Effect of Calcium on the Hydrolysis Activity of Human Butyrylcholinesterase

The aim of this experiment was to study the effects of calcium ion on the hydrolysis of cationic and anionic substrate by human butyrylcholinesterase (HuBChE). The hydrolysis of aspirin, an anionic substrate, by HuBChE was markedly increased in the presence of increasing concentrations of calcium ion (∼20 mM), as shown by the increasing k_cat (∼18-fold). Butyrylthiocholine (BTC), a cationic substrate, was biphasically hydrolyzed with substrate activation; a second BTC molecule caused a 3-fold increase in k_cat. At both lower and higher concentrations of BTC, its hydrolysis by HuBChE was slightly slowed down by the addition of calcium ion. Other cationic substrates, propranolol derivatives with butyryl and valeryl groups, were R-preferentially hydrolyzed by HuBChE; the rate of hydrolysis of these compounds was nearly the same in the absence and presence of calcium ion. These data indicate differential effects of calcium ion on HuBChE activity with anionic and cationic substrates. Furthermore, during the hydrolysis of aspirin in the presence of calcium ions, we demonstrated the existence of 2 additional binding sites for calcium, with K_m values of 1.8 and 5.9 mM. These binding sites exhibited much lower affinities than the EF-hand motif, previously identified as a high-affinity calcium-binding site.