The Effect of pH levels and Inhibitors on the Reaction Rate of a Phosphatase Enzyme
Jennifer Bayly
TA: Tian Zhang
Bio 230W, Section 24
10/26/11
Introduction:
All organisms require enzymes in order to survive. These proteins work by catalyzing
reactions without themselves being consumed or altered. Without these enzymes, metabolic
reactions would not take place and organisms would not be able to function. They are specific in
nature because each enzyme has unique active site that can only bind to certain molecules, and
thus each enzyme has a particular function. There are also many enzymes that perform the same
or similar function to one another, but they still have differences in their shape, and thus
eventually in their function.
Because enzymes are proteins, they are very sensitive to their environment. In the wrong
conditions, the enzymes can become denatured and therefore lose functionality. Many conditions
can affect the function enzymes, such as temperature, pH, substrate concentration, and the
presence of inhibitors. The pH level significantly affects enzymes because at a pH too acidic or
basic, the enzyme may change shape and therefore not function as well. Proteins may even
become completely denatured if there are extreme changes in pH (1). Substrate concentration is
also a variable that affects the rate of reaction for an enzyme. The concentration of substrate is
proportional to the rate of reaction of the enzyme up until the saturation point (2). At this point,
all the enzymes are already being used up in a reaction and therefore it is not possible for the rate
to increase, even if more substrate is added.
The presence of inhibitors will affect the functionality of enzymes (3). Some inhibitors,
competitive inhibitors, are close enough in shape to the substrate and can therefore bind to the
active site of an enzyme. This decreases the amount of enzyme available to bind to the actual
substrate, and therefore decreases the rate of reaction. Other inhibitors, noncompetitive
inhibitors, actually bind allosterically to the enzyme, therefore altering its shape so that the
substrate can no longer bind to the active site (1). As compared to enzymes that are uninhibited,
the Km value is increased when competitive inhibitors are present. However, at a high enough
substrate concentration, enough substrate can bind to the enzyme in order for the enzyme to
reach its maximum rate of reaction; thus, Vmax remains the same. In contrast, when
noncompetitive inhibitors are present, the Km value remains the same as the uninhibited enzyme
but the Vmax value will be decreased. This is due to the fact that these enzymes are permanently
disabled (2).
In this experiment, phosphatase enzymes were the model enzyme. This class of enzyme
functions by breaking the diester bond between a hydroxyl group and an inorganic phosphate
group. Every organism has phosphatase enzymes because phosphorylation and
dephosphorylating are required in order to regulate many biological processes (3). However,
that is not to say that every organism possesses the same phosphatase enzyme. Each one has an
optimum environment as well as specific molecules on which it functions. Some work best in
alkaline conditions whereas others work best in an acidic environment.
The purpose of this lab was to determine the optimum pH in which our phosphatase
enzyme of interest functioned, as well as to determine the effects of the inhibitor present and
whether it was competitive or noncompetitive in nature. To accomplish this, colorimetric assays
of enzyme activity were performed with the use of a spectrophotometer. If the optimum pH is
acidic, it is than an acidic phosphatase. Reversely, if the optimum pH is basic, than it is an
alkaline phosphatase. In addition, it is expected that the inhibitor would decrease the rate of
reaction, and the type of inhibitor could be determined by analyzing the data involving the
different inhibitor and substrate concentrations.
Materials and Methods:
In the first experiment, a milliliter each of phosphatase enzyme, buffer, the substrate, and
water were all placed into a series of cuvettes. Each cuvette was then placed into the
spectrophotometer, a Spec 20+, set at 405nm. A positive control with the optimum pH was used
to ensure the correct results could be obtained. A negative control with no enzyme was used in
order to make sure the spectrophotometer was reading correctly. Buffers with pH of 3, 4-11 were
used, with each cuvette containing a different buffer. For each cuvette, absorbance readings were
taken for every minute up to 7 minutes.
In the second experiment, a milliliter each of phosphatase enzyme, buffer, inhibitor, and
substrate were placed into a series of cuvettes. Each cuvette was then placed into the Spec 20+,
set at 405nm. A positive control of no inhibitor was used and a negative control of no enzyme
was used. Each cuvette had the same buffer of a pH 10, but each one had a different
concentration of substrate. Concentrations of 0.1 mg/ml, 0.3 mg/ml, 0.5 mg/ml, 0.8 mg/ml, and
1.0 mg/mL were used. The class was divided into three groups: no inhibitor, low inhibitor, and
high inhibitor. Absorbance readings for each cuvette were taken every minute up until five and a
half minutes.
