Probing the Active Center Gorge of Acetylcholinesterase by Fluorophores Linked to Substituted Cysteines

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. 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. acetylcholinesterase bicinchoninic acid 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide 7-[[(methylethoxy)phosphinyl]-oxyl]-1-methylquinolinium iodide methanethiosulfonate benzyl methanethiosulfonate 2-aminoethyl methanethiosulfonate hydrobromide sodium (2-sulfonatoethyl)-methanethiosulfonate 2-(trimethylammonium)ethyl]methanethiosulfonate bromide m-(N,N,N-trimethylammonio)trifluoromethyl acetophenone m-tert-butyl trifluoromethyl acetophenone Acetylcholinesterase (AChE),1 a serine hydrolase in the α/β fold protein superfamily (1.Cygler M. Schrag J.D. Sussman J.L. Harel M. Silman I. Gentry M.K. Doctor B.P. Protein Sci. 1993; 2: 366-382Crossref PubMed Scopus (539) Google Scholar), functions at cholinergic synapses to terminate nerve signals by catalyzing ester hydrolysis of the neurotransmitter acetylcholine (2.Massoulié J. Pezzementi L. Bon S. Krejci E. Valette F.-M. Prog. Neurobiol. 1993; 41: 31-91Crossref PubMed Scopus (1064) Google Scholar, 3.Taylor P. Radić Z. Annu. Rev. Pharmacol. Toxicol. 1994; 34: 281-320Crossref PubMed Scopus (613) Google Scholar). To enhance synaptic efficiency, AChE has evolved to function rapidly and can catalyze acetylcholine hydrolysis at near diffusion-limited rates (4.Rosenberry T.L. Adv. Enzymol. 1975; 43: 103-218PubMed Google Scholar, 5.Quinn D.M. Chem. Rev. 1987; 87: 955-979Crossref Scopus (961) Google Scholar). Several crystal structures of AChE have been solved, revealing notable features of the tertiary structure (6.Sussman J.L. Harel M. Frolow F. Oefner C. Goldman A. Toker L. Silman I. Science. 1991; 253: 872-879Crossref PubMed Scopus (2451) Google Scholar, 7.Bourne Y. Taylor P. Marchot P. Cell. 1995; 83: 503-512Abstract Full Text PDF PubMed Scopus (319) Google Scholar, 8.Bourne Y. Taylor P. Bougis P.E. Marchot P. J. Biol. Chem. 1999; 274: 2963-2970Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The active site serine resides at the bottom of a deep and contorted gorge lined primarily with aromatic amino acid side chains. This seemingly less accessible position of the serine raises questions regarding ease of substrate entry and product dissociation, which have been addressed, but incompletely resolved, through molecular dynamics simulations and site-directed mutagenesis studies (9.Ripoll D.R. Faerman C.H. Axelsen P.H. Silman I. Sussman J.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5128-5132Crossref PubMed Scopus (251) Google Scholar, 10.Gilson M.K. Straatsma T.P. McCammon J.A. Ripoll D.R. Faerman C.H. Axelsen P.H. Silman I. Sussman J.L. 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Chem. 1995; 270: 19694-19701Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 15.Radić Z. Quinn D.M. Vellom D.C. Camp S. Taylor P. J. Biol. Chem. 1995; 270: 20391-20399Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). These residual rates suggest either the presence of alternative entry points for these inhibitors or breathing motions that open a gap between the fasciculin and AChE interfaces. Fluorescent ligands can be used to probe structural characteristics of proteins in solution. The prototypic AChE peripheral site ligand propidium, for instance, elucidated the nature of a peripheral binding site for inhibitors, remote from the active site serine residue (16.Taylor P. Lappi S. Biochemistry. 1975; 14: 1989-1997Crossref PubMed Scopus (340) Google Scholar). Furthermore, 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.Berman H.A. Taylor P. Biochemistry. 1978; 17: 1704-1713Crossref PubMed Scopus (35) Google Scholar, 18.Berman H.A. Yguerabide J. Taylor P. Biochemistry. 