Abstract
To examine the influence of individual side chains in governing rates of ligand entry into the active center gorge of acetylcholinesterase and to characterize the dynamics and immediate environment of these residues, we have conjugated reactive groups with selected charge and fluorescence characteristics to cysteines substituted by mutagenesis at specific positions on the enzyme. Insertion of side chains larger than in the native tyrosine at position 124 near the constriction point of the active site gorge confers steric hindrance to affect maximum catalytic throughput (k(cat)/K(m)) and rates of diffusional entry of trifluoroketones to the active center. Smaller groups appear not to present steric constraints to entry; however, cationic side chains selectively and markedly reduce cation ligand entry through electrostatic repulsion in the gorge. The influence of side chain modification on ligand kinetics has been correlated with spectroscopic characteristics of fluorescent side chains and their capacity to influence the binding of a peptide, fasciculin, which inhibits catalysis peripherally by sealing the mouth of the gorge. Acrylodan conjugated to cysteine was substituted for tyrosine at position 124 within the gorge, for histidine 287 on the surface adjacent to the gorge and for alanine 262 on a mobile loop distal to the gorge. The 124 position reveals the most hydrophobic environment and the largest hypsochromic shift of the emission maximum with fasciculin binding. This finding likely reflects a sandwiching of the acrylodan in the complex with the tip of fasciculin loop II. An intermediate spectral shift is found for the 287 position, consistent with partial occlusion by loops II and III of fasciculin in the complex. Spectroscopic properties of the acrylodan at the 262 position are unaltered by fasciculin addition. Hence, combined spectroscopic and kinetic analyses reveal distinguishing characteristics in various regions of acetylcholinesterase that influence ligand association.
Highlights
Fold protein superfamily [1], functions at cholinergic synapses to terminate nerve signals by catalyzing ester hydrolysis of the neurotransmitter acetylcholine [2, 3]
Fluorescent phosphonates, which conjugate with the active site serine, were utilized to measure the hydrophobicity of the active site gorge, the contribution of charge to active center accessibility, and the distance between the reactive serine and the peripheral site years before a crystal structure was solved [17, 18]
Since competition experiments between radioactively labeled fasciculin and BW284c51 show that binding of these ligands is mutually exclusive [32], fasciculin dissociation rate constants were determined by measuring the time dependence of the decrease in fluorescence for the fasciculin-AChE-acrylodan conjugate upon addition excess BW284c51 at several concentrations
Summary
Materials—Acetylthiocholine iodide, 5,5Ј-dithio-bis(2-nitrobenzoic acid) (Ellman’s reagent), 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide (BW284c51), and dithiothreitol were products of Sigma. After transfection and rinsing with phosphate-buffered saline, cells were allowed to recover in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum for 3–5 days after which G418 (Gemini Bio-Products, Inc., Calabasas, CA) was added to the media for 2 to 3 weeks. Rate constants (kon) for fasciculin association with acrylodan-labeled mutant AChE was assessed by utilization of an Applied Photophysics SX.18MV (Leatherhead, UK) stopped-flow reaction analyzer and observation of the time-dependent increase in fluorescence above 420 nm upon excitation at 355 nm by means of a 420-nm emission cut-off filter. Since competition experiments between radioactively labeled fasciculin and BW284c51 show that binding of these ligands is mutually exclusive [32], fasciculin dissociation rate constants (koff) were determined by measuring the time dependence of the decrease in fluorescence for the fasciculin-AChE-acrylodan conjugate upon addition excess BW284c51 at several concentrations. Equilibrium dissociation constants (KD) were calculated from the ratios of koff to kon
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