Experiment #3: Enzyme Kinetics

Dela Cruz, John Carlo F.; Ocampo, Aliart Jerboe S.; Quitoriano, Raia S.
Group 3, Biochemistry 34.1, TAD, Ms. Arlou Angeles
September 16, 2014

I. Abstract
Enzyme kinetics describes the reaction rate by which enzymes catalyzes the breakdown of a substrate, a
substance specifically acted by an enzyme. In this paper, the catalysis of the human salivary amylase ptyalin, an α-
amylase, in the hydrolysis of starch was observed in varying incubation time, temperatures of 10°C, 40°C, 60°C
and 80°C, and pH conditions of 6, 6.5, 7, 7.5 and 8. The absorbance of each setup was measured at 620 nm after
the catalysis of ptyalin using the Lugol’s Solution (I 2KI) as indicator; the starch concentration was then determined
o
and plotted against the varying conditions. The optimum temperature and pH for amylase to function are 50.2 C
and 6.5, respectively. The Michaelis-Menten Constants Km and Vmax of the reaction are 0.016229 and 0.06871
respectively. Results established that enzymes work most effectively at specific concentration, temperature and pH
requirements and cease to function beyond these optimum conditions. Enzyme kinetics is essential in determining
the optimum conditions for enzymes to function at its maximum in various biological systems.

II. Keywords enzyme, enzyme kinetics, enzyme activity, michaelis-menten, lineweaver-burke, amylase

Menten equation and produces a straight line with the

III. Introduction equation y = mx + c with a y-intercept equivalent to
1/Vmax and an x-intercept of the graph representing
Enzymes are usually protein molecules that −1/KM (Walsh, 1979).
manipulate other molecules — the enzymes'
substrates. These target molecules bind to an
enzyme's active site and are transformed into
products through a series of steps known as the (Lineweaver-Burke Eq.)
enzymatic mechanism (Cornish-Bowden, 2004).
Enzymatic activity is affected by several factors such
as temperature, pressure, concentration, pH, and the This experiment also utilized the
presence of inhibitors (Cleland & Cook, 2007). spectrophotometric determination of the
Enzyme kinetics is the study of the chemical concentration of substances. This method follows the
reactions that are catalysed by enzymes. In enzyme Beer Lambert’s Law which states that the amount of
kinetics, the reaction rate is measured and the effects light absorbed is directly proportional to the
of varying the conditions of the reaction are concentration of the substance.
investigated. Studying an enzyme's kinetics in this
way can reveal the catalytic mechanism of this
enzyme, its role in metabolism, how its activity is (Beer-Lambert’s Law)
controlled, and how a drug might inhibit the enzyme
(Bugg, 2004). Knowledge of the enzyme's structure is
The Michaelis–Menten equation describes helpful in interpreting kinetic data. For example, the
how the initial reaction rate v0 depends on the structure can suggest how substrates and products
position of the substrate-binding equilibrium and the bind during catalysis; what changes occur during the
rate constant k2 (Walsh, 1979) reaction; and even the role of particular amino acid
residues in the mechanism (Cornish-Bowden, 2004).
Some enzymes change shape significantly during the
(Michaelis-Menten Equation) mechanism; in such cases, it is helpful to determine
the enzyme structure with and without bound
substrate analogues that do not undergo the
where, Km = measure of the affinity of the enzyme for enzymatic reaction (Cleland & Cook, 2007).
the substrate and Vmax = rate when all the active The objectives of this experiment are (1) to
sites of the enzyme are filled up. understand the fundamental concepts of enzyme
The Lineweaver–Burk plot or double kinetics and evaluate kinetic parameters; (2) to
reciprocal plot is a common way of illustrating kinetic determine the effects of various factors that affect
data (Bugg, 2004). This is produced by taking the enzyme kinetics; (3) to construct a standard curve for
reciprocal of both sides of the Michaelis–Menten the determination of product concentration; and (4) to
equation. This is a linear form of the Michaelis–

