Kinetic Analysis of Tyrosinase

15 January, 2017

Emily Mulder

Peter Mulder, Lauren Kim, partners

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Data and Calculations:

The concentration of tyrosinase from the absorbance at 280 nm was found to be 0.06024

mg/mL according to Beers Law. The enzyme correction factor 0.833 mol was found by first

determining concentration using Beers Law, as seen in Equation 1. According to this law,

concentration equaled absorbance divided by both the molar absorption coefficient and the path

length of the cuvet. In this case, the molar absorption coefficient for dopachrome (a product of

this reaction) at 475 nm was 3600 M-1 cm-1 and the path length of the cuvet wass 1 cm. This

product was
Assay tyrosinase (g) A/min Enzyme Activity
1 3.0120 0.0169 0.01410 multiplied by
2 6.0241 0.0308 0.02567
3 12.0482 0.0586 0.04883 the total volume
4 18.0723 0.0929 0.07741
of the enzyme
5 24.0964 0.1159 0.09656
assay (3000L) to get the value of the correction factor.

A = lC (1)

Before kinetic constants could be evaluated, it was critical to find the correct
Table 1. Experimental change in absorbance per minute and enzyme activity

observed from assays containing varying quantities of tyrosinase.

concentration of enzyme to use for the assays. According to Table 1, the concentration of

tyrosinase in assay 5 provided the desired reaction rate within the range of 0.10 to 0.15 A/min.
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The amount of tyrosinase used in assay 5 (0.40 mL) was used to replicate the desired reaction

rate in the remaining assays of the experiment. Figure 1 associates the amount of tyrosinase in

the assays with the rate of the reaction, and suggests a linear relationship between these

variables.

f(x) = 0x + 0

Figure 1. Illustrates the variation of reaction rate with the quantity of

tyrosinase present in an assay.

Assa2. Representation
Table [L-dopa], of the 1/[L-dopa], per minute Rate,
A/min
change-1 in absorbance 1/Rate,
and the reaction rate
y mM mM mol/min min/mol
1 0.2536
corresponding 3.9432 of L-dopa.
to varying concentrations 35.269 0.0294
A volume of 34.0136
0.40 mL of tyrosinase was a
1
2
component 0.5071
of each assay. 1.9720 53.959 0.0449 22.2717
1
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82.669
3 1.0140 0.9862 6 0.0689 14.5138
104.90
4 2.0289 0.4929 36 0.0874 11.4416
113.37
5 2.5360 0.3943 19 0.0944 10.5932
125.68
6 3.8030 0.2630 82 0.1047 9.5511

The rate and absorbance of an assay containing a constant quantity of tyrosinase was

observed at varying concentrations of substrate, namely L-Dopa. This data, displayed in Table 2,

was used to create both a Michaelis-Menten plot and a Lineweaver-Burk plot, which can be

found in Figures 2 and 3, respectively.

Figure 2. illustrates the Michaelis-Menten Kinetics the reaction exhibited by

associating the reaction rate with varying concentrations of L-dopa. Examining

the plot, the Vmax= 0.11 and Km=0.76.

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Looking first at the plot representing Michaelis-Menten Kinetics observed as a result of

tyrosinase reacting with varying concentrations of L-dopa, Vmax was estimated at 0.11 mol/min

and from that, Km was likewise estimated to be approximately 0.76. These numbers were found

by considering the projected plateau of the plot in Figure 2, the rate at which was estimated to be

the maximum velocity (Vmax), and by then naming the concentration of L-Dopa at half of this

velocity Km.

f(x) = 6.68x + 8.1

Figure 3. compiles the collected data on the reaction rate at varying

concentrations of L-Dopa into a Lineweaver-Burk plot. The points of intercept

indicate that Vmax= 0.1234 and Km = 0.8247.

