Lucas Man

Group members:

Andrew Kim

Paul Digiacobbe

Kristine Nale

Section 16 TA: Austin Nuschke

The Effects of pH and Inhibitor Concentration on

the Activity of a Phosphatase Enzyme

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Introduction

Enzymes are molecular machines that act as catalysts and are essential to virtually all cellular

processes. Most enzymes are proteins built from amino acid chains. There are twenty-two amino acids

found naturally, each with an amino group, a carbonyl group and a variable side group attached to a

central carbon. The intermolecular interactions between different amino acids and their side groups allow

proteins to fold into precise shapes that enable catalytic activity. Enzymes play varied roles ranging from

gene expression regulation to digestion.1, 2

Enzymes function as catalysts by lowering the activation energy of specific chemical reactions

involving one or more substrates1, 2

. This is accomplished with an active site where specific substrates can

bind to. Binding of a substrate to an enzyme will rapidly speed up the chemical reaction. As substrate

concentration increases, reaction rate will also speed up as more enzymes will be engaged. However a

maximum possible reaction velocity will be reached for a specific concentration of enzymes in solution

because reaction velocity cannot increase any further if all active sites on available enzymes are already

occupied.1, 2, 3

Plotting reaction rate versus substrate concentration will result in a graph known as a

Michaelis-Menten Plot with a limit at the maximum reaction velocity (Vmax). The X-value that results in

½ Vmax is the rate constant Km. Another important graph for enzyme kinetics is the Lineweaver-Burke plot

where the inverse of reaction rate is plotted against the inverse of substrate concentration. This plot gives

-1/Km as the x-intercept and 1/Vmax as the y-intercept.1, 3

The shape specific nature of enzymes allows different molecules that can alter the shape of an

enzyme to act as inhibitors1, 2

. Inhibitors can be reversible or irreversible. Irreversible inhibitors

permanently disable the enzyme and the functionality of an irreversibly inhibited enzyme cannot be

recovered. Reversible inhibitors only temporarily inhibit an enzyme, meaning that there is a process by

which to recover the enzyme’s catalytic ability. There are two main groups of reversible inhibitors,

competitive inhibitors and noncompetitive inhibitors1, 2

. Competitive inhibitors are molecules that can

compete with an enzyme’s substrate for the active site and shut down the activity of that enzyme.

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Noncompetitive inhibitors are molecules that bind to a site on an enzyme away from its catalytic site and

cause a conformational change that disrupts that enzyme’s activity. Competitive inhibitors affect the Km

of the Michaelis-Menten and Lineweaver-Burke plots of an enzyme but not the Vmax since competitive

inhibitors do not prevent substrate from binding to the enzyme and a sufficient substrate concentration

will still be as efficient in binding to the active site. Noncompetitive inhibitors will decrease the Vmax as

they disrupt enzymes regardless of substrate concentration.3

Another way enzyme activity can be influenced is through the pH of the system. pH can alter the

conformation of an enzyme and thus affect its activity1, 3

. In this experiment, both pH and inhibitor

presence were investigated as factors that affect enzyme activity. A phosphatase, a kind of enzyme that

removes a phosphate group from a substrate, was used3. There are many different kinds of phosphatases;

the specific phosphatase used in this experiment removes a phosphate group from p-nitrophenyl

phosphate, a colorless product, to form a yellow-colored product, p-nitrophenyl3. Its activity in different

pH’s and inhibitor concentrations was assessed by measuring the appearance this product through the use

of a spectrophotometer. It was hypothesized that one pH would result in optimal performance by the

enzyme. It was also hypothesized that the presence of an inhibitor would reduce the reaction rate of the

enzyme. Furthermore, by analyzing the Michaelis-Menten and Lineweaver-Burke plots created using the

data collected from the inhibitor study, it was hoped that the inhibitor used would be found to be either a

competitive or a noncompetitive inhibitor.

