Experiment 2: Kinetic Analysis of Tyrosinase

(The experiment can be performed in pairs with each student using either L-dopa or D-dopa as
substrate).
Tyrosinase (EC 1.14.18.1), also commonly called polyphenol oxidase, is a copper-containing
enzyme (an oxidoreductase) with two catalytic activities: orthohydroxylation of monophenols
and aerobic oxidation of o-diphenols.
Monophenol + O2
2-catechol + O2

catechol + H2O
2 o-quinone +2 H2O

Tyrosinase is present in plant and animal cells. Upon injury, many plant s such as bananas,
potatoes, and apples undergo a browning process due to the formation and further oxidation of
natural quinones. In mammalian cells, tyrosinase catalyzes two steps in the biosynthesis of
melanin pigments from tyrosine. The reaction is localized in the melanocytes and produces the
pigments that impart colour, to skin, hair and eyes. Tyrosinase located in the skin when activated
by UV rays of the sun causes greater melanin production and subsequently can produce a suntan.
The many tyrosinases in nature differ in cofactor requirement, metal ion content, substrate
specificity, molecular weight and oligomeric structure.
Mushroom tyrosinase is a tetrameric protein with a total molecular weight of 128 kDa. There are
four atoms of Cu+ associated with the active enzyme.
In this experiment, you will learn to measure tyrosinase activity and determine the Km of the
enzyme with L-dopa as substrate. You will also determine the activity of the enzyme with Ddopa as the substrate. The spectrophotometric assay involves the use of two substrates for the
tyrosinase-catalyzed reaction; the phenolic substrate (dopa) and oxygen. The conditions
described in this experiment are such that the reaction mixture is saturated with dissolved
oxygen. Therefore, the rate of the reaction depends only on the dopa concentration.
Materials
Mushroom tyrosinase (4800 U/10 ml buffer)* , L-dopa, D-dopa, (both at 3.15 mg /25 ml of
buffer), 50 mM sodium phosphate buffer, pH 7.0, VIS -spectrophotometer.

* Freshly prepared.

Procedure
Determination of Km and Vmax

Tube
Phosphate buffer (ml)
L-dopa ( ml)
Pour each solution into a
suitable cuvette
Tyrosinase (ml) added last
to start reaction

1
0.90
0.1

2
0.80
0.2

3
0.60
0.4

4
0.40
0.6

5
0.2
0.8

0.1

0.1

0.1

0.1

0.1

Set up a similar set with D-dopa instead L-dopa

After the addition of tyrosinase, mix the tubes by inversion (DO NOT SHAKE), and
immediately place in the spectrophotometer set at 475 nm. Measure the change in absorbance for
the conversion of dopa to dopachrome for 3 mins taking absorbance readings at every 30 s. From
the slope of each graph (Abs vs time), calculate the
A/min for each tube.
-1
The molar extinction coefficient of dopachrome is 3600 M cm-1 at 475 nm.
Prepare a Lineweaver-Burk plot and calculate the apparent Km and Vmax with L-dopa and with
D-dopa.
Plot the data using the Eadie-Hofstee plot and calculate the apparent Km and Vmax .
Comment on your results
Questions
Does tyrosinase exhibit stereoselectivity?
Which plots offers a more reliable estimation of the apparent Km and Vmax ? Explain your
answer.