UNIVERSITY OF WESTERN SYDNEY - HAWKESBURY

Enzyme Kinetics of Tyrosinase

[Type the document subtitle]

Subject: Biochemistry 2.1

Aim: Using the enzyme Tyrosinase, (1) examine the relationship between enzyme co
ncentration and reaction rate (2) Determine KM and Vmax values for Tyrosinase by
varying the L-Dopa concentration, and (3) determine the mode of action of two T
yrosinase inhibitors.
Introduction: Enzymes serve as catalysts for biochemical reactions in living org
anisms. They direct and regulate thousands of reactions providing for energy tra
nsformation, synthesis and metabolic degradation.
These catalysts speed up the forward and reverse reactions proportionately so th
at, although the magnitude of the rate constants of the forward and reverse reac
tions is increased, the ratio of the rate constants remains the same in the pres
ence or absence of the enzyme. Enzymes increase reaction rates by decreasing the
amount of energy required to form a complex of reactants (substrates) that is c
apable of producing reaction products.
The complex that forms when substrate (S) and enzyme (E) combine is called the e
nzyme substrate (ES) complex. Reaction products arise when the ES complex breaks
down, releasing free enzyme.
Between the binding of substrate to enzyme, and the reappearance of free enzyme
and product, a series of complex events must take place. An ES complex is formed
which passes to a transition state (ES*) and then advances to an enzyme product
(EP) that finally disassociates to a product and free enzyme.
E + S - ES - ES* - EP - E+ P
The kinetics of this type of reactions were first characterised by biochemists M
ichaelis and Menten.
Their basic equation to explain enzyme-catalysed reactions is:
vo = Vmax [S]/ KM + [S]
where: vo = initial velocity caused by substrate concentration [S]
Vmax = maximum velocity
KM = Michaelis constant.
Biochemical Function
Tyrosinase is the enzyme responsible for production of melanins and the darkenin
g of skin. Persons with a genetic deficiency of tyrosinase have no skin pigmenta
tion, i.e. albinism (Wilgram, 1967). The browning of apples, bananas and mushroo
ms when exposed to oxygen is due to uncontrolled oxygenation and polymerisation
of plant phenols catalysed by tyrosinase (Boyer, 2002)
Activity: Tyrosinase is an oxygen-transferring enzyme that is tetramer (a macrom
olecule produced by linking four identical monomers together) in form and contai
ns 4-gram atoms of copper per molecule (Jolley et al. 1974) and two binding site
s for aromatic compounds including phenolic substrates. There is also a distinct
ly different binding site for oxygen (Duckworth and Coleman 1970). Inactivation
of the enzyme is associated with an increase in copper concentration (Kertesz et
al. 1972).
Besides using O2 to catalyse the dehydrogenation of catechols to orthoquinones a
nd the hydroxylation of phenols to catechols, a peroxidase activity has been rep
orted (Strothkamp and Mason, 1974).
Optimum pH: 6.0 7.0
Inhibitors: Inhibitors are compounds that complex with copper. Also inhibited co
mpetitively by benzoic acid with respect to catechol and by cyanide with respect
to oxygen (www.worthington).
Materials:
[0.375 mL/100 mL ] Tyrosinase in 0.1 M phosphate buffer pH 7.0 in
ice filled bucket.
