Postlab Report on

Exercise No. 4
ENZYME KINETICS

Conti, Janelle Allyza P.

CHEM 161.1 – 3L
2nd Semester AY 2018-2019

Groupmates:
John Patricia Mae Centeno
Earlene Lagasca
Jose Lorenzo Manansala

Date Performed: March 1, 2019

Date Submitted: March 8, 2019

Sir Ralph Aldwin C. Briones

Laboratory Instructor
 I. Results and Discussion
Enzymes are substances that function as catalysts in biochemical reactions. Most of them
are globular proteins. Enzymes can be classified as simple or conjugated. Also, there are six major
classes of enzymes. These include the oxidoreductases, transferases, hydrolases, lyases, ligases,
and isomerases (Berg, et al., 2007).
Enzymes work by allowing the substrates to come into close contact with each other. They
catalyze reactions under mild conditions by providing an alternative reaction pathway that has a
lower activation energy (Chang, 2005). They also mediate the transformations of one form of
energy into another (Berg, et al., 2007),
Most of the time, the amount of enzyme present in a solution is unknown. It is difficult to
isolate the pure enzyme concentration from the solution. However, the amount of enzyme can
be determined using enzyme activity. Enzyme activity is described in terms of initial velocity and
is expressed as Units (U). This Unit (U) can be defined as 1 μmol of formed product or depleted
substrate per unit time. However, for impure solutions with enzyme, the concentration of the
enzyme can be expressed as U/mL enzyme solution, U/mg solid enzyme, or specific activity which
is the number of enzyme units per mg of protein. The higher the specific activity, the higher is its
purity.
Experimentally, enzyme activity is defined as the instantaneous rate of product formation
or substrate disappearance with time. It is measured at the start of the reaction where the rate
is linear with time. Mathematically, enzyme activity can be expressed as:
𝑑[𝑃] −𝑑[𝑆]
𝑣0 = =
𝑑𝑡 𝑑𝑡
Moreover, enzyme activity can be affected by several factors, such as temperature, pH,
and concentration of the substrate, enzyme, coenzyme, inhibitor, or activator.
An enzyme progress curve is constructed upon determining the amount of enzyme in a
solution. As substrate/product concentration depends on reaction time, initial velocity is
determined through the results obtained in the curve such as the concentration of product or of
the unreacted substrate remaining. Levelling off happens because of substrate depletion, enzyme
saturation, product inhibition, or coenzyme deactivation.
Enzyme assay is a method used to evaluate the biological activity of an enzyme. Among
the common methods, stopped method or fixed time assay was employed in this exercise for the
determination of the concentration of enzyme in the solution. After placing the reactants and
cofactors in the solution, the reaction was stopped at a certain time, employing the Nelson’s
method.
Nelson’s method was used to determine the concentration of reducing sugars. Standard
solutions were prepared by mixing varying amounts of 2 mM glucose with various amounts of
distilled water. In terms of dilution, 𝐶1 𝑉1 = 𝐶2 𝑉2 , the equivalent concentration of glucose solution
in mM was determined. Each prepared standard glucose solution was oxidized using 1.00 mL of
Nelson’s reagent, producing Cu2+ upon heating. Afterwards, the solutions were cooled to room
temperature. The ion formed was reduced to Cu+. A rust – colored complex, due to the formation
of Cu2O, was observed. The intensity of the rusty color increases as the concentration of glucose
is increases. Hence, higher the concentration of glucose, the higher is the amount of the sugar
reduced.
1.00 mL of Arsenomolybdate reagent was added and mixed using the vortex mixer. The
mixture changed in color from rusty to intense blue - green. This indicates that Cu+ was oxidized
to Cu2+ and arsenomolybdic acid was reduced to arsenomolybdous acid, which was responsible
for the resulting color. The reactions involved are as follows.

(4.1)

(4.2)

The solution was allowed to stand for five minutes at room temperature. Consequently,
7.00 mL distilled water was added to each test tube and was mixed using the vortex mixer.
The absorbance of the solutions was read at 510 nm.
Table 4.1. Absorbance readings of glucose solutions for standard curve
determination.
Test tube no. Volume, mL [glucose], μmol/mL Absorbance
1 0 0 0.000
2 0.05 0.1 0.011
3 0.10 0.2 0.028
4 0.20 0.4 0.062
5 0.40 0.8 0.081
6 0.60 1.2 0.125
7 0.80 1.6 0.133
8 1.00 2.0 0.259
 Based on Table 4.2, as glucose concentration increases, the absorbance of the solution
also increases since more moles of the reducing sugar can absorb light.

