LAUE STUDIES ON ACETYLCHOLINESTERASE. Mia L. Raves, (Weizmann Institute of Science), Raimond B.G. Ravelli, Jan Kroon, (Utrecht University), Michel Roth, Dominique Bourgeois, (IBS, Grenoble), Ling Peng, Maurice Goeldner, (Université Louis Pasteur, Strasbourg), Israel Silman and Joel L. Sussman, (Weizmann Institute)

In the last ten years it has become feasible to study dynamic processes in macromolecules at the atomic level. Most of these studies have used the Laue diffraction technique that permits data collection on a second to pico-second time scale by virtue of the polychromatic synchrotron radiation.

It is our aim to do time-resolved experiments on the enzyme acetyl-cholinesterase (AChE). AChE cleaves the neurotransmitter acetylcholine (ACh) in the synapse at a very high turnover rate. The enzyme has a strong dipole moment that draws the positively charged substrate towards the catalytic site, situated at the bottom of a narrow gorge lined with aromatic residues. One of the reaction products, choline, bears the same charge as the substrate, ACh, which raises the problem of its exit route from the active site. In order to attempt to observe the reaction process, photolabile precursors of choline and of carbamylcholine have been synthesized and characterized. These two probes generate choline in different ways, either by direct photocleavage (choline precursor) or by enzymatic hydrolysis of a substrate generated by photocleavage (These tools for the time-resolved crystallographic studies will hopefully permit monitoring of the exit of choline from the active site.

To test the feasibility of the use of the Laue method, trigonal crystals of Torpedo californica AChE soaked with the inhibitor edrophonium were used to collect data at the ESRF in Grenoble, beam line ID9 (BL3) on a CCD detector with an exposure time of 1 msec. The frames were indexed using a new program called that is able to determine the relative cell parameters from the Laue pattern semi-automatically. Further pro-cessing with the CCP4 Laue software package yielded a data set to 3.1 Å resolution of 80% completeness with 15,000 unique reflections. Constrained refinement of the protein coordinates with X-PLOR gave a difference map in which the density for the inhibitor can be clearly seen.

1. Peng, L. et al. submitted to Biochemistry (1996).

2. Ravelli, R.B.G.et al. accepted in J. Appl. Cryst (199