L-Dopa 15mM in buffer in foil covered tube.
L-Dopa 1.5 mM (15 mM diluted with dH2o)
Thiourea 20 mM
Sodium Benzoate 4 mM in buffer
7 Eppendorf tubes
Spectrophotometer and solution cuvettes
Method:
Part 1
7 Eppendorf tubes were placed into a tube rack with 0.25 mL L-Dopa added at a co
ncentration of 15 mM into all 7 tubes. Following, for tube 1, 0.75 mL of phospha
te buffer only was added. Tube 2, 0.65 mL phosphate buffer and 0.1 mL Tyrosinase
were added, tube 3, 0.575 mL phosphate buffer and 0.175 mL Tyrosianse were adde
d, tube 4, 0.5 mL phosphate buffer and 0.25 mL tyrosinase were added, tube 5, 0.
45 mL phosphate buffer and 0.3 mL tyrosinase were added, tube 6, 0.375 mL phosph
ate buffer and 0.375 mL tyrosinase were added and for tube 7, 0.25 mL phosphate
buffer and 0.5 mL tyrosinase were added.
Tyrosinase was brought to room temperature before addition to tubes as this prov
ides maximum reaction rate with the substrate. A spectrophotometer was zeroed, a
nd, using tube 1 as the blank, 1 tube at a time was treated by the addition of t
yrosinase in the above amounts. Tubes were mixed thoroughly by inverting with ca
ps on, then solution placed in cuvette and analysed in spectrophotometer at an a
bsorbance of 475 nm. Time was noted and absorbency values recorded every 15 seco
nds for 2 minutes.
Part 2:
A table was provided to adjust buffer and enzyme quantities, so the final volume
s of a second set of tubes (1 9) reached 1.0 mL Tube 1 was the blank, with 0.25
mL of 1.5 mM L-Dopa added and 0.75 mL of buffer. Tube 2 to 6 each had L-Dopa add
ed at a concentration of 1.5mM at varying quantities 0.05 mL, 0.125 mL, 0.175 mL
, 0.25 mL, 0.625mL respectively. Tubes 7, 8 and 9 had L-Dopa added at a concent
ration of 15 mM and amounts of 0.125, 0.175 and 0.25 mL respectively.
Final L-Dopa concentrations for tubes 1-9 were 0.0, 0.075, 0.188, 0.263, 0.375,
0.934, 1.88, and 2.63, 3.75 mM respectively.
The spectrophotometer was zeroed using tube 1 (blank). Tubes 2-9 were filled wit
h the required L-Dopa and buffer concentrations and quantities.
Tyrosinase was added at intervals of 15 seconds for 2 minutes, placed in cuvette
and analysed in the spectrophotometer with absorbances recorded.
Part 3:
In this part of the experiment we determined the mode of action of two Tyrosinas
e inhibitors, 1) Thiourea and 2) Sodium Benzoate.
1) Thiourea: Tube 1 was again the blank with 0.25 mL of 15 mM L-Dopa, 0.125
Thiourea and 0.625 buffer added. Tubes 2-5 had L-Dopa added at a concentration
of 1.5 mM and amount 0.05, 0.125, 0.175 and 0.25 mL respectively.
Tubes 6-9 had L-Dopa added at a concentration of 15.0 mM and amounts of 0.063, 0
.125, 0.175 and 0.25 mL respectively.
Over all the tubes 1-9, Thiourea was added at 0.125 mL with buffer added to make
up a total of 1.0 mL.
Tyrosinase was added at 0.375 mL in tubes 2 9.
2) In illuminating the mode of action for Sodium Benzoate, the same procedu
res
were undertaken as above, but replacing Sodium Benzoate for Thiourea.
For both inhibitors the spectrophotometer was zeroed with solution from tube 1.
The enzyme Tyrosinase was added at room temperature, covered with parafilm and m
ixed just prior to placing solution in cuvette. Each individual absorbance readi
ng was recorded at intervals of 15 seconds over 2 minutes.