1980; 19: 2226-2235Crossref PubMed Scopus (62) Google Scholar). Potentially, site-directed mutagenesis on recombinant DNA-derived AChE should render a broader range of discrete positions available for selective fluorescence labeling on the enzyme surface. Cysteine substitution mutagenesis, followed by labeling the resulting reactive thiol with methanethiosulfonate (MTS) compounds or fluorescent probes, has been utilized to identify and characterize functionally important residues in several enzyme and receptor systems (19.Kenyon G.L. Bruice T.W. Methods Enzymol. 1977; 47: 407-430Crossref PubMed Scopus (175) Google Scholar, 20.Akabas M.H. Stauffer D.A. Xu M. Karlin A. Science. 1992; 258: 307-310Crossref PubMed Scopus (597) Google Scholar, 21.Lew J. Coruh N. Tsigelny I. Garrod S. Taylor S.S. J. Biol. Chem. 1997; 272: 1507-1513Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). A monomeric form of mouse AChE, in which the C-terminal cysteine is removed, leaving the remaining six cysteines linked through three disulfide bonds in a stable structure (1.Cygler M. Schrag J.D. Sussman J.L. Harel M. Silman I. Gentry M.K. Doctor B.P. Protein Sci. 1993; 2: 366-382Crossref PubMed Scopus (539) Google Scholar), presents an ideal candidate for cysteine substitution mutagenesis. In this study, we substitute cysteine into three positions on the enzyme: within the active site gorge (Tyr124), at the gorge rim (His287), and on the enzyme surface removed from the gorge entrance (Ala262). The cysteines were then modified with cationic, neutral, and anionic substituents. Substrate and inhibitor binding kinetics were examined to assess the influence of electrostatic and steric parameters on ligand association kinetics. Acrylodan, a fluorescent ligand offering a neutral side chain substitution (22.Prendergast F.G. Meyer M. Carlson G.L. Iida S. Potter J.D. J. Biol. Chem. 1983; 258: 7541-7544Abstract Full Text PDF PubMed Google Scholar), enabled us to evaluate polarity of the probe environment and solvent accessibility to the probe for unliganded AChE and the fasciculin-AChE complex and relate these physical parameters to the kinetics of ligand access. Acetylthiocholine iodide, 5,5′-dithio-bis(2-nitrobenzoic acid) (Ellman's reagent), 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide (BW284c51), and dithiothreitol were products of Sigma. Substituted methanethiosulfonates ((sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES), benzyl methanethiosulfonate (MTSBn), (2-(trimethylammonium)ethyl)methanethiosulfonate bromide (MTSET), and 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA)), and acrylodan were purchased from Toronto Research Chemicals, Inc. (Toronto, Canada) or Molecular Probes (Eugene, OR), respectively. Fasciculin 2 (purified from the venom of Dendroaspis angusticeps) was a gift of Dr. Pascale Marchot (Marseille, France).m-(N,N,N-Trimethylammonio)trifluoromethyl acetophenone and m-tert-butyl trifluoromethyl acetophenone (TFK+ and TFK0, respectively) were synthesized as described (23.Nair H.K. Seravalli J. Arbuckle T. Quinn D.M. Biochemistry. 1994; 33: 8566-8576Crossref PubMed Scopus (80) Google Scholar) and kindly provided by Dr. Daniel M. Quinn (University of Iowa). 7-[[(Methylethoxy)phosphinyl]-oxyl]-1-methylquinolinium iodide (MEPQ) (24.Levy D. Ashani Y. Biochem. Pharmacol. 1986; 35: 1079-1085Crossref PubMed Scopus (86) Google Scholar) was a gift of Drs. Yacov Ashani and Bhupendra P. Doctor (Walter Reed Army Research Center, Washington, D.C.). All other chemicals were of at least reagent grade. A cDNA encoding mouse AChE truncated at position 548 and yielding a monomeric form of the enzyme has been characterized previously (25.Radić Z. Pickering N.A. Vellom D.C. Camp S. Taylor P. Biochemistry. 1993; 32: 12074-12084Crossref PubMed Scopus (426) Google Scholar). Mutant mouse AChE cDNAs encoding the monomeric form of the enzyme were generated either by Kunkel (26.Kunkel T.A. Roberts J.D. Zakour R.A. Methods Enzymol. 