Reversibly Bound and Covalently Attached Ligands Induce Conformational Changes in the Omega Loop, Cys69–Cys96, of Mouse Acetylcholinesterase
2001; Elsevier BV; Volume: 276; Issue: 45 Linguagem: Inglês
10.1074/jbc.m106896200
ISSN1083-351X
AutoresJianxin Shi, Aileen E. Boyd, Zoran Radić, Palmer Taylor,
Tópico(s)Chemical synthesis and alkaloids
ResumoWe 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 (Leu76, Glu81, Glu84, Tyr124, Ala262, and His287) 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 (Ω 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 Ω 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 Ω loop accompany ligand binding. 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 (Leu76, Glu81, Glu84, Tyr124, Ala262, and His287) 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 (Ω 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 Ω 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 Ω loop accompany ligand binding. acetylcholinesterase mouse acetylcholinesterase 5,5′-dithio-bis(2-nitrobenzoic acid) 7-[[(methylethoxy)phosphinyl]-oxyl]-1-methylquinolinium iodide m-(N,N,N-trimethylammonio)trifluoromethyl acetophenone m-tert-butyl trifluoromethylacetophenone 6-acryloyl-2-dimethylaminonaphthalene Acetylcholinesterase (AChE),1 a serine hydrolase in the α/β-fold hydrolase protein superfamily (1Cygler 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), terminates nerve signals by catalyzing hydrolysis of the neurotransmitter acetylcholine at a diffusion limited rate (2Rosenberry T.L. Adv. Enzymol. Relat. Areas Mol. Biol. 1975; 43: 103-218PubMed Google Scholar, 3Quinn D.M. Chem. Rev. 1987; 87: 955-979Crossref Scopus (955) Google Scholar). The crystallographic structure of mouse AChE reveals a catalytic triad (Ser203, Glu334, and His447) located at the bottom of a narrow active site gorge 20 Å in depth (4Sussman J.L. Harel M. Frolow F. Oefner C. Goldman A. Toker L. Silman I. Science. 1991; 253: 872-879Crossref PubMed Scopus (2440) Google Scholar, 5Bourne Y. Taylor P. Marchot P. Cell. 1995; 83: 503-512Abstract Full Text PDF PubMed Scopus (319) Google Scholar, 6Bourne 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). Because the cross-section of the physiological substrate acetylcholine is larger than the narrowest part of the gorge, the remarkably high turnover rate of AChE raises questions regarding substrate access to the catalytic site. Molecular dynamic simulations suggest that rapid fluctuations of gorge width combined with diffusion facilitated by electrostatic forces could enhance substrate accessibility (7Ripoll 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, 8Tan R.C. Truong T.N. McCammon J.A. Sussman J.L. Biochemistry. 1993; 32: 401-403Crossref PubMed Scopus (136) Google Scholar, 9Wlodek S.T. Shen T. McCammon J.A. Biopolymers. 2000; 53: 265-271Crossref PubMed Scopus (40) Google Scholar, 10Zhou H.X. Wlodek S.T. McCammon J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9280-9283Crossref PubMed Scopus (208) Google Scholar). In addition, the high affinity and slowly dissociating complex of fasciculin and AChE retains slight residual catalytic activity (11Eastman J. Wilson E.J. Cerveñansky C. Rosenberry T.L. J. Biol. Chem. 1995; 270: 19694-19701Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 12Radic 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), despite the occlusion of the active site gorge by fasciculin as shown in the crystal structures (5Bourne Y. Taylor P. Marchot P. Cell. 1995; 83: 503-512Abstract Full Text PDF PubMed Scopus (319) Google Scholar,13Harel M. Kleywegt G.J. Ravelli R.B. Silman I. Sussman J.L. Structure. 1995; 3: 1355-1366Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 14Kryger G. Harel M. Giles K. Toker L. Velan B. Lazar A. Kronman C. Barak D. Ariel N. Shafferman A. Silman I. Sussman J.L. Acta Crystallogr. Sec. D Biol. Crystallogr. 