Hi dear researchers
Is it possible that observed increase of Km for one substrate and decrease for another one (for enzymes with two substrates such as HRP) after immobilization?
Hi Soudabeh Farhadi . It is more than likely that to see changes in Km when substrate and enzyme are not freely associating in a single phase.
Thanks Dr.Stenni Stefansson for your attention.
Could you explain more?
Hi @Soudabeh Farhadi,
The chemical affinity of the carrier used for immobilization of HRP enzyme may differ which can hinder free avaialbility of substrates to enzymes, giving variable KM values. Good luck.
If there is some slight change of conformation, it may open the cavity more for one substrate, but hinder access for other more bulky substrate. Or it may not bind so efficiently smaller substrate (due to lack of interactions).
Hi Soudabeh Farhadi, Make sure that the carrier material and process used for immobization should not cause any sort of physical hindrance for free permeability of both substrates to reach the enzyme. The pore size of the the carrier material and the molecular size and shape of substrates may give rise to a difference in avialabilities to make differences in KM values.
yes, it could be, according to infinity of enzyme to the substrates.
Dear Soudabeh,
I had immobilized my enzyme and observed a change in the Km value. However, mine was a single substrate specific enzyme. I second the opinion of Tapan Kumar; the pore sizes, the immobilization technique used, etc., could play a role in the availability of the reactive site to your substrate.
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