Site-Directed Mutagenesis of Functional Residues in Torpedo Acetylcholinesterase

Abstract

Acetylcholinesterase (AChE) is remarkable for its very high catalytic activity relative to other serine hydrolases (Quinn, 1987). It displays a turnover number of the order of 100 µseconds (Vigny et al., 1978), and thus approaches a rate of activity at which substrate diffusion may become rate-limiting (Bazelyansky et al., 1986). The recent solution of the three-dimensional structure of Torpedo californica AChE has revealed that AChE displays some rather unusual, perhaps unique, structural features compared to other hydrolases in general and to serine hydrolases in particular (Sussman et al., 1991, 1992). Thus a principal structural (and presumably functional) feature of this enzyme is a deep and narrow cavity, penetrating 20 Å into it, with the catalytic triad characteristic of serine hydrolases being located close to its bottom. The function of this cavity is, as yet, obscure, but it has been named the ‘aromatic gorge’, since a substantial part of its surface is provided by the rings of fourteen highly conserved aromatic amino acids. Some of these aromatic rings, located near the catalytic triad, may be involved directly in substrate-binding, by dipole-dipole interaction with the quaternary group of the substrate, acetylcholine (ACh). However, the role of more distal aromatic rings, further up the gorge, remains to be clarified. The catalytic triad of AChE is unusual inasmuch as it contains a glutamic acid, E327 in the Torpedo numbering, rather than the aspartic acid residues found previously in serine hydrolases, although a conserved aspartic acid residue, D326, occurs adjacent to it. A feature which is restricted to Torpedo AChE is the presence of a non-conserved cysteine residue, C231, bearing a free sulfhydryl group, which is situated deep within the core of the protein, ca 8 Å from Oγ of the active-site serine. This cysteine residue is of interest because its covalent modification by several sulfhydryl reagents abolishes AChE activity (Steinberg et al., 1990), even though it is not involved in catalytic activity.