Structural and functional characterization of the interaction of the photosensitizing probe methylene blue with Torpedo californica acetylcholinesterase.
The photosensitizer, methylene blue (MB), generates singlet oxygen that irreversibly inhibits Torpedo californica acetylcholinesterase (TcAChE). In the dark, it inhibits reversibly. Binding is accompanied by a bathochromic absorption shift, used to demonstrate displacement by other acetylcholinesterase inhibitors interacting with the catalytic "anionic" subsite (CAS), the peripheral "anionic" subsite (PAS), or bridging them. MB is a noncompetitive inhibitor of TcAChE, competing with reversible inhibitors directed at both "anionic" subsites, but a single site is involved in inhibition. MB also quenches TcAChE's intrinsic fluorescence. It binds to TcAChE covalently inhibited by a small organophosphate (OP), but not an OP containing a bulky pyrene. Differential scanning calorimetry shows an ~8° increase in the denaturation temperature of the MB/TcAChE complex relative to native TcAChE, and a less than twofold increase in cooperativity of the transition. The crystal structure reveals a single MB stacked against Trp279 in the PAS, oriented down the gorge toward the CAS; it is plausible that irreversible inhibition is associated with photooxidation of this residue and others within the active-site gorge. The kinetic and spectroscopic data showing that inhibitors binding at the CAS can impede binding of MB are reconciled by docking studies showing that the conformation adopted by Phe330, midway down the gorge, in the MB/TcAChE crystal structure, precludes simultaneous binding of a second MB at the CAS. Conversely, binding of ligands at the CAS dislodges MB from its preferred locus at the PAS. The data presented demonstrate that TcAChE is a valuable model for understanding the molecular basis of local photooxidative damage.
Department of Neurobiology, Weizmann Institute of Science, Rehovot 76100, Israel.
Aviv
A
Paz
Esther
E
Roth
Yacov
Y
Ashani
Yechun
Y
Xu
Valery L
VL
Shnyrov
Joel L
JL
Sussman
Israel
I
Silman
Lev
L
Weiner
D000110:Acetylcholinesterase / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002800:Cholinesterase Inhibitors / Q000494:pharmacology*; D018360:Crystallography, X-Ray; D029941:Fish Proteins / Q000737:chemistry / Q000378:metabolism*; D008751:Methylene Blue / Q000494:pharmacology*; D008958:Models, Molecular; D017319:Photosensitizing Agents / Q000494:pharmacology*; D014101:Torpedo / Q000378:metabolism*
D002800:Cholinesterase Inhibitors; D029941:Fish Proteins; D017319:Photosensitizing Agents; D000110:Acetylcholinesterase; D008751:Methylene Blue