The reaction of acetylcholinesterase with the N-methyl-7-hydroxyquinolinium iodide ester of dimethylcarbamic acid has been followed spectrofluorometrically. In an excess of substrate the observed rate of carbamoylation of the enzyme is strictly first order and is much greater than the concentration-independent rate of decarbamoylation, allowing a titration of enzyme active sites. The method is sensitive to enzyme normalities in the nanomolar region, and from the turnover number and the enzyme normality, the number of active sites per molecule can be calculated. The effects of inhibitors on both the carbamoylation and decarbamoylation rates have been analyzed. Competitive inhibition constants for the inhibition of carbamoylation are the same as those obtained for the inhibition of enzyme-catalyzed acetic acid ester hydrolysis and support the idea that the same enzyme active site is involved in both cases. Some competitive inhibitors of acetylcholine hydrolysis and of carbamoylation are bound to the carbamoyl-enzyme as well as to the free native enzyme. These “inhibitors” are positive effectors of the rate of carbamoyl-enzyme hydrolysis and thereby support previous proposals of a second allosteric binding site.
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