Results:
The 4 tables below show the absorbance values recorded for each section of the l
aboratory practical.

Data Table 1
A475 Tube 2 Tube 3 Tube 4 Tube 5 Tube 6 Tube 7
15 s 0.029 0.033 0.057 0.061 0.073 0.106
30 s 0.047 0.053 0.080 0.088 0.111 0.156
45 s 0.057 0.071 0.106 0.121 0.148 0.206
60 s 0.069 0.090 0.130 0.148 0.185 0.254
75 s 0.079 0.109 0.153 0.178 0.220 0.297
90 s 0.088 0.126 0.175 0.203 0.248 0.336
105 s 0.097 0.142 0.196 0.230 0.253 0.366
120 s 0.109 0.157 0.219 0.256 0.314 0.380
Using Beers Law: [C] = A/ .l , A is entered to give [C] in Moles Litre-1min -1 which
is then converted to mol/min.
e.g. 0.253-0.111 = 0.142, 0.142/3600 = 3.94 x 10-5 M l-1min-1,
which =(3.94 x 10-5) x 106 = 39.4 g/mL
Conversion table 1 to determine Enzyme Concentration
Tube 2 Tube 3 Tube 4 Tube 5 Tube 6 Tube 7
V as
A/min 0.041 0.071 0.094 0.111 0.133 0.150
V as mol/min 0.11 0.018 0.026 0.0318 0.041 0.056
Enzyme Concentration
g/mL 4 7 10 12 15.8 21.6

Graph 1.
 *note: Full size graphs in Appendix

Graph 2

Data Table 2
A475 Tube 2 Tube 3 Tube 4 Tube 5 Tube 6 Tube 7 Tube 8 Tube 9
15 s 0.022 0.033 0.030 0.040 0.055 0.108 0.094 0.121
30 s 0.028 0.043 0.041 0.055 0.081 0.146 0.133 0.159
45 s 0.031 0.053 0.053 0.069 0.106 0.180 0.166 0.198
60 s 0.035 0.061 0.063 0.084 0.130 0.212 0.202 0.230
75 s 0.039 0.069 0.074 0.097 0.155 0.243 0.236 0.266
90 s 0.042 0.077 0.083 0.111 0.178 0.273 0.266 0.299
105 s 0.045 0.084 0.092 0.123 0.199 0.300 0.297 0.330
120 s 0.048 0.090 0.100 0.135 0.222 0.323 0.323 0.359

Graph 3

Conversion Table 2 to determine Reaction Rates of Control.

Tube 2 Tube 3 Tube 4 Tube 5 Tube 6 Tube 7 Tube 8 Tube 9
V as
A/min 0.013 0.031 0.035 0.059 0.097 0.122 0.127 0.154
V as mol/min 0.0037 0.0086 0.0096 0.016 0.027 0.034 0.055 0.043
Substrate Concentration
g/mL 0.075 0.188 0.263 0.375 0.934 1.88 2.63 3.75

Data Table 3
A475 Tube 2 Tube 3 Tube 4 Tube 5 Tube 6 Tube 7 Tube 8 Tube 9
15 s -0.003 -0.002 0.000 0.010 0.024 0.021 0.018 0.023
30 s -0.001 0.000 0.001 0.013 0.033 0.029 0.026 0.047
45 s 0.000 0.001 0.003 0.016 0.042 0.037 0.035 0.036
60 s 0.001 0.002 0.006 0.019 0.050 0.044 0.043 0.048
75 s 0.003 0.004 0.008 0.022 0.057 0.052 0.051 0.053
90 s 0.004 0.005 0.010 0.025 0.064 0.058 0.059 0.058
105 s 0.006 0.007 0.011 0.026 0.070 0.065 0.066 0.069
120 s 0.007 0.008 0.013 0.030 0.076 0.071 0.073 0.077

Graph 4

Conversion Table 3 to determine Reaction Rates of inhibitor Thiourea

Tube 2 Tube 3 Tube 4 Tube 5 Tube 6 Tube 7 Tube 8 Tube 9
V as
A/min 0.0004 0.0004 0.008 0.012 0.028 0.032 0.032 0.02
V as mol/min 0.0011 0.0011 0.0022 0.0033 0.0078 0.0089 0.0089 0.0056
Substrate Concentration
g/mL 0.075 0.188 0.263 0.375 0.934 1.88 2.63 3.75