2000; 56: 1385-1394Crossref PubMed Scopus (305) Google Scholar). Rapid fluctuations in residues lining the gorge walls may leave transient gaps at the fasciculin-AChE interface and may account for residual activity. The large omega loop (Ω loop), Cys69–Cys96, flanking the active site gorge in mouse AChE corresponds to the activation loop of Cys60–Cys97 in Candida rugosa lipase, a related α/β-fold hydrolase protein (15Schrag J.D. Cygler M. J. Mol. Biol. 1993; 230: 575-591Crossref PubMed Scopus (139) Google Scholar, 16Grochulski P. Li Y. Schrag J.D. Bouthillier F. Smith P. Harrison P. Rubin B. Cygler M. J. Biol. Chem. 1993; 268: 72843-72847Abstract Full Text PDF Google Scholar, 17Grochulski P. Li Y. Schrag J.D. Cygler M. Protein Sci. 1993; 3: 82-91Crossref Scopus (335) Google Scholar). Crystallographic studies of the lipase revealed that the activation loop occludes the active center in the absence of substrate but folds back in the presence of lipid substrate allowing its access. Although kinetic and structural studies of AChE have not revealed evidence for such large substrate, induced lid-like movements (18Velan B. Barak D. Ariel N. Leitner M. Bino T. Ordentlich A. Shafferman A. FEBS Lett. 1996; 395: 22-28Crossref PubMed Scopus (29) Google Scholar, 19Faerman C. Ripoll D. Bon S. Lefeuvre Y. Morel N. Massoulie J. Sussman J. Silman I. FEBS Lett. 1996; 386: 65-71Crossref PubMed Scopus (57) Google Scholar), high catalytic turnover rates for the cholinesterases might indicate that small amplitude motions along the Ω loop allow rapid access of incoming substrate and release of reaction product (19Faerman C. Ripoll D. Bon S. Lefeuvre Y. Morel N. Massoulie J. Sussman J. Silman I. FEBS Lett. 1996; 386: 65-71Crossref PubMed Scopus (57) Google Scholar). To elucidate the nature of the ligand-dependent conformational changes of AChE, we have employed cysteine substitution mutagenesis and site-directed labeling with an environmentally sensitive fluorophore, acrylodan. The emission spectrum and quantum yield of the fluorophore are dependent on the effective dielectric constant and thus reflect the degree of solvent exposure and the local polarity experienced by the fluorophore (20Boyd A.E. Marnett A.B. Wong L. Taylor P. J. Biol. Chem. 2000; 275: 22401-22408Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). For example, when acrylodan is conjugated to a cysteine lining the gorge, upon fasciculin binding, it becomes sandwiched between the fasciculin loop and wall of the gorge, thereby becoming protected from solvent (20Boyd A.E. Marnett A.B. Wong L. Taylor P. J. Biol. Chem. 2000; 275: 22401-22408Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). To examine further the role of the Ω loop in ligand binding, we have conjugated cysteines at various positions on the Ω loop and opposing gorge wall. Six single cysteine mutants were prepared for acrylodan conjugation (Fig. 1). Three were on the Ω loop as follows: L76C near the tip of the loop and E81C and E84C on their outer surface not lining the gorge. Two residues on the opposing face of the gorge H287C and Y124C were selected, along with a distal residue A262C whose temperature coefficient (B factor) would indicate flexible movement of another disulfide loop on which it resides (5Bourne Y. Taylor P. Marchot P. Cell. 1995; 83: 503-512Abstract Full Text PDF PubMed Scopus (319) Google Scholar, 6Bourne 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). We examined the kinetics of substrate catalysis and inhibitor association with the modified enzymes, and we correlate these kinetic parameters with the spectroscopic changes in the conjugated acrylodan upon ligand association. Fluorescence measurements reveal changes in conformation reflected in the substituted side chains well removed from the active center gorge. The results suggest that ligand binding at the catalytic site allosterically alters the conformation of a specific segment of the Ω loop whereby gorge closure occurs and residue side chain positions distal to the binding site are affected. Acetylthiocholine iodide, 5,5′-dithiobis(2-nitrobenzoic acid) (Ellman's reagent), dithiothreitol, tacrine (9-amino-1,2,3,4-tetrahydroacridine hydrochloride hydrate), BW286c51, decamethonium, and edrophonium were purchased from Sigma.m-(N,N,N-trimethylammonio)trifluoromethylacetophenone (TFK+) and (−)-huperzine A were purchased from Calbiochem. Acrylodan was obtained from Molecular Probes (Eugene, OR). Fasciculin 2 (purified from the venom of Dendroaspis angusticeps) was a gift of Dr. Pascale Marchot (University of Marseille, France). Drs. Yacov Ashani and Bhupendra P. Doctor (Walter Reed Army Research Center, Washington, D. C.) kindly provided 7-[[methylethoxy)phosphinyl]-oxyl]-1-methylquinolinium iodide (MEPQ) and procainamide-linked Sepharose CL-4B resin.m-tert-Butyl trifluoromethylacetophenone (TFK0) was synthesized as described (21Nair 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 Quinn, University of Iowa, Iowa City, IA. All other chemicals were of the highest grade commercially available. 