Investigating Enzyme Activity in Response to Temperature and Inhibition

J35856
enzyme by changing its configuration and allowing
the sucrose to be exposed to a H2O molecule, where it
Introduction breaks down the oxygen bridge as seen in figure 2.
This allows for the enzyme to release the two
The aim of the practical was to investigate how monosaccharide proteins glucose and fructose.
the change in environment can affect the activity of
enzymes. J.B.S Haldane stated that enzymes are not
alive, they are tools used by the cell, where their
actions are similar of inorganic catalysts thus,
enzymes are also known as a biological catalyst.
Where necessary biochemical reactions within the
body have high activation energy so enzymes act as
catalysts to lower the activation energy of the
reaction; allowing for the reaction to take place. This
can be displayed with a reaction coordinate diagram
as seen in figure 1:

Figure 2: Shows the process in which sucrose is

catabolised into its two monosaccharides glucose and
fructose.

The surface of the active site is lined with amino acid

residue which binds the substrate to the enzyme
creating the enzyme-substrate transient
complex[CITATION Nel \l 2057 ].

As the Enzyme affects reaction rates, a simple

reaction can be written as follows:
Figure 1 shows a reaction coordinate diagram of
respiration.
E+ S ⇌ ES ⇌ EP ⇋ E+ P (1)

Where ‘E’, ‘S’ and ‘P’ represent the enzyme,

In this reaction, glucose is the enzyme and both
substrate and product respectively; ‘ES’ and ‘EP’ are
reactions with and without the enzyme are exergonic
reaction intermediates that represent transient
as there is a net release of free energy.
complexes.
In this practical, the enzyme sucrase catabolizes the
Method
sucrose substrate into its monosaccharides
A series of test tubes were labelled containing
varying amounts of each of the chemical
compositions shown in table 1 below.

glucose and fructose via hydrolysis, the sucrose

substrate acts upon the active site of the sucrase
Table 1: Shows the varying amount of each of the
chemicals in each test tube. Results
Tub 0.5ml of substrate Buff Aniline
e concentration er inhibitor (only
no. (mM) (ml) for 30oC) Table 2: Shows the absorbance reading for the
1 0 1 0 different test tubes at 30oC and 50oC.
2 10 0.5 0 Tu Absorbance Absorbance 0.5ml of
be @540 nm @540 nm Substrate
3 25 0.5 0 No (in 30oC (in 50oC concentratio
4 50 0.5 0 . bath) bath) n (mM)
5 90 0.5 0 1 0 0 0
6 150 0.5 0 2 0.376 0.349 10
7 300 0.5 0 3 0.666 0.551 25
8 450 0.5 0 4 1.229 0.899 50
9 450 0 0.5
5 1.698 1.18 90
6 0.841 1.198 150
The contents of each tube were then thoroughly 7 0.964 1.339 300
swirled and were all placed in the 30oC water bath. 8 0.93 1.442 450
9 0.274  - 450
Once the tubes were placed in the bath, 0.5 ml of the
enzyme were added to each test tube in a sequence as Assuming the absorbance was the velocity, mmol s -1,
such that each tube would have spent exactly 10 a Michaelis-Menten (MM) plot and Lineweaver-Burk
minutes in the water bath. (LB) can be plotted to find VMAX and KM.
Using reaction (1) the reaction can be re-written as
After 10 minutes, the reaction was brought to a stop follows:
and 1 ml of DNS solution was added to each tube in
such a way that they have 5-second intervals between
adding the DNS. When adding the DNS to the tubes
they were also removed and transferred over to an ice
bath.

The tubes were then transferred into a beaker full of Where k1 is the rate constant for the forward reaction
boiling water for 10 minutes and covered in tin foil to of E+S->ES and k-1 is the rate constant of the reverse
prevent evaporation so that the colour of the solution reaction. K2 also known as kcat is the rate constant of
can develop. the forward reaction ES->E+P.

After 10 minutes, the tubes were transferred into an The three rate constants can be combined into one
ice bath and then returned to the test racks once term, known as the Michaelis-Menten constant as
cooled. 5mls of distilled water were added to each follows:
tube.
k −1 +k cat
K M= (3)
The absorbance of each solution was recorded with a k1
bench top spectrometer at 540 nm, by using test-tube
1 as the blank.

