specified by default.
1.4. Initial set-up for incubation temperature
In this experiment, an incubation temperature range from 0-100 °C is used on the
incubation of enzymes 4. The optimum value was obtained from 1.3. the amount of Enzymes
which is 250µL, optimum incubation time of 12 min, and the optimum pH level of 8.80 is used
to obtain the optimum incubation temperature as the other condition is specified default
1.5. Initial set-up for the concentration of substrates
A concentration of substrates ranging from 0-250mmol/Lon the incubation of enzyme
4 is used in the experiment. the optimum value of 1.1., 1.2, 1.3, and 1.4 is used to obtain the
optimum condition of the concentration of substrates.

2. With inhibitor
2.1 Modification on pH incubation
The group performs three modifications in this set-up which includes three different pH
ranges. The first modification, has a pH range of 1-6, the second modification with a 6-8 range,
and the third modification with 8-14 pH ranges. This includes the addition of 125-1000 mmol/L
inhibitor B and a different set of condition that obtained from the optimum value in the initial set-
up of 1.2, 1.3, 1.4 and 1.5. It also shows that the optimum value of pH in this setting is 9.2 that
differs from the initial set up which is 8.8.
2.2. Modification of incubation time
Three modification is performed in this experiment with three different ranges of
incubation time. The first modification has an incubation time of 0-20min, the second
modification with 20-40 min, and the third modification with 40-60min incubation time. This
includes the addition of 125-1000 mmol/L inhibitor B and a different set of condition that
obtained from the optimum value in the initial set-up of 1.1, 1.3, 1.4 and 1.5.
2.3. Modification of Amount of Enzymes
The group performs three modifications in this set-up which includes three different
amounts of enzymes. The first modification, have an enzyme concentration of 0-80µL, the
second modification with 80µL-160µL, and the third modification with the 160µL-250µL amount
of enzymes. This includes the addition of 125-1000 mmol/L inhibitor B and a different set of
condition that obtained from the optimum value in the initial set-up of 1.1, 1.2, 1.4 and 1.5.
2.4. Modification of Incubation Temperature
Three modification is performed in this experiment with three different ranges of
incubation temperature. The first modification has incubation temperature of 0°C-33°C, second
modification with 33°C-66°C and third modification of 66°C-100°C incubation temperature. This

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includes the addition of 125-1000 mmol/L inhibitor B and a different set of condition that
obtained from the optimum value in the initial set-up of 1.1, 1.2, 1.3 and 1.5.
2.5. modification of concentration of substrates
Three modification is performed in this experiment with three different ranges of
concentration of substrates. The first modification has a concentration of substrates of 0mM-
80mM, the second modification with 80mM-160mM, and the third modification of 160mM-
1250mM concentration of substrates. This includes the addition of 125-1000 mmol/L inhibitor
B and a different set of condition that obtained from the optimum value in the initial set-up of
1.1, 1.2, 1.3 and 1.4.

This method will prove that in a different range of inhibitor concentrations, there is an enzymatic
reaction that occurs. Sometimes it will give a little difference in the result if the inhibitor is too little and
a big difference if it is simulated with higher ranges, thus the student always remembers that to yield a
big difference in inhibitor they should choose a higher range of incubation.

Results

The effect of pH on enzymatic activity

To investigate the effect of pH of incubation in enzyme activity, 250 μL of enzyme 4 was
incubated with 250 mmol/L of the substrate at 70 ℃ for 12 min. at varying pH. The initial setup with no
inhibitor, consisting of 11 incubations whose pH ranges from 1-14, shows the maximum enzyme activity
is at 8.80 pH, which produced 213.43 μmol of product.
The pH of incubation ranging from 1-6, showed little to no enzyme activity. With the pH of
incubation ranging from 6-8 with an inhibitor, an increasing enzyme activity as the pH of incubation
approaches alkalinity was observed. Moreover, the third modified setup depicted in Figure 1 (left), with
pH of incubations ranging from 8-14 showed the pH optimum of enzyme 4 is at 9.2. At the optimum pH,
219.42 μmol of the product was formed, the highest among other incubations. However, at pH levels
9.8-11, enzyme activity has started to decrease. Beyond pH 11, where alkalinity is becoming more
extreme, there was no enzyme activity at all.

