A biochemist obtains the following set of data for an enzyme purified from Caenorhabditis elegans that is known to follow Michaelis-Menten kinetics. The experimental conditions were with 0.1 μmol of enzyme and pH 7.4.
Substrate Initial
concentration velocity
(μM) (μmol/min)
1 49
2 96
8 350
50 621
100 676
1,000 698
Question 1. Plot V0 vs [ S ]. (Hint, the plotting is quickly done with a scatter plot in excel).
Estimate what would be the initial velocity in μmol/min at a substrate concentration of 5,000 μM. Do not put units in blackboard.
Question 2. What is the approximate Vmax for this enzyme in μmol/min? Do not put units in blackboard.
Question 3. Explain how you came up with the answer to question 2.
Question 4. What is the approximate Km for this enzyme in μM? Do not put units in blackboard.
Question 5. Explain how you came up with the answer to Question 4.
Question 6. What is the approximate Kcat for this enzyme? Do not put units in blackboard.
Question 7. Explain how you came up with the answer to question 6.
Question 8. Using the last 3 substrate concentrations above, make a Lineweaver-Burk plot and determine the Vmax and Km. (Hint graph in excel, add a trendline to get an equation of the line).
8a. What is the Vmax
8b. What is the km.
Question 9. Is the Vmax and Km similar or different to that determined from the plot of V0 vs [ S ]? Explain why this may be?
Question 10. 0.01 μM of the compound salicylic acid was added to all samples in the experiment above. At substrate concentrations less than 50 μM the initial rate of the enzyme was greatly reduced, however at concentrations greater than 1,000 μM there was little effect on the initial rate. What is the name of the phenomenon cause by salicylic acid?
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