Acetylcholinesterase. Two types of inhibition by an organophosphorus compound: one the formation of phosphorylated enzyme and the other analogous to inhibition by substrate

• Published in: Biochemical journal 1969-11, Vol.115 (2), p.147-162
• Main Authors: Aldridge, W N, Reiner, E
• Format: Article
• Language: English
• Subjects: Acetylcholine - metabolism; Anthelmintics - pharmacology; Binding Sites - drug effects; Cholinesterase Inhibitors; Coumarins - pharmacology; Kinetics; Models, Chemical; Phosphates - pharmacology
• ISSN: 0264-6021; 0306-3283; 1470-8728
• DOI: 10.1042/bj1150147

1. The kinetics of the reaction of di-(2-chloroethyl) 3-chloro-4-methylcoumarin-7-yl phosphate (haloxon) and related compounds with acetylcholinesterase were studied and found to be unusual. 2. By a progressive reaction haloxon produces a di-(2-chloroethyl)phosphorylated enzyme. The influence of substrate on this reaction leading to a phosphorylated active centre was studied. From competition experiments between inhibitor and substrate values of K(m) for acetylcholine and acetylthiocholine of 0.79mm and 0.23mm respectively were derived. 3. Haloxon also combines with acetylcholinesterase by a non-progressive reaction, producing a complex that is reversible by dilution and by high concentrations of acetylcholine and acetylthiocholine. From this non-progressive reaction the competition between haloxon and substrate was studied, and it was shown that haloxon combines with a site involved in inhibition by substrate. From competition experiments the following dissociation constants were derived: for combination of haloxon and this site K(i) is 4.9mum and for the combination of substrates with this site K(88) values are 12mm and 3.3mm for acetylcholine and acetylthiocholine respectively. 4. The non-phosphorus-containing compound 3-chloro-7-hydroxy-4-methylcoumarin was shown to be a good reagent for the site involved in inhibition by substrate; its dissociation constant for the combination with this site is 30mum. 5. In order to interpret the experimental results, theoretical equations were derived for an enzyme with two binding sites to both of which substrate and inhibitor can combine. The equations correlate the activity of the enzyme with the concentration of substrate and inhibitor, for both progressive and non-progressive inhibition. These equations are applicable to reactions of acetylcholinesterase with organophosphorus compounds, carbamates etc. and may be applicable to other enzymes possessing two binding sites.