Introduction to Data Gathering and Analysis Using Enzyme Kinetics Data as

Experimental Models
Remiel N. Alquileta
Jamica Romaine B. Ambion
Ma. Celline Victoria Ancheta
De La Salle University-Dasmarias
Dasmarias, Cavite Philippines

INTRODUCTION
Enzyme kinetics is the study of the chemical reactions produced or catalyzed by the enzymes. It
describes the quantitative aspects of enzyme catalysis and the rate of substrate concentration into products.
Chiefly, enzymes are globular proteins that act as a catalyst of a biochemical reaction; virtually all cellular
reactions are mediated by protein catalysts that manipulate other molecules. The study of enzyme kinetics can
reveal the catalytic mechanism of a specific enzyme, how a drug can inhibit an enzyme, how the activity is
controlled, and the role in metabolism.
An equation that describes the kinetics of enzyme-catalyzed reaction is known as Michaelis-Menten
equation in which the velocity of reaction multiplied to the substrate concentration, divided to the MichaelisMenten constant plus the substrate concentration. This equation yields to a hyperbolic curve and the velocity of
the reaction is the asymptote.
Another equation that can be helpful in the enzyme kinetics is the Lineweaver-Burke equation. It is
derived from the Michaelis-Menten equation and can be transformed into a linear equation that provides a faster
and easier identification of reaction than the hyperbolic curve which results from the double reciprocal of the
equation. There are three types of inhibitor under the Lineweaver-Burke equation. A competitive inhibitor is
almost closely related to the substrate. This competition results to higher saturation point and the velocity of
reaction remains unchanged. The noncompetitive inhibitor binds to the site in enzyme other than the active site.
It yields to lower velocity reaction while saturation point remains unchanged. The graph of noncompetitive is
always intersecting at the x axis. The uncompetitive inhibitor binds to the enzyme substrate complex. This
results both to lower velocity reaction and saturation point. The graph is always parallel.

MATERIALS AND METHODS

This experiment aims to gather data and analyze how enzyme kinetics work and through the procedures
which will be elaborated throughout, the goal is fulfilled.
Preparation of Catalase Extract
A 10g pork liver was used for this, weighed and then rinsed momentarily in cold distilled water and was
then transferred to a blender for homogenization. Before homogenization, 100 mL of phosphate buffer was added
to the blender and was blended for 30 seconds. The product which was the homogenate was filtered through a
filter paper and the resulting suspension was thinned down with cold buffer to final concentration of 8g per liter of
buffer. The end product was labeled Catalyze Enzyme Stock.

Preparation of Hydrogen Peroxide Substrate

This procedure focuses on the preparing of 0.25 M hydrogen peroxide with different concentrations of
phosphate buffer. The following concentrations of phosphate buffer were 0.010, 0.015, 0.020, 0.025, 0.05, 0.075,
0.10, 0.15 and 0.20 M. The end product of each mixture must have the measurement of 20 mL and the following
formula was used.
C1V1

= C2V2

Wherein C1 is the stock concentration, V 1 is the volume of stock needed, C 2 is the desired concentration
and V2 stands for the desired volume.
Catalase Assay
After preparing the hydrogen peroxide concentration and the enzyme stock, this last procedure will
determine the enzyme kinetics. 20 mL of hydrogen peroxide were prepared in ten 50-ml beakers each and were
immersed in the water bath with a temperature of 30C for 10 minutes. When the water bath is done, the assay
procedure was prepared. Using forceps, a filter paper disc was immersed in the enzyme stock for 10 seconds,
blotted both sides of the disc and was placed at the bottom of the beaker were the 0.2 M substrate was contained.
A timer was used and the paper disc was timed the moment it reached the bottom of the beaker. Once the disc
reached the surface of the solution, the time was determined and recorded. The assay requires three duplicates.
The remaining hydrogen peroxide concentrations are then used to repeat the assay procedure.

RESULTS AND DISCUSSION

Table 3.1. Catalase Reaction on Different Substrate Concentration in Relation to Time and Velocity

Figure 3.1. MM Plot of Reaction Velocity versus Substrate Concentration in an Enzyme-Catalyzed

Reaction

Figure 3.1. MM Plot of Reaction Velocity versus Substrate Concentration in an Enzyme-Catalyzed Reaction
shows that the reaction rate is correlated to substrate concentration where the reaction rate speeds up as the
substrate concentration increases but only to a certain point. Enzyme-Catalyzed reaction limits the increase of
velocity through the presence of the finite number of enzymes available.

1/[S]

1/V

10

909.09

156.25

3.3

50

2.5

20

16.67

1.67

8.33

1.43

6.67

1.25

1.11

3.57
Table 3.2. Reciprocal Data

Figure 3.2. LB Plot of Reaction Velocity versus Substrate Concentration in an Enzyme- Catalyzed
Reaction
The LineweaverBurk plot in Figure 3.2. along with the equation of the linear regression line and the correlation
index of r2 shows a non-linear relationship wherein values ranging from 0 to 1.0 has no relationship, while there is
a relationship with values ranging 100. On the other hand, Lineweaver-Burk plot is used to determine significant
terms in enzyme kinetics, such as Km and Vmax,.The y-intercept of such a graph is equivalent to the inverse of Vmax;
the x-intercept of the graph represents 1/Km. In other words, it is simply the reciprocal of both sides of the
Michaelis-menten equation. It also gives a quick, visual impression of the different forms of enzyme inhibition.

Figure 3.3. SIGNIFICANT RELATIONSHIP BETWEEN THE MORALITY OF THE HYDROGEN PEROXIDE
SOLUTION AND THE MEAN TIME REQUIRED FOR THE DISC TO FLOAT USING AN APPROPRIATE
STATISTICAL TOOL
The molarity of hydrogen peroxide serves as the independent variable while its mean time is the dependent
variable. It simply shows that the rate of reaction varies due to the fixed concentrations of hydrogen peroxide.

Vmax is the maximum velocity of the reaction. It has the same units as the reaction velocity (V). It is the
highest reaction rate that can be achieved at saturating substrate concentrations while Km is inversely related
to the apparent affinity of the enzyme for its substrate. Result shows that the Km obtained from this
experiment has a low numerical of 0.58M t herefore, a low numerical value of Km refers to a very high
affinity of interaction between the protein and its substrate. It takes a very small amount (i.e., low concentration)
of the substrate to reach 50% of the saturating concentration. Conversely, a high numerical value of Km is
indicative of a low affinity of the enzyme/transporter for its substrate. It takes a large amount (i.e., high
concentration) of the substrate to reach 50% of the saturating concentration. Thus, Km is a very useful parameter
by which the affinity of the protein for various substrates can be compared.

REFERENCES
(1) Retrieved September 16, 2016 from: http://www.biology-pages.info/E/EnzymeKinetics.htm
(2)
Retrieved
September
16,
2016
http://www.physiologyweb.com/calculators/michaelis_menten_equation_interactive_graph.html
(3)
Retrieved
September
16,
2016
https://en.wikibooks.org/wiki/Structural_Biochemistry/Enzyme/Michaelis_and_Menten_Equation
(4)
Retrieved
September
16,
2016
http://laurenperryman.weebly.com/uploads/7/0/7/3/7073304/cell_lab_report.pdf

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