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Kinetic Analysis of Tyrosinase

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					                               Kinetic Analysis of Tyrosinase


                                       Safety Precautions
Wearing goggles, lab coats, and gloves are required in this lab. L-DOPA is very hazardous in
case of ingestion, and is slightly hazardous in case of skin contact (irritant), of eye contact
(irritant), or of inhalation. Benzoic acid may be harmful if swallowed, may act as an eye or
respiratory irritant, and may cause allergic respiratory or skin reactions. Enzymes/proteins may
cause allergic reactions in certain sensitive individuals. Therefore, it is important in this
experiment to wear gloves.


Introduction:

        Enzymology is the study of enzyme catalytic mechanisms, the most noble of all
biochemical pursuits. Enzymes have the amazing ability to accelerate the rates of biochemical
reactions by a factor of 105 to 1017 over the non-catalyzed rate. This ability allows life to exist as
we know it today. Not surprisingly, understanding the mechanisms by which enzymes catalyze
reactions has been a topic of basic research for many years. With the advent of modern
genomics, and all of the information it has to offer, the field of enzymology continues to flourish.
        An understanding of enzyme kinetics begins with the ideas of Michaelis and Menten as
well as Briggs and Haldane. Michaelis and Menten postulated that the first step in an enzymatic
reaction is the formation of a complex between substrate and enzyme, the ES complex.
Additionally, they postulated that the slow step in the reaction sequence is formation of product,
k2:

                          k1              k2
                E+S               ES               E+P
                          k-1

The overall reaction rate is therefore proportional to [ES]. Briggs and Haldane realized that the
concentration of ES builds up quickly, and then reaches a constant level in the steady state.
These assumptions allowed for the derivation of the Michaelis-Menten equation:

               0 = Vmax[S]/Km + [S]
where
        0 = the initial reaction velocity
        Vmax = the maximal velocity, obtained when all of E is in the ES complex
        [S] = the substrate concentration
        Km = the Michaelis constant = (k2 +k-1)/k1

A plot of observed velocity vs substrate concentration yields a hyperbolic curve approaching the
maximal velocity. Additionally, Km has the incidental property of being equal to the substrate
concentration at which 0 is one-half Vmax.
        Enzyme activity can be measured by following the change in concentration of any
substrate or product. Ideally, this is done in a direct manner, is very sensitive, and involves a
change in absorbance in the UV-Vis range. One can then measure the reaction velocity at
different substrate concentrations and make a plot of 0 vs [S] to verify that the enzyme follows
Michaelis-Menten kinetics. The kinetic parameters Vmax and Km can be obtained by a fit of the
data to the Michaelis-Menten equation, or they can be derived from a double reciprocal plot.
Taking the reciprocal of both sides of the Michaelis-Menten equation yields the Lineweaver-
Burk relationship:

               1/0 = (Km / Vmax*[S]) + 1/ Vmax

A plot of 1/0 vs 1/[S] yields a straight line with a slope of Km / Vmax, an intercept on the 1/0
axis of 1/ Vmax, and an intercept on the 1/[S] axis of –1/ Km.
        A second type of plot is the Eadie-Hofstee plot. In this case, the Lineweaver-Burk
relationship is rearranged to yield:

               0 = Vmax – Km*(0/[S])

A plot of 0 vs 0/[S] gives a straight line with the intercept on the x-axis of Vmax/ Km, a y-
intercept of Vmax, and a slope of – Km.
        A third type of plot is the Hanes-Woolf plot. In this case, the Lineweaver-Burk equation
is rearranged to yield:

               [S]/0 = Km / Vmax + [S]/ Vmax

A plot of [S]/0 vs [S] gives a straight line with a slope of 1/ Vmax, and x-intercept of – Km, and a
y-intercept of Km / Vmax.
         What do the kinetic parameters Km and Vmax mean? Vmax is simply the maximal velocity
of the enzyme, which occurs when all of the enzyme is in the ES complex. This occurs when
substrate is at a saturating level, and in the simple example above is equal to k2[Et]. The
meaning of Km can get more complicated. For a two step reaction with k2<<k-1, Km reduces to
(k-1/k1). This is the dissociation constant of the ES complex, and Km measures an affinity of the
enzyme for substrate. Unfortunately, this is untrue for most enzymes, and Km cannot be
considered a simple measure of substrate affinity. A third important kinetic parameter is kcat, the
turnover number. It is equivalent to the number of substrate molecules converted to product in a
given unit of time on a single enzyme molecule when the enzyme is saturated with substrate. In
the Michaelis-Menten equation kcat = Vmax /[Et]. One can compare the catalytic efficiencies of
enzymes using the parameter kcat / Km, also called the specificity constant. This ratio relates the
maximal rate at which an enzyme can function to the concentration of substrate needed to reach
that rate.
         The activity of an enzyme depends on many parameters, such as pH and the presence of
inhibitors. One way enzymes catalyze reactions is through the use of general acid-base catalysis.
This involves the transfer of a proton between an enzyme group and the substrate or product.
Therefore, the protonation state of catalytic residues can dramatically affect enzyme activity.
For example, if an enzymatic reaction requires the abstraction of a proton from the substrate by a
basic residue on the enzyme, this residue needs to be unprotonated for catalysis to occur. If the
pH is lowered to the point where this residue is now in its protonated state, the reaction rate will
be negatively affected. The presence of an inhibitor will also have an effect on the reaction rate,
providing the inhibitor is at a high enough concentration.
        In this experiment we will determine the Km and Vmax of the enzyme tyrosinase with its
substrate 3,4-dihydroxyphenylalanine (L-DOPA). Tyrosinase catalyzes the oxidation of L–
DOPA to dopachrome, using molecular oxygen (O2) as the second substrate. Mushroom
tyrosinase is a tetrameric enzyme with a total molecular weight of 128,000. Each active enzyme
binds four molecules of Cu+, which interact with the bound oxygen.
        To monitor the enzyme activity we will follow oxidation of DOPA at 475 nm. The
extinction coefficient of dopachrome is 3600 M-1cm-1. To convert the raw data (change in
absorbance per minute) to moles of product formed per minute per liter, each A/min is divided
by 3600. This is then be converted to moles/min*mg by making the appropriate conversions.

