Enzyme Kinetic Analysis of Alkaline Phosphatase

Enzyme Kinetic Analysis of Alkaline Phosphatase
Introduction

Alkaline phosphatase catalyzes the cleavage of esters of phosphoric acid.

In this experiment, the kinetics of hydrolysis of 4-nitrophenolphosphate by alkaline phosphatase will be measured.

To measure the activity of these enzymes, one can follow the liberation of phosphate or of the other product released by hydrolysis. The assay can be simplified by using a substrate whose phosphate-free product is highly colored. In this experiment, we will utilize 4-nitrophenylphosphate as the substrate, which upon hydrolysis releases phosphate to genterate 4-nitrophenolate under alkaline conditions. 4-nitrophenolate has a high molar absorptivity at 405 nm (e₄₀₅ = 18.8 x 10³ M^-1cm^-1).

This experiment will examine the effects of 1) enzyme concentration, 2) substrate concentration, and 3) an inhibitor on the rate of alkaline phosphatase catalyzed hydrolysis of 4-nitrophenylphosphate. You should review the enzyme kinetics section of your biochemistry textbook. It will give a good explanation of Michaelis-Menten kinetics, the plots that you will make with your data, the method for determining Vmax and Km of alkaline phosphatase from your data, and how to interpret the effects of the inhibitor on your enzyme.

Experimental Procedure
Part 1. Effects of enzyme concentration on the velocity of the reaction.

1. Take 400 µL of the 1 mg/mL alkaline phosphatase stock solution that is provided and place it in a 1.5 mL microcentrifuge tube. This solution contains 1 mg/mL alkaline phosphatase and 1 mg/mL BSA in distilled water. The BSA is added to stabilize the enzyme.

2. Make two serial dilutions of your 1 mg/mL alkaline phosphatase stock into 1.5 mL microcentrifuge tubes to make 0.5 and 0.25 mg/mL alkaline phosphatase solutions. Use a solution of 1 mg/mL BSA to dilute your stock. Invert your microcentrifuge tubes genltly to mix. DO NOT SHAKE! This will denature the protein. Bubbles or foam in your solution indicate that you have denatured protein. You should end up with three solutions that contain 0.25, 0.5, and 1 mg/mL alkaline phosphatase. Each solution will also contain 1mg/mL BSA. You want to have at least 200 µL of each alkaline phosphatase solution. KEEP ENZYME SOLUTIONS ON ICE!

3. Solutions of 4 mM p-nitrophenylphosphate and 0.05 M amidiol buffer pH 9.0 will be provided. Make 15 mL of 0.2 mM p-nitrophenylphosphate in 0.05 M amidiol buffer pH 9.0. Keep the substrate on ice until your ready to place it in the spectrophotometer. The substrate will slowly hydrolyze with time at room temperature.

4. Adjust the temperature on the Shimadzu spectrophotometer to 20.0 °C for enzyme kinetics. See directions at the end of this procedure for setting up the spectrophotometer to measure kinetics.

5. The spectrophotometer has 6 spaces to hold sample cuvettes and 1 space to hold a reference cuvette. Place 4 cuvettes in the first 4 sample positions and 1 cuvette in the reference position. Wipe cuvettes with a Kimwipe before placing them in the spectrophotometer to remove fingerprints. Use clean cuvettes.

6. To cuvettes 1 - 4 (sample cuvettes), add 2.94 mL of 0.2 mM p-nitrophenylphosphate in amidiol buffer. To the reference cuvette add, 2.94 mL of 0.05 M amidiol buffer and 60 µL of 1 mg/mL BSA. Let the substrate come to room temperature before adding enzyme (ie let it incubate in the spectrophotometer for 2 - 3 minutes before adding enzyme).

7. When you are ready to initiate enzyme reactions, add

60 µL of 1 mg/mL BSA to sample cuvette #1

60 µL of 1 mg/mL alkaline phosphatase to sample cuvette #2

60 µL of 0.5 mg/mL alkaline phosphatase to sample cuvette #3

60 µL of 0.25 mg/mL alkaline phosphatase to sample cuvette #4.

Suggested procedure for making additions quickly:

General procedure:

One student adds enzyme to cuvette and the second student mixes the samples after enzyme addition.

For example:

Student 1: Add 60 µL of BSA to cuvette #1. Remove pipet tip & get a clean tip.

Student 2: Quickly mix solutions in cuvette # 1 using plastic stirring rod or mix by drawing solution into and out of 1 mL pipet tip on a pipetman with the volume set to 1 mL. Change the tip before mixing the next solution.

Student 1: With the clean pipet tip, add 60 µL of 1 mg/mL alkaline phosphatase to cuvette #2. Remove pipet tip and get a clean tip.

Student 2: Quickly mix solutions in cuvette #2 as before.

Student 1: With the clean pipet tip, add 60 µL of 0.5 mg/mL alkaline phosphatase to cuvette #3. Remove pipet tip and get a clean tip.

Student 2: Quickly mix solutions in cuvette #2 as before.

