title: Chemical modification of Torpedo acetylcholinesterase by disulfides: appearance of a "molten globule" state.
abstract: 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".
authors.affiliation: Department of Neurobiology, Weizmann Institute of Science, Rehovot, Israel.
authors.forename: E A
authors.initials: EA
authors.lastname: Dolginova
authors.forename: E
authors.initials: E
authors.lastname: Roth
authors.forename: I
authors.initials: I
authors.lastname: Silman
authors.forename: L M
authors.initials: LM
authors.lastname: Weiner
mesh_terms: D000110:Acetylcholinesterase / Q000737:chemistry*; D000818:Animals; D002800:Cholinesterase Inhibitors; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D011487:Protein Conformation; D014101:Torpedo; D014357:Trypsin
chemical_list: D002800:Cholinesterase Inhibitors; D004220:Disulfides; D000110:Acetylcholinesterase; D014357:Trypsin