Aliphatic ketones are acetylcholinesterase inhibitors but not transition state analogs.

A number of C4--C9 aliphatic ketones are acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) inhibitors, with Ki values in the 0.7--5 mM range. Comparison to analogous substrates would suggest that these ketones are transition state analogs; e.g. 2-pentanone binds to the enzyme approx. 550 times more tightly than ethylacetate. However, a number of other criteria contradict this conclusions: (1) the binding is insensitive to ketone structure: isomeric ketones, cycloalkanones, and sterically hindered ketones have similar inhibitory potencies. (2) Analogous alcohols are also good inhibitors even though they cannot form hemiketals with the enzyme. (3) Representative ketones are relatively ineffective at blocking inactivation of the enzyme by methylsulfonyl fluoride, indicating that ketones do not bind principally at the hydrolytic site. (4) A competition experiment shows that binding of tetramethylammonium chloride excludes binding of 2-pentanone, suggesting that ketones bind to the anionic rather than the hydrolytic site. Thus, observation of tight binding relative to a substrate is not a sufficient criterion to establish that an inhibitor is a transition state analog.

The mode of binding of potential transition-state analogs to acetylcholinesterase.

Phenylacetone, 4-phenyl-2-butanone, and 4-oxopentyltrimethylammonium chloride were tested as potential transition state analogs for eel acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7). Phenylacetone is a competitive inhibitor of the enzyme but not a transition state analog, since its binding constant is similar to that for the substrate phenyl acetate. 4-Phenyl-2-butanone binds 6-18 times more tightly than the inhibitors 4-phenyl-2-butanol and N-benzylacetamide and the substrate benzyl acetate and also blocks inactivation of the enzyme with methanesulfonyl fluoride. However, its binding is independent of pH in the range 5-7.5, whereas both V and V/Km for benzyl acetate hydrolysis decrease with decreasing pH in this range. These data indicate a specific but weak interaction between the ketone carbonyl and the enzyme, but probably do not justify considering this compound a transition state analog. 4-oxopentyltrimethylammonium iodide has previously been shown to bind about 125 times more strongly than the substrate acetylcholamine. It also binds about 375 times more strongly than the alcohol 4-hydroxypentyltrimethylammonium iodide. Furthermore, the ketone protects the enzyme from inactivation by methansulfony fluoride, while the corresponding quaternary ammonium alcohol accelerates this inactivation reaction. This additional information confirms that the ketone is a transition state analog.