title: Targeted oxidation of Torpedo californica acetylcholinesterase by singlet oxygen.
abstract: The photosensitizer, methylene blue (MB), is a strong reversible inhibitor of Torpedo californica acetylcholinesterase (AChE) in the dark. Under illumination it causes irreversible inactivation. Loss of fluorescence of the singlet oxygen ((1)O(2)) trap, 9,10-dimethylanthracene, was retarded in the presence of AChE, and the rate of photo-inactivation was increased in the presence of D(2)O, indicating that inactivation was due to (1)O(2) generated by the photosensitizer. CD revealed slightly reduced far-UV ellipticity, and slightly enhanced binding of an amphiphilic probe, indicating limited unfolding of the photo-oxidized AChE. However, both near-UV ellipticity and intrinsic fluorescence were markedly reduced, suggesting photo-oxidative damage to tryptophans, (Trp) supported by appearance of novel emission peaks ascribed to N'-formylkynurenine and/or kynurenine. Like other partially unfolded forms, the photo-oxidized AChE was sensitive to proteolysis. Photosensitized inactivation produced exclusively chemically cross-linked dimers, whereas irradiation of a partially unfolded state generated higher-order oligomers. The active-site gorge of AChE contains Trp in inhibitor-binding sites that might be targets for photo-oxidation. Indeed, reversible inhibitors retard photo-inactivation, and photo-inactivation destroys their binding sites. An excess of AChE protects paraoxonase from photo-inactivation by sequestering the photosensitizer. Affinity photo-oxidation of AChE by MB thus provides a valuable model for studying site-specific photo-inactivation of enzymes in both fundamental and clinical contexts.
authors.affiliation: Department of Chemical Research Support, Weizmann Institute of Science, Rehovoth, Israel. Lev.Weiner@weizmann.ac.il
authors.forename: Lev
authors.initials: L
authors.lastname: Weiner
authors.forename: Esther
authors.initials: E
authors.lastname: Roth
authors.forename: Israel
authors.initials: I
authors.lastname: Silman
mesh_terms: D000110:Acetylcholinesterase / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D020134:Catalytic Domain; D004791:Enzyme Inhibitors / Q000737:chemistry; D008751:Methylene Blue / Q000737:chemistry; D008954:Models, Biological; D010084:Oxidation-Reduction; D026082:Singlet Oxygen; D014101:Torpedo / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D026082:Singlet Oxygen; D000110:Acetylcholinesterase; D008751:Methylene Blue