A second estimation for Vmax and Km for varying concentrations of L-Dopa was found

using a Lineweaver-Burk plot, as seen in Figure 3. The equation of the resulting trend line was
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used to find the x and y intercepts of the plot. Considering that the y intercept equaled the inverse

of Vmax, a Vmax of 0.1234 was found. Likewise, the x-intercept equaled the negative inverse of Km,

according to the Lineweaver-Burk representation of enzyme kinetics, resulting in a Km of 0.8247.

[D-dopa), 1/[D-dopa], Rate, 1/Rate,

Assay mM mM -1
A/min mol/min min/mol
1 0.2536 3.9432 11.4946 0.0096 104.1667
2 0.5071 1.9720 20.8803 0.0174 57.4713
3 1.0140 0.9862 38.4485 0.0320 31.2500
4 2.0289 0.4929 61.8753 0.0515 19.4175
5 2.5360 0.3943 75.1873 0.0626 15.9744
6 3.8030 0.2630 95.5037 0.0796 12.5628
The rate and absorbance of a constant quantity of tyrosinase was observed at varying

concentrations of substrate, namely D-dopa. This data, displayed in Table 3, was used to create

both a Michaelis-Menten plot and a Lineweaver-Burk plot, which can be found in Figures 4 and

5, respectively.

Table 3. Representation of the change in absorbance per minute and the reaction rate corresponding to

varying concentrations of D-dopa. A volume of 0.40 mL of tyrosinase was a component of each

assay.

From Figure 4, Vmax was estimated at 0.14 mol/min and from that, Km was likewise estimated to

be approximately 3.17. These values were found by following the same process outlined in the

discussion of Figure 2.
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Figure 4. illustrates the Michaelis-Menten Kinetics the reaction exhibited by

associating the reaction rate with varying concentrations of D-dopa.

Examining the plot, the Vmax=0.14 and Km=3.17.

Just as was done for varying concentrations of L-Dopa in Figure 3, a second estimation

for Vmax and Km for varying concentrations of D-dopa was found using a Lineweaver-Burk plot,

as seen in Figure 5. The equation of the resulting trend line was used to find the x and y

intercepts of the plot. A Vmax of 0.1488 and a Km of 3.7064 were found by a similar method to

that outlined in the discussion of Figure 3.

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f(x) = 24.91x + 6.72

Figure 5. compiles the collected data on the reaction rate at varying

concentrations of D-dopa into a Lineweaver-Burk plot. The points of intercept

indicate that Vmax=0.1488 and Km=3.7064.

Table 4 summarizes the results found from taking assays of varying concentrations of L-

Dopa both in the presence of the inhibitor, cinnamic acid, as well as without the inhibitor. The

inhibited reaction rate changed very slowly in correlation with more rapidly increasing

concentrations of L-dopa. The inhibitor kept the reaction rate nearly constant, whereas the

uninhibited reaction showed marked changes in reaction rate in response to the same changes in

substrate concentration.

Assa [L- 1/[L- A/min Rate, 1/Rate, Inhibited 1/ of

Table 4. Representation of tyrosinase activity and the change of absorbance in the presence
y dopa], dopa], mol/mi min/m Rate, Inhibited
mM mM-1 n ol mol/min Rate,
an inhibitor and at varying concentrations of L-dopa. The data for the uninhibited rate of each
min/mol
assay is the mean of the rates found by multiple groups for each assay.1
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1 0.2536 3.9432 0.0247 0.0356 28.1294 0.0206 48.5437

2 0.5071 1.9720 0.0373 0.0598 16.7364 0.0311 32.1543
3 1.0140 0.9862 0.0484 0.0738 13.5538 0.0403 24.8139
4 2.0289 0.4929 0.0552 0.0908 11.0156 0.0460 21.7391
5 2.5360 0.3943 0.0591 0.0971 10.2955 0.0492 20.3252
6 3.8030 0.2630 0.0702 0.1082 9.2421 0.0585 17.0940