2
1.8
1.6 y = 0.8278x + 0.1643
1.4 R² = 0.9813
Absorbance

1.2
1
0.8
0.6
0.4
0.2
0
0 0.5 1 1.5 2 2.5
Concentration, umol/mL

Figure 4.1. Standard curve for the determination of sugar content using Nelson’s
assay.

The standard curve shown was used for the determination of the sugar content (in μmol
glucose/mL) once absorbance of samples is known and calculated. The relationship established
among the variables was somewhat linear due to the value of the Pearson coefficient (R2).
Table 4.2. Linear regression data obtained in the standard curve for the determination
of reducing sugar content.
Parameter Value
Slope, mL/μmol 0.82783028
y-intercept 0.164333655
R2 0.981314844

For the effect of incubation time on product formation, ten test tubes were prepared with
acetate buffer, sucrose solution, amounts of distilled water, and invertase solution. The enzyme
used in this experiment was invertase (or sucrase). Invertase catalyzes the hydrolysis of internal
α-1  β-2 glycosidic bond in sucrose to yield glucose and fructose (Chang, 2005).

(4.3)
 The solutions were incubated, with a two-minute interval at room temperature. 1.00 mL
of Nelson’s reagent was added to stop the reaction. Afterwards, the absorbance of each solution
was determined at 510 nm. The amounts of reducing sugar were determined using the standard
curve.
Table 4.3. Absorbance readings of reducing sugars with varying incubation time.
Test tube no. Incubation time, min Amount of reducing sugar, μmol Absorbance
1 0 0 0.006
2 2 0.308839084 0.42
3 4 0.747334762 0.783
4 6 1.449169443 1.364
5 8 1.720964285 1.589
6* 10 2.05
7* 12 2.241
8* 15 2.552
9* 20 2.709
10* - 0.000
*disregarded
Data for test tubes 6 to 9 were disregarded since the absorbance readings obtained were
out of the standard curve. Test tube 10 served as the control. Based on Table 4.2, as the
incubation time increases, the amount of µmol reducing sugars formed also increases.
The enzyme activity was determined based on Figure 4.2. The enzyme activity, or initial
velocity, Vo (slope), was determined by getting the linear portion of the graph. From 0 to 5
minutes, a linear part can be observed; thus, an equation of the line can be formulated. The
enzyme activity in terms of initial velocity is equal to 0.2291 μmol/min.
Expressing it as 1 U or 0.2291 μmol/min, is the amount of enzyme needed to produce 1
μmol of product/mL or to deplete 1μmol of substrate/mL per minute at a specific pH, temperature,
and substrate concentration. It is also possible to express invertase activity in terms of μmol
sucrose utilized/minute. The calculated value μmol product formed per minute will become the
negative of the amount of product formed per minute since the rate at which the product formed
is equal to the sucrose depleted or utilized from the reaction (Cooper, 1973).
 2

y = 0.2291x - 0.0712
1.5 R² = 0.9796

umol reducing sugar

1

0.5

0
0 1 2 3 4 5 6 7 8 9

-0.5
Incubation Time, min

Figure 4.2. Amount of reducing sugar, in μmoles, at varying incubation time.

Table 4.4. Linear regression data obtained in the plot of μmol reducing sugar versus
incubation time.
Parameter Value
Slope, μmol/min 0.229112946
y-intercept, µmol -0.071190271
R2 0.97963197

The graph generated is somewhat linear throughout the incubation time. Typically, the
amount of product formed increases with time, since enzymes help in catalysis of biological
reactions. However, a time is reached when there is hardly a net change in the concentration of
substrate or product. This is known as the lag phase which is due to settling of particles, long
duration of enzyme-substrate complex and enzyme-product before they reach a steady state. The
enzyme (invertase) still actively converts the substrate into product. Kinetically, the reaction
equilibrium has already been attained; thus, it will not be linear throughout the incubation time.
For the effect of substrate concentration, similar procedure as in effect of incubation time
on product formation was performed except that varying concentrations of sucrose were present
in each test tube. Same amounts of invertase solution were added to each sucrose solution,
except for three test tubes. Test tubes 9 to 11 served as correction for non – enzymatic sucrose
hydrolysis. The mixtures were then subjected to Nelson’s method of analysis after 5 minutes prior
to the addition of the enzyme.
Table 4.5. Concentration of reducing sugars, in μmol/mL, with its corrected
absorbance.

