Factors Influencing Enzyme-catalyzed Reaction Rate

Annia. I Niusulu

BIO-2051

06 November 2017

Hawai’i Pacific University

ENZYME-CATALYZED REACTIONS 2

Factors Influencing Enzyme-catalyzed Reaction Rate

I. Introduction

Enzymes are protein molecules fundamental for all living organisms, an essential power

that keeps our bodies functioning day after day. Enzymes assist chemical reactions by acting as

catalysts lowering the activation energy required for reactions to start. They also control cellular

effectiveness by synthesizing biological molecules. Enzymes bind with molecules that are called

substrates with which they form a “lock and key” type of structure. Understanding enzymatic

activity and the factors that can affect it are vital in research. In fact, research has led to the

addition of enzymes, like a-amylase, into wheat flour to provide better quality bread among other

baked goods (Kara 2005).

It is important to acknowledge the impact certain factors can have in enzyme-catalyzed

reactions. Physical influential factors like temperature and pH, and molecular factors like added

enzyme concentration and substrate concentration were measured in a lab experiment in order to

understand any effect in the rate at which the enzyme-catalyzed reactions took place. The

purpose of these experiments was to understand the environmental and molecular influences on

enzyme-catalyzed reaction rates. The enzyme Catecholase, which is an extract made from

potatoes, was added to the substrate Catechol (a colorless substance), for the formation of the

product Benzoquinone (reddish brown substance). Color absorbance and product formation have

a direct correlation, a relationship vital for the experiments conducted. Absorbance was measured

using an instrument called a Spectrophotometer (Spec 20D+). Spectrophotometers measure the

intensity of light absorbed or detected after its passage throughout a solution; therefore, given the

relationship between color absorbance and product formation, the rate of reaction was evaluated.
ENZYME-CATALYZED REACTIONS 3

In the first two experiments, enzyme concentration and substrate concentration were

altered in order to determine their influences on product production. The increase in enzyme

concentration was expected to have a direct proportional effect on the increase in reaction rate;

given that all other factors remained constant, an increase in enzyme availability would expedite

a faster changeover from substrate to product. For this experiment, it was predicted that the rate

of reaction would increase if concentration of either reactants were to be increased. In the case of

added substrate concentration, it was anticipated that the rate of the enzyme catalyzed reaction

would increase as substrate concentration increased, but it would slow down at a certain point

where the enzymes became saturated with substrate and the reaction would reach its maximum

rate and product formation. For this experiment, a rise in the reaction rate with the increase of

substrate concentration was predicted as well.

Furthermore, physical factors such as temperature and pH were measured. Temperature

can alter the shape of the enzyme molecule at its active site and possibly prevent it from

functioning properly. In this experiment, reaction rates were expected to increase as the

temperature rose between 30 0C and 40 0C. It was predicted that the rate of reaction would

decrease if the reactions were exposed to temperatures past 50 0C due to the enzyme becoming

denatured by the heat, inhibiting its function as a catalyst. Lastly, pH tests were conducted to

determine the effects different conditions would have on enzymes. A hypothesis was made, that

a decrease in reaction rates would be observed if enzymes were exposed to acidic or basic

environment. This is because these circumstances can cause enzymes to become deformed,

losing their shape, making it difficult for the substrate to bind at the active site. It was predicted

that enzymes would perform their best at around a neutral pH 7.

ENZYME-CATALYZED REACTIONS 4

II. Methods

Throughout the experiments, a test tube containing 0.5 ml of enzyme and 5.5 ml of water

was used as a blank, controlled variable, to zero the Spec 20D+. The Spec 20D+ was zeroed,

utilizing the blank tube, every time after each experimental test tube was measured. Initially, the

wavelength knob on the Spec 20D+ was set to 540 nm. Using the left knob, the 0 % T knob was

set to zero. The outside of the blank tube was cleaned with Kim-wipe and inserted into the

sample holder of the Spec 20D+ with the vertical mark facing to the front. Operating the right

knob, while the blank was inside the Spec 20D+, the absorbance reading was set to zero.

Enzyme concentration

The experiment consisted of four test tubes, labeled 1-4. The concentration of the

substrate Catechol was kept constant in all the test tubes adding 1.0 ml to each; also as a control

measure, the enzyme was added last to each test tube to prevent the starting of the reaction. Tube

1 contained 5.0 ml of water and 1.0 ml of the substrate Catechol; no enzyme was added to this

tube. For tube 2, 4.5 ml of water was added as well as 1.0 ml of substrate and 0.5 ml of the

potato enzyme extract. Tube 3 contained 4.0 ml of water, 1.0 ml of substrate and 1.0 ml of

enzyme, and tube 4 was filled with 3.5 ml of water, 1.0 ml of substrate and 1.5 ml of enzyme.