1987; 154: 367-382Crossref PubMed Scopus (4563) Google Scholar) or polymerase chain reaction-mediated (Stratagene Quik Change Kit) standard mutagenesis procedures. The presence of the mutation was detected by restriction enzyme digestion, and cassettes containing the mutation were subcloned into the mammalian expression vector, pCDNA3 (Invitrogen, San Diego, CA). The nucleotide sequences of the cassettes were confirmed by double stranded sequencing to ensure that spurious mutations were not inadvertently introduced into the coding sequence. The plasmids were purified by standard protocols involving alkali cell lysis, phenol/chloroform extraction, poly(ethylene glycol) precipitation, and CsCl gradients. Human embryonic kidney (HEK 293) cells were purchased from American Type Culture Collection (Atlanta, GA) and, 24 h before transfection, plated in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at a density of 1.5 × 106 cells per 10-cm dish. Ten μg of plasmid DNA per plate were added to the cells using standard HEPES/calcium phosphate precipitation methods. Approximately 16 h after transfection, the cells were rinsed with phosphate-buffered saline. For transient expression of mutant enzymes, cells were supplied with serum-free media for 48 to 72 h. The media containing the secreted monomeric enzyme was periodically collected and the cells were resupplied with fresh serum-free media. This process continued until at most three harvests of enzyme were obtained. In some cases, enzymes were concentrated for kinetic experiments by use of Centriprep or Centricon 30 concentrators (Millipore Corp., Bedford, MA). For large scale productions of enzyme, stable transfectants were selected by G418 resistance following co-transfection with a neomycin resistance gene. 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. Stable transfectants were pooled and grown to confluency prior to replacement of the fetal bovine serum supplemented Dulbecco's modified Eagle's medium with serum-free media. Harvests of the media containing the AChE continued for several weeks or until the expression levels began to decline. Fluorescence studies required purified AChE, both mutant and wild type. Inhibitors (trimethyl-(m-aminophenyl)-ammonium chloride hydrochloride or procainamide hydrochloride), linked through an extended chain to Sepharose CL-4B resin (Sigma), were used to affinity purify the cysteine-substituted enzymes, typically in amounts between 10 and 25 mg, as described previously (27.Berman J.D. Young M. Proc. Natl. Acad. Sci. U. S. A. 1971; 68: 395-398Crossref PubMed Scopus (110) Google Scholar, 28.De La Hoz D. Doctor B.P. Ralston J.S. Rush R.S. Wolfe A.D. Life Sci. 1986; 39: 195-199Crossref PubMed Scopus (79) Google Scholar, 29.Marchot P. Ravelli R.B.G. Raves M.L. Bourne Y. Vellom D.C. Kanter J. Camp S. Sussman J.L. Taylor P. Protein Sci. 1996; 5: 672-679Crossref PubMed Scopus (59) Google Scholar). Levels of purity were determined by SDS-polyacrylamide gel electrophoresis, and by comparisons of specific activity using absorbance at 280 nm or BCA assay for estimating protein content. Enzyme activity was measured spectrophotometrically by the method of Ellman (30.Ellman G.L. Courtney K.D. Andres Jr., V. Featherstone R.M. Biochem. Pharmacol. 1961; 7: 88-95Crossref PubMed Scopus (21913) Google Scholar). Kinetic constants for acetylthiocholine hydrolysis were determined by fitting the observed rates as described previously (25.Radić Z. Pickering N.A. Vellom D.C. Camp S. Taylor P. Biochemistry. 1993; 32: 12074-12084Crossref PubMed Scopus (426) Google Scholar). Known concentrations of MEPQ were utilized to titrate the number of active sites according to Levy and Ashani (24.Levy D. Ashani Y. Biochem. Pharmacol. 1986; 35: 1079-1085Crossref PubMed Scopus (86) Google Scholar). 