Is the EC50 of an activating protein for an enzyme a good indicator for the binding affinity Kd?

Question

We work with a membrane protein system where measuring the affinity between the enzyme and the upstream activating protein has been difficult, and when measured in detergent solution, it is almost 100 fold lesser (ie ~100nM) whereas the EC50 in an enzymatic assay using vesicles in ~1-2nM. Would it be reasonable to say that the "real" affinity is ~1nM than 100s of nM based on the biochemical assay?Alternately, is there a documented system where a huge discrepancy exists between measurements from direct binding and biochemical assays?

Answer 1

Your question is not very clear, and you are using EC50 and not IC50. Is there a reason for that? Also, you ask about Kd, but is that the Kd of the drug (activating protein) or the substrate? Also, measuring enzyme activity as you do in the vesicle assay is much more relevant than results of binding assays. At any rate, I would say that the EC50 of any agent is NOT a good indicator of the binding Kd, mainly because the EC50 will vary with the concentration of the concentration of the substrate, or other ligands. And yes, the literature is replete with examples of large discrepancies between functional biochemical assays and direct binding assays.