engleski

Amino acids involved in the inhibition of acetylcholinesterase and butyrylcholinesterase by Ro 02-0683 and bambuterol

In order to identify amino acids involved in interaction of acetylcholinesterase (AChE ; EC 3.1.1.7) and butyrylcholinesterase (BChE ; EC 3.1.1.8) with Ro 02-0683 (dimethylcarbamate of (2-hydroxy-5-phenylbenzyl)-trimethyl-ammonium bromide) and bambuterol (5-[-(tert-butylamino)-1-hydroxyethyl]-m-phenylene-bis (dimethylcarbamate) hydrochloride) the time course of inhibition by these two carbamates was studied. Recombinant mouse wild-type AChE and BChE, site-directed AChE mutants and native human serum BChE phenotypes were inhibited by varying concentrations of the carbamates. Both inhibitors are charged compounds ; Ro 02-0683 has a quaternary amino group while the secondary amine (pKa = 9.61) of bambuterol is almost completely quaternized at our experimental conditions. The second order rate constants of inhibition of mouse BChE and human serum BChE usual and fluoride-resistant variants were similar for both carbamates. The rate constant of inhibition of AChE by Ro 02-0683 was on average 10-times and that by bambuterol 16000-times smaller than that of BChE. D74N AChE mutant was inhibited slower by both inhibitors than the wild-type AChE. Likewise, the inhibition of atypical human BChE (natural mutation) by both inhibitors was slower than that of the usual enzyme. Both carbamates were better inhibitors of the peripheral site-directed AChE mutant Y124Q, than of the wild-type AChE. Bambuterol was a better and Ro 02-0683 was a worse inhibitor of the choline binding site mutant Y337A than of the wild-type AChE. The latter two amino acid substitutions mimic elements of BChE structure critical for its specificity on the AChE template.

cholinesterases; inhibition; Ro 02-0683; bambuterol; terbutaline; modelling of enzyme/carbamate complexes

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