Module 7: Enzyme Kinetics and Alkaline Phosphatase

REPORT

Include in the following order:

- Results (raw data, table, plots)

- Answers to Questions

- Summary (objective and at least one thing that you learned)

Processing Data and Reporting Your Results: Enzymatic rates are commonly measured in terms of the amount of product produced per time. Depending on the enzyme and the study of interest, published  enzymatic rates may be described in terms of µM/min, µmoles/s, etc. For this experiment, the alkaline phosphatase-catalyzed hydrolysis of p-nitrophenyl phosphate (PNPP) was measured by monitoring the production of p-nitrophenol/ p-nitrophenolate ion. To quantitate the rate at which this product was produced, we measured the observed change in absorbance at 410 nm over a period of time (ΔA410/min).

Results:

1. (1 mark) Include a clear, readable photo of your data collected in lab and signed by a TA.

2. (6 marks) Plot the following for the data collected:

(a) enzymatic rate, vi (Abs(410nm)/min) vs initial substrate concentration in the reaction, [PNPP] (µM) - this is a Michaelis-Menten Plot – a hyperbolic plot… do not connect the dots.***

You will have one curve for the “Alk.Phos. control” data and another curve for the “Alk.Phos. + inhibitor” data. Fit both datasets on one graph.

Use the non-linear regression program, Solver (an add-in for Excel), to solve for the best fit hyperbolic curve. (Refer to the respective experiment module on LEARN for help and Excel sheets that have already been set-up for you).

Indicate the final solved equation used for each curve (i.e. include the final calculated KM  and Vmax values).

Clearly label parts of the plot that relate to the determination of KM and Vmax, i.e. similar to the examples in the introduction.

Make sure that you have a legend identifying each curve.

(b) 1/vi versus 1/[S] - this is a Lineweaver-Burk Plot – Use the Scatter Plot format on Excel; Do not connect the dots.

Perform a separate linear regression for both the control and the inhibited data set (“trendline” or the “LINEST” function on Excel)

You should have one line (linear regression) for the “Alk.Phos. control” and another line for the “Alk.Phos. + inhibitor”.  Fit both lines on one plot.

Make sure that you have a legend identifying each data set.

Indicate the equations of the lines and theirrespective R2  value on the plot

Clearly label the parts of the plot that relate to the determination of KM and Vmax, i.e. similar to the examples in the introduction.

NOTE: Additional file submission: In addition to submitting a complete PDF for your lab report, you will be required to submit your Excel file that includes Solver non-linear regression/calculations. Non-linear regression will not be graded if Excel file is not accompanied.

Questions:

1. (1 mark) For the first part of the experiment (Part A), you monitored the absorbance at 410 nm in a reaction that consisted of 1.00 mL of 200 µM PNPP and 0.100 mL of just enzyme buffer (with no enzyme). What was the resulting rate? What was the importance of this part of the experiment and what did you learn from it?

2. (4 marks)

(a) In your own words, describe how KM is related but different to enzyme affinity.

(b) In your own words, describe how Vmax is related to but different to the enzyme’sturnover number (or turnover rate).

(c) Assuming that substrate is always in excess concentration relative to enzyme, which of the above parameters is expected to change as enzyme concentration is varied? Briefly explain.

3. (5 marks) Determine and then record the solved parameters, KM  and Vmax  (abs/min) derived from your plots by providing a table similar to that below.

To convert the solved Vmax  values for both control and inhibited data (from both the Michaelis- Menten and Lineweaver Burk plots) to “µM/min”, use the ε = 16,000 au M-1cm-1  as the molar    extinction coefficient for the p-nitrophenol/ p-nitrophenolate ion product. Show sample

calculations! (Helpful tip: Use the Beer-Lambert Law and assume a 1.0 cm pathlength for the cuvettes used).

Make sure to also show your calculations for when determining the kinetics parameters from the Lineweaver-Burk plot.

4. (2 marks) How do the solved parameters (Vmax  and KM) compare between the two plots (i.e. when comparing the same set of data - inhibited or non-inhibited)? Are they similar between plot types or very different? Explain.

5. (6 marks)

(a) Explain how the Lineweaver-Burk plot is prone to error?

(b) In your Excel Sheet - for your Lineweaver-Burk plot ONLY, remove the data point for the lowest substrate concentration (for both control and inhibited sets). Include the resulting plot.

(c) For this Revised Lineweaver-Burk plot, solve for the inhibited KM  and Vmax

(d) How do the revised inhibited parameters compare with parameters determined from the respective Michaelis-Menten plot (that includes all data points). Is there no difference or do they become more or  less similar to the results derived from the Michaelis-Menten plot? Explain what it is about your collected data that would cause for the difference seen (or not seen) when removing the inhibited data point at the lowest substrate concentration.

6. (5 marks)

(a) What was the concentration of the inorganic phosphate inhibitor in the reaction? (show calc.)

(b) From your graph interpretations, which parameter (Vmax  and KM) changed significantly when you included the sodium phosphate inhibitor in the reaction? Describe how it changed.

(c) Sodium Phosphate is a competitive inhibitor of alkaline phosphatase. Do your results agree with this? Briefly explain why it is reasonable to expect phosphate to cause such a change in the kinetic parameter  as mentioned in part (b) - what is phosphate doing to cause this parameter to change.

(d) Assuming competitive inhibition, determine the “α” factor observed for the sodium phosphate

inhibition by comparing the control, KM and inhibited KM' values derived from the Michaelis-Menten Plot only. (Helpful tip: KM' = αKM)

(e) In your own words, what information is received by calculating this α factor?

7. (6 marks)

(a) What is KI? What property does it portray?

(b) Determine the KI with the above solved “α” factor. Helpful tip: α = 1 +

(c) What is the equation for Kd (in terms of the relationship between substrate and enzyme) and how would you define it for the interaction between Alkaline Phosphatase and PNPP?

(d) Assume that the KM  value for PNPP (from your Michaelis Menten plot) is similar to its respective Kd value. Compare the determined KM  value for PNPP with the determined KI  for the inorganic

phosphate. How do they compare and what does it mean with respect to comparable affinity towards the enzyme?

8 (3 marks)

(a) Convert the enzyme concentration used in the reaction volume from “µg/mL to “µM” by using the molecular weight of 150 kDa (or 150,000 µg/µmol).

(b) Determine the control and inhibited kcat values (in units of “s-1”) for alkaline phosphatase. Note: Use the parameters derived from the Michaelis-Menten plot for both the control and the inhibited set.

(c) Should the turnover rate (kcat value) of an enzyme change in the presence of a competitive inhibitor? Briefly explain why. Does your reasoning agree with your results?