CHM 2612 (Biochemistry Lab)

Introduction

Tyrosinase is an enzyme involved with that catalysis of monophenols and catechols. Specifically in mammals, tyrosinase catalyzes two steps in the biosynthesis of melanin pigments from tyrosine. The pigment produced from this reaction is used in eyes, hair, and skin. In this laboratory experiment, the kinetics of mushroom tyrosinase is observed by monitoring the oxidation of L and D-3,4-dihydroxyphenyl alanine (Dopa). A crimson colored complex forms from due in part to the oxidoreductase and copper containing functionality of the tyrosinase. The K_M and V_max for tyrosinase can be calculated from resulting data obtained by monitoring the kinetics of the tyrosinase-DOPA solution with a UV-vis spectrophotometer. The enzymatic activity of tyrosinase can then be inhibited and followed via inhibitors such as thiourea and cinnamic acid.

Experimental

During the first week of the experiment, the enzyme kinetics of tyrosine in the presence of L-Dopa and D-Dopa were observed using a UV-vis spectrophotometer at 475 nm. To begin, six solutions were prepared using varying amounts of phosphate buffer and L-Dopa, but an unwavering amount of tyrosinase. The buffer-L-Dopa solutions were prepared in 1 mL cuvettes, and the tyrosinase, kept on ice, was added immediately before subjecting the solutions to UV-vis spectrophotometry. The cuvettes were inverted using paraffin as a cover, in order to mix the enzyme and substrate together, and thus begin the reactions, which was of kinetic interest. The UV-vis was used to monitor the kinetics for one minute. The recorded data could then used to determine the K_M and V_max of tyrosinase. This procedure was then repeated, only using D-Dopa in lieu of L-Dopa.

During the second week of the experiment, the enzyme kinetics of tyrosinase were observed in the presence of L-DOPA and the inhibitors thiourea and cinnamic acid, and were again monitored using a UV-vis spectrophotometer. As in the aforementioned procedure used during the first week of the experiment, the enzyme kinetics of tyrosinase in the presence of varying amounts of L-Dopa and phosphate buffer was monitored using a UV-vis spectrophotometer. For the next two trials, varying amounts of inhibitor was added along with the L-Dopa and phosphate buffer. The inhibitors used were thiourea and trans-cinnamic acid. Again, the enzyme kinetics were followed using a UV-vis spectrophotometer and by comparing the Michaelis-Menten and Lineweaver-Burk plots of the trial without inhibitor to the trials with inhibitor, it could be deciphered as to what class of inhibitors were being dealt with.

Data

L-Dopa Week 1

K_M = 479.0208
V_max = 0.1321

D-Dopa Week 1

K_M = 6616.7
V_max = 0.4886

L-Dopa Week 2

K_M = 512.77
V_max = 0.1529

Thiourea

* Samples 5 and 6 discounted

K_M = 283.6
V_max = 0.1181

Trans-Cinnamic Acid

* Samples 3, 5, and 6 discounted

K_M = 279.8
V_max = 0.1172

Results

In order to find the K_M and V_max, the raw data was first graphed as absorbance versus time. The slopes elicited from the linear regression of these plots were representative of velocity in terms of A/min. These velocities were then converted to umol/min using the equation A = Elc. Absorbance was divided by 3600 M^-1 cm^-1 and multiplied by 1 cm to give M/min, which was then converted to moles/min by multiplying by 0.001 L, the volume of the cuvette, and finally this value was converted to umol/min by multiplying by 10⁶ umol/mol.

The concentration of the substrates was found by taking the known mg/mL concentration, dividing by the formula weight of the molecule to obtain mol/L, then multiplying by 10⁶ umol/mol to obtain units in uM. These values were then multiplied by the percentage they comprised of the mL solution. The reciprocal values were graphed, 1/V versus 1/[S], with the y-intercept being equal to 1/V_max and the x-intercept being equal to -1/K_M.

As far as the results go, the K_M and V_max for L-Dopa are both significantly lower than that of D-Dopa found during the first week. This shows that tyrosinase exhibits stereoselectivity, otherwise the values would be exactly the same. It should have been expected that L-Dopa would have a higher K_M and V_max than that of D-Dopa however, because the naturally occurring Dopa molecule has L configuration. It seems more likely that the naturally occurring molecule would have fast enzyme kinetics than the synthesized molecule.

In regards to the inhibitors, the produced strikingly similar K_M and V_max values, both of which are lower than that of the reaction without either inhibitor. This suggests that both thiourea and cinnamic acid are uncompetitive inhibitors. The V_max values for the runs with the inhibitors is around 0.12 for each, which is fairly close to that of the run without inhibitor, 0.15, but because the K_M values for the inhibitor runs are nearly half that of the K­_M for the trial without inhibitor, I am not sure how to decipher that. Having equal V_max values could potentially make the inhibitors competitive, but the K_M values should be greater, not lower, than that of the enzyme kinetics without inhibitor.

Conclusion

There is undoubtedly some error in the raw data which affected the K_M and V_max values for all the trials. I had to cut out a lot of data points in order to obtain linear regression lines with reasonable R² values for the original absorbance versus time graphs, the slope of which was the velocity of the reaction. Even then, I still had to cut out more points for the Michaelis-Menten and Lineweaver-Burk plots in order to have reasonable looking graphs and values. Because the K_M and V_max values are not as expected, I would have to say the results are inconclusive. The inhibitors had nearly identical K_M and V_max values and had to be classified as uncompetitive. I would have expected the inhibitors to be competitive or noncompetitive, just because for a laboratory experiment I doubt the professor would have us analyze an uncompetitive inhibitor; it does not show much significance. The K_M and V_max for L-Dopa compared to D-Dopa from week one also seem odd; I would have expected L-Dopa to have the higher enzymatic activity.

I am guessing most of the error came from not being able to keep the solutions cold. There was a lot of waiting around in order to use the UV-vis spectrophotometer, and once the solutions of buffer and L-dopa were concocted, there was no real way to keep them chilled. This is probably what interfered with the ability to obtain valid data. It was also somewhat difficult to add the tyrosinase to each cuvette at the very same time, as for some solutions the droplet would gravitate down into the solution before the rest, so this cause error in the UV-vis spectrophotometer readings as well.

Questions

Question 1

Sodium azide – NaN₃

Non-competitive inhibitor because of its ability to bind to copper.

 

Sodium cyanide – NaCN

Non-competitive inhibitor because of its ability to bind to copper.

 

L-phenylalanine – HO₂CCH(NH₂)CH₂C₆H₅

Competitive inhibitor because of its inability to bind to copper.

 

8-hydroxyquinoline – C₉H₇NO

Competitive inhibitor because of its inability to bind to copper.

 

Tryptophan – C₁₁H₁₂N₂O₂

Competitive inhibitor because of its inability to bind to copper.

 

Diethyldithiocarbamate – S₂CN(C₂H₅)₂^–

Non-competitive inhibitor because of its ability to bind to copper.

 

Cysteine – HO₂CCH(NH₂)CH₂SH

Non-competitive inhibitor because of its ability to bind to copper.

 

Thiourea – CH₄N₂S

Non-competitive inhibitor because of its ability to bind to copper.

 

4-chlororesorcinol – C₆H₃(OH)₂Cl

Competitive inhibitor because of its inability to bind to copper.

 

Phenylacetate – CH3COOC₆H₅

Competitive inhibitor because of its inability to bind to copper.

 

Question 2

a. Increase

b. Decrease

c. Decrease

d. Increase

e. Increase

f. No change