Dear colleagues!
Due to the strong irreversible inhibition of the enzyme I have difficulties with the calculation of Kobs.
The FRET scan measurement units changes only by 30-60 units during full reaction time, while the noice is +/- 25 units.
How to check kobs in case of so strong inhibitory activity?
Can you reduce the inhibitor and enzyme concentrations to slow down the inhibitor reaction? Can you increase the substrate concentration as a competitor with the inhibitor?
Adam B Shapiro , thank you for your help!
The range of the concentration of the inhibitor have been changed, but the graphic still wasn't proper enough for the evaluation of kobs depending on the inhibitor concentration. The concentration of inhibitors ranges from 0,3 mM till 100 mM ( the solution of 0,3 mM of the potential inhibitors gives no inhibitory effect) - 0.3, 1, 3, 10, 30, 100 mM.
The rate of the reaction doesn't look like it is an exponential function in its nature. It seems that it has a strong inhibitory activity at the beginning (during 1-2 hours no change in the intensity units) an after that it shows a weak linear regression in stead of exponential association, comparing with negative control (only protein and substrate occur in the solution). The graphs of the negative control always increases strongly enough.
Svjatoslavs Kistkins , when you say "irreversible" do you really mean that your inhibitor is covalently binding to your enzyme? Also, due to a difference in terminology between our countries, I am not sure if you are talking about the rate constant kobs, or the equilibrium substrate binding constant Kobs. Do you stopped-flow techniques to measure the kinetics of kobs? If your inhibitor effect is kicking in so very quickly after the enzyme starts its catalytic mechanism, then you might want to try this technique instead of a normal fluorometer setup. That is... you might want to try it if you are studying the kinetics instead of the equilibrium. I have references if you need them. Good luck, solving what sounds like quite the headache of a problem.
The progress curve you described sounds like you have nearly complete inhibition from the start, with just enough activity remaining to see a slow linear increase. The slow linear increase could also be a background uncatalyzed rate. Sometimes, inhibitors, even covalent ones, are reversible, which results in a slow residual rate. This could be because the enzyme slowly destroys the inhibitor, or because the inhibitor dissociates from the enzyme intact.
It might be helpful if you share some images of your progress curves.
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