Amino acid residues involved in the interaction of acetylcholinesterase and butyrylcholinesterase with the carbamates Ro 02-0683 and bambuterol, and with terbutaline

In order to identify amino acids involved in the interaction of acetylcholi nesterase (AChE; EC 3.1.1.7) and butyrylcholinesterase (BChE; EC 3.1.1.8) w ith carbamates, the time course of inhibition of the recombinant mouse enzy mes BChE wild-type (w.t.), AChE w.t. and of 11 site-directed AChE mutants b y Ro 02-0683 and bambuterol was studied. In addition, the reversible inhibi tion of cholinesterases by terbutaline, the leaving group of bambuterol, wa s studied. The bimolecular rate constant of AChE w.t. inhibition was 6.8 ti mes smaller by Ro 02-0683 and 16 000 times smaller by bambuterol than that of BChE w.t. The two carbamates were equipotent BChE inhibitors. Replacemen t of tyrosine-337 in AChE with alanine (resembling the choline binding site of BChE) resulted in 630 times faster inhibition by bambuterol. The same r eplacement decreased the inhibition by Ro 02-0683 ten times. The difference in size of the choline binding site in the two w.t. enzymes appeared criti cal for the selectivity of bambuterol and terbutaline binding. Removal of t he charge with the mutation D74N caused a reduction in the reaction rate co nstants for Ro 02-0683 and bambuterol. Substitution of tyrosine-124 with gl utamine in the AChE peripheral site significantly increased the inhibition rate for both carbamates. Substitution of phenylalanine-297 with alanine in the AChE acyl pocket decreased the inhibition rate by Ro 02-0683. Computat ional docking of carbamates provided plausible orientations of the inhibito rs inside the active site gorge of mouse AChE and human BChE, thus substant iating involvement of amino acid residues in the enzyme active sites critic al for the carbamate binding as derived from kinetic studies. (C) 1999 Else vier Science B.V. All rights reserved.