This was all repeated again for a water bath at a

temperature of 50oC. But the test-tube solutions for
the 50oC were diluted down by 50/50 so that the
absorbance was in the range of the spectrometer.
 Using the following equation and comparing it to the
equation of a straight line:

Figure 3: Shows a Michaelis-Menten plot of the Thus, KM and VMAX can be calculated using the
results at 30oC. equations of the line in figure 4 and equation (4). The
equations for 30oC and 50oC are y = 17.667x +
0.8038 and y = 22.608x + 0.6815 respectively.

Table 4: Shows the VMAX and KM for both reactions at

different temperatures using LB plot.

Bath Half
Temperature/oC Vmax Vmax Km
1.24409 0.62204 21.9793
30 1 5 5
1.46735 0.73367 33.1738
Figure 4: Shows a Michaelis-Menten plot of the 50 1 6 8
results at 50oC.
Discussion
Using the equations of the trendlines in both figures,
KM can be calculated by rearranging for x for the y- The KM values represent the rates of reaction for the
value that is half the VMAX value. ES transient complex to E + P, therefore, the results
Table 3: Shows the VMAX and KM for both reactions at are shown in both table 3 and 4 show that the
different temperatures using MM plot. temperature does interfere with the activity of the
enzyme as the KM values have different readings.
Bath Vmax/ Half ln KM/
Temperat (mmols- Vmax/ K (mm Though there was not enough data collected to plot
ure/oC 1
) (mmols-1) m ols-1) another line to show the effect of an inhibitor,
2. comparing test tubes 8 and 9 readings from table 2,
30 1 0.5 7 14.4 the absorbance is 0.930 and 0.274 respectively. Thus,
3. inhibitors have an impact on the activity of enzymes
50 1.6 0.8 7 42.2
as there are three types of inhabitance; competitive,
noncompetitive and uncompetitive with respect to
their relationship to a substrate of the
enzyme[ CITATION Lei \l 2057 ]. Where
competitive inhibitors compete with substrates for the
binding site on an enzyme and noncompetitive
enzymes do not. The type of inhabitance also
influences the values gained to calculate VMAX and
KM, Competitive inhabitance changes Km whilst the
VMAX is constant, noncompetitive and uncompetitive
inhabitance changes both KM and VMAX[ CITATION
Nel \l 2057 ].
Figure 4: Shows a Lineweaver-Burk plot for
temperatures 30oC and 50oC.
Within industries, Sucrase is used on a large scale by fructose which can be used in foods such as creams,
the food industry, such as sweeteners as sucrase splits mints,
sucrose into its monosaccharides glucose and
marshmallows and etc; to preserve shelf lives of
these products as well as enhance the taste. They can
also be used in pharmaceuticals, as unpalatable drugs
can be flavoured so they are more palatable for
consumers such as children[ CITATION Suc \l
2057 ].
down into glucose and fructose. Therefore, studying
the activity of enzymes
Conclusion
Sucrase acts upon the lumen of the intestines where
food is digested and the sugars such as sucrose act
upon the binding site of the enzyme sucrase to break
References
Haldane, J. B. S. (1927). Possible Worlds and Other Essays. London: Chatto and Windus.

Leiberman, M., Marks, A. D., Smith, C. M., & Marks, D. B. (2007). Marks' Essential Medical Biochemistry.
Lippincott Williams & Wilkins.

Nelson, D. L., & Cox, M. M. (2013). Lehninger Principles of Biochemistry (6th ed.). W. H. Freeman.

Sucrose (Inactive Ingredient). (n.d.). Retrieved from Drugs.com:

https://www.drugs.com/inactive/sucrose-74.html
 Questions

1. The effect of an inhibitor of an enzyme was tested and the experiment gave the results below. Plot the data
as a Line Weaver Burke plot; then determine, by inspection of the graph, what type of inhibition is
involved.

