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my enzyme shows suicide inhibition, how do i identify the inhibitor?

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#1 question

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Posted 21 December 2016 - 10:42 PM

I have a non-natural enzyme which takes a non-natural substrate and catalyze it.

 

the reaction rate goes up then down with time, before substrate depletion is reached. when you take the reacted enzyme, dialyze it fully, then give it the substrate the activity is low or null. this does not happen with the wt enzyme with wt substrate.

 

we have tried various mutations to try to stabilize the catalysis of this enzyme but nothing seems to help. the inactivation seems to be caused by multimerization of the enzyme because after the reaction when you run the sample on an SDS PAGE, under denaturing condition, you see a ladder of multimers which is not seen in the sample before the reaction. could some one help me out on what might be the cause of this inhibition and if it is a suicide inhibition by a byproduct or substrate or product what is the experiment that i can do to figure out the cause and how do i identify the inhibitor?

thank you so much for your help in advance!!!!

 



#2 bob1

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Posted 22 December 2016 - 09:24 AM

It sounds like the enzyme is binding irreversibly to the substrate, and it might be that there is more than one binding site of the enzyme on the substrate or a product of the reaction - hence the multimerization. If you have structures of the enzyme and substrate you might be able to model this and see if it is feasible.

 

With regards to the inhibition, you could take the initial reaction to depletion, or generate the end products by synthesis, and apply these systematically to the enzyme, looking for inhibition of reaction in the presence of.

 

Another possibility is that the enzyme is multimerizing itself and that this is sterically hindering the active site. You would need crystal structures to confirm this probably.



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Posted 25 December 2016 - 06:35 PM

thank you for your answer bob1, is there a kinetic method by plotting the change in the reaction that i can do to get an idea whether this is caused by substrate or product or byproduct? could i figure this out by plotting a lineburke weaver plot or some other plotting method?

 

by looking for inhibition systematically are you mentioning i should separate by some method, the components in the reaction solution, apply those samples to the enzyme and see which fraction inhibits the enzyme and then analyze the fraction and see what components are in it? that sounds pretty time and labor consuming.......

 

thank you for your help



#4 bob1

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Posted 25 December 2016 - 07:38 PM

It's been a long time since I did any biochemical of this sort, so I can't give you any advice regarding plotting data.

With regards the inhibition: I was thinking that if you know the substrate then you should be able to determine the end product(s), which you could then synthesize or even take a reaction to completion and mix stoichiometrically with unique reacted enzyme to see the levels of inhibition





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