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. The plasmid was transfected into HEK293 cells. Cells were selected with G418 to obtain stable producing cell lines, and AChE was expressed as a secreted soluble enzyme in serum-free media (20Boyd A.E. Marnett A.B. Wong L. Taylor P. J. Biol. Chem. 2000; 275: 22401-22408Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Mutant enzymes were generated by standard mutagenesis procedures, and cassettes containing the mutation were subcloned into pCDNA 3 (20Boyd A.E. Marnett A.B. Wong L. Taylor P. J. Biol. Chem. 2000; 275: 22401-22408Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Nucleotide sequences of the cassettes were confirmed by double-stranded sequencing to ensure that spurious mutations were not introduced into the coding sequence. Affinity chromatography using (m-aminophenyl)trimethylammonium linked through a long chain to Sepharose CL-4B resin (Sigma) permitted one-step purification of AChE. From 4 to 6 liters of media, mutant and wild type enzyme were purified in quantities ranging between 5 and 25 mg, as described previously (22Marchot P. Ravelli R.B. 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, 23Berman J.D. Young M. Proc. Natl. Acad. Sci. U. S. A. 1971; 68: 395-398Crossref PubMed Scopus (109) Google Scholar, 24De 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). Purity was ascertained by SDS-PAGE and by measurements of specific activity. The spectrophotometric method of Ellman was used (25Ellman G.L. Courtney K.D. Andres V.J. Featherstone R.M. Biochem. Pharmacol. 1961; 7: 88-95Crossref PubMed Scopus (21719) Google Scholar), and kinetic constants for acetylthiocholine hydrolysis were determined by fitting the observed rates as described (26Radic Z. Pickering N.A. Vellom D.C. Camp S. Taylor P. Biochemistry. 1993; 32: 12074-12084Crossref PubMed Scopus (425) Google Scholar). Titration of active sites with known concentrations of the irreversible phosphorylating agent, MEPQ, was accomplished by the method of Levy and Ashani (27Levy D. Ashani Y. Biochem. Pharmacol. 1986; 35: 1079-1085Crossref PubMed Scopus (86) Google Scholar). Mutant enzymes were pretreated with 0.25 mm dithiothreitol for 30 min at room temperature to ensure reduction of the introduced cysteine. Excess dithiothreitol was removed by use of a G-50 Sephadex spin column (Roche Molecular Biochemicals) equilibrated in 10 mm Tris, 100 mm NaCl, 40 mm MgCl2, pH 8.0. A volume of 1 μl of acrylodan at 100 times the enzyme concentration was slowly mixed with the enzyme to achieve an ∼5-fold molar excess of acrylodan to mutant enzyme. Labeling was allowed to proceed for at least 12 h at 4 °C, and unreacted acrylodan was removed by size exclusion chromatography using Sephadex G-25 (Amersham Pharmacia Biotech) in 0.1m sodium phosphate buffer, pH 7. Concentrations of acrylodan-labeled enzyme were determined from the maximal acrylodan absorbance found between 360 and 380 nm (ε ∼16,400m−1 cm−1). Stoichiometry of labeling of the various preparations, estimated from a comparison of enzyme concentration by protein (280 nm) to acrylodan (360–380 nm) absorbance, ranged as follows: L76C, 0.7–0.8; E81C, 0.79–1.0; E84C, 0.77–1.0; Y124C, 0.79–1.0; A262C, 0.69–0.85; and H287C, 0.82–0.88. Specificity of labeling was assessed by comparison of areas under the fluorescence emission curves for acrylodan-treated mutant and wild type enzymes. Specific labeling for each mutant was as follows: L76C, 70–85%; E81C, 81–91%; E84C, 85–93%; Y124C, 83–90%; A262C, 80–90%; H287C, 70–76%. Picomolar amounts of enzyme in 0.01% bovine serum albumin in 0.1 m sodium phosphate buffer, pH 7.0, were reacted with TFK+ 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 fits of the data (28Radic Z. Kirchhoff 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). Steady-state emission spectra were measured at room temperature using a Jobin Yvon/Spex FluoroMax II