[S] µM V (µmol/min) with 0.0 nM V (µmol/min) with 25 nM V (µmol/min) with 50 nM

Inhibitor Inhibitor Inhibitor

0.4 0.22 0.21 0.20

0.67 0.29 0.26 0.24

1.00 0.32 0.30 0.28

2.0 0.4 0.36 0.32

4
1/V (min/µmol)

0nM Inhibitor
3 0nM Inhibitor Trendline
25nM Inhibitor
25nM Inhibtor Trendline
2 50nM Inhibitor
50nM Inhibitor Trendline

0
0 0.5 1 1.5 2 2.5 3

1/[S] ( µM-1)

As the plot shows no crossing of the lines, it can be concluded that the inhabitance is of an
uncompetitive nature.
 2. You perform a kinetics experiment on the enzyme phosphatidylinositol synthase (PI synthase) using as
substrate radiolabeled inositol in a tracer amount mixed with unlabeled inositol.

PI Synthase

CDP-DAG + Inositol ----------------------------- Phosphatidylinositol

Using a scintillation counter, you have determined that the inositol substrate has a specific radioactivity of 100 dpm
(disintegrations per minute) / 1 nmol of inositol. You collect the following data that represent the amount of
radiolabeled phosphatidylinositol formed (data in dpm) after a 10 min reaction using 5 µM enzyme.

[S] nmol/100 µl Reaction Volume dpm Recovered in Product Formed

250 10000

500 16000

750 17500

1000 21700

i) Determine n mol product formed per min for each substrate concentration used.
The substrate nmol= [S]x100 µl reaction volume; as moles= concentration x volume.

Therefore, for 250, 500, 750 and 1000, the nmol are; 0.025nmol, 0.0500nmol, 0.0750nmol and 0.100nmol
respectively.

ii) Prepare a Lineweaver-Burk plot of the data.

0
f(x) = 0.02 x + 0
1V (min/disintergration)

0
0 0 0 0 0 0 0 0 0
1/[S] (100 µl Reaction Volume/nmol)
iii) Determine Km and Vmax for PI synthase.
Using equation (4) and the equation of the trend line the Km and V max are calculated to be 576.7 and
33333.3dpm respectively.

3. A Hanes-Woolf plot is yet another method of linearizing the data from a Michaelis-Menten enzyme. In this
type of graph, you make a plot of v against v/[S]. In this plot Vmax is the y-intercept, Vmax/Km is the x-
intercept, and Km is the negative slope. Many scientists consider this a less error-prone method than the
Lineweaver-Burke plot.

The Hanes-Woolf Plot

Using the data from question 1, plot a Hanes-Woolf plot and again calculate values for Km and Vmax.
Comment on any differences.

6 f(x) = 2.64 x + 0.97

f(x) = 2.27 x + 1.04
5
[S]/V (µMmin/µmol))

f(x) = 2 x + 1.03
4 0nM Inhibitor
Linear (0nM Inhibitor)
3 25nM Inhibitor
Linear (25nM Inhibitor)
2 50nM Inhibitor
Linear (50nM Inhibitor)
1

0
0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2
[S] (µM)

With the same principle as equation (4) the Hanes-Woolf plot can be compared to the equation of a
straight line as follows:
 There KM and VMAX can be calculated through the equation of the lines as follows:

Inhibitor/nM VMAX KM
0 0.501 0.516
25 0.441 0.456
50 0.379 0.367

As there isn’t much of a change between the VMAX and KM values of the three inhibitors, the reaction can
still be safely assumed as an uncompetitive inhabitance.

4. Two different enzymes are capable of catalysing the reaction A -> B. The enzymes have the same Vmax, but
different Km values for the substrate A. Enzyme X has a Km of 2.0 µM, enzyme Y has a Km of 0.5 µM. The
plot below shows the kinetics of two reactions carried out with the same concentration of each enzyme and
with [A] 1.0 µM. Determine which curve corresponds to each enzyme, explain the rationale underpinning
your selection.

2
A

It can be assumed that the rate of formation of product and concentration of the substrate are in a linear
relationship; as X has a KM value of 2.0 µM whilst Y is lower at 0.5µM, the rate of reaction of X must be
bigger than Y therefore, line 1 is for the enzyme X and line 2 is for the enzyme Y.