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RND For Exer 4 Enzyme Kinetics-Kim
RND For Exer 4 Enzyme Kinetics-Kim
Exercise No. 4
ENZYME KINETICS
Groupmate:
Joel Amado
Giorgia Escalante
CJ Carlos
Pamela Macalagay
However, the concentration of enzymes cannot be easily determined since its purity is
unknown; thus the amount of enzyme present in a sample cannot be expressed in common
concentration unit. The amount of enzyme is described in terms of its activity that is
measured by the amount of substrate consumed or product formed during the reaction.
Usually, Unit is the commonly used quantity, sometimes referred as International Unit or
Enzyme Unit (Nelson and Cox, 2008).
In this exercise, the enzymatic activity of invertase was determined using the Nelson’s
method for analysis of reducing sugar. This enzyme catalyzes the hydrolysis of sucrose to
fructose and glucose.
Reaction 4.3.
Nelson’s method involves heating the sugar with alkaline cupric reagent (Nelson’s
reagent) that results in the formation of cuprous oxide, a rust colored precipitate. In the
experiment, glucose and fructose reduced cupric copper to cuprous copper. Arsenomolybdic
acid is then added to the solution which is quantitatively reduced to arsenomolybdous acid
by cuprous ion producing an intense blue color. The intensity of the blue color depends on
the amount of glucose and fructose formed. Therefore, higher intensity of the blue color
indicates that more sucrose were hydrolyzed resulting to a higher amount of reducing sugars
produced, and the higher the enzymatic activity. Also, solution with high enzymatic activities
gave a higher absorbance reading since more species were present to absorb light.
𝛥
Reducing sugar + Cu2+→ Cu2O (rust colored) Reaction 4.4.
Cu2O + arsenomolybdic acid → arsenomolybdous acid (blue) + Cu2+ Reaction 4.5.
Table 4.1. Data on the determination of the calibration curve for the standard glucose.
µmol Corrected
Test tube number glucose/mL Absorbance Absorbance
0.0 0
1 0.025
0.1 0.101
2 0.126
0.2 0.164
3 0.189
0.4 0.354
4 0.379
0.8 0.638
5 0.663
1.2 0.951
6 0.976
1.6 1.319
7 1.344
2.0 1.569
8 1.594
2
Absorbance
1.5
1
y = 0.7898x + 0.015
0.5 R² = 0.9986
0
0 0.5 1 1.5 2 2.5
µmol glucose/mL
Glucose standards were made in order to produce the standard curve prior to the
analyses of enzyme activity. Eight test tubes were prepared with varying concentrations of
glucose solution. Nelson’s method was then used to determine the amount of glucose
present in each solution. The absorbance of the solutions at 510 nm was shown in Table 4.1
while the standard curve is shown in Figure 4.1. The linear curve obtained was used in the
determination of concentrations of reducing sugars in the succeeding analyses with their
corresponding absorbance. The slope of the curved generated by the standard curve was
0.7898 with a y-intercept of equal to 0.015. The resulting r2 value was equal to 0.9986.
Based on the data obtained, the absorbance is directly proportional to concentration since
the absorbance reading increases as glucose concentration increases and it shows a strong
linear relationship due to the value of r2.
To determine the effect of incubation time, 10 test tubes were prepared containing
equal amounts of 0.1M acetate buffer at pH 4.50, 50mM sucrose solution, distilled water,
and invertase. The test tubes were subjected to different incubation time except for the test
tubes 1 and 10 which serve as blank and control, respectively. Sucrose serves as substrate
for the enzyme invertase. The pH was set at 4.50 since this is the optimal pH for the
invertase. The acetate buffer was added in order to prevent drastic changes in pH that may
cause deactivation or denaturation of the enzyme. Upon changes on the pH, the enzyme
might possible to lose its activity. After completing the said incubation time for each test
tube, the reaction was continued upon the addition of Nelson’s reagent and Nelson’s method
was used to determine the amount of reducing sugar produced from each incubation time.
Table 4.2. Data on the effect of incubation time on the enzyme activity.
