A METASTABLE STATE OF TORPEDO-CALIFORNICA ACETYLCHOLINESTERASE GENERATED BY MODIFICATION WITH ORGANOMERCURIALS

Chemical modification of Torpedo californica acetylcholinesterase by v arious sulfhydryl reagents results in its conversion to one of two pri ncipal states. One of these states, viz., that produced by disulfides and alkylating agents, is stable. The second state, produced by mercur y derivatives, is metastable. At room temperature, it converts spontan eously, with a half-life of ca. 1 h, to a stable state similar to that produced by the disulfides and alkylating agents. Demodification of a cetylcholinesterase freshly modified by mercurials, by its exposure to reduced glutathione, causes rapid release of the bound mercurial, wit h concomitant recovery of most of the enzymic activity of the native e nzyme. In contrast, similar demodification of acetylcholinesterase mod ified by disulfides yields no detectable recovery of enzymic activity. Spectroscopic measurements, employing CD, intrinsic fluoresence, and binding of 1-anilino-8-naphthalenesulfonate, show that the state produ ced initially by mercurials is ''native-like'', whereas that produced by disulfides and alkylating agents, and after prolonged incubation of the mercurial-modified enzyme, is partially unfolded and displays man y of the features of the ''molten globule'' state. Arrhenius plots sho w that the quasi-native state produced by organomercurials is separate d by a low (5 kcal/mol) energy barrier from the native state, whereas the partially unfolded state is separated from the quasi-native state by a high energy barrier (ca. 50 kcal/mol). Comparison of the 3D struc tures of native acetylcholinesterase and of a heavy-atom derivative ob tained with HgAc2 suggests that the mercurial modified enzyme may be s tabilized by additional interactions of the mercury atom attached to t he free thiol group of Cys(231), specifically with Ser(228)O gamma and with the main-chain nitrogen and carbonyl oxygen of the same serine r esidue.