Abstract
We have used a combination of cysteine substitution mutagenesis and site-specific labeling to characterize the structural dynamics of mouse acetylcholinesterase (mAChE). Six cysteine-substituted sites of mAChE (Leu(76), Glu(81), Glu(84), Tyr(124), Ala(262), and His(287)) were labeled with the environmentally sensitive fluorophore, acrylodan, and the kinetics of substrate hydrolysis and inhibitor association were examined along with spectroscopic characteristics of the acrylodan-conjugated, cysteine-substituted enzymes. Residue 262, being well removed from the active center, appears unaffected by inhibitor binding. Following the binding of ligand, hypsochromic shifts in emission of acrylodan at residues 124 and 287, located near the perimeter of the gorge, reflect the exclusion of solvent and a hydrophobic environment created by the associated ligand. By contrast, the bathochromic shifts upon inhibitor binding seen for acrylodan conjugated to three omega loop (Omega loop) residues 76, 81, and 84 reveal that the acrylodan side chains at these positions are displaced from a hydrophobic environment and become exposed to solvent. The magnitude of fluorescence emission shift is largest at position 84 and smallest at position 76, indicating that a concerted movement of residues on the Omega loop accompanies gorge closure upon ligand binding. Acrylodan modification of substituted cysteine at position 84 reduces ligand binding and steady-state kinetic parameters between 1 and 2 orders of magnitude, but a similar substitution at position 81 only minimally alters the kinetics. Thus, combined kinetic and spectroscopic analyses provide strong evidence that conformational changes of the Omega loop accompany ligand binding.
Highlights
Nals by catalyzing hydrolysis of the neurotransmitter acetylcholine at a diffusion limited rate [2, 3]
We have used a combination of cysteine substitution mutagenesis and site-specific labeling to characterize the structural dynamics of mouse acetylcholinesterase
The bathochromic shifts upon inhibitor binding seen for acrylodan conjugated to three omega loop (⍀ loop) residues 76, 81, and 84 reveal that the acrylodan side chains at these positions are displaced from a hydrophobic environment and become exposed to solvent
Summary
Inhibitors and Substrates—Acetylthiocholine iodide, 5,5Ј-dithiobis(2nitrobenzoic acid) (Ellman’s reagent), dithiothreitol, tacrine (9-amino1,2,3,4-tetrahydroacridine hydrochloride hydrate), BW286c51, decamethonium, and edrophonium were purchased from Sigma. m-(N,N,Ntrimethylammonio)trifluoromethylacetophenone (TFKϩ) and (Ϫ)-huperzine A were purchased from Calbiochem. C.) kindly provided 7-[[methylethoxy)phosphinyl]-oxyl]-1-methylquinolinium iodide (MEPQ) and procainamide-linked Sepharose CL-4B resin. M-tertButyl trifluoromethylacetophenone (TFK0) was synthesized as described [21] and kindly provided by Dr Daniel Quinn, University of Iowa, Iowa City, IA. Expression, Mutagenesis, and Purification of mAChE—Mouse AChE was produced by transfection of expression plasmid (pCDNA3, Invitrogen, San Diego, CA) containing an encoding cDNA where the AChE sequence was terminated at position 548. Cells were selected with G418 to obtain stable producing cell lines, and AChE was expressed as a secreted soluble enzyme in serum-free media [20]. Mutant enzymes were generated by standard mutagenesis procedures, and cassettes containing the mutation were subcloned into pCDNA 3 [20]. Purity was ascertained by SDS-PAGE and by measurements of specific activity
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