Corrected Reducing
Incubation Absorbance Sugar
Test tube number TIme Absorbance (µmol/mL)
0 0 0
1 0.010
2 0.025 0.0126
2 0.035
4 0.048 0.0418
3 0.058
6 0.072 0.0721
4 0.082
8 0.113 0.124
5 0.123
10 0.128 0.143
6 0.138
12 0.157 0.180
7 0.167
15 0.158 0.181
8 0.168
20 0.162 0.186
9 0.172
------- 0.163 0.187
10 0.173
0.2
0.15
(µmol/mL)
0.1
0.05
0
0 5 10 15 20 25
Time (min.)
Figure 4.2. The plot for the effect of the incubation time on the enzyme activity.
The effect of incubation time on product formation was shown in Table 4.2. and the
concentration of the reducing sugar was plotted against the incubation time as shown in
Figure 3.3. As seen on the graph, as incubation time was increased, there was also an
increase in the formation of the product. This happens because a longer incubation time
means more substrate was consumed or able to react with the enzyme, hence more
products formed. Though, the rate of formation of product is not a simple linear function of
the time of incubation. As seen on figure 4.2, there is a leveling off of the curved observed in
the graph since eventually a time is reached wherein there will be no net change in the
concentration of substrate and product. The enzyme is still actively converting substrate into
product and vice versa during this time, but equilibrium has been achieved (Berg et al.,
2012). With the data, the leveling off of the curved was observed.
In an enzyme catalyzed reaction, the reaction starts at a high rate and slows down
over time at the presence of limited substrate. This may be due to different factors such as
decrease in substrate concentration, denaturation of enzyme, and product inhibition of the
enzyme. When the experimental rate is still linear, the fixed-time assay can be improved by
changing the time of measurement (Boyer, 2012).
0.2
0.1
0.05
0
0 2 4 6 8 10 12
-0.05
Time (min.)
The linear portion of the graph in Figure 4.3 was considered and plotted in Figure 4.4
in order to obtain the activity of the invertase. The slope of the line in figure 4.4 indicates the
velocity reaction designated as Vo. Hence, the initial velocity of the invertase was 0.015
µmol/min. It is possible to express invertase activity in terms of µmol sucrose utilized/min
since one International unit (I.U.) is defined as that catalyzing the 1 µmol of substrate
(sucrose) or the formation of 1 µmol product (reducing sugars) per minute.
The concentration of substrate is a key factor affecting the rate of enzyme catalyzed
reaction. The effect of substrate concentration to enzyme activity was determined by
preparing 11 test tubes containing varying concentrations of sucrose solution together with
0.1M acetate buffer at pH 4.50. Invertase was added only to test tubes 1 to 8 since test
tubes 9 to 11 were supposed to be used to correct for the non-enzymatic sucrose hydrolysis.
The test tubes were incubated for 5 minutes. Afterwards, Nelson’s reagent was added to
stop the reaction. The Nelson’s method was again used to determine the amount of reducing
sugars formed.
Table 4.3. Data on the effect of substrate concentration on the enzyme activity.
[Sucros Corrected [S] Vo 1/[S] 1/Vo
e] Absorbanc Absorbanc µmol/ µmol/min mL/µm min-
Test tube mM e e ml -mL ol mL/µm
number ol
1 0 0.000 0 0 0 0 0
2 10 0.118 0.103 0.111 0.022 9.009 45.455
3 20 0.288 0.257 0.306 0.061 3.268 16.393
4 30 0.392 0.345 0.418 0.084 2.392 11.905
5 40 0.582 0.519 0.638 0.128 1.567 7.813
6 60 0.835 0.740 0.918 0.184 1.089 5.435
7 80 0.902 0.774 0.961 0.192 1.041 5.208
8 100 1.034 0.874 1.088 0.218 0.919 4.587
9 10 0.015 --- --- --- --- ---
10 40 0.063 --- --- --- --- ---
11 100 0.160 --- --- --- --- ---
Vmax, min- -13.715
mL/µmol
KM -69.251
60
y = 5.0493x - 0.0724
40
R² = 1
1/Vo
20
0
0 2 4 6 8 10
-20
1/[s]
Figure 4.5. Plot on the effect of the substrate concentration using Lineweaver-Burk plot for
data analysis.
0.25
y = 0.2003x - 6E-05
0.2
R² = 1
0.15
Vo
0.1
0.05
0
0 0.2 0.4 0.6 0.8 1 1.2
-0.05
[S]
Figure 4.6. Michaelis-Menten plot on the effect of substrate concentration on the enzyme
activity,
The results of the effect of substrate concentration is summarized in Table 4.3.
