Rate of enzyme activity decreases with increasing concentration of substrate?

01 January 2019 0 8K Report

I am working on an enzyme which has a feedback inhibition. I have observed that with increasing substrate concentration (it is a uni-reactant enzyme that has three products), the rate of enzyme decreases. I have varied the substrate concentration from 10uM to 1mM. I have even varied my enzyme concentration, time of incubation but the phenomenon is observed in each case. Due to this issue I am unable to perform a steady state kinetic assay to determine Km, kcat. In such scenario, what should one do to have a steady state curve? Any suggestions on the assay method or how would I characterize my enzyme? Any suggestion is welcome.

Thanking you in advance.

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