POSTLAB Report on

Exercise No. 4
ENZYME KINETICS

Bolandos, Kim Leonard C.

CHEM 161.1 – 2L
1st Semester AY 2018-2019

Groupmate:
Joel Amado
Giorgia Escalante
CJ Carlos
Pamela Macalagay

Date performed: October 2, 2018

Date submitted: October 09, 2018

Sir Bong Remillion

Results and Discussion
Enzymes are essential to every biochemical processes. They act in organized
sequences catalyzing hundreds of stepwise reactions that make biological macromolecules
from simple precursors, conserve and transform chemical energy, and degrade nutrient
molecules by lowering the activation energy of these reactions. Almost all known enzymes
are proteins. However, proteins do not have the absolute control on catalysis; the findings
about catalytically active RNA molecules give forcing confirmation that RNA was a
biocatalyst early in evolution. The high specificity of the catalytic function of an enzyme is
because of its protein nature. Due to the capacity of proteins to specifically bind a very wind
range of molecules, they act as highly effective catalyst for a vast diversity of chemical
reactions (Berg et al, 2012).

The main function of enzymes is to enhance rates of reactions depending on the

needs of the organism. Enzyme activities are being studied in order to understand how
enzymes work. The interaction between a substrate molecule with an enzyme molecule
initiates enzyme reactions.

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

Figure 4.1. Calibration curve for the glucose standard.

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.

Enzyme activity is affected by different factors such as incubation time on product

formation, substrate concentration, presence of inhibitor, pH, and temperature. Changes that
occur to the environment of an enzyme greatly affects its activity. Each factors were
observed in the exercise to see the differences it contributes to the enzyme activity.

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

0.15 y = 0.0154x - 0.0115

R² = 0.9723
(µmol/mL)

0.1

0.05

0
0 2 4 6 8 10 12
-0.05
Time (min.)

Figure 4.4. The linear relationship for the incubation time.

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).

Figure 4.7. Effect o substrate concentration on enzyme activity.

The Michaelis-Menten equation was derived in order to accountfor the kinetic
properties of the enzymes. Vmax is the maximum velocity wherein all the enzymes are
bound to the substrates. On the other hand, the Michaelis constant, KM, used to determine
the affinity of the enzyme to the substrate. This equation shows the quantitative relationship
between Vo, Vmax, and initial substrate concentration through the Michaelis constant. The
Michaelis-Menten equation can be transformed into a Lineweaver-Burk equation that is more
useful in plotting experimental data. In Figure 4.5, it shows the graph of 1/Vo vs 1/[S] and the
equation of the line obtained was used to calculate for the Vmax and KM(Nelson and Cox,
2008). The calculated Vmax and KM for the effect of substrate concentration were -13.715
µmol/min and -69.251, respectively. Theoretically, the values must be positive. This signifies
that there is error committed on the experimentation, this could be possible due to the errors
in sample preparations and in reading the absorbance.
Inhibitors are group of substances that decrease the rates of enzyme catalyzed
reaction, or in some cases, stop the catalysis. In boiological systems, inhibiting enzyme
activity serves as a control mechanism. Enzyme inhibition may be classified as competitive,
non-competitive, and uncompetitive inhibition (Nelson and Cox, 2008).
In a competitive inhibition, an enzyme can bind a substrate or a an inhibitor, but not
both. A competitive inhibitor can compete with the substrate for occupancy in the active site
of the enzyme since its resembles th subtrate’s shape and charge distribution. Although the
inhibitor remains unchanged, its physical presence on the active site prevents the normal
substrate molecule to bind to the enzyme. This inhibition does not affect the Vmax of the
system but increases the value to Km. Increasing the concentration of the substrate can
decrease the competitive inhibition (Berg et al., 2012).
Non-competitive inhibition is wherein the substrate can still bind to the active site of
the enzyme, but the presence of the inhibitor changes the shape of the enzyme. This
prevents the catalytic group in the activity site from performing their normal catalyzing
reaction. The Vmax of the system decreases while the KM remains the same. By lowering the
cocncentration of the noncompetitive inhibitor, the enzyme can return to its normal activity as
enzyme molecules become free and in its normal shape (Stoker, 2010).
Enzyme inhibition in which the enzyme binds only to enzyme substrate (ES) complex
is known as uncompetitve inhibition. This type of inhibition will only influence the enzyme
activity when susbtrate cocncetrations and ES concentrations are high. This inhibition
decreases both Vmax and the Km of the system. (Stoker, 2010).
The inhibitior used in the exercise was urea. The procedure done in determining the
effect os substrate concentration was also performed in this part of the experiment except
that urea was added to the solution.
Table 4.4. Effect of adding an inhibitor to the enzyme activity.

Test [Sucros Absorban Corrected [S] Vo 1/[S] 1/Vo

Tub e] ce Absorbanc µmol/ml µmol/min- mL/µmol min-
No. mM e mL mL/µmol

1 0 0 0 0 0 0 0

2 10 0.025 0.0196 5.80 x 1.16 x 10 - 172.41 826.07

10-3 3

3 20 0.12 0.0924 0.0980 0.0196 10.20 51.02

4 30 0.196 0.146 0.166 0.0332 6.02 30.12

5 40 0.278 0.206 0.242 0.0484 4.13 20.66

6 60 0.55 0.434 0.530 0.106 1.89 9.43

7 80 0.732 0.572 0.705 0.141 1.42 7.09

8 100 0.942 0.737 0.914 0.183 1.09 5.46

9 10 0.002 ----- ----- ----- ----- -----

10 40 0.077 ----- ----- ----- ----- -----

11 100 0.203 ----- ----- ----- ----- -----

Vmax, min-mL/µmol 1.349
KM 6.457

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.

