Hydrolysis by acetylcholinesterase. Apparent molal volumes and trimethyl and methyl subsites.

A study has been made of hydrolysis by acetylcholinesterase (EC 3.1.1.7)and by hydroxide of acetate ester RCH V, CHSNHZ"; VI, CH3CH2"; VII, h 3 -; Br; XIV, N s -.Acylation rate constants, kz, are normalized for reactivity in hydrolysis by hydroxide, OH).Comparison of K. within the pairs, Compounds I11 and IV, Compounds V and VI, Compounds VI1 and VIII, and probably Compounds I and LI, and of K. for Compounds X to X I V with Compounds V and M I indicate that positive charge makes little if any contribution to binding.Compounds I and 11 have similar normalized values of kz and bimolecular constants, k2(n)/K,.Compounds IV, VI, and VIII have greater normalized values than the charged analogues 111, V, and W. The effect of positive charge on kcat and k2 is attributed to the effect on intrinsic reactivity, k(OH).Positive charge is not specifically activating in acylation and is absent in the very efficient deacylation.The generally accepted "anionic" site is better considered an uncharged Trimethyl site, complementary to substrate (CH 11, (CHS)3C-; 111, (CH3),h-; N, W I , CH3-; IX, H; X, H l t ; XI, CH3W; XII, C1; XIII,The major features considered to be involved in the interactions of acetylcholine, (CH3)3&CH&H20COCH3 (Compound I), with the enzyme which hydrolyzes it and the receptors on which it acts are the three N-methyl groups, the positive charge, and the ester group.The active site of acetylcholinesterase is generally characterized as comprising (1, 2) an anionic site at which the quaternary ammonium group binds and an esteratic site which is acetylated (3) at serine (4) in the course of the hydrolysis.In the receptor there would be a similar anionic site, and an esterophilic site (5).