After solutions were added each test tube contained 6.0 ml of total volume. Each was covered

with para-film paper in order to keep substances from leaking out. The reaction time was

staggered in order to keep the enzyme from reacting and the reaction from starting. The timer

was set for three minutes, with each tube being inverted at every 1 minute interval to keep the

reaction mixed.
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Substrate concentration

For this experiment, several test tubes were labeled 1-6, where concentration of the

enzyme was kept constant in all, adding 3.0 ml to each. Also, as a control measure, the enzyme

was added last to each test tube to prevent the starting of the reaction. Tube 1 contained 4.0 ml of

water and 3.0 ml of enzyme, no substrate was added to this tube. For tube 2, 3.9 ml of water was

added as well as 0.1 ml of substrate and 3.0 ml of the potato enzyme extract. Tube 3 contained

3.8 ml of water, 0.2 ml of substrate and 3.0 ml of enzyme. Tube 4 was filled with 3.6 ml of water,

0.4 ml of substrate and 3.0 ml of enzyme. Tube 5 had 3.2 ml of water, 0.8 ml of substrate and 3.0

ml of enzyme. Lastly, tube 6 had 2.4 ml of water, 1.6 ml of substrate and 3.0 ml of enzyme. The

timer was set for three minutes, inverting each tube at every 1 minute interval to keep the

reaction mixed. The reaction time was staggered in order to keep the enzyme from reacting and

the reaction from star. After all solutions were added, each test tube contained 7.0 ml of total

volume and each was covered with para-film paper in order to prevent leakage when being

mixed.

Temperature

For this experiment four clean test tubes were used, each containing a mixture of 1 ml of

Catechol substrate and 4 ml of water. Tube 1 was placed in ice water at approximately 00C. Tube

2 was left at a room temperature of approximately 220C. Tube 3 was placed in a warm water bath

at approximately 500C and tube 4 was placed in a boiling water bath at approximately 1000C.

Each tube incubated at their respective temperature for 5 minutes. After 5 minutes, 1 ml of

enzyme Catecholase was added to each test tube. Tubes 1-3, were covered with para-film and
ENZYME-CATALYZED REACTIONS 6

inverted to mix, while tube 4 was stirred carefully to avoid injuries. The tubes were incubated for

another 5 minutes; once done incubating, the temperature on each respective test tube was taken.

After 10 minutes, each tube was inserted into the Spec 20D+ and the absorbance reading was

recorded

pH

Procedures for pH testing involved five clean test tubes labeled, pH4, pH6, pH7, pH8,

and pH10. Several buffers were used in this experiment to create the various pH environments.

Initially, 4 ml of the appropriate buffer was added to each tube and mixed with 1 ml of the potato

enzyme. The test tubes were covered with para-film and inverted every 1minute to keep the

content mixed for a duration of 3 minutes. After zeroing the Spec 20D+ with a blank test tube, the

absorbance of each solution was recorded (Table 2).

III. Results

Enzyme concentration

The results indicated that enzyme concentration is directly proportional to absorbance,

affecting product formation and reaction rate. Data collected supports the hypothesis and is

consistent with the prediction, demonstrating a steady increase in absorbance, as enzyme

concentration increased to 1.5 ml so did the absorbance increase to 0.494 au (Figure 1).

Substrate concentration

In this experiment, the results did not support the hypothesis. It was hypothesized that the

rate of reaction would increase with increased substrate concentration, but that, at one point, the

rate of reaction would be constant when enzymes reached saturation. Data regarding correlation

between substrate concentration and absorbance displayed an inconsistent linear curve (Figure
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2). A decrease in absorbance was observed in the second trial when concentration was increased

from 0 ml to 0.1 ml. Subsequently, there was an increase in the third trial, containing 0.2 ml,

reaching 0.546 au. Furthermore, absorbance decreased again to 0.432 au with trial 4, which

contained 0.5 ml of mixture. The linear curve slightly increased to 0.436 with trial 5 which

contained 0.8 ml of mixture. Lastly, trial 6 demonstrated the highest absorbance detected,

reaching 0.756 au (Figure 2). The point of saturation was never reached in this experiment.

Temperature

In this test, reaction rates were expected to decrease at a higher temperature due to the

enzyme becoming denatured by the heat, inhibiting its function to catalyze the reaction. The

hypothesis was supported. Absorbance increased linearly as temperatures were in the minimum

ranges, between 200C to 400C (Figure 3). The prediction was also supported; there was a drop to

0.134 au once temperature passed 500C (Figure 3).

pH

The results of this experiment supported the hypothesis, as it was predicted. The graph

showed low absorbance levels around pH 4, 5 and 6. A peak was observed on pH 7 where

absorbance was recorded at 1.07 au (Figure 4). A decrease in absorbance was detected when

exposed to pH conditions past pH 7. Absorbance decreased to 0.281 when exposed to pH 10

