Histochemical method for characterization of enzyme crystals: application to crystals ofTorpedo californicaacetylcholinesterase

Abstract

Histochemical methods are employed to detect and localize a wide range of enzymes. Even though protein crystallographers do not commonly use this technique, the extensively used colorimetric reaction of Karnovsky was successfully adapted for easy and quick identi®cation of acetylcholinesterase crystals. The method relies on the reduction of ferricyanide to ferrocyanide by thiocholine, released from acetylthiocholine by enzymatic hydrolysis, followed by formation of a cupric ferrocyanide precipitate, and allows rapid differentiation between salt and enzyme crystals and between native and inhibited crystals of the enzyme.

Structural insights into substrate traffic and inhibition in acetylcholinesterase

“…Native Tc AChE crystals obtained from the same enzyme batch did not display any electron density adjacent to Ser200 (not shown). Consequently, and as enzymatic activity within the crystal has been demonstrated (Nicolas et al , 2001), we conclude that Ser200 had been acetylated by the ATCh in the soaking solution.…”

Evidence for Subdomain Flexibility in Drosophila melanogaster Acetylcholinesterase

“…Kinetic models can describe the data without assuming classical conformational motions , . Resolved crystal structures in the absence and presence of ligands do not reveal backbone variations: a low AChE activity has been recorded within the crystal, where most of the backbone motions are prevented by the tight molecular packing and despite a substantially decreased level of diffusion, due to reduced water content. Omega loop fixation by the introduction of an additional disulfide bond (G80C/V431C in Torpedo marmorata AChE) did not impair substrate hydrolysis − .…”