Enzyme Kineticskcat km
Posted 08 November 2012 - 01:23 PM
Both substrates have the same Km
Substrate X has a Kcat of 100
Substrate Y has a Kcat of 1
I am trying to figure out why Substrate Y has such a lower enzyme turnover (Kcat). I have determined the Ki for substrate Y in a competition experiment with Substrate X and it has the same value as the Km's. I am thinking it could be non-productive binding, i.e. substrate Y binds the enzyme in the wrong configuration and is not catalyzed, thus reducing the observed Kcat.
My problem is, I don't know how to show this. I have been going through textbooks and I have not seen an experiment, or graphical methods to determine if non-productive binding is occurring. I have ruled out product inhibition through studies where I added more enzyme after saturation and i did not see a increase in cleavage.
Any advice on experiments/analysis would be greatly appreciated. I have tons of kinetic data (Michaelis-Menten experiments) for these substrates and enzyme, so if there is a way to interpret it for non-productive binding, that would be great.
Posted 11 November 2012 - 03:20 PM
Will this line of thought help?: If you are assuming that both substrates ought to have similar Kms; and that substrate Y has an additional competitive Ki; then the apparent-Km for Y will be significantly smaller than the Km for X. Hence, because you have an apparent-Km for Y equal to the Km for X either you do not have unproductive binding or the initial assumption of similar Kms is wrong.
I wonder too if stopped-flow experiments could help tease out the rate limiting step for each substrate.
Posted 12 November 2012 - 09:20 AM
I am looking into Surface Plasmon Resonance (SPR) to look at the binding of these substrates with my enzyme. It might be able to confirm if they have the same binding affinities (Km's)