spectrofluorometer (Instrument S.A., Inc., Edison, NJ) with the excitation and emission bandwidths set at 5 nm. The excitation wavelength for acrylodan was set at 359 nm, and emission was monitored between 420 and 600 nm. Equilibrium dissociation constants,Kd, for BW286c51 and edrophonium with the acrylodan-labeled enzyme were obtained by titration of a fixed quantity of labeled enzyme (54–120 nm) with various concentrations of indicated inhibitors. Kd values were obtained by monitoring the fractional decrease in the total area under the fluorescence emission curves from 420 to 600 nm for the acrylodan-labeled E84C or a limited segment of the emission between 450 and 485 nm for the acrylodan-labeled E81C. For ligands of high affinity such as BW286c51, where binding is nearly stoichiometric, data were fitted to Equation 1. ΔF=ΔFmax(Et+It+Kd−{(Et+It+Kd)2−4EtIt}0.5)(2Et)−1(Eq.1)Eq. 1 ΔF and ΔFmax are the change and maximum change in fluorescence, respectively; Et is the total enzyme concentration, and It is the total inhibitor concentration. Association of TFK+ with acrylodan-labeled E81C and E84C was assessed from the kinetics of decrease in fluorescence at 470 and 477 nm respectively, following addition of a stoichiometric excess TFK+ at several concentrations. Data were fitted to a single exponential approach to equilibrium. Association and dissociation rate constants of edrophonium and BW286c51 with E81C and E84C AChEs were determined from changes in the tryptophan fluorescence using a stopped-flow spectrophotometer as described previously (29Radic Z. Taylor P. J. Biol. Chem. 2001; 276: 4622-4633Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Time-dependent decreases in tryptophan fluorescence were observed upon excitation at 276 nm by means of a 305-nm emission cut-off filter. The cysteine-substituted enzymes show kinetics of acetylthiocholine hydrolysis similar to wild type enzyme (Table I and Scheme 1) suggesting that all mutant enzymes fold correctly despite the presence of the newly introduced cysteine. The Km value of E84C shows slightly less than a 4-fold increase, whereas the change in turnover rate,kcat, is minimal. Similar changes in kinetic constants were observed previously for E84Q mAChE (28Radic Z. Kirchhoff 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). Since Km, in diffusion limited catalysis, depicts the initial encounter between substrate and enzyme, an increase in Km likely arises from the reduction of negative charge that electrostatically steers the cationic substrate into the active center gorge. Interestingly, a similar E81C mutation has little or no effect on substrate hydrolysis. Not all negatively charged residues around the active center appear to be involved equivalently in electrostatic steering.Table IConstants for acetylthiocholine hydrolysis by wild type and mutant mouse AChEsEnzymeKmKSSbkcatkcat/Kmμmmm105/min109/m · minWTaData are from Ref. 20.54 ± 1614 ± 50.2 ± 0.071.6 ± 0.43.0Y124CaData are from Ref. 20.65 ± 1720 ± 140.2 ± 0.091.4 ± 0.32.2H287CaData are from Ref. 20.58 ± 712 ± 60.2 ± 0.061.8 ± 0.23.1A262CaData are from Ref. 20.59 ± 411 ± 30.2 ± 0.041.6 ± 0.12.7L76C97 ± 1917 ± 10.2 ± 0.031.8 ± 0.11.9E81C57 ± 611 ± 10.2 ± 0.031.6 ± 0.12.9E84C190 ± 926 ± 20.2 ± 0.051.9 ± 0.41.0Data shown as means ± S.D. typically from three measurements. Data were fit to the Equation, v = (1 +b[S]/KSS)Vmax /(1 + [S]/KSS) (1 +Km/[S]), where [S] is substrate concentration, KSS is the substrate inhibition or activation constant, and b is the relative catalytic turnover of the ternary complex (12Radic 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).a Data are from Ref. 20Boyd A.E. Marnett A.B. Wong L. Taylor P. J. Biol. Chem. 2000; 275: 22401-22408Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar. Open table in a new tab Data shown as means ± S.D. typically from three measurements. Data were fit to the Equation, v = (1 +b[S]/KSS)Vmax /(1 + [S]/KSS) (1 +Km/[S]), where [S] is substrate concentration, KSS is the substrate inhibition or activation constant, and b is the relative catalytic turnover of the ternary complex (12Radic 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). Association and dissociation rates of fasciculin with A262C, H287C, and Y124C mutant enzymes were also found