Figure 6 uses the Lineweaver-Burk plot method to demonstrate the type of reversible competition

which cinnamic acid imposed. The way the trend lines representing the inhibited and non-inhibited

enzymes meet on the left x-axis suggests that cinnamic acid brought about noncompetitive inhibition. The

two trend lines met slightly below the x-axis and therefore did not perfectly reflect the pattern expected

from a noncompetitive inhibitor due to experimental error and small sample size. The data for uninhibited

reaction rate in Table 4 was compiled and averaged from the work of multiple peers. 1

f(x) = 8.1x + 16.58

f(x) = 4.95x + 8.19

Figure 6. expresses the difference in rate of tyrosinase between an assay containing the

inhibitor cinnamic acid and an assay without the inhibitor present. Based on this

Lineweaver-Burk plot, the inhibition appeared to be noncompetitive. The Vmax with

inhibition was 0.0603, and the Km with inhibition was 0.4882 mM. The Vmax of the

uninhibited enzyme was 0.1221 and the uninhibited Km was 0.6036 mM.
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Results and Discussion:

The KM and Vmax values for L-dopa found with the Lineweaver-Burk graph were higher

than those found with the Michaelis-Menten graph. The Vmax for D-dopa found with the

Lineweaver-Burk graph was greater than that found using the Michaelis-Menten graph, though

the reverse trend was observed when comparing the Km values found using the respective graphs.

The Lineweaver-Burk plot appeared to provide values for Km and Vmax which more closely

paralleled the literature values than those found with the Michaelis- Menten plot.2 The Michaelis

Menten plots which were crafted in Figures 2 and 4 did not have enough data points to

accutarely show where Vmax would be. This obscured our estimation of Vmax and Km where these

figures were concerned. The literature values also showed a nearly constant Vmax value with a Km

value that varied widely based on the isomer present in the assay,2 as was observed in our data.

Figure 1 expressed the rate of tyrosinase (mol/min) versus the amount of tyrosinase

(g). The figure suggested a definite linear relationship between these two variables, and would

therefore allow for the quantification of an unknown level of tyrosinase. If the rate of the

reaction could be determined using spectrophotometry, it could be compared with an established

plot, similar to Figure 1, to find the approximate quantity of tyrosinase in the sample.

The Km and Vmax for L-dopa were both lower than that of D-dopa. One interpretation of

this observation was that tyrosinase did indeed exhibit stereoselectivity. If tyrosinase was not

stereoselective, the values would be nearly identical when comparing the values for L-Dopa and

D-dopa. This is surprising considering that the naturally occurring dopa molecule has L

configuration, and it seems more likely that the naturally occurring molecule would have more

rapid enzyme kinetics than the synthetic molecule. The lower Km values for L-isomers than for

D-isomers were hypothesized to be a result of the spatial orientation of the ring substituents.2
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Conversely, spatial orientation did not have a similar effect on the values of Vmax, 2 which

remained relatively constant for the assays of both L-dopa and D-dopa, especially in comparison

with the more major fluctuations in Km values collected for the same assays.

According to Figure 6, cinnamic acid appeared to be a noncompetitive inhibitor of

tyrosinase. The inhibitor bound tyrosinase with or without a substrate, and changed the

conformation of tyrosinase as well as its active site, which made the substrate unable to bind to

the enzyme effectively. Thus the efficiency decreased and Vmax was reduced and Km remained

nearly constant because the enzyme substrate reaction still proceeded. Figure 6 supports this

because it shows Km only changing slightly with inhibition, while Vmax decreased by over one-

half from the original, uninhibited Vmax value.

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References:

1. Students in CHEM 362-L. Kinetic Analysis of Tyrosinase. Southern Adventist University,

United States of America, Unpublished research, 2017.

2. Boyer, Rodney (2000) Modern Experimental Biochemistry, 3rd ed. Benjamin/Cummings: San

Francisco, CA; pp 289-298. Kinetic Analysis of tyrosinase.

3. Espn, J. C., Garca-Ruiz, P. A., Tudela, J., & Garca-Cnovas, F. (1998). Study of

stereospecificity in mushroom tyrosinase. Biochemical Journal, 331(Pt 2), 547551.