I've found online tools to predict protein cleavage sites within unknown proteins, but I'd like to know whether it's possible to do the opposite: given the sequence of an unknown protease, is there a way for me to predict the amino acid(s) it should cleave after?
Protein structure prediction made big progress recently using the AI-based program Alphafold. However, I doubt that it can reach the accuracy and reliability required for predicting the substrate specificity of an unknown protease, even though the general reaction mechanism might be identified. Cleavage sites on protein substrates can be identified because the cleavage specificity of a number of proteases has been determined experimentally.
Hi Michelle M Leger . I agree with Pierre Béguin. The substrate specificity of a proteinase depends on the 3D configuration of its substrate binding pocket.
This pocket can be modeled using homologous proteinase structures, but substrate specificity is easier to determine experimentally if you have the proteinase in hand.
Dear Michelle
it depends also from what do you mean for unknown oroteiase.
If it means with low homology with other known proteases, i think that the prediction is quite difficult, while if it means poorly characterized (from the specific organism) but with high homology with other characterized proteases there are some probablity.
However as Steingrimur Stefansson suggest, it you are able to purifiy it, there are many in vitro tools (for example the : https://shop.jpt.com/2-Enzyme-Substrates/9-Enzyme-Substrate-Sets/101-Protease-Substrate-Set.html) that may allow to you to readily test the protease specificity. However is important to understand if your proteinase require the addiction of some cofactor (eg is a metallo proteases) to become active or not. because otherwise you will obtain false negative results.
Ciao
Manuele
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