(Figure 4).
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IV. Discussion

The experiments set out to test the impact certain factors can have in enzyme catalyzed

reaction rates. The research tested the possible effects different concentrations would have in

reaction rates. An increase in product formation was observed when the reaction was exposed to

higher enzyme concentration. The experiment indicated that enzyme concentration and reaction

rate were directly proportional. The outcome was expected, since enzymes have a tendency to

keep working and not get consumed by the reaction as long as there is a vast supply of substrate

available to continue the reaction going. If instead, substrate supply stopped, enzyme activity

would decay, as well as reaction rate, which was not the case in this experiment. In the second

experiment, the results of increased substrate concentration appeared to be inconsistent and

formulated erroneous data. It was expected that, with increased substrate concentration, enzyme

activity would increase and, so therefore, would product formation; this would be until enzyme

activity would reach plateau and a constant line would be displayed in the graph. This constant

would be indication that, at this point, any added concentration would have no effect on enzyme

activity, since the enzymes became saturated. The experiment did not support the hypothesis.

There were several factors that could have caused the erroneous outcome, such as, human error

when recording the time, adding incorrect concentrations of enzyme or substrate, or when

zeroing the Spec 2D+.

Temperature can alter the shape of the enzyme molecule at its active site and possibly

prevent it from functioning properly. High temperatures can also break hydrogen bonds,
ENZYME-CATALYZED REACTIONS 9

denaturing the shape of the enzyme. The results from the experiment conducted, implied that

enzyme activity was at its best in an optimum temperature, ranging from 20 0C to 40 0C; as

temperature increased so did the rate of reaction (Figure 3). It was also observed that, when

exposed to temperatures higher than approximately 50 0C, product formation started falling,

indicating a decrease in reaction rate due to the denaturing of enzymes when exposed to high

temperature. The pH experiment resulted as expected, displaying an energetic increase in

enzyme activity while exposed to pH levels between 4-7 (Figure 4). Also, as it was predicted,

when exposed to acidic levels where pH is lower than 7, or basic levels when pH is greater than

7, there was a decay in product formation and, the reaction rate dropped. This effect can be due

to the nature of enzymes in which they can become denatured when pH levels are too basic or

acidic. The tertiary structure of enzymes that is held together by ionic bonds can be disrupted and

lose its shape when exposed to low or high pH levels, preventing the enzymes from binding

(chem.com 2017).

In conclusion, the experiments provided useful insights on the effects certain factors

can have on enzyme-catalyzed reactions. Enzymes are proteins fundamental for everyday life. It

is imperative that we understand how enzymes work and the factors that can potentially harm

their functioning. It is important that enzymes function properly and support cellular functions.

Without the enzyme’s ability to act as catalysts, lowering the activation energy, reactions would

not be able to happen fast enough to support human life. The results offered valuable

understanding in the effects factors like temperature and pH have on enzyme-catalyzed reaction

rates; it was visible that reaction rates would slow down when exposed to high temperature

levels and, acidic or basic conditions, where pH was lower or higher than pH 7. Moreover, tests

conducted to determine the effects of molecular factors, like added enzyme concentration,
ENZYME-CATALYZED REACTIONS 10

provided useful results reflecting a direct correlation between enzyme concentration and reaction

rates. With the exception of the substrate concentration experiment, which did not support the

hypothesis and exhibited erroneous information, it is safe to deem the experiment a successful

tool in aiding students to understand the powerful world of enzymes.

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References

Kara, M., Sivri, D., & Köksel, H. (2005). Effects of high protease-activity flours and commercial

proteases on cookie quality. Food Research International, 38(5), 479-486.

The Effect of Changing Conditions on Enzyme Catalysis. Retrieved November 06, 2017, from

https://chem.libretexts.org/Core/Biological_Chemistry/Catalysts/The_Effect_of_Changin

g_Conditions_on_Enzyme_Catalysis
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Figures

0.6

0.5

0.4
Absorbance (au)

0.3

0.2

0.1

-0.1
0 0.05 0.1 0.15 0.2 0.25 0.3

Enzyme concentration (mL)

Figure #1. Absorbance vs. Enzyme concentration (mL). Proportional correlations

between enzyme concentration, absorbance and product formation.

0.8

0.7

0.6

0.5
Absorbance (au)

0.4

0.3

0.2

0.1

0
0 0.05 0.1 0.15 0.2 0.25
Substrate Concentration (mL)

Figure #2. Absorbance vs. Substrate Concentration (mL). Effect of substrate

concentration on product absorbance.

ENZYME-CATALYZED REACTIONS 13

0.6

0.5

0.4
Absorbance (au)

0.3

0.2

0.1

0
0 20 40 60 80 100
Temperature ( ºC)

Figure #3. Absorbance vs. Temperature (ºC). Effects of different temperature on

absorbance.

1.2

1
Absorbance (au)

0.8

0.6

0.4

0.2

0
4 5 6 7 8 9 10
pH

Figure #4. Absorbance vs. pH values. Effects different pH values have on absorbance.