Based from the results, there was an increasing enzyme activity indicated by an increase in
the concentration of reducing sugars formed as substrate concentration increases with a
fixed amount of enzyme. Theoretically, the rate of enzyme catalyzed reaction rises linearly
as substrate concentration increases and starts to level off and eventually reached a point
where enzyme capabilities are used to their maximum extent. This was not observed on the
experiment and a linear graph was obtained. Each enzyme active site must be filled up by a
substrate for a finite amount of time, and the products formed must leave the site before the
cycle can be repeated (Berg et al., 2012). As substrate concentration increases, the enzyme
concentration becomes a limiting factor. As the number of substrate molecules increases,
the sites are covered and enzymes beome saturated. On the other hand, at low substrate
concentrations, the active sites on the enzyme molecules are not fully filled up by substrate,
thus the enzyme rate varies with substrate concentration (Stoker, 2010).
1 0 0 0 0 0 0 0
1000
800
600
1/Vo
400
200
0
-50 -200 0 50 100 150 200
1/[S]
Figure 4.8. Plot on the effect of the substrate concentration using Lineweaver-Burk plot for
data analysis with inhibitor.
WIth inhibitor
0.2
Vo, µmol/min-mL
y = 0.2002x - 4E-05
0.1 R² = 1
0
0 0.2 0.4 0.6 0.8 1
[S], µmol/mL
Figure 4.9. Plot for the substrate vs the initial velocity with the presence of an inhibitor.
Table 4.4 summarized the results for the effect of the inhibitor on enzyme activity. The
Micahelis-Menten plot and Lineweaver plot are shown in Figure 4.8.Comparing the
computed values of Km and Vmax with and without inhibitor, the type of inhibition urea
perform cannot be determined. Since for without the inhibitor, a negative Vmax and Km was
obtained, it cannot use to distinguish with the obtained value with an inhibitor. Theoretically,
urea is a noncompetitive inhibitor of invertase. Urea is a very strong denaturing reagent due
to its highly polar structure. It binds to the non-active site of the enzyme, thereby affecting its
structure and activity. Upon affecting the structure and activity, the active site for the
substrate can be possible to deform or rearrange causing it to lose its activity to the specific
enzyme. The graph for the determination for Vmax and Km for with and without inhibitor was
not provided since it cannot show the relationship of the plot for a type of inhibition.
Another factor that affects enzyme activity is the pH. Enzymes be on an environment
with an optimum pH in order for it to reach maximum activity. Since enzymes are proteins,
pH change affects the ionic character of the amino and carboxylic acid groups on the protein
thereby affecting enzyme catalysis. Deviation from the optimum pH can cause enzyme
denaturation and consequent loss of enzyme activity (Wilson and Woker, 2010). In the
exercise, acetate buffers with pH 3.7, 4.5 and 5.0 and phosphate buffer with pH 6.0, 7.0 and
8.0 were prepared. Seven test tubes were prepared with test tube 1 serving as the control
while test tubes 2 to 7 contained the buffers, sucrose solution and the invertase. The
solutions were incubated for 5 minutes before performing the Nelson’ method.
Table 4.5. Data on the effect of pH on the enzyme activity.
[Reducing Enzyme
pH sugar ] activity
Test tube number Absorbance µmol/ml µmol/ml-min
0 6.306 x 10 - 1.261 x 10 -
3 3
1 0.020
2 3.7 0.805 1.000 0.200
3 4.5 0.838 1.042 0.208
4 6.0 1.218 1.523 0.304
5 7.0 1.069 1.334 0.267
6 8.0 0.525 0.646 0.129
0.35
0.3
Enzyme activity
µmol/ml-min
0.25
0.2
0.15
0.1
0.05
0
0 2 4 6 8 10
pH
Temperature also affects the enzyme activity. At higher temperature, molecules are
moving faster and colliding more frequently. The rate of the enzyme catalyzed reaction
increases as temperature increases. However, beyond optimum temperature, the enzyme is
denatured because the increased energy destroys the tertiary structure of the enzyme
(Stoker, 2010). To determine the effect of temperature on enzyme activity, five test tubes
were prepared all containing acetate buffer, sucrose solution, water and the invertase
solution except for test tube 1 that acted as the control. The test tubes were subjected to
different temperatures: 30°C, room temperature, 23°C for the cold treatment, 39°C and
50°C. Table 4.6 summarizes the results of effect of temperature on enzyme activity. While
Figure 4.11. shows the relationship between temperature and enzyme activity for invertase.
