This week your written assignment will consist of your Excel spreadsheet, Figures, Tables, one sentence of the Discussion section, and a Reference for your literature K_m value. You will eventually incorporate most of this writing into a full report. See requirements below for detailed requirements.

Excel Spreadsheet

Note: You now have enough experience with kinetics that the correctness of your calculations will be worth more points than in past assignments. In the instructions below, you will be asked to highlight the cells containing specific sample calculations by filling the cell in yellow. The purpose of this is to help the grader find the required information. Neglecting to do this will result in points deductions.

• The respective substrate concentrations should be clearly labeled in your organized raw kinetics data.
• Clearly label the slope calculations and highlight the cell of one sample calculation in yellow.
• Clearly label the relative activity calculations and highlight the one sample calcuation in yellow.
• Clearly label the values for your Eadie-Hofstee plot and highlight one sample calculation fot the x-axis in yellow.
• In your LINEST array, the four cells for slope, slope standard error, y-intercept, and y-intercept standard error must be highlighted in yellow.
• Clearly label the cell containing any equation(s) used to propagate error for V_max/K_m and highlight this cell in yellow.
• Create an organized summary of your estimates for V_max, V_max/K_m, and K_m with estimated uncertainty. If you organize these data into a formal table, you can simply cut and paste it into your writing assignment as Table 2 (see below).

Results and Discussion

• Figure 1 (Illustrate the quality of your kinetics data)
  • Plot A₄₀₀ vs. time for all of your raw kinetics data from Part C (This should be one graph)
  • Fit trendlines to each data set.
  • Make sure that each dataset is distinguishable (use different marker shapes)
  • You are not required to provide the linear equations and R² values for any of these data because you will compile the slopes as v_o values in Table 1.
• Table 1
  • Summarize your [catechol] and v_o (relative activity) data.
  • Create a table with columns for the catechol concentration [S] of each reaction sample in Part C (in mM) and Relative Activity v_o (in CU/μL).
• Figure 2
  • Plot the values in Table 1 with catechol concentration [S] on the x-axis and Relative Activity v_o on the y-axis.
  • Do not attempt to fit a trendline to these data (none of the built-in trendline equations in Excel are appropriate).
• Figure 3
  • Prepare an Eadie-Hofstee plot of your kinetics data from Figure 2.
  • Plot a linear trend line through your data. Include the equation and R² in the caption.
• Table 2
  • Create a table summarizing your estimates for V_max, V_max/K_m, and K_m.
  • Include uncertainty estimates as explained in Step 6 of the post-lab Analysis.
• Literature Comparison
  • Use the "Functional Parameters" category in BRENDA to locate a literature K_m value for catechol with Agaricus bisporus tyrosinase. Select a value that has given conditions.
  • In one sentence, state this literature K_m value for catechol with Agaricus bisporus tyrosinase and the conditions (pH, temperature, etc.) of the measurement.
  • Using the appropriate format, provide a citation for the primary literature for your K_m value. Note that BRENDA is secondary literature, cite the paper that BRENDA cited, not the database itself.
  • In lab next week, we will discuss how to appropriately compare this value to your measured value.

References

• Report the full citation of the primary literature source for your published Km reference value.

Submit by next lab meeting

• Your Excel spreadsheet to the online Submissions folder. Highlight the cells for each requested calculation in yellow to make it easier to grade. 

• A printed copy of the Lab 5 Assignment with the rubric stapled to the top.

• A digital copy of the Lab 5 Assignment to the Submissions folder.

Post your data online

• Post your groups's V_max, K_m, and V_max/K_m results to your section's data spreadsheet.

 

Start working on the upcoming full report

You will write a full report after Lab 6. If you want to get started early, write-up the following this week. It will not be graded, though you are, of course, free to get feedback from your instructor or TA.

Material and Methods

• Kinetics measurements: Modify your Assignment 3 Kinetics method but change the identity of the substrate. Be sure to identify catechol concentrations, SDS concentration, buffer composition, temperature, spectrophotometer model, wavelength observed, what was recorded.

• Estimation of kinetics parameters: For the analysis of Part C from this week's exercise, describe your linear analysis method for obtaining initial rates, identify the software used, define CU activity units, definition of relative activity, include the Eadie-Hofstee transform of the Michaelis-Menten equation, describe the linear analysis method for obtaining V_max and K_m parameters, state the nature of uncertainty measurements (identify the software and function used to obtain them).

Introduction

• Provide background for the system studied (mushroom tyrosinase inhibition).

• Make sure your facts are supported by references to literature. You may not cite the lab handouts as references. However, you are encouraged to use the references given in the handouts themselves.

• Important background should include a description of tyrosinase function, substrates and products of the reaction catalyzed, features of the enzyme active site, at least one substrate and inhibitor, existence of isozymes, and how specific chemicals/enzymes regulate activity.

• You should at least mention the source species for tyrosinase in this study.

• Assume that your reader understands the elementary theory behind your methods.

• Next week, we will articulate the purpose and goals.