Test tube [reducing Volume, Corrected

Absorbance NE Hydrolysis
no. sugar], μmol mL Absorbance
1 0 0 0.081 0.06647619 0
2 10 0.050 0.778 0.085714286 0.692285714
3 20 0.100 1.122 0.104952381 1.017047619
4 30 0.150 1.615 0.124190476 1.490809524
5 40 0.200 1.628 0.143428571 1.484571429
6 60 0.300 1.834 0.181904762 1.652095238
7 80 0.400 2.056 0.220380952 1.835619048
8 100 0.500 2.114 0.258857143 1.855142857
9 10 0.080 0.050
10 40 0.152 0.200
11 100 0.256 0.500

0.3

0.25
y = 0.0019x + 0.0665
0.2 R² = 0.9927
Absrobance

0.15

0.1

0.05

0
0 20 40 60 80 100 120
Concentration, umol/mL

Figure 4.3. Sucrose correction in the absence of urea.

Table 4.6. Linear regression data obtained in the sucrose correction (without urea).
Parameter Value
Slope, mL/μmol 0.00192381
y-intercept 0.06647619
R2 0.99270144
 From the standard curve in Figure 4.1, the corrected absorbance was used to calculate
the equivalent amount of reducing sugar as shown in Table 4.7. To calculate the initial velocity,
the concentration per minute was determined by dividing the interpolated concentration by five
minutes.

Table 4.7. Initial velocities of substrate concentration after five minutes.

Test tube no. Corrected Absorbance [reducing sugar], μmol/mL Vo, μmol/mL-min
1* 0 0 0
2 0.692285714 0.637753985 0.127550797
3 1.017047619 1.030058921 0.206011784
4 1.490809524 1.602352441 0.320470488
5 1.484571429 1.594816965 0.318963393
6 1.652095238 1.797181886 0.359436377
7* 1.835619048 2.018874441 0.403774888
8* 1.855142857 2.042458755 0.408491751
*disregarded

Table 4.8. Parameters 1/Vo and 1/[S] for Lineweaver-Burke plot construction
(without urea).

Test Tube No. 1/Vo , mL-min/μmol 1/[S], mL/µmol

1* 0 0
2 7.840013736 0.100000
3 4.854091256 0.050000
4 3.120412135 0.033333
5 3.135156015 0.025000
6 2.782133539 0.016667
7* 2.47662752 0.012500
8* 2.44802985 0.010000
*disregarded

To observe the effect of adding an inhibitor, 2 M urea was used. Urea is an enzyme
inhibitor that abolish or decrease enzyme activity (Khanna, 2008). Inhibition can either be
reversible or irreversible. The removal of the inhibitor in a reversible inhibition restores enzyme
activity while the inhibitor in an irreversible inhibition permanently inactivates the enzyme ().
Table 4.9. Corrected Absorbance of varying sucrose concentrations with the addition
of inhibitor, urea.

Test [reducing sugar], Volume, Corrected

Absorbance NE Hydrolysis
tube no. μmol mL Absorbance
1 0 0 0.0204 0.021390476 0
2 10 0.05 1.1848 0.026085714 1.158714286
3 20 0.1 1.7579 0.030780952 1.727119048
4 30 0.15 1.8306 0.03547619 1.79512381
5 40 0.2 1.9738 0.044866667 1.928933333
6 60 0.3 2.062 0.049561905 2.012438095
7 80 0.4 2.0212 0.058952381 1.962247619
8 100 0.5 2.0212 0.068342857 1.952857143
9 10 0.08 0.0292
10 40 0.152 0.0355
11 100 0.256 0.0699

0.08
0.07 y = 0.0005x + 0.0214
R² = 0.9646
0.06
Absorbance

0.05
0.04
0.03
0.02
0.01
0
0 20 40 60 80 100 120
Concentration, umol/mL

Figure 4.4. Sucrose correction in the presence of urea.

