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Kinetic Mechanisms of Tyrosinase Inhibitors in the Common White

Mushroom (Agaricus bisporus) with Tropolone.


Edward B. Yokana
CHE 341 Section: 702
Department of Chemistry, DePaul University, 1110 W. Belden Avenue, Chicago, IL 60614
November 5th, 2014

Introduction
A kinetic study of the inhibition of the enzyme tyrosinase, as found in the different parts of the common
white mushroom Agaricus bisporus, by tropolone was conducted. The enzyme kinetic

parameters , , and were determined by varying catechol concentrations, and thus,

substrate concentrations with the enzyme-catalyzed reaction. The effect of changing either enzyme or
substrate concentrations on initial reaction rate was analyzed. Modern scientists estimate kinetics
parameters from experimental kinetics data using non-linear regression. However, the kinetic
parameters that described the inhibition by tropolone were evaluated by using the Eadie-Hofstee
equation. Linear transformation was used to study the inhibition mechanism of the tropolone in
tyrosinase from the specific mushroom tissue and compare it to a published Ki value for the same
inhibitor. The Km of tyrosinase from the specific mushroom tissue was compared to its literature value.
Tyrosinase is a copper-containing enzyme present in microorganisms, plants and animals. [1] In fungi,
tyrosinase catalyzes the rate-limiting step in the formation of the pigment melanin from tyrosine. [2]
Tyrosinase catalyzes oxidations of tyrosinase to L-3,4-dihydroxyphenylalanine (L-DOPA), the substrate,
to o-dopaquinone then to dopachrome, the primary pigment found in human red hair. [1]

Tyrosinase is also responsible for the browning of potatoes and many other foods. This is because
tyrosinases catalyze the oxidation of monophenols and o-diphenols to o-quinones, the precursor
compounds of the brown-colored pigment melanin.[3] Melanins are heterogeneous polymers of
polyphenolic character and little defined structure with color typically ranging from yellow to dark
brown. [1] Melanins originate the enzymatic browning in fruits and vegetables as well as the
pigmentation of animals. [1]] Tyrosinase has been ascribed other functions apart from melanin
production, including the detoxification of host plant defensive phenols for symbiotic bacteria, the
sclerotisation of insect cuticles, and the syntheses of amino acid based antibiotics. [2]
A complication of working with enzymes is that they can exist in different forms: isomers. It is unknown
how many isomers of tyrosinase exist in the common white button mushroom, and multiple isomers of
tyrosinase exists in Agaricus bisporus. However, the protein concentration of the mushroom extract for
the experiment was measured and the tyrosinase isomers were identified using gel electrophoresis. In
the cell, tyrosinase is activated by hydrolysis catalyzed by an unknown protease. Thus, tyrosinase is
activated by the detergent sodium dodecyl sulfate (SDS) for this experiment.

Materials and Methods


Preparation of Extracts:
The characterization of the localization of tyrosinase isozymes was conducted using the common white
button mushroom, Agaricus bisporus. The mass of mushroom collected from the other dissected
mushrooms was 7.466 g of the skin region (K1 ) and was used for the analysis of this experiment. Prior
to the lab, a buffer solution of pH 6.5 50 mM phosphate extraction buffer was prepared by the lab
instructor for the protein extraction. For every gram of mushroom tissue weighed, 3 mL of pH 6.5
phosphate extraction buffer was used (22.4 mL of buffer solution).
The collected mushroom tissue was chopped into small pieces. The mushroom sample was transferred
to a mortar and frozen with liquid nitrogen and grinded with a pestle to pulverize it. The buffer was
added to the mortar and the solution was allowed to homogenize for 1 minute. The extract was to
undergo gravity filtration. Upon, filtration, six 1.5 mL aliquots of the filtrate were collected and labeled
(702, 1, DS and EY). Five of those samples were stored in a freezer at -20C to maintain cell activity for
later use.
Kinetics Measurements:
The enzymatic activity of the mushroom extracts were measured under the conditions of a 0.1 % SDS
solution, 15 mM catechol, 50 mM pH 6.5 phosphate buffer and enzyme at 23C. The reactions with an
inhibitor used a 100 M solution of tropolone. The spectrophotometer used for this experiment was
Genysys-20, manufactured by Thermo Scientific. The spectrophotometer was warmed up for about 25
minutes at a wavelength of 400 nm, the wavelength that the reactions were observed at.
For each tube, the respective volume of catechol, SDS solution and buffer solution were used as a blank
for each trial in the spectrometer. Then the extract would be added to the cuvette to start the reaction.
After mixing the solution, the cuvette was placed in the spectrometer and the initial absorbance would
be recorded. The timer was started and the absorbance was recorded at 10-second intervals for one
minute. The previous steps were repeated for the 8 different concentrations. The definition of CU
activity units is one CU equals the increase in absorbance at 400 nm per minute in one microliter of
assay volume (1 1400 min1 ) when catechol is the substrate. The definition of relative
activity is the activity (in CU) in the amount of enzyme added (L).