Materials and Methods

The procedures for this experiment were taken from the Enzyme Action: Effects of Environmental

Conditions lab manual from the Department of Biology at Penn State University. For the pH assay

portion of the experiment, 10 cuvettes numbered 1 through 10 were used. Cuvette 1 contained the positive

control, a 4 mL mixture of 1 mL of buffer of an unknown pH that guaranteed enzyme activity, 1 mL of

the substrate, 1 mL of H2O and 1 mL of Enzyme B in solution. Cuvette 2 contained the negative control

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with the same mixture as in cuvette 1 but with 2 mL of H2O and no enzyme. Cuvettes 3 through 10

contained them same mixture as in cuvette 1 but instead of a buffer of unknown pH, buffers ranging from

pH 3 through 11 (skipping pH 4) were used. After loading each cuvette with its respective mixture,

reaction progress was measured by placing each cuvette into a spectrophotometer. This instrument

measures the amount of light of a specific wavelength that is absorbed by the sample in each cuvette. As

the chemical reaction in the cuvettes produces a colored product, the measure of the amount of light of the

complementary color that is absorbed is a direct measure of the amount of that product. In this case, the

wavelength of light that corresponds to the complementary color of the color of the product is 405 nm. As

the reaction progressed in each cuvette, the absorbance value for each cuvette was recorded at specified

time intervals. A graph of absorbance value plotted against time was made for each cuvette (Figure 1).

The slope of each line gives the rate of reaction in each cuvette.3 The rates of reactions were then plotted

against pH (Figure 2).

The inhibitor assay portion of the experiment was performed with similar procedures. Seven

cuvettes were used, a positive control with enzyme, substrate, buffer and H2O, a negative control with

substrate, buffer, inhibitor and H2O , and 5 cuvettes with enzyme, buffer, inhibitor and varying

concentrations of substrate. The substrate-enzyme reaction was allowed to run in each cuvette and the

absorbance values were recorded along with the times at which those readings were taken. These steps

were performed by 6 different lab groups. Two groups used 0 inhibitor concentration, two used low

inhibitor concentration (10 millimolar), and the last two groups used a high inhibitor concentration (20

millimolar). Absorbance vs. Time was plotted for each cuvette and the slopes and reaction rates were

calculated. As 2 groups recorded absorbance values for each inhibitor concentration, the reaction rates

calculated from the 2 data sets for each inhibitor concentration as averaged together. These average

reaction rates were then plotted against substrate concentration to create Michaelis-Menten graphs.

Lineweaver-Burke plots were also created by plotting the inverse of reaction rate against the inverse of

substrate concentration.

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00.010.020.030.040.050.060.070.080.09

0 1 2 3 4 5 6 7 8 9 10 11 12

Rea

ctio

n V

elo

city

pH

Reaction Velocity vs. pH

Results

The pH assay produced the following data. Figure 1 shows absorbance value versus time.

The slopes of the trend lines created from Figure 1 were used for the values of reaction velocities

in Figure 2.

As expected, in Figure 1, the positive

control has the largest slope and reaction velocity

while the negative control has a slope and

reaction velocity of 0. Furthermore, Figure 2

shows that at pH 10, the enzyme demonstrates

the fastest reaction velocity. It was hypothesized

that the enzyme would have one optimal pH that

would yield the highest reaction velocity; figure 2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0 2 4 6 8 10 12 14 16 18

Ab

sorb

ance

(A40

5)

Time (Minutes)

Absorbance Value vs. Time (pH Assay)

Positive Control

Negative Control

pH 3

pH 5

pH 6

pH 7

pH 8

pH 9

pH 10

pH 11

Figure 1 - Absorbance Value vs. Time (pH Assay) - absorbance value of each cuvette in the pH assay plotted against the time at which that reading was taken; all 10 cuvettes are plotted on this graph

Figure 2 - Reaction Velocity vs. pH - reaction velocities calculated from Figure 1 plotted against pH

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supports these hypotheses and shows that pH 10 is the optimal pH. This means that Enzyme B is

a basic enzyme and functions best in basic conditions.

Data from the inhibitor assay was collected by 6 groups and pooled together. For each

group, a plot of absorbance value versus time was made for each cuvette (example Figure 3).

