Reference no: EM132990952

Enzymes: Activation Energy

Learning Objectives:

1. Gain experience with laboratory techniques and practice performing calculations;
2. To perform a controlled experiment and discuss results obtained by comparing two sets of data;
3. To learn about reaction rates and activation energy and the effect that enzymes have on these;
4. To practice using a computer to produce graphs;
5. To become familiar with the elements of a practical report.

Part A: Enzyme-Catalysed Hydrolysis of Phenylphosphate

1. Label 9 microfuge tubes and pipette into each 60 μl of acetate buffer and 15 μl of acid phosphatase (see Table 1).

2. To tube 1 add 75 μl of phenyl disodium orthophosphate (phenyl phosphate) and place immediately on ice. Add 150 μl of FC reagent and MIX WELL. This is your control. Acid hydrolysis has been stopped.

3. Place: tubes 2 & 3 in the 24°C dry heater block tubes 4 & 5 in the 28°C dry heater block tubes 6 & 7 in the 34°C dry heater block tubes 8 & 9 in the 45°C dry heater block

Take note of the actual temperatures on the thermometers, these will be the temperatures you use on your graph. Even one or two degrees difference between what you graph and the actual temperature will significantly affect your result.

- Allow 3 - 4 minutes for temperature equilibration.
- Add 75 μl of phenyl phosphate solution to tubes 2 and 3 and start timing your reaction. You have started the reaction by adding the substrate.
- Repeat for each of the other 6 tubes, noting the time. It is easiest to wait one minute between each heater block. Things will run more smoothly when you take the reactions off.
4. Fifteen minutes after adding substrate remove tube 2 to ice and immediately add 150 μl of FC reagent, MIX well. At this point the reaction has been stopped.

Repeat for tubes 4, 6 and 8 (the even numbered tubes are the fifteen minute time-points).

5. Thirty minutes after adding substrate remove tube 3 to ice and immediately add 150 μl of FC reagent, MIX well. At this point the reaction has been stopped.

Repeat for tubes 5, 7 and 9 (the odd numbered tubes are the thirty minute time-points).

6. This is done to all tubes at the end of the assay:
Add 750 μl of 0.6 M sodium carbonate to all nine tubes. MIX thoroughly and incubate in a dry heater block 37 °C /10 min. This step allows the phenol to react with the FC reagent to produce a blue colour.

7. Read the absorbance at 500 nm using the solution from tube 1 as a blank. Please use only two cuvettes, one for your blank and one for your samples.

Part B: Non-Enzymic Hydrolysis of Phenylphosphate

1. Label 9 microfuge tubes and pipette into each 60 μl of acetate buffer and 15 µl dH2O (Table 2).

2. To tube 1 add 75 μl of phenyl disodium orthophosphate (phenylphosphate) and place immediately on ice.

Add 150 μl of FC reagent and MIX WELL. This is your control. Acid hydrolysis has been stopped.

3. Pipette 75 μl of phenylphosphate into each tube. MIX. You have now added the substrate. Add to dry heater block immediately (step 4) and start timing.

4. Note the time and place reaction mixtures in dry heater block as follows:
tubes 2 & 3 in the 65°C dry heater block tubes 4 & 5 in the 75°C dry heater block tubes 6 & 7 in the 80°C dry heater block tubes 8 & 9 in the 88°C dry heater block

5. Fifteen minutes after adding substrate remove tube 2 to ice and immediately add 150 μl of FC reagent, MIX well. At this point the reaction has been stopped
Repeat for tubes 4, 6 and 8 (the even numbered tubes are the fifteen minute time-points)..

6. Thirty minutes after adding substrate remove tube 3 to ice and immediately add 150 μl of FC reagent, MIX well. At this point the reaction has been stopped.
Repeat for tubes 5, 7 and 9 (the odd numbered tubes are the thirty minute time-points).

7. This is done to all tubes at the end of the assay:

Add 750 μl of 0.6 M sodium carbonate to all nine tubes. MIX thoroughly and incubate in a dry heater block 37 °C / 10 min. This step allows the phenol to react with the FC reagent to produce a blue colour.

8. Remove the tubes from the heater block. Which ones have the strongest colour development?

Read the absorbance at 500 nm using the solution from tube 1 as a blank. Please use only two cuvettes, one for your blank and one for your samples.

Methods

Q1. In UV-Vis spectrophotometry, why is it necessary to dilute the sample if its absorbance exceeds 0.95?
Hint: you need to talk about the instrument.

Results (Analysis / Calculations)

Q2. Plot the data as an Arrhenius plot, use one graph for both data sets. It should generate two straight lines with negative slope. Use the slopes to calculate the activation energy (Ea) for the reaction under the two conditions according to Eq 1. Compare these values. Show where all initial values came from and all working.

Q3. What would be the hydrolytic rates at 37 °C of the non-enzymic acid-catalysed and enzyme-catalysed reactions (in AU min-1 and AU mg-1 min-1 respectively)?
Hints: use the two equations that fit the lines on the Arrhenius plot and remember that temperature is in Kelvin; the reaction rate is directly proportional to the amount of enzyme present in the enzymic reaction (you did not use 1 mg of enzyme).

Q4. How much faster is the enzyme-catalysed reaction at 37 °C when 1 mg of enzyme is present than the
acid-catalysed, non-enzymic one? Comment on the action of the enzyme.
Hint: Compare the reaction rates. Is one a number of times faster than the other?

Discussion

Q5. The bond energy of a phosphorous-oxygen bond is on the order of 340 kJ mol-1. You most likely did not see this bond energy in your results. From your knowledge of activation energy what happens in the transition state and how does this relate to bond energy in a non-enzymic (acid-catalysed) and enzymic reaction. Did you see what was expected? Explain why your results are/are not consistent with what was expected.
Hint: What happens in the transition state? What do you expect in an enzyme catalysed reaction? Did you see or not see what was expected?

Q6. Compare the decrease in the activation energy you observed by the enzyme catalysed reaction (e.g.
5-fold) with the increase in the rate of reaction at 37°C. Discuss your result.
Hint: How efficient is the enzyme-catalysed reaction verses the acid catalysed reaction?

Q7. Name two ways in which the rate of the acid catalysed non-enzymic reaction could be increased (other than by adding enzyme). What would happen to an enzyme under these conditions?
Hint: Remember to note the conditions of this experiment - see introduction.

Attachment:- Activation Energy.rar