I am looking for some expert advice on enzyme kinetics but for an assay in plate format which has the substrate bound. This is different that the usual homogeneous plate assays or heterogeneous assays where the enzyme (catalyst) is plate bound.
In short I am trying to look at the Ki of a compound and wish to use a plate assay as the compound absorbs at the same wavelength as the typical para-nitroanaline substrate that is usually used. Is it possible to do normal Michaelis Menten kinetics in a plate format given the substrate is bound and not homogeneous despite rigorous shaking? We believe the inhibitor is mixed in its mechanism, it binds non covalently but does seem to react, albeit poorly with the Cys nucleophile in the protease. We have shown this through a few different studies but wish to obtain a Ki and generate a graph that will provide evidence of either competitive or mixed inhibition. Hoping to hear form you all!!! Thanks - Steve - Email:conne...@scripps.edu