For the pH experiment, a graph of the absorbance vs. time for each pH level portrayed the
rate of reactions, as determined by the slopes, which could be used to graph the rate vs pH. This
could then be used to determine the optimum pH. For the inhibitor experiment, high inhibitor
data from Group 6 (4) and no inhibitor data from Group 2 (5) were used to create a lineweaverburke graph, which was used to determine the type of inhibitor (3).
Results:
Table 1: Absorbance readings for pH experiment
Time
0
2.5
4
5.5
7
3
0.045
0.04
0.04
0.04
0.04
5
0.11
0.29
0.39
0.5
0.6
6
0.12
0.3
0.38
0.5
0.68
pH
7
0.13
0.4
0.58
0.7
0.85
8
0.11
0.19
0.43
0.58
0.7
9
0.13
0.3
0.21
0.22
0.38
10
0.13
0.17
0.18
0.21
0.22
11
0.16
0.2
0.22
0.23
0.25
Data of the absorbance values over time at various pH levels.
Figure 1: Absorbance vs. time at various pH levels
This is a graph of the absorbance vs. time of the phosphatase enzyme when placed in buffers of various pHs.
Figure 2: Reaction Rate at Different pH Levels
Reaction Rate vs. pH
0.12
Reaction Rate
0.1
0.08
0.06
0.04
0.02
0
0
2
4
6
8
10
12
pH
This graph portrays the reaction rates of the phosphatase enzyme when put in buffers of different pH levels.
The first graph shows the absorbance vs. time of the phosphatase enzyme at different pH levels.
The rate of reaction was derived from the slopes of the lines of best fit. These are portrayed in
Figure 2. The rate for pH 3 was omitted because it had a negative value and was therefore an
outlier. This was most likely a result of an error of the spectrometer or a reading thereof. As
seen from Figure 2, the phosphatase enzyme had much greater reaction rates when in the acidic
buffers than it did for the basic buffers.
Figure 3: Absorbance vs. Time graph for No Inhibitor
This graph shows the absorbance vs. time for the enzyme when in a solution with no inhibitor. The equations go in
order of concentration.
Figure 4: Absorbance vs. Time for Low Inhibitor
This graph shows the absorbance vs. Time for the enzyme when with a low concentration of inhibitor. The
equations are listed in order of the concentrations.
Figure 5: Absorbance vs. Time for High Inhibitor
This graph shows the absorbance vs. time for the enzyme when with a high concentration of inhibitor.
Figures 3-5 show the absorbance vs. time of the enzyme in three different conditions:
with no inhibitor, with a low concentration of inhibitor, and with a high concentration of
inhibitor. The slope of each line of best fit portrays the rate of reaction for the enzyme under
each set of conditions. Data points seen to be outliers were omitted. These were mostly readings
of 0 at the beginning time points.
Figure 6: Lineweaver-Burke Plot
This graph shows the lineweaver-burke plot for the enzyme under three different conditions: no
inhibitor, low inhibitor, high inhibitor.
The 1/Vmax values are the y-intercepts from the lines of best fit of the Lineweaver-Burke
plot. From these values, the Vmax values could be derived. For example:
1/Vmax = 21.188
therefore
Vmax = 1 / (1/Vmax) = 1/21.188 = 0.047197
The Km/Vmax values are the slopes of each respective line of best fit. From both this
value, as well as the y-intercept, the Km could be calculated. For example:
Km/Vmax = 0.2798
therefore
Km = (Km/Vmax)(Vmax) = (0.2798) x (0.047197) = 0.169319
Table 2: Vmax and Km values for all conditions
Vmax
Km
High Inhibitor
0.004201 0.169319
Low Inhibitor
0.013412 -0.0336
No inhibitor0.047197 0.013206
This table lists the Vmax and Km values for the three different conditions. These values were derived as shown above
from the values in the lineweaver-burke plot.
