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!!!!