Indophenyl acetate and acetylcholinesterase: binding of a non-specific substrate on the margin of the active center.

1. Indophenyl acetate is a very poor substrate of eel or bovine acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7), with a V less than 5% of that of phenyl acetate, but it is a labile ester and in imidazole buffer is hydrolyzed, non-enzymically, even faster than phenyl acetate. 2. Indophenyl acetate completely protects the enzymes against inhibition by diisopropylphosphorofluoridate but promotes inhibition by methanesulfonyl fluoride. 3. With either of these inhibitors the measured rate of inactivation of eel acetylcholinesterase is the same whether activity is determined with this poor substrate or with a good substrate, acetylthiocholine. With bovine enzyme the inactivation rate is 25% lower when assayed with the former substrate. However this preparation contains a minor enzyme component which is involved in hydrolysis of indophenyl acetate but not good substrates, and which is not readily inhibited. When this is taken into account the inactivation rates for bovine acetylcholinesterase, too, are found to be the same in either assay. These and other observations in the literature can be explained if indophenyl acetate, because of its size, cannot fully penetrate into the active center and is bound in adjoining non-polar regions of the protein. From this external position it makes only intermittent contact with the esteratic site. Hence it is slowly hydrolyzed and fails to protect the enzyme against methanesulfonyl fluoride, though it does protect, possibly sterically, against the larger inhibitor diisopropylphosphorofluoridate.