Trend lines were drawn for the cuvettes and the

slope of each was calculated. Since there were 2

groups working on each inhibitor concentration,

2 plots of absorbance value versus time were

made for each inhibitor concentration. The 2

slopes calculated for each substrate

concentration were averaged (Table 1). These

averages are what are used as the values for

reaction velocities for the rest of the figures in

this report.

Table 1. Reaction Velocities at Different Substrate Concentrations – values taken from the

slopes of absorbance vs. time plots

Positive

Control

Negative

Control

0.1

mg/mL

0.3

mg/mL

0.5

mg/mL

0.8

mg/mL

1.0

mg/mL

Uninhibited (1) 0.1558 0.0003 0.0539 0.0969 0.1172 0.1169 0.1417

Uninhibited (2) 0.0958 0 0.0396 0.0619 0.0495 0.0754 0.0898

Uninhibited (Avg) 0.1258 0.00015 0.04675 0.0794 0.08335 0.09615 0.11575

Low (1) 0.1421 0 0.0011 0.0189 0.0269 0.0361 0.049

Low (2) 0.0975 0 0.0022 0.0068 0.0106 0.016 0.0182

Low (Avg) 0.1198 0 0.00165 0.01285 0.01875 0.02605 0.0336

High (1) 0.082 0 0 0.0037 0.0045 0.0074 0.0073

High (2) 0.0873 0.0003 0.0003 0.004 0.0056 0.0107 0.0065

High (Avg) 0.08465 0.00015 0.00015 0.00385 0.00505 0.00905 0.0069

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 2 4 6Ab

sorb

ance

Val

ue

(A40

5)

Time (Minutes)

Absorbance Value vs. Time (Uninhibited)

Positive Control Negative Control

0.1 mg/mL 0.3 mg/mL

0.5 mg/mL 0.8 mg/mL

1.0 mg/mL

Figure 3 - Absorbance Value vs. Time (Uninhibited) - absorbance value readings were plotted against the time at which they were taken; readings from all 7 cuvettes are plotted

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In the uninhibited cuvettes, rate of reaction increases as substrate concentration increases,

as Figure 4 shows. Figure 5, the Lineweaver-Burke plot of the uninhibited enzyme shows that

the x-intercept is at -8.450. As the x-intercept is equal to -1/Km, Km of the uninhibited enzyme

Table 2. Km and Vmax of Enzyme at Different Inhibitor Concentrations – calculated using values from Lineweaver-Burke Plots

Km Vmax % Difference from no inhibitor

No inhibitor 0.1183 0.1188 Km Vmax

Low inhibitor concentration 1.684 0.08410 13.23 0.29

High inhibitor concentration 0.8673 0.01483 6.33 0.875

-10

-5

0

5

10

15

20

25

30

35

40

-20 -10 0 10 20 30

1/V

1/[S]

1/V vs. 1/[S] (Uninhibited)

Uninhibited

-0.02

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0 0.5 1 1.5

Rat

e o

f R

eact

ion

V

Substrate Concentration [S] (mg)

Rates of Reaction vs. Substrate Concentration (Uninhibited)

Uninhibited

Figure 4 - Rates of Reaction vs. Substrate Concentration (Uninhibited) - Michaelis-Menten plot of uninhibited enzyme

Figure 5 - 1/V vs. 1/[S] (Uninhibited) - Lineweaver-Burke plot of uninhibited enzyme; x-intercept = -8.450, y-intercept = 8.416

can be calculated to be 0.1183. Vmax can also be found since 1/Vmax is equal to the y-intercept.

Vmax for the uninhibited enzyme is calculated to be 0.1188. The same can be done to find the Km

and Vmax of the enzyme at low and high inhibitor concentrations. This was done using Figures 6, 7,

8, and 9. Table 2 shows the Km and Vmax values calculated for the enzyme at 0, low and high

inhibitor concentrations

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0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0 5 10 15

Rat

e o

f R

eact

ion

V

Substrate Concentration [S] (mg/mL)

Rates of Reaction vs. Substrate Concentration (Low inhibitor

concentration)

Low Inhibitor Concentration

-40

-20

0

20

40

60

80

100

-4 -2 0 2 4

1/V

1/[S]

1/V vs. 1/[S] (Low Inhibitor Concentration)