The effect of Incubation time on enzyme activity

Assessment of enzyme activity at varying incubation times revealed that most products were
formed at the 12-minute duration of incubation. As seen on the initial setup in Figure 2 (right), enzyme
activity increased at 0 minutes and peaked at 12 mins, wherein 213.18 μmol of products were formed.
However, beyond 12 minutes, enzyme activity has started to decrease, and at 24 minutes of incubation,
enzyme activity has ceased. No enzyme activity was observed at the 40-60 minutes of incubation.

The effect of the Amount of Enzyme

Results from the present experiment showed a linear relationship between the amount of
enzyme and the number of products formed, at optimal conditions. As the amount of enzyme increases,
the product formed has increased as well. Figure 3 shows that the least amount of product formed was
at 0 μL of enzyme 4, while the most amount of product formed was at 250.0 μL of enzyme
4.

The effect of Incubation Temperature

Assessment on the effect of incubation of temperature revealed that 66°C is the optimum
incubation temperature for enzyme 4. According to Figure 4, which shows the initial setup (without an
inhibitor), an increase in enzyme activity is observed as temperature increases from 0 to 60°C.
However, there is a drastic decrease in enzyme activity as the temperature increased beyond 60°C.
The second modified setup, with temperatures ranging from 60 - 80°C and has an inhibitor, allowing for

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the precise identification of the optimum temperature. As seen in Figure 4, the greatest number of
products produced, and thereby the optimum temperature, is at 66° C.

The effect of the concentration of substrate

The investigation on the effect of concentration showed a linear relationship between the
concentration of substrate and the number of products formed at optimal pH and temperature. As seen
in Figure 5, an increase in the concentration of substrate had also marked an increase in the formation
of products as well. Figure 5 shows that the least amount of product formed was at 0mM of the
substrate, while the greatest amount of product formed was at 250.0mM of the substrate.

The effect of adding inhibitor B

It was observed from all the experimented conditions that the addition of inhibitor B had
significantly limited the product yield of enzyme 4. Moreover, the addition of a higher concentration of
inhibitor also elicits higher limitations to the enzyme activity of enzyme 4. As seen in Figures 1-5, the
product yield of 125 mM of inhibitor B is higher compared to that of 1000 mM of inhibitor B.

Figure 1. Products formed at varying levels of pH. (Right) The numerical data and graph from the initial
setup of varying pH (1-14) without an inhibitor. (Left) The numerical data and graph from the third modified
setup with varying pH (8-14) and the amount of product yield. Both showing the increase in enzyme activity
as pH levels reach the optimum and decrease in activity as the pH exceeds the optimum.

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Figure 2. Products formed at varying incubation times. (Left) The numerical data and graph from the
initial setup, whose incubation time ranges from 0-60 mins., with no inhibitor. (Right) The data from the
modified setup with an inhibitor whose incubation time ranges from 0-60 mins. Both show that maximum
products are formed at 12 minutes.

Figure 3. Products are formed at varying amounts of enzymes. (Left) The numerical data and graph from
the initial setup, with varying amounts of enzyme (0-250 μL), with no inhibitor. (Right) The data from the
modified setup with an inhibitor whose amount of enzyme ranges from 80-60 μL. Both show the linear
relationship between the amount of enzyme and the number of products formed.

Figure 4. Products formed at varying incubation temperatures. (Left) The numerical data and graph from
the initial setup, whose incubation temperature ranges from 0-100°C., with no inhibitor. (Right) The data from
the modified setup with an inhibitor whose incubation temperature ranges from 33-66 °C. Both show that
enzyme 4 has an optimum temperature, at which it works best.

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Figure 5. Products formed at varying substrate concentrations. (Left) The numerical data and graph
from the initial setup, whose substrate concentration ranges from 0-250 mM, with no inhibitor. (Right) The
data from the modified setup with an inhibitor whose substrate concentration ranges from 160-250 mM. Both
show that maximum product yield is formed at 250 mM of a substrate and the linear relationship between
substrate concentration and the number of products formed at optimum conditions.

