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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) approximately 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(2+), Mg(2+), 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 degrees 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.

Original publication

DOI

10.1110/ps.03110703

Type

Journal article

Journal

Protein Sci

Publication Date

10/2003

Volume

12

Pages

2337 - 2347

Keywords

Acetylcholinesterase, Algorithms, Anilino Naphthalenesulfonates, Animals, Calorimetry, Differential Scanning, Catalysis, Circular Dichroism, Cross-Linking Reagents, Cysteine, Electrophoresis, Polyacrylamide Gel, Enzyme Inhibitors, Enzyme Stability, Glutathione, Glycerol, Hot Temperature, Hydrolysis, Kinetics, Magnesium, Methylamines, Models, Theoretical, Perylene, Protein Conformation, Protein Denaturation, Spectrometry, Fluorescence, Sulfhydryl Reagents, Sulfinic Acids, Thermodynamics, Torpedo, Trypsin