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

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
and Km were computed as follows:
The absorbance of each solution was
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
pepsinogen, is another enzyme found in the stomach
which works at pH of 2.0 (Campbell, 2005).
The α-amylase catalyzes the breakdown of
starch or polysaccharides into disaccharides and
eventually converting them into monosaccharide like
glucose and maltose (Enzymes Essentials, 2014).
These polysaccharides are connected with α-1,4-
glucosidic linkages. These bonds are the specific
structures in the starch by which the α-amylase
hydrolyzes (Wang, n.d.).
Another enzyme assay to detect the
presence of reducing sugars is done with 3,5-
dinitrosalicylic acid (DNS). In a basic solution, the
reducing sugar forms an aldehyde (glucose) or
ketone (fructose) group. The aldehyde or ketone
group then reduces the DNS to form 3-amino-5-
nitrosalicylic acid, which has a different measured
light absorbance with DNS at 540 nm (Miller, 1958)
The reaction rate of enzyme catalysis can be
altered with the use of inhibitors, substances that
interferes with the reaction between the enzyme and
the substrate. The inhibitors compete with the
substrate either to the active site (competitive
inhibitors) or to the other side of the enzyme (non-
competitive inhibitors) (Campbell, 2012).
A standard curve was prepared by using
varying starch concentration while keeping the iodine
concentration constant. The reaction of starch and
iodine, I3 + starch  starch-iodine complex,
produced a dark blue solution. Higher concentration
of the starch yields higher product concentration in
order to maintain its equilibrium constant. Thus
solutions that had higher amounts of starch had
higher absorbance reading using spectrophotometer
due to the intensity of the dark blue color at 620 nm,
the wavelength at which the absorptivity of the color
of the complex is highest. The plot of the starch
concentration vs absorbance had a positive slope
because of their direct proportionality.
The addition of saliva decreased the
concentration of starch in parts B, C, and D of the
experiment. The enzyme amylase in the saliva
facilitates the break down by hydrolysis of starch into
either glucose, a carbohydrate with one subunit, or
maltose, a carbohydrate with two subunits. These
subunits, called monosaccharide and disaccharide
respectively, do not form complex with iodine. At
higher enzyme activity, more starch is hydrolyzed.
Therefore, at same initial starch concentration,
solutions with higher enzyme activity yields lower
concentration of remaining starch to form complex
with iodine, and, as a consequence, lower
absorbance readings.
The effect of concentration of substrate, the
reactant that is activated by the enzyme, on the
velocity of enzyme activity was determined by
measuring the concentrations of different solutions of
Biochemistry 34.1 Enzyme Kinetics
starch and amylase incubated at different amounts of
time. The graph of starch concentration vs time
showed that at time=0, the slope is most negative,
and it becomes less and less negative as it
approaches time=infinity. This is due to the high
velocity of the enzyme activity or the high decrease in
starch concentration per unit time when the
concentration of the starch is high. As the starch is
consumed, the starch concentration and the velocity
become lower. More relationship was observed by
plotting the starch concentration against velocity. As
the concentration becomes higher, the velocity also
becomes higher. The initial increase in velocity is
high and the velocity stabilizes at a certain value
known as the Vmax, or the highest velocity an enzyme
is capable of in a given temperature and pH. Ideally,
linear relationship is observed from the graph of the
reciprocal of the starch concentration against the
reciprocal of the velocity, and the equation of this line
may be used to obtain the Km and the Vmax. Km is
the dissociation constant and a measure of the
affinity of an enzyme to a substrate which is also
equal to the negative reciprocal of the x-intercept of
the equation. The Vmax is the reciprocal of the y-
intercept of the said equation.
The effect of temperature and pH on the
enzyme activity was also tested by measuring the
concentration of the starch-iodine complex after
reacting starch and amylase with iodine solution at
different temperature and pH conditions. This way,
the optimum conditions was determined by
calculating the minima of the temperature vs starch
concentration and pH vs starch concentration plots.
The minima indicate the conditions at which the
starch-iodine complex concentration is minimal,
which are the temperature and pH at which the
enzyme activities are highest.
VII. Conclusions and Recommendations
Using the Lineweaver-Burke equation, the
data were plotted to determine the relationship
between the factors affecting the enzyme activity and
its kinetics. It can be concluded that the activity on an
enzyme to function is affected by concentration,
temperature, and the pH level of the solution.
It can also be said that there is an optimum
temperature and pH level for an enzyme to function
at its maximum. Any value above or below the
optimum level would greatly affect the enzyme
activity. From the experiment, the optimum
o
temperature and pH level are 50.2 C and 6.5
respectively.
The Michaelis-Menten constants (Km and
Vmax) were also determined having values of
0.016229 and 0.06871 respectively after plotting the
1/[S] vs. 1/v.
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