Kinetic Analysis of Tyrosinase __________________________________________________________________________________ Abstract: The purpose of this experiment was to study the stereo-specific kinetics of mushroom

tyrosinase with stereo-isomers D-Dopa and L-Dopa. The calculated values for D-Dopa substrate is, Km 1409.97 M and Vmax 135.92 M/min. For L-Dopa substrate Km is 676.01 M and Vmax 111.85 M/min. The Km value of D-Dopa is 2 times the Km value of L-Dopa. The inhibition mechanism of sodium benzoate was studied using L-Dopa as the substrate of tyrosinase. For inhibited tyrosine with L-Dopa substrate Km 437.31 M and Vmax 56.82 M/min. Na-benzoate is an uncompetitive inhibitor according to experimental data, but the inhibition mechanism of sodium benzoate was expected to be competitive in nature. Introduction: Biological catalysts are known as enzymes. Enzymes are proteins with the exception of some RNA. Compared to chemical catalysts enzymes are highly specific, tightly regulated and several times faster (1). Unlike chemical catalysts enzymes work in mild conditions such as physiological pH, temperature and pressure. One interesting feature of enzymes is stereo-specificity, meaning the enzyme will bind and react with only one stereo-isomer. An example is mushroom tyrosinase. This enzyme has a copper-containing prosthetic group and catalyzes sequential oxidation of phenolic substrates. Mushroom tyrosinase is also known as polyphenol oxidase or catechol oxidase. The primary function the enzyme is to catalyze the first two step of pigments production such as melanin(2). Figure 1 shows the reaction.

Figure 1: Tyrosinase catalyzed reactions The common biological substrate for this enzyme is L-Dopa as shown in figure 2. The other stereo-isomer D-Dopa is also shown in figure 3.

Figure 2: D-Dopa

Figure 3: L-Dopa

The stereo-specificity can be quantitatively measured using enzyme kinetics. Enzyme kinetics is the study of reactions catalyzed by enzymes. The one of the simple models to study enzyme catalyzed

reactions is Michaelis-Menten kinetics. Michaelis-Menten model gives the reaction rate for the following enzyme catalyzed reaction(3),

E, S and P represents enzyme, substrate and product respectively. kf is the rate constant for the forward reaction of substrate binding enzyme while kr is the rate constant for the reverse reaction. kcat is the rate constant for the product formation step. For the above reaction the reaction rate is given below,

Here is the reaction rate, Vmax is the maximum rate achieved when all the enzyme is saturated by substrate and Km is the Michaelis-Menten constant. Km is the concentration of substrate when reaction rate is half of Vmax. Michaelis-Menten kinetics gives two important measure, km dictates the amount of substrate needed to saturate enzyme and kcat dictates how efficiently the substrate bound to the enzyme is converted to product. The plot of Michaelis-Menten equation is a hyperbolic curve, thus it is not adequate to determine the maximum or Vmax of the hyperbola accurately. Thus a linear equation is needed. Moreover, Michaelis-Menten model is unable to predict the effect of a kinetic inhibitor on reaction rate. Lineweaver-Burk transformation is more suitable to study the kinetics of enzyme inhibition. By taking the reciprocals of Michaelis-Menten equation the Lineweaver-Burk equation is obtained(4),

Commonly studied enzyme inhibitions are competitive, noncompetitive, mixed and uncompetitive(5). Competitive enzyme inhibition means the inhibitor competes with the substrate to bind to the active site of the enzyme. As a result of competitive inhibition, km is increased as more substrate is needed to saturate the enzyme. But the Vmax remains same as the inhibitor is displaces by substrate from enzyme-inhibitor complex at higher substrate concentration. Noncompetitive inhibitors binds to the enzyme at a site other than active site leaving the active site free for substrate-binding, as a result km remains the same. But when bound to the enzyme-substrate complex, inhibitor causes a configuration change in the enzyme causing loss of functionality and lowering Vmax.