to be close to the rates with wild type enzyme (20Boyd A.E. Marnett A.B. Wong L. Taylor P. J. Biol. Chem. 2000; 275: 22401-22408Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Fasciculin, at low concentrations, is also capable of associating with the mutant enzymes after acrylodan conjugation (Fig. 2). In addition, enzyme activity measurements of fasciculin-bound acrylodan conjugates show greater than 99% inhibition (data not shown). TFK+binding to cysteine-substituted enzymes, both free and modified with acrylodan, was also examined (Table II). For E81C and E84C, the association rate constants (kon) for TFK+ were obtained from measurements of enzyme activity. Although positions 81 and 84 are both spatially removed from TFK+-binding site,kon for E84C is slightly slower than that for wild type enzyme. By contrast, E81C shows no difference in the kinetic constants. Conjugation of acrylodan, a neutral naphthalene derivative, with E84C reduces kon of TFK+ 7-fold compared with unconjugated E84C, whereas conjugation of E81C with acrylodan only reduces kon of TFK+slightly. For acrylodan-labeled mutants, kon was measured from the time-dependent decrease of fluorescence signal (Fig. 3).Table IIKinetic and equilibrium constants for reaction of enzymes with TFK+, edrophonium, and BW284c51 in the presence and absence of fluorescent (acrylodan) cysteine labeling compoundEnzymeTFK+EdrophoniumBW284c51konkon WTKdKd mutantKdKd mutantkon mutantKd WTKd WT109m−1min−1nmnmWild type150 250aData are from Ref. 29.2.0aData are from Ref. 29.E81C1501 260bEquilibrium dissociation constants are derived from the ratio of koff/kon using stopped-flow measurement of tryptophan fluorescence quenching.12.6bEquilibrium dissociation constants are derived from the ratio of koff/kon using stopped-flow measurement of tryptophan fluorescence quenching.1.3E81C-acrylodan941.6 6402.66.93.5E84C931.6 550bEquilibrium dissociation constants are derived from the ratio of koff/kon using stopped-flow measurement of tryptophan fluorescence quenching.2.2 35bEquilibrium dissociation constants are derived from the ratio of koff/kon using stopped-flow measurement of tryptophan fluorescence quenching.18E84C-acrylodan131163002513065Data are shown as means from two to three measurements. Individual determinations are within 33% of the mean. Rates for TFK+ are calculated based on ratios of the hydrated and unhydrated ketone (21Nair H.K. Seravalli J. Arbuckle T. Quinn D.M. Biochemistry. 1994; 33: 8566-8576Crossref PubMed Scopus (80) Google Scholar).a Data are from Ref. 29Radic Z. Taylor P. J. Biol. Chem. 2001; 276: 4622-4633Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar.b Equilibrium dissociation constants are derived from the ratio of koff/kon using stopped-flow measurement of tryptophan fluorescence quenching. Open table in a new tab Data are shown as means from two to three measurements. Individual determinations are within 33% of the mean. Rates for TFK+ are calculated based on ratios of the hydrated and unhydrated ketone (21Nair H.K. Seravalli J. Arbuckle T. Quinn D.M. Biochemistry. 1994; 33: 8566-8576Crossref PubMed Scopus (80) Google Scholar). A similar trend in inhibition kinetics was seen with noncovalent active site inhibitors such as edrophonium and BW286c51 (Table II). An increase over wild type Kdof 2-fold occurs for edrophonium binding to E84C, and an 18-fold increase in Kd is observed for BW286c51 binding. Similar increases in Kd of edrophonium and BW286c51 were seen for E84Q human AChE (18Velan B. Barak D. Ariel N. Leitner M. Bino T. Ordentlich A. Shafferman A. FEBS Lett. 1996; 395: 22-28Crossref PubMed Scopus (29) Google Scholar). By comparison, E81C showed no alterations in ligand binding constants. For acrylodan-labeled mutants,Kd was measured from the fluorescence signals of an equilibrium titration (Fig. 4). Acrylodan-labeled E84C shows Kd increases of 10-fold for edrophonium and 3-fold for BW286c51 compared with unreacted E84C. For acrylodan-labeled E81C, only a slight increase in Kd is seen for both ligands. The high concentration of acrylodan-labeled E81C required for equilibrium titrations precludes an accurate estimate of Kd for high affinity ligands such as BW286c51. The peptide toxin, fasciculin, inhibits AChE by tightly capping the mouth of active center gorge (Fig. 1) (11Eastman J. Wilson E.J. Cerveñansky C. Rosenberry T.L. J. Biol. Chem. 1995; 270: 19694-19701Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 30Radic Z. Duran R. Vellom D.C. Li Y. Cervenansky C. Taylor P. J. Biol. Chem. 1994; 269: 11233-11239Abstract Full Text PDF PubMed Google Scholar, 31Taylor P. Radic Z. Annu. Rev. Pharmacol. Toxicol. 