Discussion

pH of incubation
Enzyme activity varies with pH changes. The amino acids regulate substrate specificity and
limit enzyme activity to certain pH ranges. Enzymes have an optimum pH at which most enzymes work
best, often in slightly acidic or basic conditions. However, some enzymes are native to extremely acidic
or basic environments; thus, they are most active in these pH ranges (Voet, D., Voet, J. G., and Pratt,
C.W., 2006).
Based on the results shown in figure 1 (left), enzyme 4 shows maximum activity at 8.80, the
optimum pH. The observed downward trend, in terms of products produced, at acidic levels and extreme
alkaline conditions is due to the denaturation of the enzyme. At the extremes of pH, the tertiary structure
of the enzyme, particularly, the active site is disrupted. This explains the little to no activity observed at
pH ranges 1-6 and 11-14. However, upon modifying the pH ranges, particularly in the third modification
with pH level ranges 8-14, the result presented in figure 1 (right) reveals an optimum pH at 9.2. This
is contrary to the results in the initial setup shown in figure 1 right. Modification of parameters helps to
reveal the real optimum pH value as it stretches the ranges and emphasizes each value, producing
more precise results.

Incubation time
The longer time an enzyme spends in contact with its substrate, the more product it produces.
However, the enzyme activity would not always produce a linear function at the time of incubation. All
proteins lose their catalytic function due to denaturation. In addition, if the incubation time continues to
proceed, the enzymes will be fully saturated by the substrates over time; thus, decreasing the amount
of product yield. This was observed in the results shown in figure 2 (left), which shows a remarkable
increase in enzyme activity at 0-12 minutes of incubation. However, the product yield decreases when
it reaches 24-60 minutes of incubation.
Furthermore, in figure 2 both left and right shows the same optimum time at 12 minutes.
Nevertheless, the initial setup at the left produces more products than the modified setup at the right
due to the inhibitor B added to the modified setup.

Amount of enzyme
The amount of enzyme affects its rate of activity. As long as there are substrates, higher
enzyme concentration will increase the rate of reaction. More enzyme molecules are accessible to
process the substrate when the enzyme concentration is higher. Because of the high quantities of the
enzyme-substrate complex, the reaction has a faster initial catalytic rate, giving it a head start in the
shift toward reactant-product equilibrium (Voet, D., Voet, J. G., and Pratt, C. W., 2006). In figure 3, both
the initial and modified setups show the same optimum concentration of enzyme at 250uL. This reveals
that the higher the concentrations of enzyme, the more product it produces.

Incubation Temperature
An increase in temperature increases the rate of enzyme activity as the molecules are moving
more quickly and make contact with one another. Changing the temperature outside of the optimal
range, on the other hand, can disrupt chemical bonding inside the enzyme and modify its shape (Daniel,
R. M. et al., 2008). If the enzyme's shape changes, the active site may lose its ability to bind to the
correct substrate, slowing the reaction rate. In addition, each enzyme operates best at its optimal

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temperature, thus; any temperature change affects an enzyme's activity, and it can also lead to enzyme
denaturation (Vitolo, M., 2020).
In the experiment, it was observed that an increase in temperature also marked an increase in
the rate of enzyme activity. Based on the result shown in figure 4 left (the initial setup), the optimum
temperature falls at 60℃. However, when the setup was modified with ranges 33-66℃ shown in figure
4 (right), the results revealed a more precise optimum temperature at 66℃. In addition, enzyme activity
started to decrease as the temperature continued to increase at 80℃, which is beyond the optimum
temperature of the enzyme. This means that the enzymes are starting to modify the shape of their active
site, losing their ability to bind with the correct substrate, thus; decreasing the reaction. Hence, enzyme
4 loses its ability to bind with the correct substrate, which decreases the rate of reaction and the amount
of product yield.

Concentration of substrate
When a geometrically and electrically complementary substrate can access the active site,
enzyme activity occurs. The active residues form a temporary bond with the substrate, accelerating the
substrate's transition into a product. As a result, the higher the enzyme activity and the faster the shift
toward enzyme-product equilibrium, the more substrate-occupied active sites there are (Boyer, R.,
2006).
Based on the result in figure 5 (left and right), both the initial and modified setups show the
same optimum concentration of substrates at 250mM. Noticeably, the product yield increases when the
concentration of substrate also increases. This thus reveals that the higher the concentration of
substrates, the higher the enzyme activity as well.

