Masson_2026_Biochim.Biophys.Acta.Proteins.Proteom_1874_141152

The existence of multiple molecular forms of enzymes, genetic polymorphism and functional promiscuity raise the question of the identity of active center(s) responsible for several activities. In the present review, we recapitulate the general strategy, implementing the simple and rigorous inhibition kinetic method for probing the existence of single or multiple active sites on enzyme molecules. The model enzyme we chose to illustrate this approach is human butyrylcholinesterase, an enzyme that shows a complex functional (promiscuity), structural (multiple oligomeric forms) and genetic polymorphisms (numerous allelozymes and isoenzymes). This classical active site discrimination method is based on the analysis of enzyme irreversible inhibition profiles of enzymes under first-order conditions by monitoring the progressive enzyme activity decay with two reporter substrates of different specificity. The use of chiral irreversible inhibitors and/or chiral reporter substrates provides additional kinetic information about preferential enantioselectivity or binding complementarity of the target enzyme, allowing selection of the best inhibitors or substrates. Then, additional investigations, using structural methods (X-ray structure analysis, mass spectrometry), in silico simulations and classical biochemical methods (electrophoresis, PCR) provide definitive answers.