# 1998 International Union of Crystallography Acta Crystallographica Section D Printed in Great Britain ± all rights reserved ISSN 0907-4449 # 1998 1359 Acta Cryst. (1998). D54, 1359±1366 Static Laue Diffraction Studies on Acetylcholinesterase Raimond B. G. Ravelli, a ² Mia L. Raves, b ² Zhong Ren, c Dominique Bourgeois, d Michel Roth, d Jan Kroon, a Israel Silman e and Joel L. Sussman b,f * a Department of Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands, b Department of Structural Biology, Weizmann Institute of Science, 76100 Rehovot, Israel, c Department of Biochemistry and Molecular Biology, The University of Chicago, 920 East 58th Street, Chicago, IL 60637, USA, d Institut de Biologie Structurale Jean-Pierre Ebel, 41 Avenue des Martyrs, 38027 Grenoble CEDEX 1, France, e Department of Neurobiology, Weizmann Institute of Science, 76100 Rehovot, Israel, and f Biology Department, Brookhaven National Laboratory, Upton NY 11973-5000, USA. E-mail: joel.sussman@weizmann.ac.il (Received 16 October 1997; accepted 6 April 1998 ) Abstract Acetylcholinesterase (AChE) is one of nature's fastest enzymes, despite the fact that its three-dimensional structure reveals its active site to be deeply sequestered within the molecule. This raises questions with respect to traf®c of substrate to, and products from, the active site, which may be investigated by time-resolved crystal- lography. In order to address one aspect of the feasibility of performing time-resolved studies on AChE, a data set has been collected using the Laue technique on a trigonal crystal of Torpedo californica AChE soaked with the reversible inhibitor edrophonium, using a total X-ray exposure time of 24 ms. Electron-density maps obtained from the Laue data, which are of surprisingly good quality compared with similar maps from mono- chromatic data, show essentially the same features. They clearly reveal the bound ligand, as well as a structural change in the conformation of the active-site Ser200 induced upon binding. 1. Introduction 1.1. Reaction of AChE The principal function of the enzyme acetyl- cholinesterase (AChE; E.C. 3.1.1.7) is rapid hydrolysis of the neurotransmitter acetylcholine (ACh) in the central nervous system and at vertebrate neuromuscular junctions (Massoulie  et al. , 1993). As demanded by this biological role, it is an extremely ef®cient catalyst, approaching a reaction velocity at which substrate diffusion becomes rate limiting, with a turnover number of 20000 s 1 (Quinn, 1987). The X-ray structure of T. californica AChE (TcAChE) (Sussman et al., 1991) unexpectedly revealed that the active-site residues are buried within the protein, apparently accessible only via a long and narrow gorge lined with aromatic residues. The AChE molecule displays an asymmetric charge distribution, resulting in a strong dipole moment aligned approximately along the active-site gorge (Ripoll et al., 1993; Tan et al., 1993). It has been suggested that the electric ®eld so generated attracts the positively charged ACh to the rim of the gorge (Ripoll et al., 1993). The substrate is then guided by the electric ®eld, using the aromatic residues in the gorge as a series of low-af®nity sites, towards the active site. The side chains of two aromatic residues, Trp84 and Phe330, serve as the so- called anionic site for binding the quarternary moiety of ACh via cation± interactions (Verdonk et al., 1993; Dougherty, 1996). Hydrolysis of ACh to choline and acetate is accomplished by the catalytic triad consisting of Ser200, His440 and Glu327, with Ser200 being tran- siently acetylated (Quinn, 1987). The narrow dimensions of the active-site gorge, together with the potential gradient along it, raise cogent questions regarding the traf®c of substrate and products to and from the active site. Thus, in addition to steric considerations, the potential along the gorge would be expected to accelerate expulsion of acetate but retard release of choline from the active site. Further- more, movement of water between the bulk phase and the gorge must be taken into account. Theoretical calculations, however, showed that it is still possible for choline to go back through the gorge suf®ciently rapidly if there is no steric hindrance (S. Lifson, S. Botti and C. Felder, personal communications). A second possibility that has been proposed (Ripoll et al., 1993) is that choline leaves through a back door ± a transient opening in the thin aspect of the gorge wall arising by movement of one or more residues (Gilson et al., 1994). The resi- dues that contribute to the opening of the putative back door are expected to be located on the -loop between Cys67 and Cys94 in TcAChE (Axelsen et al., 1994), a loop which is known to adopt an open and closed conformation in the structurally similar enzyme Candida rugosa lipase (Grochulski et al., 1994). Further evidence ² Both authors contributed equally to this work.