Table 4.10. Linear regression data obtained in the sucrose correction (with urea).
Parameter Value
Slope, mL/μmol 0.000469524
y-intercept 0.021390476
R2 0.964634232

The corresponding μmol/mL reducing sugar was determined using the corrected
absorbance derived from the standard curve. Table 4.7 shows the interpolated concentration of
reducing sugar using the corrected absorbance. The initial velocity in the presence of the inhibitor
urea was also calculated by dividing the concentrations of the reducing sugar by five minutes.

Table 4.11. Initial velocities of sucrose samples at varying concentrations in the

presence of urea.
Test tube [reducing sugar],
Corrected Absorbance Vo, μmol/mL-min
no. μmol/mL
1 0 0 0
2 1.158714286 1.201189006 0.240237801
3 1.727119048 1.887808928 0.377561786
4 1.79512381 1.969957121 0.393991424
5 1.928933333 2.131595959 0.426319192
6 2.012438095 2.232467797 0.446493559
7 1.962247619 2.171838852 0.43436777
8 1.952857143 2.160495372 0.432099074

Table 4.12. Parameters 1/Vo and 1/[S] for the Lineweaver-Burke plot construction in
the presence of urea.
Test Tube No. 1/Vo , mL-min/μmol 1/[S], mL/µmol
1* 0 0
2 4.162542259 0.1
3 2.648573129 0.05
4 2.53812631 0.033333333
5 2.345660293 0.02
6 2.239673964 0.016666667
7* 2.30219659 0.0125
8* 2.314284059 0.01
*disregarded
 0.4

0.35

0.3
Vo 0.25

0.2
y = 0.2x
0.15 R² = 1

0.1

0.05

0
0 0.5 1 1.5 2
[S], umol/mL

Figure 4.5. Substrate-saturation curve in the absence of inhibitor, urea.

0.5
0.45
0.4
0.35
0.3
0.25
Vo

y = 0.2x + 2E-15
0.2 R² = 1

0.15
0.1
0.05
0
0 0.5 1 1.5 2 2.5
[S], umol/mL

Figure 4.6. Substrate-saturation curve in the presence of inhibitor, urea.

The substrate-saturation curve is a plot of substrate concentration against initial velocity.

Based on Figure 4.5 and 4.6, the initial velocities increase linearly with substrate concentration,
reaching a stable line, approaching Vmax. Vmax relates to the fastest rate at which certain amount
of enzyme can catalyze the reaction.
Lineweaver-Burke equation is a double reciprocal plot obtained by plotting 1/Vo as a
function of 1/[S] for both conditions, in the presence and absence of urea.
 9
8
7
6
1/Vo 5
4 y = 63.236x + 1.5007
R² = 0.9816
3
2
1
0
0 0.02 0.04 0.06 0.08 0.1 0.12
1/[S]

Figure 4.7. Lineweaver-Burke plot in the absence of inhibitor, urea.

4.5
4
3.5
3
2.5
1/Vo

2 y = 22.653x + 1.7902
R² = 0.9585
1.5
1
0.5
0
0 0.02 0.04 0.06 0.08 0.1 0.12
1/[S]

Figure 4.8 Lineweaver-Burke plot in the presence of inhibitor, urea.

After constructing the Lineweaver-Burke plot, the kinetic parameters were calculated and
determined. In the presence of urea, the Vmax calculated was 0.5586 mL/μmol while Km was
12.6543. In the absence of urea, the Vmax calculated was 0.6663 mL/μmol while Km was 42.1367.
The Michaelis constant or Km is essential in the field of enzyme kinetics. High value of Km means
that it needs a lot of substrate to reach half of the maximum velocity. This indicates weak binding
for enzyme-substrate system. Meanwhile, low Km value means that half of the maximum velocity
has been reached, thus implying that it indicates strong binding between the enzyme-substrate
systems. Km is equal to the substrate concentration at which the reaction rate is half its maximum
value. Moreover, Km can also measure the strength of enzyme-Substrate complex. It simply
measures the activity of enzymes, or the affinity of enzymes to substrates (Berg, et al., 2007).
Michaelis-Menten equation describes the relationship between initial velocity, substrate
concentration, maximum velocity and Michaelis constant.