Estimation of Kinetics Parameters:


The relationship between and [S] is most commonly expressed as the Michaelis-Menten Equation:

[]
( )
+ []

1
+ []
( + []) + [] []
( ) = (
)
=
=
=
+
[]
[] []

[]

[]



=
+ 1 =
+ =
+
[]
[]
[]

+ ( )
[]

Due to not having access to a scientific data processing software, the Eadie-Hofstee method was the
linear analysis method used to obtain parameters. Microsoft Excel software was used for linear analysis
by creating a calculated column of v0 divided by catechol concentration (v0/[S]) to then plot a linear
trend through the values. The mechanism for 3.0 mM tropolone and tyrosinase enzyme was confirmed
to be competitive inhibition. The equilibrium constant for inhibitor binding for competitive inhibition
was defined as =

[][]
[]

. The equilibrium constant modifies the apparent / , but not the .

Thus, was determined by using the equation:


V
( Kmax )
Vmax
m
(
)
=
[I]
K m app
1+
Ki
Microsoft Excel software was used to use the LINEST function to calculate the respective uncertainty
values of the parameters.

Results
Opening:
A kinetic study of the inhibition of the enzyme tyrosinase, as found in the different parts of the common
white mushroom Agaricus bisporus, by tropolone was conducted. The enzyme kinetic

parameters , , and were determined. The kinetic parameters that described the tyrosinase

reaction of catechol, with and without 3.0 mM tropolone, were evaluated by using the Eadie-Hofstee
equation. This method of linear transformation was used to study the inhibition mechanism of the
tropolone in tyrosinase from the specific mushroom tissue and compare it to a published Ki value for the
same inhibitor. The Km of tyrosinase from the specific mushroom tissue was compared to its literature
value. The goals of the experiment were successfully achieved, but lacks validity. The mechanism of the
tyrosinase inhibition was determined to be competitive inhibition. The inhibitor binding for competitive

inhibition modified , but not which held true. However, the uncertainty for the experimentally

determined parameters was very high and the plotted data shown in Figure 2, the Eadie-Hofstee plot,
lacked the linearity to be able trust the results.

0.8

Absorbance at 400 nm

0.7

Series2

0.6

Series3

0.5

Series4

0.4

Series5

0.3

Series6

0.2

Series7
Series8

0.1

Series9

0
0

10

20

30

40

50

60

70

Series1

Time (s)

Figure 1. Absorbance vs Time plot. This absorbance vs time plot shows the kinetics raw data obtained
during the experiment. Absorbance readings were observed at a wavelength of 400 nm. The quality of
the raw kinetics data was determined from each of the trendlines r-squared value. Some data points
were removed from plot to improve linearity. The legend distinguishes the trendlines that correspond
with each trial. Series refers to the data series of its respective trial (i.e. Series 1 corresponds to Trial
1).
Eight samples were prepared with an initial concentration series of catechol, the substrate, respectively
of 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, and 0.39 mM. Eight samples were prepared with and without an
inhibitor. The rate measurements of each sample served as its and was determined by converting
slope values of the trendlines, shown in Figure 1, to relative activity (U/L). Each slope had a linear
relationship as determined through the r-squared value. The initial catechol concentration for each
sample in Part C (in mM) and Relative Activity (U/L), with and without an inhibitor, are shown in Table
1.
Table 1: Relative Activity and Initial Catechol Concentration
Sample
1
2
3
4
5
6
7
8

Initial Catechol Relative Activity Relative Activity


Concentration Without inhibitor With inhibitor
(mM)
(U/L)
(U/L)
50.0
1.95
20.2
25.0
5.39
7.34
12.5
13.2
5.49
6.25
6.86
4.12
3.13
4.24
4.74
1.56
1.03
1.22
0.780
0.807
1.76
0.390
0.379
0.557

