Question

Sphingosine 1-phophate (SPP) is important for cell survival. The synthesis of SPP from sphingosine and ATP is catalyzed by the enzyme sphingosine kinase. An understanding of the kinetics of the sphingosine kinase reaction may be important in the development of drugs to treat cancer. The velocity of the spingosine kinase reaction was measured inthe presence and absence of threo-sphingosine, a sterioisomer of sphingosine that inhibits the enzyme. The results are next.

Contruct a lineweaver-Burk plot to answer the following questions:
a. What are the apparent Km and Vmax values in the presence and absence of the inhibitor.
b. What kind of inhibitor is threo-sphingosine? Explain.

-Do problem 12-25 (the one shown above) from book and show ALL calculations and a data table and provide a good graph with labels using a graphing program such as Excel. Explain your answer and show sample calculations where necessary. Additional part to problem. Calculate the KI (as a dissociation constant) if the concentration of inhibitor was 10 uM.

Answer 1

a. From the graph given above Km can calculated using the intercept made on the X-axis. The intercept made on the X-axis will give -1/Km. So the Km would be 8.333uM without inhibitor and 25uM with inhibitor.

Vmax can calculated from the intercept made on the Y-axis. The intercept on the Y-axis will give a value of 1/Vmax. So the Vmax would be 111.11 mg/ min without inhibitor and 83.33 mg/min with inhibitor.

b. The type of inhibition is known as mixed type inhibition where both Km and Vmax are changed with the addition of the inhibitor.