Discuss your data in terms of the effect that the mutation may have had on activity

Chemistry by Geoffrey

Writing Assignment:

Write an Experimental Procedures, Results, and Discussion sections for labs 6 and 7 in the format of Journal of Biological Chemistry (JBC). This should include details such that an experienced biochemist could reproduce the experiments from the kinetics and inhibition experiments you performed. Also, be sure to discuss the data as though you’re convincing someone of the significance of what you’ve done.  Treat this as novel research that you want to publish. Like before, find a JBC article that includes enzyme kinetics and/or enzyme inhibition to help guide you. Be sure to include either the printed article or cite the article with your report.

 

What should be included in the paper?

 

Experimental Procedures:

  • All details for performing the experiments performed in Lab 6 and 7 (enzyme kinetics and inhibition, respectively)

Results: (make sure to label all figures, graphs, and tables as seen in publication)

  • From Lab 6
    • Prepare a figure with your graphs from the experiment.
      • Graph 1: Michaelis plot for the WT and mutant enzyme.
      • Graph 2: Lineweaver-Burk plot for the WT and mutant enzyme
    • Table showing x-intercept from Lineweaver-Burk, y-intercept from Lineweaver-Burk, KM, Vmax, and turnover number for both enzymes. (see table example below)

 

  wild-type mutant (                     )
x-intercept from LB    
y-intercept from LB    
KM    
Vmax    
Turnover (kcat)    

 

  • From Lab 7
    • Michaelis-Menton graphs for sodium phosphate and L-phenylalanine inhibition (separate graph for each, new line for each concentration)
    • Lineweaver-Burk for sodium phosphate and L-phenylalanine inhibition (separate graph for each, new line for each concentration).

 

  • Table showing Km, Vmax, turnover and Ki for all samples tested (see table example below)

 

  uninhibited 100 µM NaPO4 500 µM NaPO4 1 mM L-Phe 5 mM L-Phe
KM          
Vmax          
Turnover          
Ki          

 

 

Discussion: (this is not a limited list- be sure to discuss all the important findings of your experiments)

  • Discuss your data in terms of the effect that the mutation may have had on activity
  • Discuss types of inhibition and differences of Ki between the different inhibitors. For example:
    • Based on your data and your knowledge of the enzyme, what kind of inhibitor is NaPO4?
    • Based on your data and your knowledge of the enzyme, what kind of inhibitor is L-phenylalanine?

 

 

Supporting Information:

In addition, please show a sample calculation for the following:

  • Conversion of the initial rate in absorbance/second to concentration/second.
  • KM­
  • Vmax
  • Turnover
  • Ki