10–250 mm solutions of MTS compounds were added to enzymes to result in 1–10 mmconcentrations of labeling agent. The reaction was allowed to proceed at least 30 min, after which labeled enzymes were separated from MTS compounds by means of G-50 Sephadex spin columns (Roche Molecular Biochemicals). Enzymes were assayed for kinetic changes immediately thereafter. One to 2 mg of acrylodan were dissolved in dimethylformamide to a stock concentration of 3–12 mm, as measured by absorbance at 387 nm (ε = 16,400m−1 cm−1 in ethanol; Ref. 22.Prendergast F.G. Meyer M. Carlson G.L. Iida S. Potter J.D. J. Biol. Chem. 1983; 258: 7541-7544Abstract Full Text PDF PubMed Google Scholar). The A262C and H287C mutant enzymes were pretreated with 0.5–1 mm dithiothreitol for 1 h at room temperature to ensure that the free cysteine had not partially oxidized. Free dithiothreitol was removed by use of a G-50 Sephadex spin column (Roche Molecular Biochemicals). A volume of 0.5 to 1 μl of acrylodan at 100 times the enzyme concentration was slowly added to the enzyme to achieve a 5–10-fold molar excess. Labeling was allowed to proceed at least 12 h at 4 °C. Unreacted acrylodan was removed by size exclusion chromatography using G-25 Sephadex (Amersham Pharmacia Biotech AB, Uppsala, Sweden) equilibrated in 0.1 m sodium phosphate buffer, pH 7. Labeled enzyme concentrations were determined from the maximal acrylodan absorbance found between 360 and 380 nm (ε ≅ 16,400 m−1 cm−1). Stoichiometry of labeling, estimated from a comparison of enzyme concentration by protein (280 nm) with acrylodan (360–380 nm) absorbance, ranged as follows: Y124C, 0.6–0.9; A262C, 0.75–1.0; H287C, 0.6–0.85. Since acrylodan absorbance for the wild type enzyme was virtually undetectable, specificity of labeling was assessed by comparison of areas of the fluorescence emission curves for acrylodan-treated mutant and wild type enzymes. In the absence of dithiothreitol, labeling of Y124C-acrylodan was ≥95% selective. Treatment of enzymes with 0.5–1 mm dithiothreitol increased wild type acrylodan fluorescence. For A262C-acrylodan, specific labeling was 85% whereas for H287C-acrylodan it was 70%. Picomolar amounts of enzyme in 0.01% bovine serum albumin in 0.1 m sodium phosphate buffer, pH 7, were reacted with the charged or uncharged substituted trifluoroacetophenone or fasciculin 2 in the absence of substrate. Inhibition was monitored by measuring residual enzyme activity by removal of aliquots during the course of the reaction. Bimolecular rate constants of inhibition were determined by nonlinear fit of the data (31.Radić Z. Kirchoff P.D. Quinn D.M. McCammon J.A. Taylor P. J. Biol. Chem. 1997; 272: 23265-23277Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Fluorescence spectra were collected using a Jobin Yvon-Spex Fluoromax fluorometer (Instruments S.A., Inc., Edison, NJ) and analyzed with GRAMS/386 data analysis software (Galactic Industries Corp., Salem, NH). The excitation wavelength for acrylodan was set at 355 nm and emission was detected between 420 and 600 nm. Emission and excitation slits were set at 5 nm. Rate constants (k on) for fasciculin association with acrylodan-labeled mutant AChE was assessed by utilization of anApplied 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. Data were fit according to a single exponential rise to maximum fluorescence. Since competition experiments between radioactively labeled fasciculin and BW284c51 show that binding of these ligands is mutually exclusive (32.Marchot P. Khélif A. Ji Y.