FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2045542952> ?p ?o ?g. }

• W2045542952 endingPage "38955" @default.
• W2045542952 startingPage "38948" @default.
• W2045542952 abstract "The acetylcholinesterase (AChE) active site consists of a narrow gorge with two separate ligand binding sites: an acylation site (or A-site) at the bottom of the gorge where substrate hydrolysis occurs and a peripheral site (or P-site) at the gorge mouth. AChE is inactivated by organophosphates as they pass through the P-site and phosphorylate the catalytic serine in the A-site. One strategy to protect against organophosphate inactivation is to design cyclic ligands that will bind specifically to the P-site and block the passage of organophosphates but not acetylcholine. To accelerate the process of identifying cyclic compounds with high affinity for the AChE P-site, we introduced a cysteine residue near the rim of the P-site by site-specific mutagenesis to generate recombinant human H287C AChE. Compounds were synthesized with a highly reactive methanethiosulfonyl substituent and linked to this cysteine through a disulfide bond. The advantages of this tethering were demonstrated with H287C AChE modified with six compounds, consisting of cationic trialkylammonium, acridinium, and tacrine ligands with tethers of varying length. Modification by ligands with short tethers had little effect on catalytic properties, but longer tethering resulted in shifts in substrate hydrolysis profiles and reduced affinity for acridinium affinity resin. Molecular modeling calculations indicated that cationic ligands with tethers of intermediate length bound to the P-site, whereas those with long tethers reached the A-site. These binding locations were confirmed experimentally by measuring competitive inhibition constants K I2 for propidium and tacrine, inhibitors specific for the P- and A-sites, respectively. Values of K I2 for propidium increased 30- to 100-fold when ligands had either intermediate or long tethers. In contrast, the value of K I2 for tacrine increased substantially only when ligands had long tethers. These relative changes in propidium and tacrine affinities thus provided a sensitive molecular ruler for assigning the binding locations of the tethered cations. The acetylcholinesterase (AChE) active site consists of a narrow gorge with two separate ligand binding sites: an acylation site (or A-site) at the bottom of the gorge where substrate hydrolysis occurs and a peripheral site (or P-site) at the gorge mouth. AChE is inactivated by organophosphates as they pass through the P-site and phosphorylate the catalytic serine in the A-site. One strategy to protect against organophosphate inactivation is to design cyclic ligands that will bind specifically to the P-site and block the passage of organophosphates but not acetylcholine. To accelerate the process of identifying cyclic compounds with high affinity for the AChE P-site, we introduced a cysteine residue near the rim of the P-site by site-specific mutagenesis to generate recombinant human H287C AChE. Compounds were synthesized with a highly reactive methanethiosulfonyl substituent and linked to this cysteine through a disulfide bond. The advantages of this tethering were demonstrated with H287C AChE modified with six compounds, consisting of cationic trialkylammonium, acridinium, and tacrine ligands with tethers of varying length. Modification by ligands with short tethers had little effect on catalytic properties, but longer tethering resulted in shifts in substrate hydrolysis profiles and reduced affinity for acridinium affinity resin. Molecular modeling calculations indicated that cationic ligands with tethers of intermediate length bound to the P-site, whereas those with long tethers reached the A-site. These binding locations were confirmed experimentally by measuring competitive inhibition constants K I2 for propidium and tacrine, inhibitors specific for the P- and A-sites, respectively. Values of K I2 for propidium