Enzyme Kinetics - (Nov/08/2012 )

Hi, I have an enzyme that cleaves 2 substrates very differently.

Background:
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.

Thanks,

Jared

Cornish-Bowden (in Fundamentals of Enzyme Kinetics) has this description for competitive substrate inhibition (I assume you are not seeing uncompetitive substrate inhibition with the characteristic drop in rate) “the measured values V and Km may be less, by an unknown and unmeasurable amount”. So it is likely that for a M-M reaction there is no steady-state method applicable other than to compare a series of related substrates and/or inhibitors with careful consideration of what else is known about the reaction chemistry.

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.
Good luck

Thanks for the input. I actually have this book on my desk and as you said, if this is competitive substrate inhibition the actual Km of substrate Y is higher than what I have observed. Thus, my initial assumption of similar Km's could very well be wrong, but I just dont know how to show it. The stopped-flow experiments would definitely provide insight into pre-steady state.

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)