ACTIVITY OF TORPEDO CALIFORNICA ACETYLCHOLINESTERASE IN THE CRYSTALLINE STATE

Nicolas, Anne1, Millard, Charles B. 2, Raves, Mia L. 3, Ravelli, Raimond B.G. 1, Kroon, Jan 1, Silman, Israel 2, and Sussman, Joel L. 3,4

1 Dept. of Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Utrecht University, The Netherlands.
Depts. of 2 Neurobiology and 3 Structural Biology, Weizmann Institute of Science, Rehovot, Israel.
4 Biology Department, Brookhaven National Laboratory, Upton NY, USA.

Keywords: Acetylcholinesterase, Enzymatic avtivity in crystals

There is evidence that enzymes display catalytic activity in the crystalline state [1]. This activity may be influenced by:

  1. Ability of substrates to reach the active site and of products to exit,
  2. chemical composition of the crystallisation mother liquor (ML),
  3. conformational freedom of residues involved in substrate binding and hydrolysis.

Torpedo californica acetylcholinesterase (TcAChE) is one of nature's fastest enzymes, whose active site is located near the bottom of a deep and narrow gorge. In trigonal crystals of the enzyme, the entrance to the active-site gorge is blocked by a symmetry-related molecule, except for a few channels that are just large enough for water molecules [2]. We are interested in using time-resolved crystallography to study traffic of substrate and products to and from the active site. Direct evidence that crystalline AChE displays catalytic activity is obviously pertinent to pursuit of this experimental approach.

The pH of the ML we employed (34% PEG200, 50mM NaCl, 150 mM MES buffer, pH 5.8 at 4 °C) reduced the kcat for TcAChE in solution ~ 6-fold from the optimum at pH 7.5. Another ~4-fold reduction is due to the PEG200; increasing concentrations of PEG caused an exponential decrease in kcat/Km.

Trigonal crystals of TcAChE showed measurable hydrolysis of acetylthiocholine (ATCh), but the apparent rates were 50- to 100-fold lower than those obtained after dissolving the crystalline enzyme in ML. This might be due either to diffusion-limited availability of substrate for such a rapid enzyme, or to incomplete participation of the active sites in the crystal to catalysis. To address this question, we employed carbamylcholine (CCh) as a substrate analog, since the second step of its hydrolysis, decarbamylation of the enzyme, is much slower than the first step. The X-ray data collected on rapidly frozen crystals soaked with CCh revealed essentially complete carbamylation, thus establishing that the reaction takes place throughout the crystal.

  1. Rossi, G.L. Current Opinion in Structural Biology 2 816-820 (1992).
  2. Axelsen et al. Protein Science 3 188-197 (1994)