As seen from this data, the Vmax values of both the high inhibitor and the low inhibitor
were both significantly less than the Vmax of the control with no inhibitor. The Km value of the
low inhibitor, while negative, is close to zero as is that of the no inhibitor. The Km of the high
inhibitor is greater than that of the no inhibitor, however. Therefore, the data is inconclusive. If
the inhibitor was competitive, the Vmax would have remained the same in all conditions, but the
Km would have increased for the high and low concentrations. If the inhibitor was
noncompetitive, the Vmax would have decreased for the conditions with the inhibitor while the
Km would have remained the same.
Discussion:
The phosphatase enzyme used in this lab is an acidic phosphatase because its rate of
reaction was much higher in the lower pH buffers than in the higher pH buffers. This confirms
the idea that an acidic phosphatase would work best in acidic conditions. This implies that the
basic conditions alter the structure of this phosphatase, preventing it from performing optimally
and thus lowering its rate of reaction. Reversely, it requires an acidic environment in order to
retain its ideal form. The data from the second experiment was inconclusive and therefore it
cannot be stated whether the inhibitor present was a competitive or noncompetitive inhibitor.
There was no pattern to the Km and Vmax values of the conditions including the inhibitor as
compared to the control with no inhibitor. However, it was confirmed that the presence of an
inhibitor does decrease the rate of reaction of the enzyme, as seen from the reaction rates when in
the presence of the inhibitor as compared to with no inhibitor.
The inconclusiveness of the data could be a result of error. Some possible sources of
error include the spectrophotometer not being calibrated exactly or the enzyme reacting with the
substrate before readings could be taken. Furthermore, it is possible that there was not exactly a
milliliter of each substance added to the curettes because of the imprecise instruments that were
used. This could also alter the data by affecting the spectrophotometer readings. This experiment
could be improved by using more precise micropipettes in order to insure the proper amount of
each substance is being added. Furthermore, it could be improved by taking readings at smaller
intervals and for a much longer time period, which would result in more accurate data and trends.
Knowing the conditions in which an enzyme performs best can help to determine certain
illnesses. Furthermore, understanding the effects of inhibitors and substrate concentration would
be beneficial in clinical settings. For example, if an organism was ill because of some type of
toxin which was acting as a competitive inhibitor, a potential treatment could be simply
increasing the intake of the substrate of interest. Furthermore, if the issue is the rate of reaction
being too high for some process, it could be treated by adding a competitive inhibitor which
would not have permanent results.
These clinical applications can be seen all around. Many antibiotics act as inhibitors on
one enzyme or another. For example, Rifamin is an antibiotic that works by inhibiting RNA
polymerase in E. coli, thus stopping bacterial growth. It inactivates the enzyme at a very low
dose. At this dosage, RNA polymerases in mammalian tissue are not inhibited (6). Therefore, it
can be seen that bacterial infections can be treated by administering enzyme inhibitors.
Enzyme inhibitors can be administered to treat other, non-microbial diseases as well. One
such instance of this is Alzheimer’s disease. Cholinesterase inhibitors have been shown to be
effective because they prevent the inactivation of acetylcholine after it has been released from
the neuron. Consequently, its ability to stimulate nicotinic and muscarinic receptors is increased.
In this fashion, the cognitive deficits associated with the disease can be treated (7).
This experiment portrayed that there is a specific pH or pH range in which an enzyme has
the highest rate of reaction, and that different types of inhibitors affect enzymes in different
ways. These findings are significant because they can be generalized to clinical and medical
applications where enzymes are involved. Further research can be done pertaining to whether
there are both competitive and noncompetitive inhibitors which can act on this particular
enzyme, as well as determining the optimum temperature at which this phosphatase enzyme
functions.
References:
1. Campbell, N.A. Biology.8th ed, 153-158 (2008).
2. Enzyme kinetics. Department of Biology (2011).
3. Enzyme action: Effects of Environmental Conditions. Department of Biology (2011).
4. Group 6: Courtney Ettaro, Kathy Trinh, Krista Loeffler
5. Group 2 : Brad Wiekrykas, Chris Cetnar, Dan Boshinsky
6. Wehil, Walter. Reviews of Infectious Disease. Vol 5, 407-411 (1983).
7. Weinstock, Marta. CNS Drugs. Vol 4, 307-323 (1999).