Nuclear magnetic resonance studies on acetylcholinesterase. The use of atropine and eserine to probe binding sites.

Abstract

Nuclear magnetic resonance has been used to study the association of atropine and eserine with acetylcholinesterase extracted from squid head ganglia. Line width changes of the N -methyl and phenyl groups of atropine and the N -methyl and C -methyl groups of eserine resulting from association with the enzyme have been utilized to study the interactions. The observed line widths in the presence of enzyme were similar for atropine and eserine. The addition of tetraethyl pyrophosphate or diisopropyl fluorophosphate diminished the binding of eserine but not of atropine. Kinetic studies, using acetylcholine as the substrate, showed that eserine was a potent cholinesterase inhibitor whereas atropine was a poor anticholinesterase. These results indicate that there are at least two binding sites on the enzyme. One is the active center (where eserine binds), which consists of an anionic and an esteratic site; the other is an anionic site (where atropine binds), which is distinct from the catalytic site. ACKNOWLEDGMENT We are greatly indebted to Professor K. Krnjevic for his interest and encouragement throughout these studies.