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Objectives

1. Familiarize the application of enzyme kinetics in experimental study.


2. Familiarize and improve the use of computer application in solving enzyme kinetics.

Theorietical Framework

Basic Enzyme Reactions


Enzymes are catalysts and increase the speed of a chemical reaction without themselves
undergoing any permanent chemical change. They are neither used up in the reaction nor do they
appear as reaction products.

The basic enzymatic reaction can be represented as follows

where E represents the enzyme catalyzing the reaction, S the substrate, the substance being
changed, and P the product of the reaction.

Energy Levels

Chemists have known for almost a century that for most chemical reactions to proceed,
some form of energy is needed. They have termed this quantity of energy, "the energy of
activation." It is the magnitude of the activation energy which determines just how fast the reaction
will proceed. It is believed that enzymes lower the activation energy for the reaction they are
catalyzing. Figure 3 illustrates this concept.

The enzyme is thought to reduce the "path" of the reaction. This shortened path would
require less energy for each molecule of substrate converted to product. Given a total amount of
available energy, more molecules of substrate would be converted when the enzyme is present (the
shortened "path") than when it is absent. Hence, the reaction is said to go faster in a given period
of time.

Enzyme Substrate Levels

A theory to explain the catalytic action of enzymes was proposed by the Swedish chemist
Savante Arrhenius in 1888. He proposed that the substrate and enzyme formed some intermediate
substance which is known as the enzyme substrate complex. The reaction can be represented as:
If this reaction is combined with the original reaction equation [1], the following results:

The existence of an intermediate enzyme-substrate complex has been demonstrated in the


laboratory, for example, using catalase and a hydrogen peroxide derivative. At Yale University,
Kurt G. Stern observed spectral shifts in catalase as the reaction it catalyzed proceeded. This
experimental evidence indicates that the enzyme first unites in some way with the substrate and
then returns to its original form after the reaction is concluded.

Chemical Equilibrium

The study of a large number of chemical reactions reveals that most do not go to true
completion. This is likewise true of enzymatically-catalyzed reactions. This is due to the
reversibility of most reactions. In general:

where K+1 is the forward reaction rate constant and K-1 is the rate constant for the reverse reaction.

Combining the two reactions gives:

Applying this general relationship to enzymatic reactions allows the equation:

Equilbrium, a steady state condition, is reached when the forward reaction rates equal the
backward rates. This is the basic equation upon which most enzyme activity studies are based.
Factors affecting enzyme activity
Knowledge of basic enzyme kinetic theory is important in enzyme analysis in order both to
understand the basic enzymatic mechanism and to select a method for enzyme analysis. The
conditions selected to measure the activity of an enzyme would not be the same as those selected
to measure the concentration of its substrate. Several factors affect the rate at which enzymatic
reactions proceed - temperature, pH, enzyme concentration, substrate concentration, and the
presence of any inhibitors or activators.

Results and Discussions

Table 1. Raw Data for Calculation

km 42 mM
vm 10 mol/h

S V 1/s 1/v
0 0
10 1.923077 0.1 0.52
25 3.731343 0.04 0.268
50 5.434783 0.02 0.184
100 7.042254 0.01 0.142
200 8.264463 0.005 0.121
300 8.77193 0.003333 0.114
500 9.225092 0.002 0.1084
750 9.469697 0.001333 0.1056
1000 9.596929 0.001 0.1042

Here, Vmax represents the maximum velocity achieved by the system, at maximum
(saturating) substrate concentrations. KM (the Michaelis constant; sometimes represented
as KS instead) is the substrate concentration at which the reaction velocity is 50% of the Vmax. [S]
is the concentration of the substrate S. Then to be able to plot the Michealis Menten graph and
Lineweaver-Burk plot, we have to assume values of [S], V and get both their reciprocal.
Figure 1. Michaelis Menten Plot

Michaelis Menten
12

10

6
V

0
0 200 400 600 800 1000 1200
[S]

From the figure 1 above, we plot the [S] vs. V to get the Michealis Menten graph. The
above graph is true because even if we increase the substrate to infinity, the Vmax wouldnt get
beyond 10 mol/h. The Michaelis-Menten model is the one of the simplest and best-known
approaches to enzyme kinetics. It takes the form of an equation relating reaction velocity to
substrate concentration for a system where a substrate S binds reversibly to an enzyme E to form
an enzyme-substrate complex ES, which then reacts irreversibly to generate a product P and to
regenerate the free enzyme E.
Figure 2. Lineweaver-Burk Plot

LineweaverBurk plot

0.6
y = 4.2x + 0.1
0.5 R = 1
0.4

0.3
1/V

0.2

0.1

0
-0.04 -0.02 0 0.02 0.04 0.06 0.08 0.1 0.12
-0.1
1/[S]

Here in Figure 2, it showed the Lineweaver-Burk Plot. It is the linearization of Michaelis Menten
model. It is obtained by getting the double-reciprocal of both sides of the Michaelis-Menten
equation. The double-reciprocal plot is created by plotting the inverse initial velocity (1/V) as a
function of the inverse of the substrate concentration (1/[S]). The Vmax can be accurately
determined and thus KM can also be determined with accuracy because a straight line is formed.
The slope of the resulting line is KM/Vmax, the y-intercept is 1/Vmax, and the x-intercept is -1/KM.
The slope is 4.2. The y-intercept is 0.1. The x-intercept is -0.023

References

http://www.sciencedirect.com/science/article/pii/0922338X95912657
http://www.worthington-biochem.com/introbiochem/equilibrium.html
https://depts.washington.edu/wmatkins/kinetics/michaelis-menten.html