Student 1: With the clean pipet tip, add 60 µL of 0.25 mg/mL alkaline phosphatase to cuvette #4.

Student 2: Quickly mix solutions in cuvette #2 as before.

Make these additions as quickly as possible and then start measuring kinetics. Follow the reaction for 5 minutes taking points every 30 seconds. When reactions are complete, obtain a print out of your data and clearly label.

After adding enzyme to initiate kinetics, cuvettes will contain the following:

Reference ® 2.94 mL amidiol buffer + 60 µL 1 mg/mL BSA

Sample #1 ® 2.94 mL 0.2 mM p-nitrophenylphosphate + 60 µL 1 mg/mL BSA

Sample #2 ® 2.94 mL 0.2 mM p-nitrophenylphosphate + 60 µL 1 mg/mL alkaline phosphatase

Sample #3 ® 2.94 mL 0.2 mM p-nitrophenylphosphate + 60 µL 0.5 mg/mL alkaline phosphatase

Sample #4 ® 2.94 mL 0.2 mM p-nitrophenylphosphate + 60 µL 0.25 mg/mL alkaline phosphatase

The sample in cuvette #1 provides a measure of the nonenzymatic rate of hydrolysis of your substrate. Any increase in absorbance values obtained from this sample should be subtracted from the corresponding time readings in your enzyme catalyzed reactions before constructing final plots. This will allow you to determine the rate of substrate hydrolysis due to the enzyme alone.

Plot on a single graph, plot the absorbance at 405 nm vs time for reactions at each enzyme concentration. Determine the velocity for each enzyme reaction from these plots by determining the initial slope of the lines on plot 1. Note that the velocity of the reaction is equal to the change in absorbance divided by the change in time.

Part 2. Effects of substrate concentration on the reaction velocity and effect of an inhibitor (phosphate) on the reaction velocity.

1. Get 10 mL of a stock solution of 4 mM p-nitrophenylphosphate substrate in 0.05 M amidiol buffer pH 9.0. Place it in a 50-mL volumetric flask and dilute it to 50 mL with 0.05 M amidiol buffer pH 9.0.

2. Make 6 serial 1/2 dilutions of this stock by adding 25 mL of the diluted substrate prepared in step 1 with 25 mL of 0.05 M amidiol buffer pH 9.0. You should end up with dilutions containing 0.4, 0.2, 0.1, 0.05, 0.025, and 0.125 mM p-nitrophenylphosphate in amidiol buffer.

3. Make 1.2 mL of alkaline phosphatase in a 1.5 mL microcentrifuge tube. Use the results of Part 1 to determine the appropriate enzyme concentration to use. Remember to dilute your enzyme with a solution of 1 mg/mL BSA.

4. Make 1.2 mL of 8 mM Na₂HPO₄ in distilled water from the 150 mM Na₂HPO₄ stock solution provided.

5. Measure reaction kinetics using the 6 substrate dilutions ( 0.4, 0.2, 0.1, 0.05, 0.025, and 0.125 mM p-nitrophenylphosphate) that you prepared in step 2.

Enzyme assays:

• Measure the rate of the reaction at substrate dilution in the absence of the Na₂HPO₄ inhibitor and in the presence of Na₂HPO₄ inhibitor. Also measure the rate for the nonenzymatic reaction. Make these measurements "side-by-side" at each substrate concentration.

• You will need all six sample compartments and the reference compartment. You can run each of the three reactions at two different substrate concentrations in 1 run.

• For enzyme catalyzed reactions with no inhibitor, place 2.94 mL of substrate + 60 µL of enzyme + 60 µL of water in cuvette.

• For enzyme catalyzed reactions with inhibitor, place 2.94 mL of substrate + 60 µL of enzyme + 60 µL of 8 mM Na₂HPO₄ in cuvette.

• For nonenzymatic control reaction, place 2.94 mL of substrate + 60 µL of 1 mg/mL BSA + 60 µL of water in cuvette.

• For the reference, place 2.94 mL of buffer + 60 µL of 1 mg/mL BSA + 60 µL of water in cuvette.

• Add the enzyme last. Use the procedure that you used in Part 1 to quickly mix the enzyme with the substrate.

Example of samples placed in spectrophotometer:

Cell Solution

Reference 2.94 mL of buffer + 60 µL of 1 mg/mL BSA + 60 µL of water

Sample #1 2.94 mL 0.4 mM p-nitrophenylphosphate + 60 µL enzyme + 60 µL 8 mM Na₂HPO₄

Sample #2 2.94 mL 0.4 mM p-nitrophenylphosphate + 60 µL enzyme + 60 µL water

Sample #3 2.94 mL 0.4 mM p-nitrophenylphosphate + 60 µL 1 mg/mL BSA + 60 µL water

Sample #4 2.94 mL 0.2 mM p-nitrophenylphosphate + 60 µL enzyme + 60 µL 8 mM Na₂HPO₄