Vo = Vmax [Substate] (4.4)

Km + [Substrate]
At very low concentrations where [Substrate] is already far less than Km, Vo =
(Vmax/Km)([S]), thus the initial velocity is proportional to the substrate concentration. At high
concentrations where the [Substrate] is far greater than Km, Vo = Vmax, the rate is at maximum,
and thus independent of the substrate concentration. Lastly, if the [Substrate] is approximately
equal to the Michaelis constant, then Vo is just equal to half of its maximum velocity (Berg, et al.,
2007).
In the presence of urea, competitive inhibition can be observed based from the calculated
values. The value of Km greatly affects the analysis of enzyme inhibition, especially for urea.
Competitive inhibition diminishes the rate of catalysis be reducing the proportion of enzyme
molecules bound to a substrate. The most affected kinetic parameter in a competitive inhibition
is the Michaelis constant Km, not the Vmax. Change in Km from low to high value indicates
competitive inhibition and weak binding of enzyme to the substrate (ES complex).
At any given inhibitor concentration, competitive inhibition can be relieved by increasing
the substrate concentration.
Another type of enzyme inhibition is the noncompetitive inhibition, which is the
counterpart of competitive inhibition. The inhibitor and the substrate can bind simultaneously to
an enzyme molecule at different binding sites. Its effect is on Vmax, not on Km. Vmax should be
lowered from the one without the presence of inhibitor. Irreversible inhibition is also another
type. The inhibitor dissociates very slowly from its target enzyme because the inhibitor has
become tightly bound to the enzyme, either covalently or non-covalently.
Enzyme inhibitors are widely used and applied in the field of medicine. Several important
drugs are protease inhibitors. A certain drug, captopril, used in blood pressure regulation, is an
inhibitor of the angiotensin-converting enzyme (ACE) that is a metalloprotease. Other compounds
such as indinavir and Retrovir are used in the treatment of AIDS are inhibitors of HIV protease,
which is an aspartyl protease (Berg, et al., 2007). Other competitive inhibitors are commonly used
in drugs and other medicines. Drugs such as ibuprofen are competitive inhibitors of enzymes that
participate in signaling pathways in the inflammatory response. Statins are also drugs that reduce
high cholesterol levels by competitively inhibiting a key enzyme in cholesterol biosynthesis.
With the analysis of the effect of pH on enzyme activity, seven test tubes were prepared
filled with different amounts and kinds of buffer solutions for resisting the drastic changes in pH.
The solutions were subjected to Nelson’s method. The absorbance was read at 510 nm. After five
minutes, the enzyme activity was calculated at each pH value.
Table 4.13. Absorbance readings and enzyme activities of sucrose samples at various
pH.
Test [reducing sugar], Enzyme Activity, μmol/mL-
pH Absorbance
tube no. μmol/mL min
1* -0.180391632 0.015 -0.036078326
2 3.7 -0.159131235 0.0326 -0.031826247
3 4.5 -0.157923258 0.0336 -0.031584652
4 5 0.224280689 0.35 0.044856138
5 6 -0.161667987 0.0305 -0.032333597
6 7 -0.165654311 0.0272 -0.033130862
7 8 -0.12252953 0.0629 -0.024505906
*disregarded

0.05

0.04
0.03
Enzyme Activity, umol/ml-min

0.02

0.01

0
0 1 2 3 4 5 6 7 8 9
-0.01

-0.02

-0.03

-0.04

-0.05
pH

Figure 4.9. Enzyme activity of sucrose samples at different pH values.

 Enzymes have optimum pH/ pH range at which their activity is maximal. Theoretically, as
the pH of the solution increases, from acidic to basic, the reaction rate or enzyme activity should
also increase. As seen at the peak in Figure 4.9, the optimum pH is at 5 which corresponds to the
highest value of enzyme activity that is 0.0449. Afterwards, the enzyme activity should fall down
as the pH still increases. Sudden rise of the reaction rate/enzyme activity is one possible error in
this part. This may be due to some impurities in the solution which triggered the enzyme to
catalyze more, although the substrate was already depleted.
For the effect of temperature, five test tubes were prepared, with test tube 1 as the
control. Each was filled with acetate buffer solutions, sucrose, distilled water, and the enzyme
invertase. It was incubated at different temperatures. Test tubes 1 and 3 were incubated at room
temperature, test tube 2 at 20 oC, test tube 4 at 40 oC, and test tube 5 at 50 oC. After incubation
for five minutes, the reaction was stopped using the Nelson’s reagent. The absorbance of the five
test tubes were read at 510 nm. Using the data on absorbance readings, the concentration of the
reducing sugar was determined from the standard curve in Figure 4.1. These concentrations were
divided by five minutes to get the equivalent enzyme activity.
Table 4.14. Absorbance readings and enzyme activities of sucrose samples at different
temperatures.