9
8
7

v0 (U/L)

6
5

[I] = 0.0 mM

[I] = 3.0 mM

3
2
1
0
0

v0/[S] (U/L/mM)

Figure 2. Eadie-Hofstee plot. An Eadie-Hofstee plot of the kinetics data, with and without an inhibitor,
shown in Table 1 was generated. A linear trendline through the two separate groups of data was
plotted. The equation of the linear trendline corresponding to the activity in the absence of an inhibitor

was = 1.011 + 8.963 ( 2 = 0.5698). The r-squared value indicates that there was no linear
trend between v0 and v0/[S] for the activity with no inhibitor. The equation of the linear trendline
corresponding to the activity with an inhibitor was = 1.143 + 8.3441

( 2

= 0.7959). The r-

squared value indicates that there is a slight linear rend between v0 and v0/[S] for the activity with an
inhibitor, but some data was removed to increase the linearity of the relationship. The inhibitor used
was 3.0 mM tropolone in a competitive inhibition mechanism (as indicated in the legend). The inhibition
constant was determined to be = 1.2 0.3.
The estimates of the parameters, ,K m , and / for tyrosinase reaction of catechol at pH = 6.5,
were made by referencing the linear equations generated in Figure 2. For estimation of kinetics
parameters, the Eadie-Hofstee plot was used. Parameters / and could have been directly
read-off as the x- and y- intercepts of the trendline respectively. However, the slope and intercept
functions were used in Microsoft Excel to calculate the values. This was done for the tyrosinase reaction
of catechol with and without 3.0 mM tropolone inhibitor. In the absence of the 3.0 mM tropolone,
the , the y- intercept, was determined to be 9 2 U/L. The K m , equal to negative slope of the plot,
was determined to be 1.0 0.4 mM. The / , the x- intercept, was determined to be 9 4
U/L/mM. In the presence of 3.0 mM tropolone, the was determined to be 9 1 U/L. The K m was
determined to be 1.2 0.3 mM. The / was determined to be 7 2 U/L/mM.
Table 2: Relative Activity and Initial Catechol Concentration
Parameter
U
)
L
K m (mM)

Vmax

(
)
Km

Vmax (

Value
(no inhibitor)

Value
(with 3.0 mM tropolone)

92

91

1.0 0.4

1.2 0.3

94

72

Figure 1 shows an absorbance vs time plot of the kinetics raw data obtained during the experiment. The
quality of the raw kinetics data was determined from each of the trendlines r-squared value. The rsquared values of each of the trendlines were close to 1, but that was after a few data points being
removed. The data points were removed to improve the linearity of their corresponding trendline. Eight
samples were prepared with an initial concentration series of catechol, the substrate, respectively of 50,
25, 12.5, 6.25, 3.13, 1.56, 0.78, and 0.39 mM. The rate measurements of each sample served as its
and was determined by converting slope values of the trendlines, shown in Figure 1, to relative activity
(U/L). The initial catechol concentration for each sample in Part C (in mM) and Relative Activity (U/L)
are shown in Table 1 for the tyrosinase reaction in the absence of 3.0 mM tropolone and in the presence
of 3.0 mM tropolone.
An Eadie-Hofstee plot was generated of the kinetics data, shown In Figure 2. Two linear trendlines were
plotted through each set of data to generate linear equations for the determination of the kinetics
parameters. A few data points had to be dropped from each set of data to improve linearity and to even
get the slope of the equations to be negative. It was important for the slope to be negative to atleast
calculate a realistic Michaelis constant. Even then, the r-squared value of the data corresponding to the
tyrosinase reaction with no inhibitor and with an 3.0 mM tropolone was 0.5698 and 0.7959 respectively
indicating that there was no linear relationship observed. The quality of the data was very bad.
Regardless of the quality, the estimates of the parameters, ,K m , and / were made by
referencing the linear equations generated in Figure 2 . The linear equation of the generated EadieHofstee plot for the tyrosinase reaction with no inhibitor and with 3.0 mM tropolone was =

1.011 + 8.963 ( 2 = 0.5698) and = 1.143 + 8.3441 ( 2 = 0.7959) respectively.


