Hasan_1981_J.Biol.Chem_256_7781

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.