The Effects of pH and Inhibitor Concentration on the Activity of a Phosphatase Enzyme

Lucas Man Group members: Andrew Kim Paul Digiacobbe Kristine Nale Section 16 TA: Austin Nuschke

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 (V max). The X-value that results in 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 enzymes 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 enzymes 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 enzymes 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 used 3. 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 pHs 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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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. Absorbance Value vs. Time (pH Assay)
1.6 1.4 Absorbance (A405) 1.2 1 0.8 0.6 0.4 0.2 0 0 2 4 6 8 10 12 14 16 18 Positive Control Negative Control pH 3 pH 5 pH 6 pH 7 pH 8 pH 9 pH 10 pH 11

Time (Minutes)

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

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
Reaction Velocity

Reaction Velocity vs. pH

0.09 0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0 0 1 2 3 4 5 6 7 8 9 10 11 12 pH
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).
Absorbance Value vs. Time (Uninhibited)
Absorbance Value (A405) 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0 2 4 6

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

Time (Minutes) Positive Control 0.1 mg/mL 0.5 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

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.

Negative Control 0.3 mg/mL 0.8 mg/mL

Table 1. Reaction Velocities at Different Substrate Concentrations values taken from the slopes of absorbance vs. time plots Positive Negative 0.1 0.3 0.5 0.8 1.0 Control Control mg/mL mg/mL mg/mL mg/mL 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 Man 6

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

Rates of Reaction vs. Substrate Concentration (Uninhibited)

0.14 0.12 Rate of Reaction V 0.1 0.08 1/V 0.06 0.04 0.02 0 -0.02 0 0.5 1 1.5 Substrate Concentration [S] (mg) Uninhibited
Figure 4 - Rates of Reaction vs. Substrate Concentration (Uninhibited) - Michaelis-Menten plot of uninhibited enzyme

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

40 35 30 25 20 15 10

5
0 -20 -10 -5 0 -10 1/[S] Uninhibited
Figure 5 - 1/V vs. 1/[S] (Uninhibited) - Lineweaver-Burke plot of uninhibited enzyme; x-intercept = -8.450, y-intercept = 8.416

10

20

30

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 K m 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 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

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Rates of Reaction vs. Substrate Concentration (Low inhibitor concentration)

0.07 0.06 Rate of Reaction V 0.05 0.04 0.03 0.02 0.01 0 0 5 10 15 -4 1/V

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

100 80 60 40 20 0 -2 -20 0 2 4

Substrate Concentration [S] (mg/mL) Low Inhibitor Concentration

-40 1/[S] Low Inhibitor Concentration

Figure 6 - Rates of Reaction vs. Substrate Concentration (Low Figure 7 - 1/V vs. 1/[S] (Low inhibitor concentration) inhibitor concentration) - Michaelis-Menten plot of enzyme at Lineweaver-Burke plot of enzyme at low inhibitor low inhibitor concentration concentration; x-intercept = -0.594, y-intercept = 11.891

Rates of Reaction vs. Substrate Concentration (High inhibitor concentration)

0.018 0.016 0.014 0.012 0.01 0.008 0.006 0.004 0.002 0 2 7 12

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

300 250 200 150 1/V 100 50

Rate of Reaction V

0
-4 -2 -50 0 -100 1/[S] High Inhibitor Concentration 2 4

-3

Substrate Concentration [S] (mg/mL) High Inhibitor Concentration

Figure 8 - Rates of Reaction vs. Substrate Concentration (High Figure 9 - 1/V vs. 1/[S] (High inhibitor concentration) inhibitor concentration) - Michaelis-Menten plot of enzyme at Lineweaver-Burke plot of enzyme at high inhibitor high inhibitor concentration 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 Man 9

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 Man 10

was long believed that Aurovertin is a noncompetitive inhibitor 4 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 bodys 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

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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. 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. 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. Johnson, K., L. Swenson, A. W. Opipari Jr., R. Reuter, N. Zarrabi, C. Fierke, M. Brsch, and G. Glick. Mechanistic Basis for Differential Inhibition of the F1 Fo-ATPase by Aurovertin. Biopolymers. 21 May 2009. Volume 91, Issue 10. Wiley Periodicals. Pp. 830-840. 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. 69136917. Wang, F., D. Luo, J. Liu. "Aurovertin E, a New Polyene Pyrone from the Basidiomycete Albatrellus conuens. The Journal of Antibiotics. 31 May 2005. Issue 58. Pp. 412-415.

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