1994; 34: 281-320Crossref PubMed Scopus (609) Google Scholar, 32Marchot P. Khélif A. Ji Y.H. Mansuelle P. Bougis P.E. J. Biol. Chem. 1993; 268: 12458-12467Abstract Full Text PDF PubMed Google Scholar). TableIII shows changes in emission maxima of acrylodan-labeled AChE mutants in the presence of fasciculin. There is no discernible change in fluorescence emission of acrylodan-conjugated A262C (20Boyd A.E. Marnett A.B. Wong L. Taylor P. J. Biol. Chem. 2000; 275: 22401-22408Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), consistent with the position 262 being distal to the fasciculin-binding site. The large hypsochromic shifts seen at both the 124 and 287 positions reflect solvent exclusion and an increase in hydrophobicity experienced by the fluorophores in the gorge upon fasciculin binding (20Boyd A.E. Marnett A.B. Wong L. Taylor P. J. Biol. Chem. 2000; 275: 22401-22408Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). For the Ω loop mutant, L76C, fasciculin binding produces a 40% increase in quantum yield but no change in emission maximum. Bathochromic shifts are found at both the 81 and 84 positions, with position 84 producing a shift of larger magnitude (Fig. 2 and Table III).Table IIIFluorescence emission parameters of mouse AChE mutants labeled with acrylodan in the presence of fasciculinData are shown as mean values of at least three determinations. Relative quantum yields were determined by comparison of areas of the fluorescence emission curves.a Data are from Ref. 20Boyd A.E. Marnett A.B. Wong L. Taylor P. J. Biol. Chem. 2000; 275: 22401-22408Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar. Open table in a new tab Data are shown as mean values of at least three determinations. Relative quantum yields were determined by comparison of areas of the fluorescence emission curves. a Data are from Ref. 20Boyd A.E. Marnett A.B. Wong L. Taylor P. J. Biol. Chem. 2000; 275: 22401-22408Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar. Changes in emission maxima of acrylodan-labeled AChE mutants in the presence of conjugating trifluoroacetophenones are shown in TableIV. The trifluoroacetophenones inhibit the enzyme by conjugating to form a hemiketal at active site serine without dissociation of leaving group (33Harel M. Quinn D.M. Nair H.K. Silman I. Sussman J.L. J. Am. Chem. Soc. 1996; 118: 2340-2346Crossref Scopus (344) Google Scholar). Both the isosteric neutral and cationic trifluoroketones (TFK0 and TFK+) produced no discernible changes in emission spectra of acrylodan conjugated at H287C and A262C, consistent with a fluorophore position distant from gorge base and hence not in direct contact with ligand. Remarkably, both TFK0 and TFK+ produce a substantial bathochromic shift (at least 30 nm) with acrylodan-E84C. The trifluoroketones also produce spectral shift of intermediate value (20 nm) for E81C and a much smaller change (4–6 nm) for L76C. Interestingly, neutral TFK0 produces a large 22 nm of hypsochromic shift with the Y124C acrylodan conjugate.Table IVFluorescence emission parameters of mouse AChE mutants labeled with acrylodan in the presence of covalent active site inhibitorsData are shown as mean values of at least three determinations. Relative quantum yields were determined by comparison of areas of the fluorescence emission curves. Data for the unconjugated enzymes are found in Table III. Open table in a new tab Data are shown as mean values of at least three determinations. Relative quantum yields were determined by comparison of areas of the fluorescence emission curves. Data for the unconjugated enzymes are found in Table III. O,O-Dimethyl-O-(2,2-dichlorovinyl)phosphate, a small achiral organophosphonate, phosphorylates the active site serine of mAChE, with subsequent departure of the dichlorovinyloxy group (34Wilson I.B. Boyer P.D. Lardy H. Myrback K. The
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