Data Table 4
A475 Tube 2 Tube 3 Tube 4 Tube 5 Tube 6 Tube 7 Tube 8 Tube 9
15 s 0.005 0.010 0.027 0.029 0.049 0.050 0.054 0.052
30 s 0.07 0.014 0.028 0.033 0.059 0.064 0.070 0.070
45 s 0.009 0.018 0.028 0.036 0.067 0.080 0.085 0.085
60 s 0.010 0.021 0.028 0.040 0.076 0.094 0.102 0.102
75 s 0.011 0.023 0.028 0.044 0.084 0.108 0.118 0.119
90 s 0.011 0.027 0.028 0.048 0.092 0.123 0.133 0.137
105 s 0.012 0.030 0.028 0.052 0.103 0.138 0.149 0.154
120 s 0.013 0.037 0.029 0.056 0.110 0.152 0.166 0.171
Graph 5

Conversion Table 4 to determine Reaction Rates of inhibitor Sodium Benzoate

Tube 2 Tube 3 Tube 4 Tube 5 Tube 6 Tube 7 Tube 8 Tube 9
V as
A/min 0.004 0.013 0.004 0.016 0.032 0.058 0.064 0.069
V as mol/min 0.0013 0.0028 0.0011 0.0044 0.0089 0.016 0.018 0.019
Substrate Concentration
g/mL 0.075 0.188 0.263 0.375 0.934 1.88 2.63 3.75

Below are the values given by SPSS computer programme for Km and Vmax using 3 di
fferent plotting methods:
Control
Method Km Vmax
Michaelis-Menten 0.925 0.139 0.0511 0.0028
Lineweaver-Burke 0.9 0.3662 0.161 0.0641
Hanes-Woolf 1.0 0.103 0.0524 0.0026
Thiourea
Method Km Vmax
Michaelis-Menten 0.5693 0.3525 0.00959 0.0019
Lineweaver-Burke 0.4250 0.0.2498 0.0063 0.0034
Hanes-Woolf 0.436 0.349 0.0077 0.0015
Sodium Benzoate
Method Km Vmax
Michaelis-Menten 2.3535 0.7251 0.0328 0.0051
Lineweaver-Burke 0.6295 0.6778 0.0094 0.0097
Hanes-Woolf 5.0 4.4 0.0493 0.0408
Graph 6

Discussion:
Enzyme inhibitors fall into two classes. There are those which cause irreversibl
e inactivation of enzymes, and those in which their inhibitory effects can be re
versed. Inhibitors of the first type usually cause an inactivating, covalent mod
ification of the enzyme structure.
(An example of this type of inhibitor is cyanide. By covalently binding mitochon
drial cytochrome oxidase, it inhibits all the reactions associated with electron
transport.)
The kinetic effect of irreversible inhibitors is to decrease the concentration o
f active enzyme, thereby decreasing the maximum possible concentration of ES com
plex.
Reversible inhibitors can be divided into main types, Competitive inhibitors and
non-competitive inhibitors. There is also a type called uncompetitive inhibitor
s but they are not often encountered.
The common identifier of reversible inhibitors is that when the inhibitor concen
tration drops, enzyme activity is regenerated. They usually bind to enzymes by n
on-covalent forces with the inhibitor maintaining a reversible equilibrium with
the enzyme.
Competitive inhibitors have two means of reversal (1) decreasing the inhibitor c
oncentration and (2) Raising the concentration of substrate (S) while holding th
e concentration of the inhibitor constant as the substrate and competitive inhib
itors both bind at the same site they compete with each other for binding, allow
ing a second way of inhibition reversal.
High concentrations of substrate can displace nearly all competitive inhibitor b
ound to active sites. Thus, Vmax should be unchanged by competitive inhibitors.
This trait of competitive inhibitors is reflected in the identical vertical-axis
intercepts of Lineweaver-Burk plots, with and without inhibitor.
Since attaining Vmax requires appreciably higher substrate concentrations in the
presence of competitive inhibitor, Km (the substrate concentration at half maxi
mal velocity) is also higher, as demonstrated by the differing negative intercep
ts on the horizontal axis in panel B.
Similarly, panel C illustrates that non-competitive inhibitors appear to have no
effect on the intercept of the negative line implying that non-competitive inhi
bitors have no effect on Km of the enzymes they inhibit. Since non-competitive i
nhibitors do not interfere in the equilibration of enzyme, substrate and ES comp
lexes, the Km s of Michaelis-Menten type enzymes are not expected to be affected
by non-competitive inhibitors.
However, because Enzyme Substrate complexes that contain inhibitor (ESI) are inc
apable of progressing to reaction products, the effect of a non-competitive inhi
bitor is to reduce the concentration of ES complexes that can advance to product
. Since Vmax = k2 [Etotal], and the concentration of competent Etotal is diminis
hed by the amount of ESI formed, non-competitive inhibitors are expected to decr
ease Vmax, as illustrated by the ordinate intercepts in panel C.
A corresponding analysis of uncompetitive inhibition leads to the expectation th
at these inhibitors should change the apparent values of Km as well as Vmax. Cha
nging both constants leads to double reciprocal plots, in which intercepts on th
e vertical and horizontal axis are proportionately changed; this leads to the pr
oduction of parallel lines in inhibited and uninhibited reactions.
Lineweaver-Burk Plots of Inhibited Enzymes http://web.indstate.edu

