Determining the Effects of pH and Inhibitors on the Rate of Reaction using the Phosphatase Enzyme

Kevin Bobeck Group Memebers: Kelli Dunker, Michael Yacovelli, and Jessica Jankowski Biol 230W

Introduction: Enzymes are catalysts that exist only in living things. Catalysts are compounds or molecules that speed up a chemical reaction by lowering the activation energy. Catalysts cannot be altered or changed during the course of a reaction. Enzymes bind to the reactants of a chemical reaction, the substrates, at a specific site on the enzyme called the active site; therefore, forming the enzyme – substrate complex. An enzyme can only bind to specific substrates based on the conformation of the active site. The activity of an enzyme can be affected by four different factors. These factors are pH, temperature, concentration of substrate, and prescence of inhibitors. Changes in the levels of pH affect the conformation of the active site, which therefore affects the substrates that can bind to it, but if the change is pH is too drastic it could denature the enzyme and the enzyme will become inactive. Temperature also affects the activity of an enzyme by increasing the kinetic motion of the reaction. As the temperature rises, the kinetic motion increases; thus, increasing collisions between enzymes and substrates. This increase in collisions increases the rate at which the product is formed; therefore, increasing rate of the overall reaction. Concentration of substrate also influences the speed of the reaction. The higher the concentration of substrate the quicker the reaction will proceed. This is because the more available free substrates the easier the enzymes can bind to them, and thus the reaction will progress faster. (Thomas. 2009) As mentioned earlier, enzyme activity can be influenced by the presence of an inhibitor. These compounds decrease the enzyme activity by binding either directly to the active site or indirectly to another site on the enzyme. Competitive enzymes bind directly to the active site, thus blocking the ability of the substrate to bind to the enzyme. Competitive inhibition is a reversible process and can be overcome by increasing the concentration of substrates. Increasing

the concentration of the substrate overcomes competitive inhibition by outcompeting the inhibitor for the active site. There are also noncompetitive inhibitors which indirectly bind to the enzyme. These inhibitors cause inhibition by binding to a site other than the active site and changing the conformation of the active site. Since the enzyme does not bind directly to the active site, increasing the concentration of substrate will not have an effect on the inhibitor. (Thomas) Enzyme kinetics is the study of the rate at which a specific enzyme is active. The rate of a reaction is the Vmax . The rate constant (Km ) can be figured out by calculating the concentration of a substrate at which the velocity is equal to half of the Vmax. Enzyme inhibitors can also have an effect on the Vmax. For instance, noncompetitive inhibitors decrease the Vmax because they alter the conformation, which disable the enzyme. However, competitive inhibitors have no effect on the Vmax because they do not change the enzymes conformation and is a reversible process. In order to overcome a competitive inhibitor, the number of substrate molecules must increase; hence, increasing the rate constant. Although competitive inhibitors do not affect the Vmax, they do affect the rate constant of a reaction. (Thomas) In this experiment, the enzyme phosphatase will be used to catalyze the reaction. Phosphatase enzyme acts by binding the substrate and removing the phosphate group. This process of removing the phosphate group is called dephosphorylation. Two different types of phosphatase are alkaline phosphatase and acidic phosphatase. Acidic phosphatase works best under acidic conditions to neutral pH. Alkaline phosphatase works best under alkaline conditions. Through this experiment, one will be able to determine whether the phosphatase is either acidic or alkaline. The enzyme inhibitor that will be used in this experiment is disodium hydrogen phosphate, or Na2HPO4. This compound is water soluble. According to a study done

by Chen QX and Zhou HM at the Tsinghua University in1999, Na2HPO4 is a competitive inhibitor. The major objectives of this experiment were two fold. The first objective was to determine the optimum pH at which the enzyme phosphatase is most active. A second objective of this experiment is to determine whether the enzyme inhibitor Na2HPO4 is a competitive or noncompetitive inhibitor. Based on the results of the pH test, we should be able to determine the optimum pH for the unknown enzyme B. The second experiment should help us determine whether or not inhibitor number 5, low concentration, is competitive or noncompetitive. Based on the results of the Chen QX and Zhou HM experiment, I believe that inhibitor number 5 is a competitive inhibitor and as the substrate concentration increases the rate of the reaction will also decrease to overcome the inhibitor. The product of the substrates reacting with enzyme yields a product that emits a yellow color. Therefore, the more product that is formed will increase the absorbance, indicating an increase in the rate of a reaction. Material and Methods: There were two different experiments done in this lab. The first experiment was done to determine which type of phosphatase we had either alkaline or acidic by observing the pH level where the enzyme was most reactive. The second experiment was done in order to determine the effect of an inhibitor on enzyme activity and whether or not the inhibitor is competitive or noncompetitive. The first experiment was started by turning on the spectrophotometer and setting it to 405nm wavelength. The next step was labeling ten cuvettes with a marker. The cuvettes were labeled with numbers one through ten. Each cuvette must be labeled at the top so it does not

interfere with spectrophotometer reading. Then, one should place the specified measurements of each substance into the specified cuvette, making sure that each cuvette has only four millimeters of solution. One should be careful to change the pipet for different substances so there is no mixing of pH levels or other substances. One should also to be careful to always add the enzyme last to the solution, so that the enzyme can be kept on ice as long as possible. Once the enzyme has been added the group can then begin to measure the absorbance values of the products produced. The cuvettes should be measured in two minute intervals for about fifteen minutes. For instance, one should measure the absorbance for all of the cuvettes and then wait two minutes and then repeat this process for about fifteen minutes. The results should then be recorded and graphed in order to find the rate of the reaction.(Alberts, et al.) The exact measurements of the different substances can be seen in the table below:
Table 1. Treatments to determine the pH optimum of the phosphatase enzyme

