Some of the biggest changes in the balance of protonation states that we uncovered correspond to subsequent stages in the reaction or inhibition processes. They include the inhibition of hBChE by OP echothiophate and the first chemical stage of hAChE reaction with its substrate (Figure ). While the protonation balance may shift as the result of the protein adjusting to a new state, this shift may also be a part of the reaction itself. Whether it is true, and what role this coupling of the reaction to the proton transfer may have, remain open questions. In this context it is intriguing to add E197Q hBChE into consideration. This variant obviously loses the proton transfer feature and demonstrates 11-fold drop in activity towards butyrylcholine.