Sample #5 2.94 mL 0.2 mM p-nitrophenylphosphate + 60 µL enzyme + 60 µL water

Sample #6 2.94 mL 0.2 mM p-nitrophenylphosphate + 60 µL 1 mg/mL BSA + 60 µL water

Shimadzu Program for Enzyme Kinetics

Mode: Attachment

Mode: CPS Kinetics

1. wavelength = 405 nm
2. Absorbance range = +2.0
3. Cell: N=4 for Part 1

4. Factor = 1
5. Start Point (upper/lower) = lower
6. Data print = yes
7. Gain x10 = no
8. Reagent Blank = yes for Part 1 (use buffered substrate in cell #1)

9. Cell blank

Prelab Questions

1. To determine the effects of enzyme concentration on reaction kinetics, you will be measuring the rate of enzyme catalyzed reactions at a constant concentration of substrate (0.2 mM p-nitrophenyl phosphate).

2. What do you need to dilute your substrate with in Part 2 where you will measure reaction rates as a function of substrate concentration?

3. Calculate the volumes of 150 mM Na₂HPO₄ and distilled H₂O that you need to make 1.2 mL of 8 mM Na₂HPO₄.

Lab Reports
Plots to include in reports:

Before constructing plots of absorbance data (y-axis) vs time, remember to correct increases in absorbances in enzyme catalyzed reactions for increases in absorbance due to nonenzymatic reactions. Include a sample calculation in your lab reports to indicate how this was done.

Effects of enzyme concentration on reaction rates.

1. A plot of absorbance at 405 nm (y-axis) vs time. Plot the data for reactions done at each enzyme concentration on the same graph. Determine reaction velocities by measuring the initial slopes of the lines. Fit data in the linear region using a linear regression to calculate the slope. Note the velocity of the reaction is equal to the change in absorbance divided by change in time (∆A/∆t) since the absorbance is directly proportional to the concentration of product. The extinction coefficient for p-nitrophenolate at 405 nm is 18.8 x 10³ M^-1cm^-1.

2. Make a plot of the results of plot #1 by plotting the velocity of the reaction (y-axis) vs the concentration of enzyme.

Effects of substrate concentration on reaction rates.

3. Plot the absorbance at 405 nm (y-axis) vs time for enzyme catalyzed reactions at each substrate concentration. You may put all of the plots on the same graph unless plotting all six plots on the same graph makes the individual plots difficult to see clearly. In this case, you may want to make two graphs with plots for three different substrate concentrations. Determine enzyme catalyzed reaction velocities for each concentration of substrate as you did in plot #1.

4. Plot velocity (y-axis) vs. substrate concentration. Don't forget to indicate the velocity when no substrate is present. Indicate where V_max and K_m are on this plot.

5. Make a Lineweaver-Burk plot using the velocities that you determined in plot 3 and substrate concentrations. Use this plot to calculate V_max and K_m values. Indicate the equations that you used to construct your Lineweaver-Burke plot and to calculate V_max and K_m from this plot. Show a sample calculation.

Effects of an inhibitor on enzyme catalyzed reaction rates.

6. Plot the absorbance at 405 nm (y-axis) vs time for enzyme catalyzed reactions in the presence of Na₂HPO₄ at each substrate concentration. You may put all of the plots on the same graph unless plotting all six plots on the same graph makes the individual plots difficult to see clearly. In this case, you may want to make two graphs with plots for three different substrate concentrations. Determine enzyme catalyzed reaction velocities for each concentration of substrate as you did in plot #1.

7. Plot velocity (y-axis) vs. substrate concentration for enzyme catalyzed reactions in the presence of inhibitor (Na₂HPO₄). On this same plot, plot the velocity vs substrate concentration for reactions with no inhibitor (data from plot #4 above). Don't forget to indicate the velocity when no substrate is present. Indicate where V_max and K_m are on this plot.

8. Make a Lineweaver-Burk plot using the velocities that you determined in plot 6 and substrate concentrations for reactions in the presence of inhibitor (Na₂HPO₄). Also plot data from plot #5 for reactions with no inhibitor. Use this plot to calculate V_max and K_m values. Use this plot to determine what type of inhibition Na₂HPO₄ shows. Discuss how you came to this conclusion. If Na₂HPO₄ acts as a competitive inhibitor, use your data to determine its K_i value.

Questions

1. How does the reaction velocity change as a function of enzyme concentration? Does twice the enzyme concentration give twice the velocity?

2. Does the velocity of the reaction change with time? If so, why?

3. Given the following reaction

and the following equation for the initial velocity of the reaction:

_cat

₃

(k_cat is the rate constant for the reaction which forms the product from the ES complex), explain in words why the velocity is directly proportional to the amount of enzyme added in the presence of saturating substrate levels.

4. Why is K_m an important number?

Specifically consider

a) what is the velocity of the reaction when [S] = K_m?

b) what aspect of the enzymatic reaction (binding or product formation) does K_m relate most to and under what conditions will K_m measure substrate binding

c) what is the rate limiting step in the enzyme reaction when [S] is much less than K_m and when [S] is much greater than K_m. Explain your reasoning.