Inhibitor Type Binding Site on Enzyme Kinetic effect

Competitive Inhibitor Specifically at the catalytic site, where it competes wi
th substrate for binding in a dynamic equilibrium- like process. Inhibition is r
eversible by substrate. Vmax is unchanged; Km, as defined by [S] required for 1/
2 maximal activity, is increased.
Non-competitive Inhibitor Binds E or ES complex other than at the catalyti
c site. Substrate binding unaltered, but ESI complex cannot form products. Inhib
ition cannot be reversed by substrate. Km appears unaltered; Vmax is decreased
proportionately to inhibitor concentration.
Uncompetitive Inhibitor Binds only to ES complexes at locations other than the c
atalytic site. Substrate binding modifies enzyme structure, making inhibitor- bi
nding site available. Inhibition cannot be reversed by substrate. Apparent
Vmax decreased; Km, as defined by [S] required for 1/2 maximal activity, is dec
reased.
http://web.indstate.edu

The standard curve of V vs. [E] corresponds with V being directly proportional t
o E as long as S is being maintained at a high level.
The graph representing V vs. [S] corresponds with that demonstrated in the lab,
with Michaelin-Menten properties of a Saturation curve. There was only three err
ant values that could not be fitted to the curved lines. The final data point of
thiourea was lowered suggesting a error in sample preparation or recording valu
es.

Conclusion:
Thiourea demonstrated it is a non-competitive inhibitor. This is due to the fact
that Km values are similar but Vmax values have decreased. This corresponds wit
h the fact that non-competitive inhibitors bind to a place other than the active
site, so preventing the maximum rate of reaction ever being reached.
Sodium Benzoate showed that it did not respond as anticipated and recorded the h
ighest Km value. This suggests that data was incorrectly interpreted or transpos
ed.
Non-competitive inhibitors are chosen for use in situations where the role of en
zymes needs to be affected. This is achieved by selection of those with an activ
e site different in shape to that of the substrate, so that they can no longer b
ind. It is possible to predict which compounds will behave in this way by examin
ing their chemical structure and determining whether they have a form similar to
that of the substrate. Competitive inhibitors would be chosen where the rate o
f the reaction needed to be slowed down, without affecting the overall outcome o
f Vmax.
Examples of some non-competitive inhibitors are sodium azide, sodium citrate. Ex
amples of some competitive inhibitors are L-phenylanaline, tryptophan and hydroxy
quinoline.
The results in part 1 were substantiated by the close relationship of the data p
oints on the standard curve showing a direct relationship between [E] and Veloci
ty.