Biochemistry 34.1 Enzyme Kinetics Page 1 of 6

determine the values of kinetic parameters Vmax and then a graph of the starch concentration vs.
Km. incubation time was generated.
The average reaction velocity was calculated
IV. Experimental by getting the change in starch concentration and
dividing it by the change in time per interval. KM and
A. Preparation of Starch Standard Curve Vmax were determined by graphing the 1/[S] and 1/v.\

Different concentrations of starch solution in C. Effect of Temperature on Enzymatic Activity

10 mL test tubes were prepared as shown in Table 1.
Afterwards, 20.0 µL of the prepared I2 solution was In four separate 10 mL test tubes, 1.0 mL of
added to each test tube. 0.1% starch solution, 3.5 mL of 0.1 M of the prepared
phosphate buffer at pH 6.7 (or close to that), and
Table 1. Different concentrations of starch solution 0.50 mL of 0.9% NaCl solution were mixed.
Volume
Each test tube was pre-incubated in water
Volume o o o
0.1% Volume Starch baths with temperatures of 10 C, 40 C, 60 C, and 80
Test I2 o
Tube #
starch dH2O
solution
Concentration C respectively for 5 minutes. Without removing the
solution (mL) (%w/v) test tubes from the water bath, 5 drops of enzyme
(µL)
(mL)
solution was added and the whole solution was
incubated for an additional 10 minutes.
1 (blank) 0.00 3.00 0
Afterwards, the starch hydrolysis was
2 0.50 2.50 0.1656
terminated by immersing the test tubes in boiling
water for 3 minutes. The mixtures was allowed to
3 1.00 2.00 0.2311 cool down before getting 1.0 mL aliquot of the
mixture and adding 2.0 mL distilled water and 1 drop
4 1.50 1.50 20.0 0.0897 of I2 solution. The absorbance of each mixture was
measured at 620 nm.
5 2.00 1.00 0.0662 The absorbance readings were converted
into starch concentrations using the standard curve
6 2.50 0.50 0.0828 then a graph of the starch concentration vs.
temperature was generated.
7 3.00 0.00 0.0993
D. Effect of pH on Enzymatic Activity
Two mL of each standard solution was then
In five separate 10 mL test tubes, 1.0 mL of
diluted to 10 mL before reading the absorbance at
0.1% starch solution and 0.50 mL 0.9% NaCl solution
620 nm in 30 minutes. A plot of the starch
were mixed. Varying pH (6, 6.5, 7, 7.5, and 8) of the
concentration vs. absorbance was generated to give
0.1 M phosphate buffer were added in 3.5 mL
a standard curve.
volumes for each test tubes.
Next, 5 drops of the enzyme solution was
B. Reaction of Amylase to Human Saliva
added and the solutions were incubated for 10
minutes at room temperature.
A 100 mL 1:10 dilution of human saliva was
Starch hydrolysis was terminated by
prepared. In five separate 20 mL test tubes, 1.0 mL
immersing the test tubes in boiling water for 3
of the enzyme solution was mixed with 9.0 mL of 0.1
minutes. The mixtures were then allowed to cool
% starch solution.
down. Afterwards, 1.0 mL aliquot of the solution was
Each of the reaction mixtures was then
taken and 2.0 mL distilled water and 1 drop of I 2
incubated at room temperature for 0, 3, 5, 7 and 10
solution was added.
minutes respectively. After the specified number of
The absorbance of each mixture was
minutes has passed, the starch hydrolysis was
measured at 620 nm. The absorbance readings were
terminated by immersing the test tubes in boiling
converted into starch concentrations using the
water batch for 3 minutes.
standard curve then a graph of the starch
Each mixture was allowed to cool to room
concentration vs. pH was generated.
temperature before adding 2.0 mL distilled water and
1 drop of I2 solution. Two mL of each solution was
V. Results
then diluted to 5.0 mL and the absorbance of each
was measured at 620 nm. A. Starch Standard Curve
The absorbance readings were converted
into starch concentrations using the standard curve

Biochemistry 34.1 Enzyme Kinetics Page 2 of 6

 The following absorbance was obtained after
7 0.077 0.006959508
subjecting each test tube in the spectrophotomer at
620 nm (Table 2). The absorbance was also plotted 10 0.046 0.004157628
against starch concentration (Graph 1).