Figure 4: Lineweaver-Burk plot of Competitive and Noncompetitive inhibition

Figure 5: Lineweaver-Burk plot of Uncompetitive inhibition

An uncompetitive inhibitor only binds to enzyme-substrate complex. Uncompetitive inhibition increases km by increasing the apparent affinity of the enzyme and lowers Vmax, because product takes longer leave the active site. The Lineweaver-Burk plot of uncompetitive inhibition is parallel to the uninhibited plot because the catalytic efficiency of the enzyme does not change. Mixed inhibition incorporates characteristics from competitive and non-competitive inhibition. The inhibitor may bind to free enzyme or enzyme-substrate complex. It is called mixed because it acts as competitive inhibitors which bind only to the free enzyme and uncompetitive inhibitors which bind only with enzyme-substrate complex. Figure 4 and 5 represents the Lineweaver-Burk plot of different kinds of inhibition. Materials and Method: as per lab manual 59-261. Data:
120 100
Vo (M/min)

80 60 40 20 0 1 2 3
[S] M

D-Dopa L-Dopa (with Na-benzoate inhibitor)

L-dopa

Figure 6: Michaelis-Menten plot for enzyme-catalyzed reactions of D-Dopa and NA-benzoate inhibited and uninhibited L-Dopa

Table 1: Lineweaver-Burk plot data Substrate [S] (M) D-Dopa 500 1000 1500 2000 3000 L-Dopa 500 1000 1500 2000 3000 L-Dopa (with Na-benzoate inhibitor) 500 1000 1500 2000 3000

1/[S] (M-1) 0.002 0.001 0.00067 0.0005 0.00033 0.002 0.001 0.00067 0.0005 0.00033 0.002 0.001 0.00067 0.0005 0.00033

Vo (M/min) 36.2943 51.8311 71.6507 80.3359 97.8692 47.9202 66.8813 72.7522 82.341 98.132 30.5441 39.076 42.9448 45.3531 52.4782

1/Vo (min/M) 0.02755 0.01929 0.01396 0.01245 0.01022 0.0208680264 0.0149518625 0.0137452888 0.0121446181 0.0101903558 0.0327395471 0.0255911557 0.0232857063 0.0220492094 0.0190555316

0.04 0.03 0.03

1/Vo

0.02 0.02 0.01 0.01 0

1/[S]

D-Dopa L-dopa L-Dopa (with Na-benzoate inhibitor)

Linear (D-Dopa) Linear (L-dopa) Linear (L-Dopa (with Na-benzoate inhibitor))

Figure 7: Lineweaver-Burk plot for D-Dopa with equation of line y = 10.374x + 0.00736 with linear correlation R2 = 0.98. Similarly for L-Dopa, y = 6.044x + 0.00894 with R2 = 0.98 and for Na-benzoate inhibitor with L-Dopa, y = 7.702x + 0.0176 with R2 = 0.98.