increased 30- to 100-fold when ligands had either intermediate or long tethers. In contrast, the value of K I2 for tacrine increased substantially only when ligands had long tethers. These relative changes in propidium and tacrine affinities thus provided a sensitive molecular ruler for assigning the binding locations of the tethered cations. The primary physiological role of acetylcholinesterase (AChE) 1The abbreviations used are: AChE, acetylcholinesterase; Boc, tert- butyloxycarbonyl; BOP, (benzotriazol-1-yloxy)-tris(dimethylamino)phosphonium hexafluorophosphate; OP, organophosphate; MTS, methanethiosulfonyl; HPLC, high-performance liquid chromatography. is to hydrolyze the neurotransmitter acetylcholine at cholinergic synapses. The AChE structure has evolved to carry out this hydrolysis at rates that are among the highest known for enzyme-catalyzed reactions (1Rosenberry T.L. Meister A. Acetylcholinesterase. Advances in Enzymology. John Wiley & Sons, New York1975: 43Google Scholar). One feature of the AChE catalytic pathway is the formation of an intermediate acyl enzyme that is hydrolyzed by water. It has long been known that AChE forms an acyl enzyme not only with carboxyl esters like acetylcholine but also with carbamic acid esters and phosphoric acid esters (also called organophosphates or OPs) and that these intermediates differ dramatically in their deacylation rate constants (2Burgen A.S.V. Br. J. Pharmacol. 1949; 4: 219-228Google Scholar, 3Wilson I.B. J. Biol. Chem. 1951; 190: 111-117Abstract Full Text PDF PubMed Google Scholar, 4Aldridge W.N. Reiner E. Neuberger A. Tatum E.L. Enzyme Inhibitors as Substrates, Frontiers of Biology 26. North Holland, Amsterdam1972Google Scholar, 5Froede, H. C., and Wilson, I. B. (1971) in The Enzymes, 3rd Ed. (Boyer, P. D., ed) Vol. V, pp. 87-114, Academic Press, New YorkGoogle Scholar). In particular, organophosphorylated AChEs are hydrolyzed some 1010 times slower than acetylated AChE and are effectively inactivated. OP inactivation of AChE results in failure of cholinergic synaptic transmission, deterioration of neuromuscular junctions, flaccid muscle paralysis, and seizures in the central nervous system. OPs have been developed not only as pesticides targeted to insect AChEs but also, deplorably, as chemical warfare agents directed at human AChE. Therapeutic strategies against OP toxicity have been limited. Nearly 50 years ago, Wilson and Ginsburg (6Wilson I.B. Ginsburg S. Biochim. Biophys. Acta. 1955; 18: 168-170Crossref PubMed Scopus (302) Google Scholar) used the notion of complementarity in the AChE active site to introduce cationic oximes as strong nucleophiles that could reverse AChE organophosphorylation. These drugs remain a first line of defense against OP toxicity, but they act only on certain forms of inactivated AChE rather than protecting against OP inactivation itself. Important goals of our studies are to obtain new insights into the AChE catalytic mechanism and identify new strategies that may prevent OP inactivation. A number of studies have focused on the structural basis of the high catalytic efficiency of AChE. X-ray crystallography (7Sussman J.L. Harel M. Frolow F. Oefner C. Goldman A. Toker L. Silman I. Science. 1991; 253: 872-879Crossref PubMed Scopus (2426) Google Scholar) revealed a narrow active site gorge some 20 Å deep with two separate ligand binding sites. The acylation site (A-site) at the bottom of the gorge contains residues involved in a catalytic triad (His-447, Glu-334, and Ser-203) 2Throughout this report, residue numbers refer to the human AChE sequence. as well as Trp-86, which orients the trimethylammonium group of acetylcholine prior to hydrolysis. The peripheral site (P-site) consists of a binding pocket near the surface of the enzyme at the mouth of the gorge and specifically binds certain ligands like the neurotoxin fasciculin (8Karlsson E. Mbugua P.M. Rodriguez-Ithurralde D. J. Physiol. (Paris). 1984; 79: 232-240PubMed Google Scholar, 9Marchot P. Khelif A. Ji Y.