Test tube Temperature, [reducing sugar], Enzyme Activity, μmol/mL-

Absorbance
no. o
C μmol/mL min
1 29 0.2231
2 20 0.433 0.324542786 0.064908557
3 29 0.978 0.982890292 0.196578058
4 40 0.683 0.626537055 0.125307411
5 50 0.0209 -0.173264567 -0.034652913
 0.25
Optimum temperture
0.2

Enzyme Activity, umol/ml-min

0.15

0.1

0.05

0
0 10 20 30 40 50 60
-0.05
Temperature, °C

Figure 4.10. Enzyme activity as a function of varying temperature.

In the first part of the curve, as the temperature increases, the enzyme activity also
increases until it reaches the optimum temperature due to the increased collision frequency
between enzyme and substrate. Upon reaching the optimum value, the enzyme activity will fall
down, still as the temperature continues to increase. Since enzymes are proteins, there is an
upper limit beyond which the enzyme becomes denatured and ineffective. Hydrogen bonds are
easily disrupted by increasing temperature. This may disrupt the shape of the enzyme making its
affinity for its substrate diminished. Based on Figure 4.10, the optimum temperature is at 29oC.
Other factors may also affect enzyme activity. The type of enzyme may also affect its
capability of being an enzyme. There are types of enzyme that only work for a certain or specific
reaction, or there are enzymes that work faster or slower, depending on the conditions. The
amount of enzyme present can also affect its activity itself. If the amount of enzyme is small,
chances are, the observed activity will not be effective too.

II. Sample Calculations

1. Preparation of glucose solution for standard:

C1V1 = C2V2

C2 = (C1V1)/V2

C2 = [(20 mM)(5.00 mL)] / 50 mL

C2 = 2 mM glucose solution
2. Get the linear portion of the graph on Figure 3.3.

x (incubation time) y (amount of reducing sugar, μmol/mL)

2 0.308839084
4 0.747334762
6 1.449169443

a. The equation of the line is given by:

y = (0.2291)x - 0.0712

Since the slope, m is equal to the initial velocity, Vo, itself, then:

Enzyme Activity = initial velocity, Vo = 0.2291 μmol/min

b. Definition of 1 Unit for Enzyme Activity:

1 Unit = 0.2291μmol/min

3. Enzyme Concentration

Enzyme Concentration (U/mL enzyme solution) = (0.2291 μmol/min) / 1 mL

Enzyme Concentration (U/mL enzyme solution) = 0.2291 μmol/mL-min

4. Calculation of Km and Vmax in the presence and absence of urea

a. Presence of Urea: b. Absence of Urea:

Vmax = ? Vmax = ?

Get the y-intercept: Get the y-intercept:

y-int = b = 1.500737353= 1/Vmax y-int = b =1.790167 = 1/Vmax

Thus, Vmax = 0.666339 mL/μmol Thus, Vmax = 0.558607102 mL/μmol

Km = ? Km = ?

Km/Vmax = m =63.23608852 μmol/mL Km/Vmax = m = 22.65336797μmol/mL

Km = mVmax = 42.13667927 Km = mVmax = 12.65433223

 III. References/ Literature Cited

BERG, J.M., J.L. TYMOCZKO, and L. STRYER. 2007. Biochemistry, 6th ed. W.H. Freeman and
Company: New York, USA.

CHANG, R. 2005. Physical Chemistry for the Biosciences. California: University Science Books.

COOPER, T. 1973. Tools of Biochemistry. New York: McMillan Co.

LEHNINGER, A.L., D.L. NELSON, and M.M. COX. 1993. Principles of Biochemistry, 2nd ed. New
York: Worth Pub., Inc.