Low Inhibitor Concentration

-100

-50

0

50

100

150

200

250

300

-4 -2 0 2 4

1/V

1/[S]

1/V vs. 1/[S] (High inhibitor concentration)

High Inhibitor Concentration

Figure 6 - Rates of Reaction vs. Substrate Concentration (Low inhibitor concentration) - Michaelis-Menten plot of enzyme at low inhibitor concentration

Figure 7 - 1/V vs. 1/[S] (Low inhibitor concentration) - Lineweaver-Burke plot of enzyme at low inhibitor concentration; x-intercept = -0.594, y-intercept = 11.891

0

0.002

0.004

0.006

0.008

0.01

0.012

0.014

0.016

0.018

-3 2 7 12

Rat

e o

f R

eact

ion

V

Substrate Concentration [S] (mg/mL)

Rates of Reaction vs. Substrate Concentration (High inhibitor

concentration)

High Inhibitor Concentration

Figure 8 - Rates of Reaction vs. Substrate Concentration (High inhibitor concentration) - Michaelis-Menten plot of enzyme at high inhibitor concentration

Figure 9 - 1/V vs. 1/[S] (High inhibitor concentration) - Lineweaver-Burke plot of enzyme at high inhibitor concentration; x-intercept = -1.153, y-intercept = 67.423

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As hypothesized, reaction rates were decreased as inhibitor concentration was increased,

but Table 2 shows that there were large differences between both Km and Vmax values of the

enzyme at different inhibitor concentrations. This was unexpected as only one of the two values

should change due to inhibitor concentration3. However, Vmax steadily decreases as inhibitor

concentration increases while Kmax does not seem to observe any trend. Therefore, the conclusion

was made that the inhibitor used in the experiment was a noncompetitive inhibitor. The

variations in Km that cannot be explained could be attributed to errors.

A few experimental errors could have been made. There could have been inaccuracies

made while drawing up fluids to fill the cuvettes. This could have affected the ratio of enzyme,

substrate, and inhibitor in the cuvettes. The cuvettes could have been mixed inadequately

resulting in an uneven distribution of enzymes and substrate in some cuvettes and this could have

affected the reaction. The spectrophotometers could have been calibrated incorrectly or lost

calibration over the course of the experiment.

One curiosity that was observed by both groups studying the effects of high inhibitor

concentration on enzyme activity. It was expected that higher substrate concentrations would

result in higher reaction velocities. This trend was seen from substrate concentrations 0.1 mg/mL

to 0.8 mg/mL. However, at 1.0 mg/mL, one group observed a reaction velocity slightly slower

than what was observed at 0.8 mg/mL. The other group observed a reaction velocity at 1.0

mg/mL that was slightly greater than half of what was observed at 0.8 mg/mL. This trend can be

seen in Figure 8; reaction velocities increase with substrate concentration until 0.8 mg/mL where

it drops. The consistency of this observation makes experimental error less likely as an

explanation. However, since substrate solutions were provided in labeled cuvettes, it is possible

that the cuvettes were labeled incorrectly for both groups causing the unexplained observation.

Another possible explanation is a feedback regulation mechanism where high substrate

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concentrations begin to inhibit enzyme activity rather than speed it up. Further experimentation

would be needed to test this idea; for example, increasing the substrate concentrations used to see

how even higher substrate concentrations would be affected. Also future experiments can

investigate how a noncompetitive inhibitor would affect the enzyme. A different enzyme can

also be studied.

The importance of enzymes in biological processes makes understanding how enzymes

function in different environments and in the presence of different inhibitors vital to

understanding how biological processes are regulated. This experiment can be expanded to

investigate key enzymes in the blood, for example, and how they are affected by blood pH and

also how certain molecules function as inhibitors and activators to regulate the activities of an

enzyme. Furthermore, because enzymes are so vital to life, inhibition of essential enzymes can

be exploited for use in antibiotics and antiviral drugs. This experiment demonstrates that an

inhibitor can decrease the activity of an enzyme and can be used for antibiotic and antiviral

purposes. One such example of an inhibitor used as an antibiotic drug will now be discussed.

