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 approximately 8 degrees 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.
Silman I, Roth E, Paz A, Triquigneaux MM, Ehrenshaft M, Xu Y, Shnyrov VL, Sussman JL, Deterding LJ, Ashani Y, Mason RP, Weiner L (2013) The specific interaction of the photosensitizer methylene blue with acetylcholinesterase provides a model system for studying the molecular consequences of photodynamic therapy Chemico-Biological Interactions203: 63-6
Silman I, Roth E, Paz A, Triquigneaux MM, Ehrenshaft M, Xu Y, Shnyrov VL, Sussman JL, Deterding LJ, Ashani Y, Mason RP, Weiner L (2013) Chemico-Biological Interactions203: 63-6