Table 2. Absorbance at different starch concentration Table 4. Velocity of the reaction at different
incubation time
Test Tube Starch Concentration
Absorbance Time (min) v 1/v 1/[S]
# (%w/v)
1 0 0 0 initial 32.54118
3 0.003676 272.0656 50.75229
2 0.01656 0.272
7 0.003186 313.8723 143.6883
3 0.02311 0.526
10 0.000934 1070.71 240.5217
4 0.0497 0.547
Graph 2. Time vs [S]
5 0.0662 0.814 0.04
6 0.0828 1.002 Time vs. [S]
0.03
7 0.0993 1.2
0.02
Graph 1. Starch concentration vs. Absorbance

0.01

0
0 5 10 15

Graph 3. [S] vs. Velocity

0.004

0.003

0.002

From the generated equation of the line, r =

2
0.001
[S] vs. Velocity
0.9524, slope is 11.064, and the y-intercept is
0.0893.
0
B. Reaction of Amylase in Human Saliva 0 0.005 0.01 0.015 0.02 0.025

At different incubation time, the absorbance

of each solution was determined (Table 3) as well as Graph 4. 1/[S] vs. 1/v
the velocity of the reaction (Table 4). Graphs of the 1200
Time vs. [S] and [S] vs. Velocity was plotted. A plot of
1000 y = 4.2337x - 61.617
1/[S] vs. 1/v is also generated (Graph 4).
R² = 0.7987
800
Table 3. Absorbance at different incubation time
600
Time Starch Concentration
Absorbance
(min) (%w/v) 400
0 0.34 0.030730296 200 1/[S] vs. 1/V
0
3 0.218 0.019703543
0 100 200 300

Biochemistry 34.1 Enzyme Kinetics Page 3 of 6

 From the generated equation of the line, Vmax The absorbance of each solution was
and Km were computed as follows: determined at different pH levels (Table 6). A graph
of pH level was plotted against the starch
concentration (Grpah 6) to determine the optimum pH
level.

Table 6. Absorbance at different pH level

Test Starch Concentration
pH Absorbance
Tube # (%w/v)
1 6 -0.002196312 0.065
2 6.5 -0.002557845 0.061
3 7 -0.000930947 0.079

C. Effect of Temperature on Enzyme Activity 4 7.5 0.002955531 0.122

5 8 0.001328633 0.104
The absorbance of each solution was
determined at different temperature (Table 5). A
Graph 6. pH vs. [S]
graph of temperature vs. [S] was also generated
(Graph 5) to determine the optimum temperature. 0.004 y = -0.0054x4 + 0.1452x3 - 1.4658x2 +
6.5472x - 10.922
Table 5. Absorbance at different temperature 0.003
Test
Temperature
Starch
0.002
pH vs. [S]
Tube Concentration Absorbance
(C)
# (%w/v)
0.001
1 10 0.144
0
2 40 -0.003552061 0.05 0 2 4 6 8 10
-0.001
3 60 -0.006082791 0.022
-0.002
4 80 0.024105206 0.356
-0.003
Graph 5. Temperature vs. [S]
0.03 y = 5E-07x3 - 6E-05x2 + 0.0014x - 0.0039
From the graph, the minimum generated is
R² = 1 the optimum pH level having a value of 6.5.
0.025

