I'm studying the inhibition of my enzyme with an alternative substrate, i.e. a substrate of a homologous enzyme which my enzyme does not hydrolyze (so the compound is not a strong inhibitor). When I did a preliminary inhibition test, there was 20% inhibition at 1mM and increased further with concentration. However, at 0.2 mM and even smaller concentrations, the enzyme activity was enhanced >15% over the original activity. Could this be an artifact, or are there any reports on such activation and inhibition by the same compound?
It could be a lot of things, even an artifiact. You should think about the assay method, specificity of the method, inhibition by excess of substrate, competition between substrate and alternative substrate and so on. Is that pattern independent of the substrate concentration?
Hi Avinash, have you done a Lineweaver–Burk plot of your enzyme with the inhibitor?
Hi,
It could be a lot of things. When I worked with laccase, I used also different mediator of the reaction that are very effective at low concentrations, while they became inibitors at higher concentrations. I think it depends on the assay you performed, and also on the purity of your enzyme. If you purified it by yourself try to check if it's really pure, and in case try to change buffer or to dialyze the sample.
If you need more help please give us more information.
I agree with F. Solano and A. Chiado. It is common to have activation at lower concentrations, and inhibition at higher concentrations for the same molecule. But of course you have to rule out other things, like impurities, etc...as others have noted.
Thank u all for the answers. I like to clarify on certain issues raised here - my enzyme is pure, substrates are from Sigma, the assay method is specific to the substrate. My enzyme shows cooperativity and substrate inhibition so Im not able to linearize the curve (LB-plot). The preliminary inhibition assay was done using a fixed substrate concentration. @ Prof Solano: competition between substrate and alternative substrate could very well be a reason. I did a kinetics run with fixed concentration of the alternative substrate and increasing concentrations of substrate. The vmax was almost the same, but the k(0.5) was affected and the substrate inhibition was reduced a little (the curve was flat till a higher substrate concentration). I'm still working on this aspect, but I would like to know, could this be evidence of competition in binding of the natural and alternative substrates? Also, is there a connection between substrate inhibition and such competition?
OK Avinash, you should have specified in the beginning that you are not working with a simple enzyme-substrate reaction in the presence of an inhibitor.
It seems that you are working with a complex enzyme that has alternative binding sites and is showing cooperativity for substrates, products and whatever else. As such, all speculations about substrate and inhibitor concentrations are not applicable.
If you have cooperative binding, do you have a Hill or a Bohr plot?
@Stefansson: I have a Hill plot with h=2.05, but a proper interpretation of the kinetic curve is hindered by the presence of substrate inhibition. My enzyme is a homotetramer with 4 active sites per molecule. What is the possibility of the substrate/product/inhibitor binding to the active site and causing such phenomena? Or could there be a separate allosteric site? If you have time, could you pl give some pointers as to how to deal with such complex enzymes?
Hi Avinash !!! This sounds like a very cools system !!.... this is indeed a case of allostery !!! You say you have a tetramer of the protein and that the hill coeffecient is around 2. It looks like your compound is a competitive inhibitor (binds to the same site as the substrate ?)..... in such a case, at low inhibitor concentrations it allows to tip the equilibrium of the tetramer to the activated form. At these concentrations it will act like an activator.However at higher concentrations it behaves as a proper inhibitor as the tertramer has already been activated.
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