Peroxidase Kinetics and the Inhibition of P4502A6 by Ethanol
Heather Wilson, Jorge Tovar, Gio Bernardo
May 8, 2018
Dr. Gregory Raner
BCHM 451-001
Liberty University Biology and Chemistry Department
1971 University Blvd, Lynchburg, VA
Introduction
Enzymes are of critical importance for normal physiology and in chemical reactions that
happen at a cellular level every day in the human body. Cytochromes, for instance, are enzymes
that contain a heme prosthetic group (1). Many of these have been characterized and determined
to have roles in critical processes like energy production, toxin metabolism, and apoptosis (2,3).
The liver is one of the major sites in the body for heme synthesis, and 50% of the synthesized
heme will be part of a special type of cytochrome proteins, the Cytochrome P450 family. These
cytochromes are the major enzymes involved in drug metabolism and processing in the liver (1).
Cytochrome P4502A6 is commonly known for metabolizing nicotine, which leads to toxic
byproducts that can induce oxidative stress and liver damage (4). Furthermore, another important
group of enzymes are the peroxidases. Human saliva is known for its high content of peroxidases
such as salivary peroxidase (hSPO) and myeloperoxidase (hMPO) (5). These perform host
defense mechanisms by oxidizing thiocyanate in the presence of hydrogen peroxide, to create a
powerful antimicrobial solution in saliva (5). Studying the kinetics of enzymes like peroxidases
and cytochromes has become a very important topic of research due to their important medical
applications. For instance, Horseradish peroxidase is frequently used in medicine as a reporter
enzyme in histochemical staining and diagnostic assays such as ELISA (6).
Enzyme kinetics is an important area of research because there are many factors that can
influence enzyme function. These have to be taken into account so that appropriate research can
be done on the way a determined enzymatic reaction proceeds with certain conditions (7). In this
experiment, the kinetics for Horseradish peroxidase and Cytochrome P4502A6 will be analyzed
and treated with corresponding inhibitors to determine how activity of these enzymes is affected.
Methods
Peroxidase Kinetics
Three different samples were prepared from the HRP stock (0.10mg/mL). The samples
were a stock concentration, 1:5 dilution, and 1:50 dilution. These were diluted with 50 mM
phosphate buffer at a pH of 7.0. In order to determine the suitable concentration for the kinetics
reaction, these three concentrations were tested in an enzymatic assay with guaiacol. The assay
was done by using 400 μl guaiacol, 500 μl hydrogen peroxide, 50 μl buffer (pH 7.0), 5 μl
peroxidase, and 45 μl water. Once the suitable HRP concentration was determined, a guaiacol
oxidation assay was then performed in 2mM hydrogen peroxide, with guaiacol concentrations
ranging from 0.5mM to 2mM. Additionally, a 5mM concentration was included to confirm
saturation kinetics, providing an approximate V for the pure enzyme using a guaiacol as a
max
substrate.
Cytochrome P4502A6 Inhibition by Ethanol
Five solutions were prepared in duplicate containing p-Nitrophenol concentrations
ranging from 10-100 μM, phosphate buffer 50mM at a pH of 7.4, 1mM NADPH, 2μL of
Cytochrome P450, and water to achieve a final volume of 500μL. One of these sets was treated
with 3% ethanol (final concentration of518mM), an inhibitor of CytochromeP450 activity.
NADPH was not added until the start time of the reaction. The samples were heated at 30°C for
one minute and then NADPH was added. The reactions were terminated after 20 minutes by
placing them on ice and adding 200 μL 8% perchloric acid. The samples were on ice for ten
minutes and were then centrifuged for 5 minutes. The supernatant was isolated for HPLC
analysis.
Results
The suitable concentration for the guaiacol oxidation kinetics assay was a 1:50 dilution
from the provided stock. Using this 0.002 mg/mL concentration, the assay was performed with
six different concentrations of guaiacol (Table 1). The Michaelis-Menten plot from the data
provided an estimated K and V
m max values. The V was determined as1 mM/min, and this was
max
used to calculate the K value of 1.1 mM. The 5 mM guaiacol concentration exhibited saturation
m
kinetics as expected due to the 1 mM/min V observed (Figure 1a). As observed in the
max
Michaelis-Menten graph (Figure 1a), the velocity increased proportionally to the substrate
concentration until V was reached.
max
A standard curve was made from each peak that resulted in the HPLC. As shown in
Figure 2, the enzymatic activity of P4502A6 was inhibited by ethanol. In the reaction with no
inhibito present, the Vmax m
in the Michaelis-Menton plot was 0.046 μM/min, and the K was 20
μM. Using the Lineweaver-Burke plot, it was determined that ethanol acted as a noncompetitive
inhibitor because there was a reduced Vmax of cytochrome P4502A6. To calculate the K , α was
I
first determined by dividing V . The calculated concentration of ethanol was 0.25 M
max/Vmax app
from using the density of ethanol. This value was used to then calculate K as 0.30 from the
I
equation [I]/α-1.
