Cationic and uncharged substrates and reversible inhibitors in hydrolysis by acetylcholinesterase (EC 3.1.1.7). The trimethyl subsite

Abstract

Structurally related cationic and uncharged compounds have been studied as inhibitors of hydrolysis by acetylcholinesterase of acetylcholine and its uncharged carbon analog, 3,3-dimethylbutyl acetate. Similar effects of the inhibitors on hydrolysis of the two substrates indicate that the quaternary ammonium group of acetylcholine and the neopentyl group of 3,3-dimethylbutyl acetate bind at the same subsite. Comparison of (CH3)3+NCH2CH2CH2COCH3 (Compound I), Ki = 0.02 mM, and its tertiary homologue, (CH3)2-+NHCH2CH2CH2COCH3 (Compound V), Ki = 0.75 mM, with a secondary isomer of Compound I, 3-oxo-(N-tert-butyl)-butanaminium, (CH3)3C+NH2CH2CH2COCH3 (Compound II), Ki = 0.15 mM, and its lower homologue, (CH3)2CH+NH2CH2CH2COCH3 (Compound IX), Ki = 2 mM, attests to the importance of the branched trimethyl structure and the smaller effect of hydrophobicity of the quaternary ammonium structure. This is supported by competitive inhibition by tert-butyl ammonium, (CH3)3C+NH3 (Compound IV), Ki = 0.45 mM, compared with mixed inhibition by its quarternary isomer, (CH3)4+N (Compound VII), Ki = 1.5 mM, and choline (Compound VI), Ki = 1.0 mM. Uncharged analogues of Compound II, 4-tert-butylthio-2-butanone, (CH3)3CSCH2CH2COCH3 (Compound III), Ki = 0.4 mM, and 4-tert-butoxy-2-butanone, (CH3)3COCH2CH2COCH3 (Compound VIII), Ki = 1.6 mM, and of Compound VI, 3,3-dimethylbutanol (Compound XI), Ki = 7.5 mM, indicate that positive charge contributes factors of 3 to 10 to binding. This may be attributed to peripheral negative charges, present at pH 7-8 in the enzyme of isoelectric point approximately 5, indicating that the binding subsite may be explored more specifically by tert-butyl than by charged reagents.