Author/Editor		Šentjurc, Marjeta; Pečar, Slavko; Stojan, Jure; Marchot, Pascale; Radić, Zoran; Grubič, Zoran
Title		Electron paramagnetic resonance reveals altered topography of the active center gorge of acetylcholinesterase after binding of fasciculin to the peripheral site
Type		članek
Source		Biochim Biophys Acta
Vol. and No.		Letnik 1429
Publication year		1999
Volume		str. 349-58
Language		eng
Abstract		Fasciculin, a peptidic toxin from snake venom, inhibits mammalian and fish acetylcholinesterases (AChE) by binding to the peripheral site of the enzyme. This site is located at the rim of a narrow, deep gorge which leads to the active center triad, located at its base. The proposed mechanisms for AChE inhibition by fasciculin include allosteric events resulting in altered conformation of the AChE active center gorge. However, a fasciculin-induced altered topography of the active center gorge has not been directly demonstrated. Using electron paramagnetic resonance with the spin-labed organosphosphate 1-oxyl-2,2,6,6-tetramethyl-4-piperidinylethylphosphorofluoridate (EtOSL) specifically bound to the catalytic serine of mouse AChE (mAChE), we show that bound fasciculin on mAChE slows down, but does not prevent hosphorylation of the active site serine by EtOSL and protects the gorge conformation against thermal denaturation. Most importantly, a restricted freedom of motion of the spin label bound to the fasciculin-associated mAChE, compared to mAChE, is evidenced. Molecular models of mAChE and fasciculin-associated mAChE with tethered EtOSL enantiomers indicate that this restricted motion is due to greater proximity of the S-EtOSL nitroxide radical to the W86 residue in the fasciculin-associated enzyme. Our results demonstrate a topographical alteration indicative of a restricted conformation of the active center gorge of mAChE with bound fasciculin at its rim.
Descriptors		ACETYLCHOLINESTERASE ELECTRON SPIN RESONANCE SPECTROSCOPY PROPIDIUM ENZYMES TOXINS CHOLINESTERASE INHIBITORS ANTIBODIES, MONOCLONAL