Cuvette # 1 2 3 4 5 6 7 8 9 10

Buffer (pH and mL) Buffer (1mL) Buffer (1 mL) pH 3 (1mL) pH 4 (1mL) pH 5 (1mL) pH 6 (1mL) pH 8 (1mL) pH 9 (1mL) pH 10 (1mL) pH 11 (1mL)

1mg/L Substrate (mL) 1 1 1 1 1 1 1 1 1 1

H2O (mL) 1 2 1 1 1 1 1 1 1 1

Enzyme (mL) 1 0 1 1 1 1 1 1 1 1

The second experiment was started by turning on the spectrophotometer and setting it to 405nm wavelength. The next step was to label seven cuvettes with a marker. The cuvettes were to be labeled one through seven. One should begin by placing the specifically measured substances in the specified cuvette. Once again one should be careful when using the pipets not mix and that the enzyme is added last. These cuvettes should also be measured in the spectrophotometer in two minute intervals in the same way as the first experiment. The solutions

in each cuvette differ in the concentration of substrate. No inhibitor was added to cuvettes 1 and 2 because they acted as controls.(Alberts, et al.) One can view the exact amount of each substance added in the table below:
Table 2. Treatments for testing the effect of an inhibitor on enzyme activity

Cuvette # 1 2 3 4 5 6 7

Buffer (mL) 1 1 1 1 1 1 1

Inhibitor (mL and conc.) 0 0 1 1 1 1 1

Substrate (mL and conc.) 1mL 1mL .1 mg .3 mg .5 mg .8 mg 1 mg

H2O (mL) 1 2 0 0 0 0 0

Enzyme (mL) 1 0 1 1 1 1 1

As stated in the introduction, the enzyme that was used in both of these experiments was phosphatase. The range of pH’s used were three through ten. Each group was given either the acidic phosphatase or alkaline phosphatase and it is each group’s job to determine which enzyme they were given. In the second experiment, each group was given a different inhibitor, either no inhibitor, low concentration inhibitor, or high concentration inhibitor. These concentrations were 0 (none), 5mM (low), or 20mM (high). My group was given the low concentration inhibitor or 5mM concentration inhibitor. (Alberts, et al.) Results: It is possible to determine the how effective an enzyme is by determining the absorbance value because the products of this reaction absorb light. Therefore, the higher the absorbance value the more products have been formed. The more products being formed indicates the higher rate of the reaction. The results of the pH test are as shown in the table below:

Table 3. Absorbance readings for pH optimum experiment

Time(s) 0 120 240 360 480 600 720 840 960 1080 1200 1 0 .07 .28 .36 .48 .58 .62 .73 .80 .88 .95 2 0 0 0 0 0 0 0 0 0 0 0 3 0 0 0 0 0 0 0 0 0 0 0 4 0 0 0 0 0 0 0 .01 .01 .01 .01

Cuvette # 5 6 0 0 0 .01 0 .01 0 .02 0 .02 0 .03 .01 .04 .01 .05 .01 .06 .01 .07 .01 .07

7 0 .02 .04 .06 .10 .12 .17 .21 .22 .25 .26

8 0 .09 .16 .24 .32 .40 .50 .60 .66 .70 .80

9 0 .09 .19 .30 .41 .52 .68 .79 .88 .95 1.00

10 0 0 .02 .04 .07 .12 .15 .18 .21 .25 .30

Table 3 represents the optimum pH for the phosphatase enzyme. This table shows that the pH optimum for this enzyme is 10, thus indicating that the enzyme is alkaline phosphatase. This also indicates that the enzyme under pH 9 is the fastest rate of the reaction. This information can also be viewed in Figure 1, which gives one a visual way to understand the information.

Rate vs. pH Level
0.0009 0.0008 0.0007 0.0006 Rate 0.0005 0.0004 0.0003 0.0002 0.0001 0 0 2 4 6 pH Level
Figure 1. The rate of reaction in relation to the pH level of the solution that the reaction takes place in.