Table 4.6. Data on the effect of temperature on the enzyme activity.
0.1
Enzyme Activity
µmol/ml-min
0.08
0.06
0.04
0.02
0
0 10 20 30 40 50 60
Temperature, °C
Figure 4.11. Plot for the effect of temperature on the enzyme activity.
Based on the results, the optimum temperature of the invertase was at room
temperature or 30 deg-C. However, literature values showed that the optimum temperature
of the invertase is at 55 oC. At lower temperature than the optimum temperature, the enzyme
is deactivated but its conformation stays the same. On the other hand, there will be thermal
denaturation at elevated temperatures as the hydrogen bonding in the enzymes is disrupted.
The rate of enzyme catalyzed reaction decreases as enzymes are deactivated or denatured
since there will be fewer enzymes to bind for the substrate (Wilson and Walker, 2010).
Errors in the experiment may be due to the setting of the temperature for each test tubes
and erroneous reading of the absorbance.
Enzyme activity is also affected by other factors such as cofactors and coenzymes,
activator concentration, and enzyme concentration. Coenzyme and cofactors increases the
rate of enzyme catalyzed reactions. Some enzymes need coenzymes to bind to the
substrate and cause a reaction. Some enzymes need certain inorganic metallic ion such as
Mg2+, Mn2+, Ca2+, Co2+ etc. to reach their optimum activity. Moreover, as enzyme
concentration is increased, the reaction rate also increases because more substrates can be
accommodated for a given amount of time (Royal Society of Chemistry, 2004).
Summary and Conclusions
Enzymes are compounds that catalyze biochemical reactions. Enzyme activities are
being studied in order to understand how enzymes work. This exercise dealt with the study
of enzyme kinetics. Different factors affecting enzyme activity such as incubation time on
product formation, substrate concentration, presence of inhibitor, pH, and temperature were
considered on the exercise. Nelson’s method was used as the enzyme assay.
Standard solutions were first prepared using the Nelson’s method for reducing sugar.
Using this method, the enzymatic activity was measured by determining the amount of
reducing sugar produced in a reaction. The calibration curve generated was used in the
succeeding experiments of the exercise.
The effect of incubation time was determined by incubating sucrose with invertase at
different time intervals. Based on the results, it can be concluded longer incubation time
increases the production of products until such time is reached wherein there will be no net
change in the concentration of substrate and produc or a presence by the levelling off of
curve.
C1V1 = C2V2
(2 mM) (0.050 mL) = (C2) (1 mL)
C2 = 0.1 mM
*Same computation was done for the determination of the concentration of Sucrose (mM).
0.048−0.015
X= 0.7898
1 1
= = 10.20 𝑚𝐿/µ𝑚𝑜𝑙
[𝑆] 0.0980
1 1
𝑉𝑜
= 0.0196
= 51.02 min-mL/µmol
1 1 𝑀 𝐾 1
Let y = 𝑉𝑜 , x = [𝑆] , slope = 𝑉𝑚𝑎𝑥 , and y-int = 𝑉𝑚𝑎𝑥
KM = (slope)(Vmax) = 6.457
References
Balbaa, M and El Ashley E.S. 2012 Enzyme Inhibitors as Therapeutic tools.
Biochemistry Department; Alexandria University, Egypt. Retrieved from
https://www.omicsonline.org/open-access/enzyme-inhibitors-as-therapeutic-tools-
2168-9652.1000103.php?aid=9662
Berg J.M., Tymoczko J.L., Styler L. 2012. Biochemistry 7th ed. W.H. Freeman
and Company
Stoker, H.S. 2010. General, Organic, and Biological Chemistry. 5th ed. USA:
Brooks/Cole Cengage Learning.
Wilson Keith and Walker, J.. 2010. Principles and Techniques of Biochemistry
and Molecular Biology. 7thed. Cambridge: Cambridge University Press. Pp. 600-
616.