Parameters / and could have been directly read-off as the x- and y- intercepts of the
trendline respectively. However the slope and intercept of the generated linear equation was used to
estimate the parameters. The results of the estimation are shown in Table 2 with their corresponding
uncertainties. In the absence of the 3.0 mM tropolone, the , the y- intercept, was determined to be
9 2 U/L. The K m , equal to negative slope of the plot, was determined to be 1.0 0.4 mM.
The / , the x- intercept, was determined to be 9 4 U/L/mM. In the presence of 3.0 mM
tropolone, the , the y- intercept, was determined to be 9 1 U/L. The K m , equal to negative slope
of the plot, was determined to be 1.2 0.3 mM. The / , the x- intercept, was determined to be 7
2 U/L/mM. The overall quality of the kinetics data was not valid enough to have confidence in the
estimations of the kinetics parameters, especially because a few data points had to be removed to
improve linearity. The kinetics parameters were statistically different with and without inhibitor (shown

in Table 2). The inhibitor binding for competitive inhibition modified , but not which held true.

However, the uncertainty for the experimentally determined parameters is very high and the plotted
data shown in Figure 2, the Eadie-Hofstee plot, lacks the linearity to be able trust the results.

Discussion
The mechanism of the tyrosinase inhibition was determined to be competitive inhibition. Referencing
Figure 2, and the two trendlines, to determine the mechanism was difficult because the quality of data
was bad and the trendlines lacked linearity. Thus, the mechanism was determined through the
experimentally determined parameters
inhibition always modifies

, ,

and . The inhibitor binding for competitive

but not , which held true as shown in Table 2. Thus, the mechanism

of the tyrosinase inhibition was confirmed competitive. The kinetics data was analyzed using graphical
methods of analysis. Using the Eadie-Hofstee plot, the apparent kinetic constant in the presence of an
inhibitor was determined. After classifying the kinetic mechanism, the inhibition kinetic data was used
to calculate the apparent inhibitor dissociation constant Ki. The inhibition constant was determined to
be = 1.2 0.3. The value of for the mechanism of competitive inhibition was less than
literature value of 2.0 mM for for competitive inhibition. [4] This could be due to the uncertainty for
the experimentally determined parameters being very high and the plotted data shown in Figure 2, the
Eadie-Hofstee plot, lacked the linearity to be able trust the results.
The tyrosinase kinetic parameters ( ) were to be determined by constructing an EadieHofstee plot and using linear analysis. The linear equation of the Eade-Hofstee plot was still used to
determine kinetic parameters . The experimentally determined K m value of the tyrosinase
reaction in the presence of no inhibitor was 1.0 0.4 mM compared to the literature value of 5 mM
(when using catechol as the substrate). [5] The experimentally determined K m was a much lower value
but the kinetics data wasnt of good quality. However, the linear equation of the Eadie-Hofstee plot was
still used to determine the kinetic parameters. The data wasnt of good quality because the EadieHofstee plot of the enzyme reaction data with no inhibitor was not linear. Thus, the data could not be
trusted.
For future experiments I suggest more replicates of the enzyme reaction be recorded. A lot of the data
recorded in lab had to be dropped in hopes of improving the overall results of the experiment. Eight
replicates of each reaction, with inhibitor or not, was measured. However, three measurements had to
be dropped leaving only five data measurements to analyze. The data cannot be trusted when there is
barely any data. More replicates of the enzyme reactions would give more data to work with.

References
(1) Mauracher, Stephan Gerhard, et al. Latent and Active Abppo4 Mushroom Tyrosinase
Cocrystallized With Hexatungstotellurate(VI) In A Single Crystal. Acta Crystallographica: Section
D (international Union Of Crystallography-Iucr) 70.9 (2014): 2301-2315. Academic Search
Complete. Web. 13 Oct. 2014.
(2) RodrguezLpez JN, Tudela J, Varn R, GarcaCarmona F, & GarcaCnovas F (1992)
Analysis of a kinetic model for melanin biosynthesis pathway. Journal of Biological Chemistry
267(6):38013810.
(3) Chang, Tsong-Min. "Tyrosinase and Tyrosinase Inhibitors." Journal of Biocatalysis &
Biotransformation 01.02 (2012).
(4) Espn JC, Wichers HJ. Slow-binding inhibition of mushroom (Agaricus bisporus) tyrosinase
isoforms by tropolone. J. Agric. Food Chem. (1999);47:26382644.
(5) Zhang, X; Flurkley, W.H. Characterization of Tyrosinase from the Cap Flesh of Portabella
Mushrooms. J. Food Biochem. (1999).

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