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THE REVERSIBILITY OF ENZYME CATALYSED

REACTIONS IN GLYCOLYSIS
Name
Student ID
Course Code
Date

: Kelvin
: 15WAR03018I
: RBS-2 Group 2
: 28 June 2016

Objective
1. To study the reversibility of an enzyme catalysed reaction in glycolysis
2. To determine the equilibrium constant of the reaction at different temperature
3. To construct a Gibb’s graph and observe the relationship with temperature
Materials
 Beaker
 Test tubes
 Test tubes rack
 Graduating cylinder
 Pipette
 Water bath
 Spectrophotometer
 2 mM glucose-6-phosphate
 2 mM fructose-6-phosphate
 Phosphoglucose isomerase
 20% metaphosphoric acid
 0.2 M tris-HCl buffers pH 8.0
 1% resorcinol in 9% ethanol
 28% HCl
Procedure
Part A
2 test tubes were prepared and labelled with G and F indicating glucose and fructose. 8.0 ml
of glucose-6-phosphate was pipetted into test tube G. 8.0 ml of fructose-6-phosphate was
pipetted into test tube F. Both test tubes were sat on a test tube rack at room temperature. 2.0
ml of phosphoglucose isomerase was then added into each test tube and mixed well. The time
recording was started during the addition. 7 sets of test tubes were prepared and labelled with
G and F according to the time intervals of sample extraction. 1.0 ml of sample was taken
from each tube at 5, 10, 20, 30, 45, 60 and 80 minute intervals. 1.0 ml of 20%
metaphosphoric acid was then added into each sample. All the samples excluding the first 2
test tubes were assayed using the Seliwanoff Assay.

Part B
2 test tubes were prepared and labelled with G and F indicating glucose and fructose. 0.5 ml
of glucose-6-phosphate was pipetted into the test tube G. 0.5 ml of enzyme was then added
into the same test tube. 0.5 ml of fructose-6-phosphate was pipetted into test tube F. 0.5 ml of
enzyme was then added into the same test tube. Both test tubes were left in a water bath at
55°C for 80 minute. 1.0 ml of 20% metaphosphoric acid was then added into each test tube
and assayed for fructose-6-phosphate using the Seliwanoff Assay.
Part C
7 test tubes were prepared and labelled with number 1 to 7. The sample for each test tube was
prepared following the table below:
Tube #

2 mM
2 mM
0.2 M Tris20%
% FructoseGlucose-6Fructose-6- HCl buffers Metaphosphori 6-Phosphate
Phosphate
Phosphate
pH 8.0
c acid
(ml)
(ml)
#1
0
0.5
0.5
1.0
100
#2
0.1
0.4
0.5
1.0
80
#3
0.2
0.3
0.5
1.0
60
#4
0.3
0.2
0.5
1.0
40
#5
0.4
0.1
0.5
1.0
20
#6
0.5
0
0.5
1.0
0
#7
0
0
0.5
1.0
0
All the 7 test tubes were then assayed using the Seliwanoff Assay for fructose-6-phosphate.
Seliwanoff Assay
2 ml of 1% resorcinol in 95% ethanol was added into each sample. 6 ml of 28% HCl was then
added into each sample. The samples were mixed well and left in a water bath at 80°C for 8minute sharp. The samples were then immediately cooled by placing them in cold water.
Each sample was poured into the cuvette to proceed with absorbance reading at 436 nm.
Spectrophotometer was calibrated using blank solution in test tube 7.
Results
Part A
Table 1
Time intervals (min)
5
10
20
30
45
60
80

Glucose-6-Phosphate
Absorbance
0.073
0.196
0.242
0.355
0.376
0.412
0.415

%fructose-6-phosphate
5.157
29.275
38.294
60.451
64.569
71.627
72.216

Sample calculation of %fructose-6-phosphate using the standard curve equation

At 5 minute intervals
Abs = y = 0.073
y = 0.0051x + 0.0467
0.073 = 0.0051x + 0.0467
x = 5.157
At 10 minute intervals
Abs = y = 0.196
y = 0.0051x + 0.0467
0.196 = 0.0051x + 0.0467
x = 29.275
The same method is used to calculate the following %fructose-6-phosphate.

