title: The specific interaction of the photosensitizer methylene blue with acetylcholinesterase provides a model system for studying the molecular consequences of photodynamic therapy.
abstract: The photosensitizer, methylene blue (MB), generates singlet oxygen ((1)O2) that irreversibly inhibits Torpedo californica acetylcholinesterase (TcAChE). In the dark MB inhibits reversibly, binding being accompanied by a bathochromic shift that can be used to show its displacement by other reversible inhibitors binding to the catalytic 'anionic' subsite (CAS), the peripheral 'anionic' subsite (PAS), or bridging them. Data concerning both reversible and irreversible inhibition are here reviewed. MB protects TcAChE from thermal denaturation, and differential scanning calorimetry reveals a ~8 °C increase in the denaturation temperature. The crystal structure of the MB/TcAChE complex reveals a single MB stacked against W279 in the PAS, pointing down the gorge towards the CAS. The intrinsic fluorescence of the irreversibly inhibited enzyme displays new emission bands that can be ascribed to N'-formylkynurenine (NFK); this was indeed confirmed using anti-NFK antibodies. Mass spectroscopy revealed that two Trp residues, Trp84 in the CAS, and Trp279 in the PAS, were the only Trp residues, out of a total of 14, significantly modified by photo-oxidation, both being converted to NFK. In the presence of competitive inhibitors that displace MB from the gorge, their modification is completely prevented. Thus, photo-oxidative damage caused by MB involves targeted release of (1)O2 by the bound photosensitizer within the aqueous milieu of the active-site gorge.
authors.affiliation: Department of Neurobiology, Weizmann Institute of Science, Rehovot 76100, Israel. israel.silman@weizmann.ac.il
authors.forename: Israel
authors.initials: I
authors.lastname: Silman
authors.forename: Esther
authors.initials: E
authors.lastname: Roth
authors.forename: Aviv
authors.initials: A
authors.lastname: Paz
authors.forename: Mathilde M
authors.initials: MM
authors.lastname: Triquigneaux
authors.forename: Marilyn
authors.initials: M
authors.lastname: Ehrenshaft
authors.forename: Yechun
authors.initials: Y
authors.lastname: Xu
authors.forename: Valery L
authors.initials: VL
authors.lastname: Shnyrov
authors.forename: Joel L
authors.initials: JL
authors.lastname: Sussman
authors.forename: Leesa J
authors.initials: LJ
authors.lastname: Deterding
authors.forename: Yacov
authors.initials: Y
authors.lastname: Ashani
authors.forename: Ronald P
authors.initials: RP
authors.lastname: Mason
authors.forename: Lev
authors.initials: L
authors.lastname: Weiner
mesh_terms: D000110:Acetylcholinesterase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D055592:Biophysical Phenomena; D020134:Catalytic Domain; D002800:Cholinesterase Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008751:Methylene Blue / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010778:Photochemotherapy; D017319:Photosensitizing Agents / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011487:Protein Conformation; D014101:Torpedo
chemical_list: D002800:Cholinesterase Inhibitors; D017319:Photosensitizing Agents; D000110:Acetylcholinesterase; D008751:Methylene Blue