For D-Dopa, X axis intercept is equal to 1/Km. Thus from the equation of line y = 10.374x + 0.00736, Km = 10.374/0.00736 M = 1409.97 M Y axis intercept is equal to 1/Vmax. Vmax = 1/0.00736 M/min = 135.92 M/min For L-Dopa, the equation of line y = 6.044x + 0.00894, Km = 6.044/00.00894 M = 676.01 M Vmax = 1/0.00894, M/min = 111.85 M/min For L-Dopa with Na-benzoate inhibitor, the equation of line y = 7.702x + 0.0176, Km = 7.702/ 0.0176 M = 437.31 M Vmax = 1/0.0176, M/min = 56.82 M/min Discussion: Figure 6 shows the Michaelis-Menten plot of tyrosinase kinetics with different substrate. From this plot only qualitative data can be obtained. The limitation with Michaelis-Menten model is that the plot is shaped as a hyperbola. As a result the limit of the hyperbola, in other words Vmax can not be determined. Consequently, Km also can not be determined as Km is the concentration of substrate when reaction rate is half of Vmax. Figure 7 shows the Lineweaver-Burk plot of tyrosinase kinetics with different substrate. The linear plots means east extraction of Km and Vmax values. The calculated values for D-Dopa substrate is, Km 1409.97 M and Vmax 135.92 M/min. For L-Dopa substrate Km is 676.01 M and Vmax 111.85 M/min. For inhibited tyrosine with L-Dopa substrate Km 437.31 M and Vmax 56.82 M/min. The Km value of D-Dopa is 2 times the Km value of L-Dopa. This means that with L-Dopa substrate the enzyme binds with the substrate twice as fast compared to D-Dopa substrate. Thus tyrosinase is stereo-specific towards L-Dopa stereo-isomer. Interestingly the Vmax for tyrosinase for L-Dopa substrate is lower than D-Dopa substrate. The transformation rate was expected to be same since both stereoisomers have same electron density and the nucleophilic power of the oxygen from the hydroxyl group to attack the copper atoms of the active site of the enzyme is also the same(6). Therefore, Vmax should be same for both stereo-isomers. The difference in the experimental Vmax could be due to erroneous detection of the endpoint. As Michaelis-Menten equation is transformed into Lineweaver-Burk equation, small error in vo becomes larger in 1/vo. For future experimentation it is advised to use more data points for statistical accuracy. Na-benzoate is an uncompetitive inhibitor according to figure 7. The Lineweaver-Burk plot is parallel to uninhibited L-dopa plot. But the inhibition mechanism of sodium benzoate was expected to be competitive. The source of error could be the same as explained above. Additionally the color is due to the combined effect of several colorimetric compounds namely the primary and, particularly the secondary oxidation products of the substrate and their amino acids, found in the medium, compounds (2). The presence of different compounds were not compensated in the calculations and only one extinction coefficient was used. Conclusion: The calculated values for D-Dopa substrate is, Km 1409.97 M and Vmax 135.92 M/min. For L-Dopa substrate Km is 676.01 M and Vmax 111.85 M/min. For inhibited tyrosine with L-Dopa substrate Km 437.31 M and Vmax 56.82 M/min. The Km value of D-Dopa is 2 times the Km value of L-Dopa. The enzyme has higher affinity for L-Dopa but the conversion rate is same for both

stereo-isomers. Na-benzoate is an uncompetitive inhibitor according to experimental data, but the inhibition mechanism of sodium benzoate was expected to be competitive in nature. The inhibition kinetics data should be ignored and for future experimentation more data points should be acquired. References: 1. Grisham, Charles M.; Reginald H. Garrett (1999). Biochemistry. Philadelphia: Saunders College Pub. pp. 4267. 2. Warner, C. (1951). Aust. J. sci. Re8. B, 4, 554. 3. Kenneth A. Johnson and Roger S. Goody, The Original Michaelis Constant: Translation of the 1913 MichaelisMenten Paper.Biochemistry, 2011, 50 (39), pp 82648269 4. Lineweaver, H and Burk, D. (1934). "The Determination of Enzyme Dissociation Constants". Journal of the American Chemical Society 56 (3): 658666. 5. Cohen, J.A.; Oosterbaan, R.A.; Berends, F. (1967). "[81] Organophosphorus compounds". Enzyme Structure. Methods in Enzymology. 11. p. 686. 6. J C Espn, P A Garca-Ruiz, J Tudela, F Garca-Cnovas. Biochem J. 1998 April 15; 331(Pt 2): 547551. Data Analysis: (a) Figure 6. From this plot only qualitative data can be obtained. The limitation with Michaelis-Menten model is that the plot is shaped as a hyperbola. As a result the limit of the hyperbola, in other words Vmax can not be determined. Consequently, Km also can not be determined as Km is the concentration of substrate when reaction rate is half of Vmax. (b) Figure 7.For D-Dopa, X axis intercept is equal to 1/Km. Thus from the equation of line y = 10.374x + 0.00736, Km = 10.374/0.00736 M = 1409.97 M Y axis intercept is equal to 1/Vmax. Vmax = 1/0.00736 M/min = 135.92 M/min For L-Dopa, the equation of line y = 6.044x + 0.00894, Km = 6.044/00.00894 M = 676.01 M Vmax = 1/0.00894, M/min = 111.85 M/min For L-Dopa with Na-benzoate inhibitor, the equation of line y = 7.702x + 0.0176, Km = 7.702/ 0.0176 M = 437.31 M Vmax = 1/0.0176, M/min = 56.82 M/min