Dear all,
I am currently trying to design a protein mutant which is capable of binding substrate, but be unable to hydrolyse it.
I have been able to determine that some mutants are catalytically dead, but I am now at the stage to see if binding occurs. I have been thinking about using ITC. My question is: would I be able to establish Kd, since the substrate will undergo hydrolysis as soon as I inject it into the machine.
Best,
Michał
If hydrolysis occurs, the heat of reaction will be far greater than the heat of binding, so you will not be able to measure Kd. You can use ITC to measure the Km this way, but not Kd. However, if the kinetic mechanism is a rapid equilibrium one, the two constants are equal.
If you will have non-hydrolyzing yet binding mutant, ITC could be a perfect technique. However, you will need a control assay to determine that there is no catalysis (I assume it is not a problem at all).
You will see binding isotherms if the reaction is enthalpy driven, while entropy driven reaction will give no signal. Read more in "Biocalorimetry 2" handbook
https://www.wiley.com/en-us/Biocalorimetry+2:+Applications+of+Calorimetry+in+the+Biological+Sciences-p-9780470849682
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