Addition of Inhibitor
The inhibitor prevents the enzyme from binding to its substrate; thus, a decrease in the
enzyme’s activity would be observed (Bergtrom, G., 2018). This explains why the addition of an inhibitor
B to an enzyme 4 in the experiment leads to little to no enzymatic activity results at all. Before the
addition of an inhibitor, our initial setups had shown a high product yield, indicating a high enzyme
activity. However, the addition of inhibitor B in the modified setups drastically decreased the amount of
products yield, which indicates lesser enzyme activity. Since it was clear from the observations that the
initial setup which lacks an inhibitor has a higher enzymatic activity compared to the modified setups
with the addition of inhibitor B. Therefore, that adding inhibitors to the enzymes decreases the rate of
enzyme activity.

Conclusion

The addition of competitive inhibitor B decreased the rate of activity of the enzyme for each
condition. The ranges of each condition were modified at least three times to produce a more accurate
result in determining the optimum level. Altering pH level affects the rate of activity of the enzyme,
however, there is an optimal value that the enzyme works best, which is 9.2. Similar to pH, there is also
an optimum in the incubation of time. The number of products produced increased until around 12 min
of incubation. For the amount of enzyme and substrate concentration, it was observed that the rate of
enzyme activity increased as the amount of enzyme and substrate concentration increased. Lastly, in
the incubation temperature, an optimum was also observed. The rate of activity keeps increasing until
it reached 66 ℃, it decreased as the temperature went higher. Overall, it is important to perform
modifications for each condition to find out the effects of these conditions on the production and rate of
enzyme activity.

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References

Bergtrom, G. (2018). Basic Cell and Molecular Biology: What We Know & How We Found Out-4e.

Daniel, R. M., Danson, M. J., Eisenthal, R., Lee, C. K., & Peterson, M. E. (2008). The effect of
temperature on enzyme activity: new insights and their implications. Extremophiles, 12(1), 51-59.

Jibran (2021). Factors That Affect Enzyme Activity. Enzymology, Science. Retrieved October 8, 2021,
from https://conductscience.com/factors-that-affect-enzyme-activity/

John Wiley & Sons. Boyer, R. (2006). Concepts in Biochemistry, 3rd edition. New Jersey: John Wiley
& Sons.

The Effect of pH on Enzyme Kinetics. (2016, April 3). Retrieved October 8, 2021, from
https://chem.libretexts.org/@go/page/20287

Tomasik, P. and Horton, D. (2012). Chapter 2 - Enzymatic conversions of starch. Advances in

Carbohydrate Chemistry and Biochemistry, Academic Press, 68, 59-436.

Vitolo, M. (2020). Brief review on enzyme activity. World Journal of Pharmaceutical Research, 9(2), 60-
76

Voet, D., Voet, J. G., & Pratt, C. W. (2016). Fundamentals of biochemistry: life at the molecular level.

Appendices

pH of Incubation

Initial Setup: 1-14 No Inhibitor

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First Modification: 1-6, with Inhibitor B

Second Modification: 6-8, with Inhibitor B

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Third Modification: 8-14, with Inhibitor B

Incubation Time

Initial Setup: 1-14, No Inhibitor

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First Modification: 0-20 mins, with Inhibitor B

Second Modification: 20-40 mins, with Inhibitor B

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Third Modification: 40-60 mins, with Inhibitor B

Amount of Enzyme

Initial Setup: 0 – 250 μL, No Inhibitor

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First Modification: 0 – 80 μL, with Inhibitor B

Second Modification: 80 – 160 μL, with Inhibitor B

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Third Modification: 160 – 250 μL, with Inhibitor B

Incubation Temperature

Initial Setup: 0 – 100 ℃, No Inhibitor

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First Modification: 0 – 33 ℃, with Inhibitor B

Second Modification: 33 – 66 ℃, with Inhibitor B

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Third Modification: 66 – 100 ℃, with Inhibitor B

Concentration of Substrate

Initial Setup: 0 – 250 mM, No Inhibitor

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First Modification: 1 – 80 mM, with Inhibitor B

Second Modification: 80 – 160 mM, with Inhibitor B

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Third Modification: 160 – 250 mM, with Inhibitor B

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