I am getting Km value of a mutant which is similar to its wild type. How it is possible ? The residue is quite important for substrate binding. Is it possible ? please suggest !
This is very possible since you find it and I assume that you have double checked that you are working with the mutant protein.
Anyways a very important information is missing (to me): how is the maximum activity of mutant versus wild type enzyme?
Hi there,
In the general case Km is definitely not Kd. So unless you know your favorite enzyme substrate couple obeys to the fast steady state equilibrium (kcat has to be much less than k-1), you can't consider Km as Kd. As Km=(kcat+k-1)/k+1=(kcat/k+1)+Kd, if kcat/k+1 is the main contribution to Km then Kd increase due to mutation might not be detectable at the Km level.
You also say that thee mutated aa is essential for binding . What is the mutation you made and are you sure it does abolish the contribution to the binding?
In fact it might be that the contribution of the aa is not that essential for the binding or might be compensated by other contacts between enzyme ans substrate.
-Firstly how do you know that this amino acid will affect the binding ? have you run simulation and get data that supports that ?
- secondly even if your mutation has an good effect on binding it may has a bad effect on the enzyme structure may be it has bad interactions with other parts of protein structure.
can you please tell us what is your enzyme and what is your exact mutation ?
the substrate binding sites of the enzyme present in the mutant is similar and the sub. binding sights present in the enzyme are saturated the Km will not show any significant change under similar reaction conditions but its Vmax may be different
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