1
Results
Table 1: Absorbance Unit Readings for Orthoquinone Formation in
REACTION TIME (MINUTES)
Catechol-Polyphenoloxidase Reaction (Control)
SUBSTRATE CONCENTRATION
Undiluted
1/5 Conc.
1/25 Conc. 1/125 Conc.
0
1
2
3
4
5
6
7
8
9
10
.130
.165
.272
.396
.468
.582
.688
.718
.792
.802
.840
.160
.178
.196
.287
.322
.367
.412
.441
.504
.530
.548
.111
.133
.142
.162
.154
.185
.183
.199
.190
.202
.211
.120
.115
.120
.126
.126
.132
.127
.129
.129
.136
.134
As substrate concentration was reduced, the amount of orthoquinone formed and the
average rate of reaction of orthoquinone formation decreased (Table 1).
REACTION TIME (MINUTES)
Table 2: Absorbance Unit Readings for Orthoquinone Formation in
Catechol-Polyphenoloxidase Reaction with Para-hydroxybenzoic Acid (PHBA) Inhibition
SUBSTRATE CONCENTRATION
Undiluted
1/5 Conc.
1/25 Conc. 1/125 Conc.
0
1
2
3
4
5
6
7
8
9
10
.178
.220
.272
.333
.368
.434
.542
.586
.636
.656
.738
.157
.158
.157
.172
.183
.210
.227
.238
.253
.295
.314
.166
.155
.141
.128
.166
.158
.150
.162
.177
.173
.175
.120
.128
.127
.139
.138
.143
.145
.134
.141
.146
.140
As substrate concentration was reduced, the amount of orthoquinone formed and the
average rate of reaction of orthoquinone formation decreased (Table 2).
2
REACTION TIME (MINUTES)
Table 3: Absorbance Unit Readings for Orthoquinone Formation in
Catechol-Polyphenoloxidase Reaction with Potassium Arsenite Inhibition
SUBSTRATE CONCENTRATION
Undiluted
1/5 Conc.
1/25 Conc. 1/125 Conc.
0
1
2
3
4
5
6
7
8
9
10
.130
.223
.326
.438
.540
.620
.676
.748
.804
.815
.835
.123
.149
.171
.206
.232
.270
.290
.340
.368
.400
.442
.118
.122
.128
.140
.153
.157
.169
.177
.192
.204
.213
.096
.714
.109
.114
.117
.133
.140
.153
.153.
155
.162
As substrate concentration was reduced, the amount of orthoquinone formed and the
average rate of reaction of orthoquinone formation decreased (Table 3).
The control reaction and the reaction with PHBA acting as inhibitor had the same relative
Vmax while the KM of the reaction with PHBA acting as inhibitor was greater than the
control reaction (Fig.1). Due to possible error, the initial reaction rates of the reaction run
with potassium arsenite as inhibitor are inconclusive.
3
Discussion
The experiments we carried out in part five of the enzyme action laboratory
demonstrate the effectiveness of Para-hydroxybenzoic acid (PHBA) as a competitive
inhibitor of the enzyme poyphenoloxidase. The experiments also prove that potassium
arsenite is incapable of reducing orthoquinone formation. Both reactions with these two
possible inhibitors are being compared to a control reaction involving neither PHBA,
potassium arsenite, nor any other type of inhibitor.
Several conclusions can be drawn from the results of the control reaction and
reaction run with PHBA as an inhibitor. The control reaction shows an upward trend in
orthoquinone formation with time (Table 1) as does the reaction involving PHBA
(Table 2). But while both reactions have a similar Vmax (Fig.1), the KM of the reaction
involving PHBA is much greater than the KM of the control reaction (Fig.1) and
indicates that PHBA is acting as a competitive enzyme. The PHBA inhibits due to its
structure. Because PHBA has a molecular design similar to catechol, it is able to enter
the active site of polyphenoloxidase and block substrate-enzyme inhibition. The results
of such blockage could have profound effects on the survival of an organism in nature.
If a plant such as the potato, which uses the catechol-polyphenoloxidase reaction
as a defense mechanism, were to be exposed to an inhibitor such as PHBA, it would most
likely die or be damaged due to predatory attack. This could possibly occur because the
inhibitor, by blocking the active sites of polyphenoloxidase, will slow the formation of
orthoquinones. The slow production of orthoquinone, a toxic repellent, may provide
predators such as microbes, insects, or fungi with more time to damage the plant and
consume vital starches and proteins. Thus, as in the case of the potato and PHBA, it is
highly dangerous to allow certain plants to be exposed to possible competitive enzymatic
4
inhibitors. The introduction of competitive inhibitors into an ecosystem could increase
the amount of unwanted predators and likewise decrease vital plant life.
Potassium arsenite, as an inhibitor, has little or no negative affect on the rate of
catechol-polyphenoloxidase reaction (Fig.1) or amount of orthoquinone formation
(Table 2). Because potassium arsenite affects the disulfide bonds of enzymes, it may be
concluded that the breaking of disulfide bonds of polyphenoloxidase has no affect on the
conformation of its active site. Were the shape of the active site to be altered, then
product formation and rate of reaction would have both been decreased. There may not
be enough disulfide bonds holding the tertiary structure of polyphenoloxidase together
for potassium arsenite to sufficiently denature the enzyme. Also, the number of other
structures holding the tertiary structure of polyphenoloxidase together (hydrogen bonds,
ionic/covalent bonds, Van der Waals attractions) may be strong enough to hold the
enzyme together regardless of the affect of potassium arsenite on disulfide bonds.
Therefore, if an inhibitor such as potassium arsenite were to interact with a plant
species such as the potato in the wild, there would presumably be no negative affect on
the potato. However, the addition of potassium arsenite could make the potato more
susceptible to denaturization from other sources in nature.