Scribd
Upload
16 views7 pages

Sphingosine Kinase Inhibition Analysis

The document describes an experiment measuring the kinetics of the sphingosine kinase reaction in the presence and absence of the inhibitor threo-sphingosine. The results show that threo-sphingosine increases the KMapp and decreases the Vmaxapp of the reaction. Analysis of the Michaelis-Menten and Lineweaver-Burk plots reveals that threo-sphingosine has an inhibition constant (KI) of 0.77 mM and acts as a competitive inhibitor of sphingosine kinase due to its similar structure to the substrate sphingosine.

Uploaded by

Tai
Copyright
© All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
16 views7 pages

Sphingosine Kinase Inhibition Analysis

The document describes an experiment measuring the kinetics of the sphingosine kinase reaction in the presence and absence of the inhibitor threo-sphingosine. The results show that threo-sphingosine increases the KMapp and decreases the Vmaxapp of the reaction. Analysis of the Michaelis-Menten and Lineweaver-Burk plots reveals that threo-sphingosine has an inhibition constant (KI) of 0.77 mM and acts as a competitive inhibitor of sphingosine kinase due to its similar structure to the substrate sphingosine.

Uploaded by

Tai
Copyright
© All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd

SAMPLE problem 1 (answer posted on Blackboard)

Sphingosine-1-phosphate (S1P) is important for cell survival. The synthesis of S1P


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 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], mM vo (mg/min) vo (mg/min) wthreo-sphingosine


2.5 32.3 8.5
3.5 40 11.5
5 50.8 14.6
10 72 25.4
20 87.7 43.9
50 115.4 70.8
Construct M-M and L-B plots to answer the following questions:
a. What are KMapp and Vmaxapp in the presence and absence of the inhibitor?
b. The concentration of threo-sphingosine used was 2 mM. What is the inhibition
constant?
c. What kind of inhibitor is threo-sphingosine? Explain.

1
Michaelis-Menten plot

• It can be really hard, with data that look like these, to accurately decide where the
maximum velocity is!

2
Lineweaver-Burk plot

No threo-sphingosine present 2 mM threo-sphingosine present


• Vmax = 1/0.0081 = 123 mg/min • Vmaxapp = 1/0.0105 = 95 mg/min
• KM = -1/-0.1396 = 7.2 mM • KMapp = -1/-0.0387 = 26 mM

3
Sphingosine kinase inhibition
b) What is KI? c) What kind of inhibitor is threo-
• [I] = 2 mM sphingosine? Explain.
• KMapp = aKM • This is tricky. KM clearly changes, but does
• 26 mM = a(7.2 mM) Vmax also change? Or is it due to error?
• a = 3.6 • A clue can be found in the structures of the
substrate and inhibitor:
• 3.6 = 1 + [I]/KI
• 2.6KI = 2
• KI = 0.77 mM

• The similar structures support threo-


sphingosine acting as a competitive
inhibitor

4
SAMPLE problem 2 (answer posted on Blackboard)
Ethanol in the body is oxidized to acetaldehyde (CH3CHO) by liver alcohol
dehydrogenase (LADH). Other alcohols are also oxidized by LADH; methanol is
oxidized to the highly toxic formaldehyde (CH2O). The toxic effects of MeOH can
be reduced by administering EtOH. EtOH thus acts as a competitive inhibitor of
the methanol by displacing it from LADH. This gives the body sufficient time to
then excrete MeOH by the kidneys.

If an individual (assume volume of ~40 L aqueous liquids with MeOH evenly


distributed) ingested 100 mL of MeOH, how much 100 proof whisky (50% EtOH by
volume) must be ingested to reduce the activity of LADH toward MeOH to 5% of
its original value? For this problem, allow KM = KI for EtOH.

Density of EtOH and MeOH: 0.79 g/mL


KM of LADH for MeOH: 1.0 x 10-2 M
KM of LADH for EtOH: 1.0 x 10-3 M

5
How much 100 proof alcohol must be administered?

6
How much 100 proof alcohol must be administered?

You might also like