-H. Masnuelle P. Bourgis P.E. J. Biol. Chem. 1993; 268: 12458-12467Abstract Full Text PDF PubMed Google Scholar, 10Raves M.L. Harel M. Pang Y.-P. Silman I. Kozikowski A.P. Sussman J.L. Nat. Struct. Biol. 1997; 4: 57-63Crossref PubMed Scopus (392) Google Scholar, 11Harel M. Quinn D.M. Nair H.K. Silman I. Sussman J.L. J. Am. Chem. Soc. 1996; 118: 2340-2346Crossref Scopus (342) Google Scholar) and the fluorescent probes propidium (12Taylor P. Lappi S. Biochemistry. 1975; 14: 1989-1997Crossref PubMed Scopus (335) Google Scholar) and thioflavin T (13De Ferrari G.V. Mallender W.D. Inestrosa N.C. Rosenberry T.L. J. Biol. Chem. 2001; 276: 23282-23287Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). We have recently shown that catalysis is accelerated because cationic substrates transiently bind to the P-site en route to the A-site (14Szegletes T. Mallender W.D. Thomas P.J. Rosenberry T.L. Biochemistry. 1999; 38: 122-133Crossref PubMed Scopus (152) Google Scholar, 15Mallender W.D. Szegletes T. Rosenberry T.L. Biochemistry. 2000; 39: 7753-7763Crossref PubMed Scopus (125) Google Scholar). However, ligand binding to the P-site also can inhibit AChE through a process we have called steric blockade (15Mallender W.D. Szegletes T. Rosenberry T.L. Biochemistry. 2000; 39: 7753-7763Crossref PubMed Scopus (125) Google Scholar, 16Szegletes T. Mallender W.D. Rosenberry T.L. Biochemistry. 1998; 37: 4206-4216Crossref PubMed Google Scholar). This process involves a decrease in the rate constants with which substrates and their hydrolysis products enter and exit the A-site. The concept of steric blockade has led us to explore a new strategy for the design of drugs to protect AChE from OP inactivation. This strategy is to design cyclic compounds that will bind to the P-site with high affinity and selectively block the access of OPs while allowing entry of acetylcholine. Several approaches can accelerate the process of identifying cyclic compounds that have high affinity for the AChE P-site. Combinatorial chemistry in conjunction with structure-based design has been successfully employed to design high affinity ligands for therapeutic use. Molecular modeling effectively predicts good lead compounds that can then be refined using combinatorial chemistry approaches. To simplify molecular modeling calculations and to assure that cyclic compounds bind specifically to the rim of the AChE P-site in vitro, we have adopted a strategy in which we tether ligands via a disulfide linkage near the P-site. We previously showed that residue His-287 at the rim of the AChE P-site can be covalently linked to platinum(terpyridine) chloride to partially block access to the AChE active site (17Haas R. Adams E.W. Rosenberry M.A. Rosenberry T.L. Shafferman A. Velan B. Multidisciplinary Approaches to Cholinesterase Functions. Plenum Press, New York1992: 131-139Crossref Google Scholar). Because wild type human AChE has no free cysteine residues, we have mutated this histidine to a cysteine (H287C) to allow introduction of a wider range of tethered ligands at this location. Prospective compounds are synthesized with an MTS group, a highly reactive functional group that reacts with free cysteines to form a disulfide bond (18Stauffer D.A. Karlin A. Biochemistry. 1994; 33: 6840-6849Crossref PubMed Scopus (257) Google Scholar), thereby allowing their covalent linkage to H287C AChE. In this report, we test the feasibility of this approach using cationic trialkylammonium, acridinium, and tacrine ligands with demonstrated affinity for the AChE active site that are attached to MTS tethers of various lengths. We show that the length of the tether provides a molecular ruler which determines the catalytic properties of the modified AChEs. Materials—Recombinant human wild type and H287C mutant AChEs were expressed as secreted dimeric forms in Drosophila S2 cells in culture (15Mallender W.D. Szegletes T. Rosenberry T.L. Biochemistry. 