Is EC50/IC50 of a drug always directly proportional to the concentration of the enzyme it is acting on? If not, what is the relationship?
Hi there,
If the assay is correctly set up, enzyme concentration should always be far less than the agonist/antagonist concentration tested which means EC50/IC50 value should remain the same if testing several enzyme concentrations provided they remain far less than agonist/antagonist.
There is no stoichiometric relationship between enzyme and substrate since an enzyme molecule turns over several fold more substrate molecules relative to its own concentration (depending on how fast the enzyme acts). The rule of thumb is that substrate concentration should be at least 1,000x the enzyme concentration.
Hello Dominique,
Thank you for your response. This is very helpful. So I am assuming that not having an excess of drug present throughout the assay will lead to false (high?) IC50s/EC50s because there is not enough drug to have the chance of saturating all binding sites?
Would you be able to direct me to some literature about this?
Thank you!
Dear Michal,
This is dealt with in detail in Cook and Cleland's excellent book "Enzyme Kinetics and Mechanism", chapter 6, and in Williams and Morrison (1979) Methods in Enzymology 63, 439 (http://www.sciencedirect.com/science/article/pii/0076687979630197).
The summary from Cook and Cleland (p.203) is that the relationship between enzyme concentration and IC50 is:
IC50 = appKi + (Et/2)
In your case, this means that if the IC50 is approaching the enzyme concentration, then the enzyme concentration will have a significant effect on the IC50. If the IC50 is much greater than the enzyme concentration, then it is being driven by the apparent Ki, and not by the enzyme concentration.
Another obvious conclusion is that you cannot measure an IC50 of less than half the enzyme concentration.
Hope this helps!
Dear Nicholas,
Many thanks for your answer! I was looking for this equation everywhere and couldn't find it. That is most helpful!
I wonder if I can pick your brains about one last thing: would what you say above also hold for an enzyme that can only catalyse one reaction? i.e.: conversion of one molecule of substrate to one molecule of product and then does not go on to bind to another molecule of substrate ever again?
Dear Michal,
I think that in the case that you discuss, the equation would not necessarily hold. This equation is based on the assumption that a steady state is formed, which will not be the case when there is an irreversible effect on the protein. Indeed, when the protein is forming part of the reaction, it is not correct to call this catalysis (this requires that the catalyst ends in the same state as it started in), or to call the protein an enzyme in this context (as this implies that it is a catalyst).
I expect that this could be treated similarly to irreversible inhibition (where in this case the "substrate" is in fact an irreversible inhibitor). I don't currently have access to the relevant literature, but would suggest searching for the irreversible inhibitor equations in the first instance and considering adapting the relevant equations. You may well need to collect more data to satisfactorily fit these data.
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