From the graph above, the time where the concentration in equilibrium is 80 minute.
Therefore, the Ceq value for %fructose-6-phosphate is 72.216.
Table 2
Fructose-6-Phosphate
Absorbance
%fructose-6%glucose-6phosphate
phosphate
5
0.534
95.549
4.451
10
0.498
88.490
11.51
20
0.445
78.098
21.902
30
0.426
74.373
25.627
45
0.411
71.431
28.569
60
0.402
69.667
30.333
80
0.399
69.078
30.922
Sample calculation of %fructose-6-phosphate using the standard curve equation
Time intervals (min)

At 5 minute intervals
Abs = y = 0.534
y = 0.0051x + 0.0467
0.534 = 0.0051x + 0.0467
x = 95.549
At 10 minute intervals
Abs = y = 0.498
y = 0.0051x + 0.0467
0.498 = 0.0051x + 0.0467
x = 88.490
The same method is used to calculate the following %fructose-6-phosphate. Then, by
deducting the %fructose-6-phosphate results from 100%, %glucose-6-phosphate can be
determined. For example, at 5 minute intervals, 100% - 95.549% = 4.451%.

From the graph above, the time where the concentration in equilibrium is 80 minute.
Therefore, the Ceq value for %glucose-6-phosphate is 30.922.
Thus, we can proceed to the calculation of Keq as follow,
Ceq ( Fructose−6−Phosphate )
Keq( RT ° ) =
Ceq ( Glucose−6−Phosphate )
=

72.216
30.922

= 2.335

Calculation of ∆G
Assume room temperature at 27°C in this experiment.
Sample calculation of ∆G
∆G
= −2.303 × R × T × log ( K eq )
∆G

= −2.303 ×1.987 × 300× log ( 2.335 )
= −505.591 cal mol

−1

Part B
Test #

Absorbance (glucose-6Absorbance (fructose-6phosphate)
phosphate)
#1
0.143
0.249
#2
0.146
0.253
#3
0.140
0.250
3 test were done to obtain the average of the reading; the aim is to improve accuracy.
Glucose-6-Phosphate
Average Abs = 0.143
Abs = y = 0.143
y = 0.0051x + 0.0467
0.143 = 0.0051x + 0.0467
x = 18.882
The final Ceq for %fructose-6-phosphate is 18.882.
Fructose-6-Phosphate
Average Abs = 0.251
Abs = y = 0.251
y = 0.0051x + 0.0467
0.251 = 0.0051x + 0.0467
x = 40.059
The %fructose-6-phosphate is 40.059. Therefore, the Ceq for %glucose-6-phosphate is 100%
- 40.059% = 59.941.
Calculation of Keq
18.882
Keq(55 ° C )
= 59.941
= 0.315
Calculation of ∆G at 55°C
∆G
= −2.303 × R × T × log ( K eq )
= −2.303 ×1.987 × 328× log ( 0.315 )

−1

= 753.010 cal mol

Calculation of ∆S using simultaneous equation method
∆H – (300) ∆S = -505.591
∆H – (328) ∆S = 753.010 (-)
28 ∆S = -1258.601
∆S
= -44.95 cal mol-1 K-1
Calculation of ∆H
Keq 2
−∆ H
1
1

log
=
2.303 × R T 2 T 1
Keq1

(

log

0.315
2.335

∆H

=

)

−∆ H
1
1

2.303 ×1.987 328 300

(

)

0.315
24600
= −log 2.335 × 2.303 ×1.987 ×− 7
−1
= −13991 cal mol

Graph for ∆G

From the linear equation obtained from the graph, we can prove that ∆S = -slope of the linear
equation which is -44.95. As for the intersect which is -13991 is equal to the ∆H calculated.
Part C
Table

Tube#
#1
#2
#3
#4
#5
#6

%fructose-6-phosphate
100
80
60
40
20
0

Absorbance
0.56
0.47
0.32
0.27
0.13
0.06

The linear equation obtained from the graph is y = 0.0051x + 0.0467
Discussion
Glycolysis uses enzyme to catalyse the reaction in a reversible manner. In this case,
we were using phosphoglucose isomerase which convert glucose-6-phosphate into fructose6-phosphate. This reaction catalysed by the enzyme is reversible, meaning that the enzyme
catalysed both forward and back reaction. So, the rates of reactions will become equal or in a
state of equilibrium in terms of concentration. Thus, producing an equation such as
[ C ][ D ]
K eq is equilibrium constant where it will not be
K eq =
[ A ] [ B ] in A + B ↔ C + D.
affected in a presence of enzyme but depend on temperature. [1] Higher temperature
increases the rate of reaction in glycolysis conversion. Higher temperature favour reactant,
meaning that the equilibrium constant will be lower. Other than that, a test or assay called
Seliwanoff Assay was done to determine the fructose-6-phosphate. Seliwanoff Assay able to
distinguishes between aldose and ketose sugars. It able to differentiate them because of their
ketone/aldehyde group. In this test, a resorcinol reagent was used to react with the dehydrated
ketose to give pale coloured compound. [2]
In this experiment, we obtained data from the reading of absorbance in
spectrophotometer. From the absorbance obtained, we are able to construct a standard curve
in part C to get the linear equation. All the sample in section C experiment was used to
determine the % fructose-6-phosphate which will be constructed into a standard curve. The