Enzyme inhibition is important in the field of medicine. Antibiotics prevents bacterial

growth through inhibition of enzymes that is critical to their life processes. For instance,
penicillin is a type of antibiotic that inhibits transpeptidase. This enzyme catalyzes the
formation of peptide cross links between polysaccharide strands in bacterial cell wall that
gives it the necessary strength. Penicillin prevents the formation of a strong cell wall as
inhibits the transpepsidase. The bacterium is killed by any osmotic or mechanical shock that
causes lysis (Ophardt, 2003).

Other application for medicine is the inhibition of glycosidases, an enzyme involved in

carbohydrate-processing which has important roles in biological processes. This types of
enzymes have elevated activity upon presence of human tumors. The inhibitors regulate the
enzymes such as alpha-glucosidases by competitive inhibition to the glycoside-processing
enzyme and gathered interest as a therapeutic agent. (Ashry and Balbaa, 2012).

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

Figure 4.10. Plot of Enzyme activity vs pH.

Buffers were used to prevent drastic changes in pH. Different pH of buffer systems
were used to determine the effect of varying pH level on the invertase activity. Based on the
results shown in Table 4.4 and Figure 4.10, the optimum pH of the invertase was at pH 6.0.
Theoretically, the pH range of the invertase is at 3.5-5.5 with an optimum pH of 4.5. Results
also showed that as the pH increases, the enzymatic activity decreases. Decrease
enzymatic activity is due to the reaction of the amino acid chains in the active site of the
enzyme which can act as a weak acid or base. These side chains are necessary in
maintaining the structure of the enzyme. Hence, the enzyme loses its originals structure and
decreases its catalytic ability at pH that deviates greatly from the optimum pH (Nelson and
Cox, 2008). Errors that may affected the results of the experiment include inaccurate
preparation of the buffer solutions and erroneous absorbance reading.

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.

Test tube number Absorbance Corrected Temperatur [Reducin Enzyme

 Absorbanc e g sugar] Activity
e °C µmol/ml µmol/ml
-min
1 0.031 0.000 30 0 0
2 0.269 0.238 23 0.2823 0.0565
3 0.381 0.350 30 0.4241 0.0848
4 0.273 0.242 39 0.2874 0.0575
5 0.170 0.139 50 0.1570 0.0314

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.

In the effect of substrate concentration, different concentration of sucrose solutions

with the same amount of invertase were prepared and incubated at the same time. Results
showed that increasing the concentration of the substrate also increases the enzyme
activity. However, too high concentration can slow down the rate of catalysis as the enzyme
becomes saturated.

The inhibitor effect on enzyme activity was determined by preparing different

concentration of sucrose solutions with the same amount of invertase and addition of urea.
The solutions were incubated at the same time. The rate of enzymatic activity was slowed
down with the presence of the inhibitor. Since the data on Km and Vmax values on the
sample with inhibitors was not good, it is impossible to conclude what type of inhibition
happened. Theoretically, urea is a noncompetitive inhibitor.

The effect of pH was determined by subjecting sucrose solutions to different pH levels

and incubating them at same time. It was observed that the optimum pH of the invertase was
at approximately 6.0. Theoretically, the pH range of the invertase is at 3.5-5.5 with an
optimum pH of 4.5. When value of pH exceed the optimum pH of the invertase, there will be
a decrease in the rate of reaction due to denaturation.

In determining the effect of temperature, sucrose solutions with invertase was

subjected to different incubation temperature. Based on the results, the optimum
temperature in the experiment was 30°C and it can be concluded that the higher the
temperature, the higher the enzyme activity. However, beyond optimum temperature, the
enzyme is denatured because the increased energy destroys the tertiary structure of the
enzyme.
Sample Calculations
1.) Determination of the concentration of the glucose (mM) used in standard 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).

2.) Determination of the concentration of reducing sugars, µmol/mL.

From the equation of the line in Figure 3.2, y = 0.7898x + 0.015, the concentration of
reducing sugar can be determine: Let y=A510 and x=concentration of the reducing sugar,
µmol/mL.
At A510= 0.048 (for incubation time):
y = 0.7898x + 0.015

0.048 = 0.7898x + 0.015

0.048−0.015
X= 0.7898

X = [reducing sugars] = 0.0418

*Same computation was done for the determination of the concentration of product form.
3.) Calculation of Vo (For the effect of adding an inhibitor)
[𝑆] 0.0980 µ𝑚𝑜𝑙/𝑚𝐿
Vo = 𝑖𝑛𝑐𝑢𝑏𝑎𝑡𝑖𝑜𝑛 𝑡𝑖𝑚𝑒= 5 𝑚𝑖𝑛
= 0.0196 µmol/min-mL

*Same computation for enzyme activity in temperature and pH effect.

Calculation of 1/[S] and 1/[Vo]

1 1
= = 10.20 𝑚𝐿/µ𝑚𝑜𝑙
[𝑆] 0.0980

1 1
𝑉𝑜
= 0.0196
= 51.02 min-mL/µmol

4.) Detrmination of Vmax and KM

1 1
Plot 𝑣𝑠
𝑉𝑜 [𝑆]

1 1 𝑀 𝐾 1
Let y = 𝑉𝑜 , x = [𝑆] , slope = 𝑉𝑚𝑎𝑥 , and y-int = 𝑉𝑚𝑎𝑥

Vmax = 1.349 µmol/min-mL

KM = (slope)(Vmax) = 6.457
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