Substrate-assisted catalysis is the process by which one of the functional groups needed to make an enzyme active is provided by the substrate rather than by the enzyme itself. The clearest examples are provided by site-directed mutagenesis of proteases and their substrates. Removing histidine from the Asp, His, Ser catalytic triad of the serine protease subtilisin, and replacing it with alanine, inactivates the enzyme. Modifying the substrate, so that it supplies the His in the right position when enzyme binds substrate, reactivates the enzyme, and proteolysis occurs [PMID:3299704]. In both engineered and naturally occurring examples, substrate-assisted catalysis can make an enzyme highly specific.

The importance for Bioinfotropes is the warning that a protein shown by a multiple sequence alignment to lack a critical active site residue is not necessarily inactive. The enzyme in question may instead, in rare cases, be active through substrate-assisted catalysis.