Table 1. Working concentrations used for the guaiacol oxidation assay
[Guaiacol]
(mM)
Buffer (μl) Guaiacol
(μl)
Hydrogen
Peroxidase
(μl)
Enzyme
(μl)
Water ( Slope μl)
(μM/min)
0.5 50 20 200 5 725 0.249
0.75 50 30 200 5 715 0.382
1.0 50 40 200 5 705 0.466
1.5 50 60 200 5 685 0.654
2.0 50 80 200 5 665 0.803
5.0 50 200 200 5 545 0.990
Figure 1. Michaelis-Menten plot (A) and Lineweaver-Burke plot (B) of the enzymatic
activity of HRP with guaiacol and peroxide. Using an enzyme concentration of 0.002mg/mL,
it was determined that the V for HRP in Figure a was 1 mM/min, and the K was 1.1 mM. The
max m
rate of conversion of guaiacol into tetraguaiacol proportionally increased as substrate increased,
until the saturation concentration was achieved.
Figure 2. Michaelis Menten plot (A) and Lineweaver-Burke plot (B) of Cytochrome
P450A26 kinetics with and without inhibitor. The activity of Cytochrome P4502A6 was
hindered by ethanol in a non-competitive manner. In Figure A, the Vmax of the sample without
inhibitor was 0.046 μM/min, and the K was 20 μM. The V
m max of the inhibited sample was 0.025
μM/min, and the K was 35 μM. In Figure B, noncompetitive inhibition was observed. The
m
sample without inhibitor also shows a higher V as opposed to the lower V as seen in the
max max
inhibited solution.
Discussion
In the HRP kinetics experiment, the 5 mM concentration resulted in a 0.99 mM/min V ,
max
meaning it converted guaiacol to tetraguaiacol the quickest. Saturation kinetics was observed as
seen in Figure 1 because there was no further increase in rate as the substrate concentration
increased. The stock concentration and the 1:5 dilution of HRP enzyme generated an absorbance
reading out of the linear range of the guaiacol oxidation assay. Therefore, the 1:50 concentration
of the stock was used in order to determine the substrate concentration at which HRP is fully
saturated. If a more concentrated enzyme solution would have been used, this rate would not
have been achieved as quickly.
It was determined that ethanol acted as a non-competitive inhibitor of the cytochrome
P4502A6 enzyme. The Lineweaver-Burke plot was used to make this conclusion; however, the
1/Km values did not overlap at the x-intercept. The differing 1/V was accurate in depicting non-
max
competitive inhibition. The solution with no inhibitor displayed a higher V because
max
paranitrophenol was able to bind to the enzyme. In the samples with ethanol, the enzyme could
have experienced conformational change due to allosteric binding of the inhibitor. Therefore, this
explains the reduced enzymatic activity and reduced V of the solutions with ethanol. The
max
calculated K for ethanol was 616.6 mM, indicating that there needs to be a higher concentration
I
of ethanol to have a more significant influence on the enzymatic activity of Cytochrome
P4502A6.
Conclusion
Determining the kinetics for HRP has high importance in the scientific community
because HRP is commonly used as a reporter enzyme (6). In this experiment, it was determined
that a 1:50 dilution of concentrated HRP enzyme worked best for determining the maximum
velocity of the enzyme. Therefore, this result allows future research on the kinetics of HRP with
other substrates rather than guaiacol to see if other substrates influence the V of the enzyme.
max
The importance of understanding enzymatic inhibition can be seen through experiments with a
noncompetitive inhibition such as ethanol and its effects on cytochrome P4502A6. According to
this experiment, ethanol affected the maximum rate at which the enzyme was able to convert
paranitrophenol to paranitrocatechol. However, the molecular mechanism for this process was
not investigated in this experiment.
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