Abstract
Hydrolysis of acetylcholine catalyzed by acetylcholinesterase (AChE), one of the most efficient enzymes in nature, occurs at the base of a deep and narrow active center gorge. At the entrance of the gorge, the peripheral anionic site provides a binding locus for allosteric ligands, including substrates. To date, no structural information on substrate entry to the active center from the peripheral site of AChE or its subsequent egress has been reported. Complementary crystal structures of mouse AChE and an inactive mouse AChE mutant with a substituted catalytic serine (S203A), in various complexes with four substrates (acetylcholine, acetylthiocholine, succinyldicholine, and butyrylthiocholine), two non-hydrolyzable substrate analogues (m-(N,N,N-trimethylammonio)-trifluoroacetophenone and 4-ketoamyltrimethylammonium), and one reaction product (choline) were solved in the 2.05-2.65-A resolution range. These structures, supported by binding and inhibition data obtained on the same complexes, reveal the successive positions and orientations of the substrates bound to the peripheral site and proceeding within the gorge toward the active site, the conformations of the presumed transition state for acylation and the acyl-enzyme intermediate, and the positions and orientations of the dissociating and egressing products. Moreover, the structures of the AChE mutant in complexes with acetylthiocholine and succinyldicholine reveal additional substrate binding sites on the enzyme surface, distal to the gorge entry. Hence, we provide a comprehensive set of structural snapshots of the steps leading to the intermediates of catalysis and the potential regulation by substrate binding to various allosteric sites at the enzyme surface.
Highlights
AChE-catalyzed hydrolysis of ACh and other carboxyl esters proceeds via formation of an initial noncovalent enzyme-substrate complex
Inhibitors of AChE bind to the active site or to the peripheral anionic site (PAS), an allosteric site located at the active center gorge entrance, or they span the two sites thereby occupying much of the active center gorge (7–9)
Electrostatic calculations based on the TcAChE structure and subsequent molecular dynamics simulation suggested that AChE may have a “back door,” distinct from the gorge entrance and whose transient opening would contribute to the high rate of traffic of substrates, products, and water into and out of the active center gorge (27, 28)
Summary
Chemicals— 4K-TMA iodide was from ICN and ambenonium dichloride from Tocris Cookson. ACh, ATCh, SCh, BTCh, and choline, as chloride or iodine salts, were from Sigma. The enzyme and mutant were extensively dialyzed against 1 mM Mes, pH 6.5, 1 M NaCl, 40 mM MgCl2, 0.01% NaN3 (w/v), and desalted by gel-filtration FPLC on Superdex-200 (Amersham Biosciences) in 1 mM Mes, pH 6.5, 50 mM NaCl, 0.01% NaN3 (w/v) (crystallization buffer), or by extensive dialysis against this buffer. They were concentrated to about 10 mg/ml by ultrafiltration. The other seven complexes were generated by crystal soaking, carried out at 4 °C in sitting drops (20 l) made of the well solution supplemented with the ligand (250 mM) and polyethylene glycol up to 35%
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