I have come across an enzyme, AKR1C3, which posseses both an active site and an allosteric site.
The substrate I am using (9,10-PQ) is able to bind both the active site and the allosteric site.
This leads to the enzyme exhibiting substrate inhibition. In order for me to fit initial velocity/[substrate] data to different mathematical models of substrate inhibition, I need to quantify the affinity of the substrate for both the active site and the allosteric site.
How would one go about identifying the affinities for each site when in a given system, the substrate is able to bind active site and allosteric site at any given time, which makes it difficult to be certain of whether the substrate is binding the active site or the allosteric site?
The mathematical models describing the kinetics of substrate inhibition already contain the Km (and the Ki in some models) of the substrate binding site and the Ki of the inhibitor as parameters. All you have to do is collect high-quality initial rate data over a range of substrate concentrations and fit the data to the model using a nonlinear regression program, such as Grafit. This will give estimates of the parameters in the model.
The intensity of the color is the best options to determine the binding.
See the attached sections from the Biological Engineering Textbook that I'm writing. The first section is how to do the curve fitting correctly, including how to calculate the confidence limits on the parameters, you will have to expand this for the Ki parameter. The second section deals with the model for allosteric regulation.
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