Abstract
Kinetic and crystallographic studies reveal that the binding of the thiocholine-thionitrobenzoic acid product, released during the measurement of thioester-analog substrates hydrolysis according to Ellman's method, inhibits cholinesterases by a pure competitive mechanism. This can only be recorded as the progressive accumulation of the product upon subsequent additions of substrate aliquots. A wide affinity variation was observed among several tested enzymes, with the highest values found in human butyrylcholinesterase and Torpedo acetylcholinesterase. Nearly two orders of magnitude lower affinities were determined with human, mouse, and electrophorus acetylcholinesterases, and human atypical butyrylcholinesterase. These findings can be explained by the unexpected accommodation of the thiocholine-thionitrobenzoic acid in the active site of human butyrylcholinesterase, with the positively charged trimethylammonium choline pointing to the enzyme's peripheral site. At the same time, the carboxyl group of the nitrobenzoic moiety interacts with the enzyme's oxyanion hole. This explains the virtual absence of product inhibition in atypical human butyrylcholinesterase (D70G), purified or in plasma.