0.02
Temp. vs. [S] VI. Discussion

0.015 Enzymes function only in optimum conditions

of pH and temperature. Once outside this specific
0.01 range of pH and temperature, enzymes are rendered
to be inactive or denatured. This will result to a
0.005 decrease in enzyme activity. In higher temperature,
intramolecular bonds break resulting to the misfolding
0 of the proteins in the enzymes. The change in pH will
0 20 40 60 80 100 also result in the disturbance of the polar and non-
-0.005 polar parts which can alter the shape of the protein
(Garret and Grisham, 2013).
-0.01 Theoretically, the optimum conditions for
salivary amylase to work are in the pH range of 5.6 –
From the graph, the minimum generated is 6.9 and the human physiological temperature (37˚).
o
the optimum temperature having a value of 50.2 C. Since the temperature and pH conditions are different
between the mouth and the stomach, salivary
D. Effect of pH on Enzyme Activity amylase is expected to cease its function in the

Biochemistry 34.1 Enzyme Kinetics Page 4 of 6

stomach, where the pH is near 2.0 units. Pepsin, or starch and amylase incubated at different amounts of
pepsinogen, is another enzyme found in the stomach time. The graph of starch concentration vs time
which works at pH of 2.0 (Campbell, 2005). showed that at time=0, the slope is most negative,
The α-amylase catalyzes the breakdown of and it becomes less and less negative as it
starch or polysaccharides into disaccharides and approaches time=infinity. This is due to the high
eventually converting them into monosaccharide like velocity of the enzyme activity or the high decrease in
glucose and maltose (Enzymes Essentials, 2014). starch concentration per unit time when the
These polysaccharides are connected with α-1,4- concentration of the starch is high. As the starch is
glucosidic linkages. These bonds are the specific consumed, the starch concentration and the velocity
structures in the starch by which the α-amylase become lower. More relationship was observed by
hydrolyzes (Wang, n.d.). plotting the starch concentration against velocity. As
Another enzyme assay to detect the the concentration becomes higher, the velocity also
presence of reducing sugars is done with 3,5- becomes higher. The initial increase in velocity is
dinitrosalicylic acid (DNS). In a basic solution, the high and the velocity stabilizes at a certain value
reducing sugar forms an aldehyde (glucose) or known as the Vmax, or the highest velocity an enzyme
ketone (fructose) group. The aldehyde or ketone is capable of in a given temperature and pH. Ideally,
group then reduces the DNS to form 3-amino-5- linear relationship is observed from the graph of the
nitrosalicylic acid, which has a different measured reciprocal of the starch concentration against the
light absorbance with DNS at 540 nm (Miller, 1958) reciprocal of the velocity, and the equation of this line
The reaction rate of enzyme catalysis can be may be used to obtain the Km and the Vmax. Km is
altered with the use of inhibitors, substances that the dissociation constant and a measure of the
interferes with the reaction between the enzyme and affinity of an enzyme to a substrate which is also
the substrate. The inhibitors compete with the equal to the negative reciprocal of the x-intercept of
substrate either to the active site (competitive the equation. The Vmax is the reciprocal of the y-
inhibitors) or to the other side of the enzyme (non- intercept of the said equation.
competitive inhibitors) (Campbell, 2012). The effect of temperature and pH on the
A standard curve was prepared by using enzyme activity was also tested by measuring the
varying starch concentration while keeping the iodine concentration of the starch-iodine complex after
concentration constant. The reaction of starch and reacting starch and amylase with iodine solution at
iodine, I3 + starch  starch-iodine complex, different temperature and pH conditions. This way,
produced a dark blue solution. Higher concentration the optimum conditions was determined by
of the starch yields higher product concentration in calculating the minima of the temperature vs starch
order to maintain its equilibrium constant. Thus concentration and pH vs starch concentration plots.
solutions that had higher amounts of starch had The minima indicate the conditions at which the
higher absorbance reading using spectrophotometer starch-iodine complex concentration is minimal,
due to the intensity of the dark blue color at 620 nm, which are the temperature and pH at which the
the wavelength at which the absorptivity of the color enzyme activities are highest.
of the complex is highest. The plot of the starch
concentration vs absorbance had a positive slope VII. Conclusions and Recommendations
because of their direct proportionality.
The addition of saliva decreased the Using the Lineweaver-Burke equation, the
concentration of starch in parts B, C, and D of the data were plotted to determine the relationship
experiment. The enzyme amylase in the saliva between the factors affecting the enzyme activity and
facilitates the break down by hydrolysis of starch into its kinetics. It can be concluded that the activity on an
either glucose, a carbohydrate with one subunit, or enzyme to function is affected by concentration,
maltose, a carbohydrate with two subunits. These temperature, and the pH level of the solution.
subunits, called monosaccharide and disaccharide It can also be said that there is an optimum
respectively, do not form complex with iodine. At temperature and pH level for an enzyme to function
higher enzyme activity, more starch is hydrolyzed. at its maximum. Any value above or below the
Therefore, at same initial starch concentration, optimum level would greatly affect the enzyme
solutions with higher enzyme activity yields lower activity. From the experiment, the optimum
o
concentration of remaining starch to form complex temperature and pH level are 50.2 C and 6.5
with iodine, and, as a consequence, lower respectively.
absorbance readings. The Michaelis-Menten constants (Km and
The effect of concentration of substrate, the Vmax) were also determined having values of
reactant that is activated by the enzyme, on the 0.016229 and 0.06871 respectively after plotting the
velocity of enzyme activity was determined by 1/[S] vs. 1/v.
measuring the concentrations of different solutions of