2000; 39: 7753-7763Crossref PubMed Scopus (125) Google Scholar, 19Mallender W.D. Szegletes T. Rosenberry T.L. J. Biol. Chem. 1999; 274: 8491-8499Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) and purified by two cycles of affinity chromatography on acridinium resin (20Rosenberry T.L. Scoggin D.M. J. Biol. Chem. 1984; 259: 5643-5652Abstract Full Text PDF PubMed Google Scholar). The mutant AChE construction and expression methods were similar to that described previously (21Mallender W.D. Yager D. Onstead L. Nichols M.R. Eckman C. Sambamurti K. Kopcho L.M. Marcinkevicience J. Copeland R.A. Rosenberry T.L. Mol. Pharmacol. 2001; 59: 619-626Crossref PubMed Scopus (29) Google Scholar). Incorporation of the desired H287C mutation was confirmed by DNA sequencing. MTS reagents I-III (Table I) were obtained from Toronto Research Chemicals. Propidium iodide was purchased from Calbiochem and tacrine, from Sigma-Aldrich.Table ITethered ligands introduced in H287C AChE by MTS reagents 9-[N γ -(β-MTS-propionyl)-γ-aminopropylamino]acridine ( IV ) Trifluoroacetate—Compound IV was prepared in 81% yield by reacting 9-γ-aminopropylaminoacridine dihydrobromide (22Rosenberry T.L. Barnett P. Mays C. Methods Enzymol. 1982; 82: 325-339Crossref PubMed Scopus (28) Google Scholar) at room temperature with β-MTS-propionic acid (2-carboxyethyl methanethiosulfonate) (Toronto Research Chemicals, Inc.) (1.2 eq) and diisopropylethylamine (4 eq) in the presence of activating agent BOP (1.3 eq) in N,N-dimethylformamide. The resulting compound IV was purified by reverse-phase HPLC on a Vydac C-18 column (2.2 × 25 cm). 1H NMR (300 MHz, CDCl3) δ 9.71 (br t, NH), 8.11 (d, 2H, 7.4 Hz), 7.91 (br t, NH), 7.65 (br d, 2H, J = 7.2 Hz), 7.47 (t, 2H, J = 7.2 Hz), 7.16 (t, 2H, J = 7.4 Hz), 4.01 (br m, 2H), 3.53 (br m, 2H), 3.47 (t, 2H, J = 6.1 Hz), 3.36 (s, 3H), 2.87 (t, J = 6.6 Hz), 2.14 (m, 2H); (M r calc = 418; MS (FAB) [M + H]+ = C. O. 418). 9-[N γ -(N ϵ -β-MTS-propionyl-ϵ-aminocaproyl)-γ-aminopropylamino]acridine ( V ) Trifluoroacetate—Compound V was prepared in 40% yield by reacting 9-[Nγ-(ϵ-aminocaproyl)-γ-aminopropylamino]acridine dihydrobromide (22Rosenberry T.L. Barnett P. Mays C. Methods Enzymol. 1982; 82: 325-339Crossref PubMed Scopus (28) Google Scholar) at room temperature with β-MTS-propionic acid (2.0 eq) in the presence of diisopropylethylamine (4 eq) and BOP (1.3 eq) in dimethylformamide. The resulting compound V, after work-up, was purified by HPLC as described for compound IV. 1H NMR (300 MHz, CDCl3) δ 9.77 (br t, NH), 8.17 (d, 2H, 8.9 Hz), 7.73 (d, 2H, J = 8.7 Hz), 7.47 (t, 2H, J = 8.7 Hz), 7.26 (t, 2H, J = 8.9 Hz), 6.69 (br t, NH), 4.01 (br m, 2H), 3.51 (m, 2H), 3.42 (t, 2H, J = 6.6 Hz), 3.34 (s, 3H), 3.20 (m, 2H), 2.71 (t, 2H, J = 6.6 Hz), 2.27 (t, 2H, J = 6.9 Hz), 2.13 (m, 2H), 1.64 (m, 2H), 1.52 (m, 2H), 1.35 (m, 2H); (M r calc = 530; MS (FAB) [M + H]+ = 531). 9-[(4-MTS-methylphenylacetyl)glycyl-7′-amidoheptyl]amino-1,2,3,4-tetrahydroacridine ( VI )—4-MTS-methylphenylacetic acid (VII) was synthesized in 40% yield from sodium methanethiosulfonate (23Foong L.Y. You S. Jaikaran D.C. Zhang Z. Zunic V. Woolley G.A. Biochemistry. 1997; 36: 1343-1348Crossref PubMed Scopus (13) Google Scholar) and 4-bromomethylphenylacetic acid following a general method (24Bruice T.W. Kenyon G.L. J. Protein Chem. 1982; 1: 47-58Crossref Scopus (94) Google Scholar); m.p. 130-131 °C (M r calc = 260; found [M + Na]+ = 283; [M + K]+ = 299). The N-hydroxysuccinimidyl ester (VIII) of VII was prepared in 80% yield by dropwise addition of dicyclohexylcarbodiimide (1.1 eq) to VII and N-hydroxysuccinimide (1.1 eq) in acetonitrile; m.p. 129-131 °C. 9-Chloro-1,2,3,4-tetrahydroacridine (IX) was obtained from 1,2,3,4-tetrahydro-9-acridanone and POCl3 (10 eq) after boiling under reflux for 5 h (yield, 85%; m.p. 65-67 °C (lit. (25Carlier P.R. Han Y.F. Chow E.S. Li C.P. Wang H. Lieu T.X. Wong H.S. Pang Y.P. Bioorg. Med. Chem. 1999; 7: 351-357Crossref PubMed Scopus (179) Google Scholar) 67-68 °C)). Reaction of IX with 1,7-diaminoheptane (3 eq) in 1-pentanol (25Carlier P.R. Han Y.F. Chow