Tyrosinase Reaction:
                                     O                                         O

                    +                          -              +                            -
                     H3N       CH    C     O                   H3N      CH     C       O

                               CH2                                      CH2

                                                   O2   H2O




                                         OH                                        O

                               OH                                       O


                            L-DOPA                                   Dopachrome
Materials and Methods:
Materials:

500 mM         NaPhosphate, pH 6.0, 6.5, 7.0, 7.5, 8.0
10 mM          L- DOPA
10 mM          D- DOPA
3 mM           Benzoic acid (in 100 mM NaPhosphate, pH 7.0)
1.4 mg/ml      Tyrosinase

Supplies:

Pipetemen
Test tubes
Timer
Parafilm
Visible spectrophotometer


Procedure
        In this week’s lab we will first measure the Km and Vmax of tyrosinase for L- DOPA and
D- DOPA. After this we will determine the effect of an inhibitor, benzoic acid, on the rate of the
tyrosinase reaction. Finally, we will determine the reaction rate at five different pH values. In
each case we will use the same general procedure. To a test tube first add water, buffer, and
substrate. Then add the enzyme solution and mix briefly. Place the test tube in the
spectrophotometer, which should be set to monitor absorbance at 475 nm, zero the
spectrophotometer, and measure the change in absorbance over a 1 minute period. Once this has
finished, rinse the test tube 4 or 5 times with water and start the next reaction. It may be most
efficient to make up all the reaction mixtures for each set of reactions at the same time, leaving
out the enzyme. You can then add the enzyme as you are ready to start the reaction.

L- DOPA
Use the pH 7.0 buffer for these reactions. All volumes listed in the table are in l.

                                                                    Velocity                Conc.
Buffer Enzyme       L- DOPA         H2O            Abs          (mol/min*mg)          L- DOPA (M)
 300     15             24         887 x 3                                                    80
 300     15             30         880 x 3                                                   100
 300     15             40        876.7 x 3                                                  133
 300     15             60         870 x 3                                                   200
 300     15            120         855 x 3                                                   400
 300     15            240         815 x 3                                                   800
      D- DOPA
      Use the pH 7.0 buffer for these reactions. All volumes listed in the table are in l.

                                                                            Velocity              Conc.
      Buffer Enzyme        D- DOPA        H2O              Abs          (mol/min*mg)        D- DOPA (M)
       300     15              24        887 x 3                                                    80
       300     15              30        880 x 3                                                   100
       300     15              40       876.7 x 3                                                  133
       300     15              60        870 x 3                                                   200
       300     15             120        855 x 3                                                   400
       300     15             240        815 x 3                                                   800

      Inhibition by Benzoic Acid
      Use the pH 7.0 buffer for these reactions. All volumes listed in the table are in l.

                             L-      Benzoic                                  Velocity             Conc.
      Buffer   Enzyme      DOPA       Acid           H2O          Abs     (mol/min*mg)       L- DOPA (M)
       300       15          24        150          837 x 3                                          80
       300       15          30        150          835 x 3                                         100
       300       15          40        150         831.7 x 3                                        133
       300       15          60        150          825 x 3                                         200
       300       15         120        150          805 x 3                                         400
       300       15         240        150          765 x 3                                         800

      pH Curve
      All volumes listed in the table are in l.

                            L-                        Bkg                         Velocity            Conc.
pH    Buffer   Enzyme      DOPA         H2O        absorbance       Abs       (mol/min*mg)        L- DOPA
                                                                                                      (M)
6.0    300        15         150      845 x 3                                                          500
6.5    300        15         150      845 x 3                                                          500
7.0    300        15         150      845 x 3                                                          500
7.5    300        15         150      845 x 3                                                          500
8.0    300        15         150      845 x 3                                                          500
Data Analysis
        Convert all of the slopes to velocities and then make plots of velocity vs [S] for the data
with L- DOPA and D- DOPA to assure that they follow Michaelis-Menten kinetics. Also plot
1/V vs 1/[S], and from these plots determine the constants Km and Vmax. How do the values
compare between the two substrates? Now plot the data using the Eadie-Hofstee and Hanes-
Woolf plots and calculate Km and Vmax for both L- and D- DOPA using both of these plots.
Calculate kcat for tyrosinase with both L- and D- DOPA, and then calculate kcat / Km (units of s-
1 -1
 M ). Based on this data, does tyrosinase demonstrate stereospecificity?
        Plot the data from the L- DOPA experiment and the inhibition experiment (with benzoic
acid) on the same double reciprocal plot. Has any inhibition occurred? Can you tell what type
of inhibitor benzoic acid is (competitive, uncompetitive, or mixed)? Plot the observed velocity
vs pH. Is there any effect of pH on the reaction rate? What does the shape of the curve imply?

Sample Calculations
If the slope for a reaction was 0.25 min-1, then the velocity can be calculated as follows:
Velocity = (0.25 min-1/3600 M-1)  (0.003 L)  (1x106 mol/mol)  (1/0.021 mg enzyme)
Velocity = 9.9 mol/min*mg

If the maximal velocity is 100 mol/min*mg, then the kcat can be calculated as follows:
kcat = Vmax/[E] = (100 mol/min*mg)  (1 min/60 sec)  (1 mg/0.031 mol) = 32.3 s-1

				
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