Modification of Torpedo californica acetylcholinesterase (AChE) both by bis(1-oxy-2,2,5,5-tetramethyl-3-imidazolin-4-yl)disulfide (biradical) and by 4,4'-dithiopyridine, via a thiol-disulfide exchange reaction, was monitored by EPR and optical spectroscopy, respectively. Incubation with these reagents caused complete loss of enzymic activity. Treatment with glutathione of AChE modified by either of the two disulfides led to rapid release of the bound reagent with simultaneous regeneration of the single free thiol group of the enzyme. However, no concomitant recovery of catalytic activity was observed. SDS-PAGE showed that both the modified and demodified enzymes retained their structure as a disulfide-linked dimer. Circular dichroism revealed that modification of AChE by the disulfide agents with or without demodification by glutathione led to a complete disappearance of the ellipticity in the near-UV and to a much smaller decrease in ellipticity in the far-UV. The CD spectra observed are typical of the "molten globule" state of proteins. 1-Anilino-8-naphthalenesulfonate binding measurements and an enhanced susceptibility to trypsinolysis supported the supposition that chemical modification had transformed native AChE to a "molten globule".
Chemical modification of Torpedo californica acetylcholinesterase by the natural thiosulfinate allicin produces an inactive enzyme through reaction with the buried cysteine Cys 231. Optical spectroscopy shows that the modified enzyme is "native-like," and inactivation can be reversed by exposure to reduced glutathione. The allicin-modified enzyme is, however, metastable, and is converted spontaneously and irreversibly, at room temperature, with t 1/2 Ӎ 100 min, to a stable, partially unfolded state with the physicochemical characteristics of a molten globule. Osmolytes, including trimethylamine-N-oxide, glycerol, and sucrose, and the divalent cations, Ca , and Mn 2+ can prevent this transition of the native-like state for >24 h at room temperature. Trimethylamine-N-oxide and Mg 2+ can also stabilize the native enzyme, with only slight inactivation being observed over several hours at 39°C, whereas in their absence it is totally inactivated within 5 min. The stabilizing effects of the osmolytes can be explained by their differential interaction with the native and native-like states, resulting in a shift of equilibrium toward the native state. The stabilizing effects of the divalent cations can be ascribed to direct stabilization of the native state, as supported by differential scanning calorimetry.
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