Discussion

Since enzyme activity is essential for many biological processes, the inhibition of certain

enzymes can be exploited to create useful drugs that can cure many ailments. Many antibiotics

function through inhibiting key enzymes necessary for bacterial life. One such antibiotic is

Aurovertin, which functions by binding to a specific subunit of F1Fo-ATPase in certain bacteria

and eukaryotic mitochondria and inhibiting the ability of ATPase to synthesize ATP.4

Aurovertin was first discovered in the fungus Calcarisporium Arbuscula4 and a different

version, Aurovertin E was recently discovered in the basidiomycete Albatrellus confluens6. It

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was long believed that Aurovertin is a noncompetitive inhibitor4 but a recent study found that

Aurovertin alters both Km and Vmax of ATPase, contrary to enzyme kinetics predictions4. This

new study from a team based in the University of Michigan proposed that Aurovertin displays

mixed inhibition resulting from different binding states that impact ATPase activity differently.

A weak bond to ATPase will only slow while a stronger bond can completely reverse ATPase

activity, making it hydrolyze ATP instead of synthesize it.4

The ability of Aurovertin to slow or completely inhibit ATP synthesis by ATPase makes

it an effective antibiotic. ATP acts as an energy source for cells when it is hydrolyzed to ADP,

releasing a phosphate group1, 2

. ATPase is the primary generator of ATP in most cells and by

reversing the reaction it catalyzes and causing ATPase to break down ATP to ADP, Aurovertin

can deprive its target organisms of ATP and cause cellular machinery that depend on ATP

energy to grind to a halt. Due to the high specificity of enzymes and their inhibitors, Aurovertin

will only bind to specific ATPases. This allows Aurovertin to be used as an antibiotic that can

stop infections without shutting down the body’s own cells.

Many antibiotics work in similar ways and take advantage of the high level of specificity

in enzymes. The ability to target specific enzymes only found on pathogens and not in human

cells enable these drugs to stop infections without damaging the cells of the patient. Therefore,

these antibiotics have become one of our most effective weapons against microbes. However,

their strength can easily become their weakness as a single mutation can cause a change in the

shapes of the enzymes they target and cause them to become wholly ineffective. Antibiotic use

must always be carefully regulated as resistant bacteria and other pathogens are becoming more

and more prevalent.

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References

1. Alberts, B., D. Bray, K. Hopkin, A. Johnson, J. Lewis, M. Raff, K. Roberts, and P. Walter.

Essential Cell Biology, Second Edition. 2009. Garland Science, Taylor & Francis Group,

New York, N.Y. Pp. 81-114.

2.

Campbell, N., J. Reese, L. Urry, M. Cain, S. Wasserman, P. Minorksy, and R. Jackson.

Biology, 8th Edition. 2008. Pearson Education Inc., San Franciso. Pp. 149-180.

3. “Enzyme Action: Effects Of Environmental Conditions.” Edited by Nelson, K. and Burpee, D.

(2009) Department of Biology, The Pennsylvania State University, University Park, PA.

Edited by Price, M, Siegfried, E, and Burpee, D (2006). Adapted from Price, M. and

Burpee, D. 2000 Department of Biology, The Pennsylvania State University, University

Park, PA.

4.

Johnson, K., L. Swenson, A. W. Opipari Jr., R. Reuter, N. Zarrabi, C. Fierke, M. Börsch, and G.

Glick. “Mechanistic Basis for Differential Inhibition of the F1Fo-ATPase by Aurovertin”.

Biopolymers. 21 May 2009. Volume 91, Issue 10. Wiley Periodicals. Pp. 830-840.

5. Raaij, Michael J., J. P. Abrahams, A. G. W. Leslie, and J. E. Walker. “The Structure of Bovine

Fl-ATPase Complexed with the Antibiotic Inhibitor Aurovertin B”. Proceedings of the

National Academy of Science, USA: Biochemistry. July 1996. Volume 93. Pp. 6913-

6917.

6. Wang, F., D. Luo, J. Liu. "Aurovertin E, a New Polyene Pyrone from the Basidiomycete

Albatrellus confluens”. The Journal of Antibiotics. 31 May 2005. Issue 58. Pp. 412-415.