-H. Mansuelle P. Bougis P.E. J. Biol. Chem. 1993; 268: 12458-12467Abstract Full Text PDF PubMed Google Scholar), fasciculin dissociation rate constants (k off) 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. Here, data were fit to a single exponential decay to a minimal value. For the Y124C, but not the H287C, fasciculin-AChE-acrylodan this observed rate was on the concentration of In this of a of the observed rate and the concentration of BW284c51 the fasciculin dissociation rate dissociation constants were from the of All of the mutant enzymes show kinetics of acetylthiocholine hydrolysis to wild type enzyme, that catalytic activity is and the enzymes containing the introduced cysteine residues fold The m) by the the site binding is increased for the mutant Y124C, as observed previously for the mutant (25.Radić Z. Pickering N.A. Vellom D.C. Camp S. Taylor P. Biochemistry. 1993; 32: 12074-12084Crossref PubMed Scopus (426) Google Scholar). The and the of to as a measure of catalytic efficiency, and the activity of the enzyme with substrate as by the are within of the for wild type for acetylthiocholine hydrolysis by wild type and mutant mouse or conjugated with aromatic charged charged or fluorescent cysteine labeling cat/K 5 type from Ref. Kinetic of acetylthiocholine hydrolysis for wild type enzyme to MTS labeling compounds are within of the are as means typically from three Data were fit to the following is is enzyme, and In this can at discrete sites to form and in substrate For is to with and The of substrate hydrolysis of the complex as with is in the of the (25.Radić Z. Pickering N.A. Vellom D.C. Camp S. Taylor P. Biochemistry. 1993; 32: 12074-12084Crossref PubMed Scopus (426) Google Data from Ref. Z. Pickering N.A. Vellom D.C. Camp S. Taylor P. Biochemistry. 1993; 32: 12074-12084Crossref PubMed Scopus (426) Google Kinetic of acetylthiocholine hydrolysis for wild type enzyme to MTS labeling compounds are within of the in a Data are as means typically from three Data were fit to the following is is enzyme, and In this can at discrete sites to form and in substrate For is to with and The of substrate hydrolysis of the complex as with is in the of the (25.Radić Z. Pickering N.A. Vellom D.C. Camp S. Taylor P. Biochemistry. 1993; 32: 12074-12084Crossref PubMed Scopus (426) Google Scholar). of the cysteine at position 124 by the MTS compounds the kinetics of acetylthiocholine hydrolysis and Labeling of with a thiol a minimal influence on the kinetic whereas with a charged but The largest of observed with charged MTS In comparison to the mutant enzyme, with a or a m and on small changes are observed for the enzymes. The of to in the kinetics of acetylthiocholine hydrolysis required modification to of Kinetic in which was to either or MTS then to confirmed that reaction with the compounds to near 2 Acrylodan labeling of both m to m is increased is reduced resulting in a in enzyme as measured by the to dependence of acetylthiocholine hydrolysis for and the cysteine substituted of are activity be from this and I. and that a of acetylthiocholine hydrolysis in comparison to were reacted with to that the reaction to react have generated a of the enzyme yielding a reaction to followed by reaction with followed by reaction with Y124C-acrylodan followed by reaction with Rate constants for the diffusion-limited association to and dissociation from enzymes are in The association rate of the cationic inhibitor with is than that for wild type enzyme. of with either neutral or charged this but In of a charge at the position the association rate The on the it In the rate constants for with the set of enzymes are and a comparison with kinetics to the of steric and electrostatic in the inhibition shows a decrease in with wild type enzyme. The neutral benzyl side chain not decrease this rate the charged side chain it The charged side chain show a in the rate the maximum of for the not the observed for cationic with Acrylodan as the a in the association rate of a for the dissociation rate are by the presence of the mutation or dissociation rates of these rate constants for reaction of enzymes with and in the presence and absence of aromatic charged charged or fluorescent cysteine labeling on