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Exercise No. 4
ENZYME KINETICS
Groupmates:
John Patricia Mae Centeno
Earlene Lagasca
Jose Lorenzo Manansala
(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
0.5
0
0 1 2 3 4 5 6 7 8 9
-0.5
Incubation Time, min
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.
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
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).
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.
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
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 4.12. 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
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
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]
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.
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
0.1
0.05
0
0 10 20 30 40 50 60
-0.05
Temperature, °C
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.
C2 = (C1V1)/V2
C2 = 2 mM glucose solution
2. Get the linear portion of the graph on Figure 3.3.
y = (0.2291)x - 0.0712
Since the slope, m is equal to the initial velocity, Vo, itself, then:
1 Unit = 0.2291μmol/min
3. Enzyme Concentration
Vmax = ? Vmax = ?
Km = ? Km = ?
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.
LEHNINGER, A.L., D.L. NELSON, and M.M. COX. 1993. Principles of Biochemistry, 2nd ed. New
York: Worth Pub., Inc.