equation obtained from the graph will be used for all the calculation of percent sugars in the
solution. The linear equation y = 0.0051x + 0.0467 is used to sub in the absorbance which
results in the value of x. The x value determined the percent concentration of sugars. For the
first set of data, which is glucose-6-phosphate reading, % fructose-6-phosphate is needed.
This is because we wanted to know how many percent of fructose-6-phosphate is formed
from the conversion of glucose-6-phosphate in the reaction. By using the % fructose-6phosphate with the time intervals for each intake of the sugars, a graph of glucose-6phosphate can be constructed. In this graph, we are going to determine the equilibrium
concentration of the reaction for fructose-6-phosphate. It is when the data in the graph
becoming constant and hence reached the equilibrium. The same goes to the second set of
data, where fructose-6-phosphate is the sample. Using the same method, we are able to
calculate the % fructose-6-phosphate. However, in this part, we need to know how many
percent the fructose-6-phosphate has been converted to glucose-6-phosphate. Thus, we have
to calculate the % glucose-6-phosphate by deducting 100% with % fructose-6-phosphate.
With this, we can construct another graph to determine the constant data and hence be able to
determine the equilibrium concentration of glucose-6-phosphate. These 2 equilibrium
concentration, C eq , obtained from glucose and fructose will be used to calculate the value
of equilibrium constant,

K eq .

Next, after determining the equilibrium constant at room temperature, we can proceed
to the calculation of ∆G. the formula to calculate ∆G is provided in the laboratory manual,
which is ∆ G=−2.303 × R ×T ×log ( K eq ) . From this formula, we obtained -505.591 as the
∆G at room temperature. Then, we will proceed to part B where we obtained 3 sets of data for
glucose and fructose at 55°C. In this part, we were dealing with the sugars under higher
temperature which later is used to compare. We repeated 3 times of the experiment in order to
obtain a constant data by calculating the average from the 3 sets of data. Same methodology
of calculation is used in this part where the absorbance reading substituted into the y and
determine the x which is percent concentration of the sugars. No graph needed in this section
as we are able to obtain a constant data for equilibrium concentration. Then, the constant
equilibrium in this part is also determined. Hence, the ∆G value is obtained. With the
presence of both ∆G at room temperature and 55°C, entropy change, ∆S, can be determined.
From the equation ∆G = ∆H - T∆S, entropy change is determined by substituting the data in a
simultaneous equation to eliminate enthalpy change which is still an unknown. Enthalpy
change can be determined by comparing the equilibrium constant from both temperature,
room temperature and 55°C. To show and prove that the enthalpy change, entropy change and
free energy change is proportional to the temperature, a Gibb’s graph is constructed at
different temperature. From the graph, it is positive to assume that temperature affects the
reaction.
The results obtained from the experiment is positive results. However, there are some
deviation found in the results. It is due to some practical errors during the experiment. One of
suspected error is when transferring the chemical, graduated cylinder was used instead of
pipette. Measuring cylinder is not accurate compare to graduated pipette, it may affect the
outcome of the experiment. Another error is suspected to be an error in pipetting technique
where transferring different chemical using a single pipette without rinsing it. It may have
minor effect on the concentration of the sample. Other than that, during the absorbance

reading procedure, cuvette was placed wrongly or the clear side of the cuvette accidentally
touched causes the visible light fail to pass through.
Conclusion
The objective of this experiment is to study the reversibility reaction in glycolysis by
the help of equilibrium constant and Gibb’s graph. From the results, equilibrium constant tells
us that glycolysis catalysed by an enzyme is a reversible reaction where after some time, the
net movement of reaction slowed down and stop. It is where the equilibrium constant
reached. Lastly, the effect of temperature on the reaction can be observed through the changes
in Gibb’s graph.

References
1. The laboratory Manual. (2016). The Reversibility of Enzyme Catalysed Reactions in
Glycolysis. [Accessed 1 Jul. 2016].
2. The Wall of Biochemistry. (2012). Carbohydrate’s Test. [online] Available at:
https://fulltimes.wordpress.com/carbohydrates-test/ [Accessed 1 Jul. 2016].
3. Clark, J. (2013). equilibrium constants and changing conditions. [online]
Chemguide.co.uk. Available at:
http://www.chemguide.co.uk/physical/equilibria/change.html [Accessed 1 Jul. 2016].
4. Pulido, K. and Chappell, C. (2013). Effect of Temperature on Equilibrium - Chemwiki.

[online] Chemwiki.ucdavis.edu. Available at:
http://chemwiki.ucdavis.edu/Core/Physical_Chemistry/Equilibria/Le_Chatelier's_Principl
e/Effect_Of_Temperature_On_Equilibrium_Composition [Accessed 1 Jul. 2016].