Question

1) Spingospine 1-phosphate (SPP) has recently been discovered to be important for the cell survival. The synthesis of SPP from Spingospine and ATP is catalyzed by the enzymes Spingospine kinase. An understanding of the kinetics of the Spingospine kinase reaction may be important in the development of drugs to treat cancer. The velocity of Spingospine kinase reaction was measured in the presence and absence of threospingospine, a stereoisomer of singspiel. The results are shown below.

[Spingospine] (uM)	Vo (umol/min) (No inhibitor)	Vo (umol/min) (With threospingospine)
2.5	32.3	8.5
3.5	42	11.5
5	48	16
10	68	28
20	87.7	44
50	120	74

Construct a Lineweaver –Burk plot to answer the following questions:
a) What are the Km value and Vmax in the presence and absence of the inhibitor?
b) What kind of an inhibitor is threospingospine? Explain.
c) Determine the Ki for the inhibitor ([I] = 5 μM)

Answer 1

a) From the plot shown below

without inhibitor

Km = 1/0.02 = 50 uM

Vmax = 1/0.034 = 29.41 umol/min

With inhibitor

Km = 1/.01 = 100 uM

Vmax = 1/0.12 = 8.33 umol/min

b) type of inhibition = Mixed inhibition

c) For Ki

Vmax,apparent = Vmax/(1 + [I]/Ki)

8.33 = 29.41/(1 + 5/Ki)

Ki = 1.98 uM

Data for plot

1/[S]	1/Vo	1/Vo [I]
0.4	0.031	0.1176
0.286	0.024	0.087
0.2	0.021	0.0625
0.1	0.0147	0.0357
0.05	0.0114	0.0227
0.02	0.0083	0.0135

Plot