Biochemistry 34.1 Enzyme Kinetics Page 5 of 6

 It is recommended that this should be done John Carlo F. Dela Cruz
quickly especially after the addition of the I 2 solution
since the color of the solution fades after some time __________________________
due to the enzymes working on the solution. Also, it Aliart Jerboe S. Ocampo
is recommended that the reagents be freshly
prepared to avoid contaminants and the starch __________________________
solution be boiled before using it for the experiment. Raia S. Quitoriano

VIII. References

Bugg, T. (2004). Introduction to Enzyme and

Coenzyme Chemistry. Cambridge, MA:
Blackwell Publishers. ISBN 1-4051-1452-5.

Campbell, M.K.; Farrell, S.O., 2012. Biochemistry,

7th Edition. Brooks/Cole, 20 Davis Drive,
Belmont, CA 94002-3098, USA.

Campbell, N.A., Reece, J.B., et al., (2011). Campbell

Biology Ninth Edition. Pearson Education,
Inc., 1301 Sansome St., San Francsico,
California 94111.

Cleland, W. W., Cook, P. (2007). Enzyme kinetics

and mechanism. New York: Garland
Science. ISBN 0-8153-4140-7.

Cornish-Bowden, A. (2004). Fundamentals of

enzyme kinetics (3rd ed.). London: Portland
Press. ISBN 1-85578-158-1.

Garret, R.H.; Grisham, C.M., (2013). Biochemistry

5th Edition. Brooks/Cole, Cengage Learning,
20 Davis Drive , Belmont, CA 94002-3098,
USA

Miller, G.L., (1959). Analytical Chemistry: Use of

Dinitrosalicylic Acid Reagent for
Determination of Reducing Sugar. Retrieved
from
http://download.bioon.com.cn/upload/month_
1002/20100202_79e2638a4a8db64734c5Qy
CZjgBzadbY.attach.pdf

Walsh, C. (1979). Enzymatic reaction mechanisms.

San Francisco: W. H. Freeman. ISBN 0-
7167-0070-0.

Wang, N.S., (n.d.). Starch Hydrolysis by Amylase.

Retrived from
http://www.eng.umd.edu/~nsw/ench485/lab5.
htm

I hereby certify that I have given substantial

contribution to this report.

__________________________

Biochemistry 34.1 Enzyme Kinetics Page 6 of 6