General occurrence of binding to acetylcholinesterase-substrate complex in noncompetitive inhibition and in inhibition by substrate

Abstract

To assess the relative importance of binding to enzyme-substrate complex (E·S) and to acetylenzyme (EA), noncompetitive inhibition has been studied in hydrolysis by acetylcholinesterase (AcChE) of cationic and uncharged substrates - acetylcholine (AcCh), 3,3-dimethylbutyl acetate, n-butyl acetate, 2-(methylammonio)ethyl acetate, 2-(N,N-diethyl-N-n-butylammonio)ethyl acetate (DEBAAc) and 2-(methylsulfonyl)ethyl acetate. For the N-trimethyl quaternary ions related to AcCh, tetramethylammonium ion, choline and choline ethyl ether, noncompetitive inhibition (Kl(nonc)) is more favorable with the slower substrates than with AcCh, i.e., when E·S > EA, and is attributed to formation of enzyme-substrate-inhibitor complexes, E·S·I′. Noncompetitive inhibition by tetraethyl-, tert-butyl- and isopropylammonium ions, and acetamidocholine and its lower dimethyl analogue, is also attributed to E·S·I′ complexes. Peripheral binding of these inhibitors decreases acylation more than deacylation. Some tertiary dimethylammonio ions have more favorable Ki(nonc) values with AcCh, decreasing deacylation more than acylation. The substrate DEBAAc is a more effective noncompetitive than competitive inhibitor in hydrolysis in AcCh, indicating that it binds more strongly in a peripheral site than in the active site of the free enzyme. In its hydrolysis by AcChE, it acts as its own noncompetitive inhibitor, by this non-productive binding. Formation of E·S·I′ complexes is a general characteristic of hydrolysis by AcChE and decrease in rates at high concentrations of AcCh and related substrates is attributed to peripheral regulatory site binding, formation of E·S·S′ complexes, rather than to binding to the acetylenzyme. © 1991.