Question

30. Sphingosine-1-phosphate (S1P) is important for cell survival. The synthesis of S1P from sphingosine and ATP is catalyzed by the enzyme sphingosine kinase. The velocity of the sphingosine kinase reaction was measured in the presence and absence of threo-sphingosine, a stereoisomer of sphingosine that inhibits the enzyme. The results are shown below. Sphingosine (UM)v. (mg/min) v. (mg/min) with inhibitor 2.5 32.3 8.5 3.5 40 11.5 50.8 14.6 25.4 87.7 43.9 115.4 70.8 Construct 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 an inhibitor is threo-sphingosine? Explain.

Answer 1

                                        1/V = Km/Vmax [1/S] + 1/Vmax

V is the reaction velocity (rate of the reaction), Km is the Michaelis-Menten constant, Vmax is the reaction maximum velocity and [S] is the substrate concentration

In absence of Inhibitor    

                                                                    

0.035 0.03 y = 0.058x + 0.0081 R2 = 0.998. 0.025 0.02 3 0.015 0.01 ........ Linear (0) 0.005 O + O 0.1 0.2 0.3 0.4 0.5 1/[S]

In presence of Inhibitor

                   

0.14 0.12 y = 0.2711x +0.0105 R= 0.9969 0.1 0.08 NI 0.06 0.04 ....... Linear (0) 0.02 0.1 0.2 0.3 0.4 0.5 1/[S]

a)

From the graph

In absence of Inhibitor, Vmax = 123.4568 ; Km = 7.1604 , Slope (Km/Vmax = 0.058)

In presence of Inhibitor, Vmax = 95.2381 ; Km = 25.819, Slope (Km/Vmax = 0.2711)

To distinguish the type of inhibition like competitive, non-competitive and uncompetitive the Lineweaver-Burk plot will be helpful.

If the y-intercept is same for two data sets (in presence and absence of inhibitor) and different slopes and x-intercepts it is competitive inhibition. (Vmax is unaffected by competitive inhibitors and the inverse of Vmax also doesn’t change).

If the x-intercepts are same with different slopes and y-intercepts it is Non-competitive inhibition (Km is unaffected).

If the x-intercepts, slope and y-intercepts are different then it is Uncompetitive inhibition.

b) Threo-sphingosine is a uncompetitive inhibitor. Neither y-intercept nor x-intercept values doesn’t match in absence of inhibitor.

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