8

10

12

The results of the second experiment to determine the effect of an inhibitor the enzyme and if the inhibitor is competitive or noncompetitive are shown below in Table 4. Our group was given the low concentration inhibitor (5mM).
Table 4. Absorbance readings for the low concentration inhibitor

Time (s) 1 positive control .02 .15 .39 .51 .69 .81 2 Negative control 0 0 0 0 0 0 3 .1 mg 0 0 .01 .01 .01 .02

Cuvette # 4 .3 mg 0 .01 .02 .03 .04 .05

5 .5 mg 0 0 .04 .06 .09 .13

6 .8 mg 0 .02 .07 .13 .17 .22

7 1.0 mg 0 .01 .06 .09 .13 .18

0 120 240 360 480 600

These results show that the cuvette # 6 had the highest rate of reaction. This is due to the fact that it contains a high concentration of substrate. However, there must be an error in the course of this experiment because cuvette # 7 should have the highest rate because it has the highest substrate concentration to overcome the competitive inhibitor, Na2HPO4. This experiment also backs up the conclusion of Chen and Zhou that Na2HPO4 is a competitive inhibitor because as the concentration of the substrate increased the inhibitor lost its effect and the enzyme became more active. This can also be seen in Figures 2 and 3.

Rate v. Substrate Concentration
0.001 0.0008 0.0006 Rate No Inhibitor 0.0004 0.0002 0 0 -0.0002 0.2 0.4 0.6 0.8 1 1.2 Substrate Concentration (mg)
Figure 2. This graph represents the Michaelis - Menten Kinetics Plot

Low Concentration High Concentration

1/ Rate v. 1/ Substrate Concentration
50,000.00 45,000.00 40,000.00 35,000.00 30,000.00 25,000.00 20,000.00 15,000.00 10,000.00 5,000.00 0.00 0 2 4 y = 4125.6x + 4589.9

1/ Rate

y = 2471.2x + 747.9

No Inhibitor Low Concentration High Concentration

y = 564.67x + 535.85 6 8 10 12

1/ Substrate Concentration (mg)
Figure 3. This graph depicts the Lineweaver - Burke Double - Reciprocal Plot

There were a few minor difficulties with this experiment. The most obvious one can be seen in the effect of inhibitor experiment. Cuvette #6 has a higher absorbance than cuvette #7, which should not be the case because cuvette #7 has a higher substrate concentration. Based on the results of the first experiment, the enzyme reached its optimum pH at pH 10. According to this result, the enzyme must be basic and is alkaline phosphatase because it

performed best under basic conditions. The experiment that was conducted to determine whether or not Na2HPO4 is competitive or noncompetitive indicated that the inhibitor was competitive. This logic is based on the fact that as the substrate concentration increased, the rate of the reaction also increased due to the more substrate molecules being able to overcome the inhibitor for binding at the active site. Discussion: There are multiple diseases that are caused by faulty enzymes. One such disease is called Fabry’s Disease. This disease is also called a lysosomal storage disease. The disease is caused by a defective enzyme that is involved in the metabolization of lipids. This enzyme is agalactosidase A. A gene that regulates this enzyme causes an insufficient breakdown of lipids. The gene that causes this insufficient breakdown is carried on the X chromosome of the mother. The defective enzyme leads to the accumulation of lipids on the vascular tissue and the skin, heart, kidneys, and central nervous system organs. This disease is an inborn of glycosphingolipid metabolism formed by mutations of the gene that codify the lysosome enzyme α-galactosidase A, or also known as A – Gal. There are many various defects of the gene, over 300, but in families the same defect of the gene is usually passed down. Homozygote males often express the classical phenotype of this reaction, but heterozygote females can also express the disease with milder effects. The relationship between genotype and phenotype is controversial. Various genotypes have the possibility to express the same genotype. The disease can only be homozygote in males because they have only one X chromosome. In homozygote males, there is a total loss of function in the enzyme with symptoms first appearing in childhood and early adolescence. In heterozygote females, the range

of expression of the disease can vary from very mild symptoms to full blown symptoms seen in men. This is due to the process of random inactivation of the X chromosome.(Boggio, P., et al. 2009) There are some treatments for this disease but no definitive cure. For instance, enzyme replacement may slow the progression of the disease but not get rid of it. Some of the pain from the disease can be eased by medications. Although most patients survive into adulthood, these individuals become at a greater risk for strokes, heart attacks, heart disease, and renal failure. (National Institutes of Health, 2009)

References: Alberts, B., D. Bray, K. Hopkin, A. Johnson, J. Lewis, M. Raff, K. Roberts, and P. Walter. 2009.ESSENTIAL CELL BIOLOGY, Second Edition. Garland Science, Taylor & Francis Group, New York, N.Y. Anonymous. "Enzyme Kinetics and Catalysis." Ed. Graham H. Thomas. 21 Aug. 2009. Web. 5 Oct. 2009. <http://wikispaces.psu.edu/display/Biol230WFall09/Enzyme+Kinetics+and+Catalysis>. Boggio, Paula, Paula C. Luna, Maria E. Abad, and Margarita Larralde. "Fabry disease." Anais Brasileiros de Dermatologia 84.4 (2009): 367+ Chen, Q. X., and H. M. Zhou. "Inhibitors of green crab (Scylla serrata) alkaline phosphatase." Journal of Enzyme Inhibiton (1999). National Institutes of Health; National Institute of Neurological Disorders and Stroke. "Fabrys Disease." Clevelan Clinic. 5 June 2009. Web. 20 Oct. 2009.