E.S. Li C.P. Wang H. Lieu T.X. Wong H.S. Pang Y.P. Bioorg. Med. Chem. 1999; 7: 351-357Crossref PubMed Scopus (179) Google Scholar) gave 9-(7′-aminoheptyl)amino-1,2,3,4-tetrahydroacridine (X), which was successfully separated from the disubstituted product and isolated after reverse phase HPLC purification as an oil (M r calc = 311; [M + H]+ = 312). Reaction of X with the N-hydroxysuccinimidyl ester of Boc-glycine in Me2SO (1 eq) provided 9-(Boc-glycyl-7′-amidoheptyl)amino-1,2,3,4-tetrahydroacridine (XI)asanoil(M r calc = 468; found [M + H]+ = 469). The Boc-group was cleaved with 50% trifluoroacetic acid in dichloromethane, and purification provided 9-(glycyl-7′-amidoheptyl)amino-1,2,3,4-tetrahydroacridine (XII) as a light oil (M r calc = 368; found [M + H]+ = 369). Condensation of XII with VIII (1 eq) provided the desired compound VI, which was isolated after reverse phase HPLC purification and freeze-drying. Purity of the product by analytical reverse-phase HPLC was >76% (M r calc = 610; found [M + H]+ = 611). Reaction of MTS Reagents I-VI with Radiomethylated H287C AChE—Preparations of H287C were reductively radiomethylated with [3H]HCHO and sodium cyanoborohydride to allow better quantification of AChE protein following MTS labeling (26Haas R. Rosenberry T.L. Anal. Biochem. 1985; 148: 154-162Crossref PubMed Scopus (32) Google Scholar). This procedure converts the primary amino groups at the N terminus and seven lysine residues in each AChE subunit to di-[3H]methylamines but has no effect on enzyme activity. Dialyzed H287C AChE was pretreated with 1 mm dithiothreitol in 20 mm sodium phosphate (pH 8.0) for 1 h at room temperature to ensure that the cysteine-free sulfhydryl group had not partially oxidized (27Boyd A.E. Marnett A.B. Wong L. Taylor P. J. Biol. Chem. 2000; 275: 22401-22408Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), and excess dithiothreitol was removed by dialysis against 10 mm buffer (sodium phosphate, pH 7.0) in a Slide-a-Lyzer (Pierce) with 10,000 M r cutoff. MTS reagents corresponding to I-V were dissolved at 80-120 mm in 50 mm acetic acid. VI was dissolved at 1.8 mm because of lower solubility. The concentrations of IV-VI were established by absorbance (acridinium, ϵ410 = 8.08 mm-1 cm-1; tacrine, ϵ323 = 11.6 mm-1 cm-1). The compounds were then diluted to 6 mm (190 μm for VI) with AChE (3-20 μm) in 10 mm buffer and incubated for 30 min at room temperature. The AChE sulfhydryl group displaced methanesulfinic acid from the reagents to form a disulfide bond, and the modified enzymes were dialyzed against 10 mm buffer and applied to an acridinium resin (20Rosenberry T.L. Scoggin D.M. J. Biol. Chem. 1984; 259: 5643-5652Abstract Full Text PDF PubMed Google Scholar) affinity column (1-5 ml). The column was washed sequentially with 10 mm buffer, 10 mm buffer containing 0.5 m NaCl, and 5 mm buffer containing 0.5 m NaCl and 5 mm decamethonium bromide. Collected fractions were monitored for enzyme activity with the Ellman assay and for protein content by liquid scintillation counting. Steady-state Measurements of AChE-catalyzed Substrate Hydrolysis—Hydrolysis of acetylthiocholine was monitored with a spectrophotometric Ellman assay (28Ellman G.L. Courtney K.D. Andres J.V. Featherstone R.M. Biochem. Pharmacol. 1961; 7: 88-95Crossref PubMed Scopus (21516) Google Scholar). Assay solutions included 0.33 mm 5,5′-dithiobis(2-nitrobenzoic acid), and hydrolysis was monitored by formation of the thiolate dianion 3-carboxy-4-nitrothiophenol at 412 nm (Δϵ412nm = 14.15 mm-1 cm-1 (29Riddles P.W. Blakeley R.L. Zerner B. Anal. Biochem. 1979; 94: 75-81Crossref PubMed Scopus (927) Google Scholar)). Hydrolysis rates v were measured at various substrate concentrations [S] in 20 mm sodium phosphate, 0.02% Triton X-100 (pH 7.0) at 25 °C, and constant ionic strength was maintained with 0-60 mm NaCl (14Szegletes T. Mallender W.D. Thomas P.J. Rosenberry T.L. Biochemistry. 1999; 38: 122-133Crossref PubMed Scopus (152) Google Scholar). The dependence of v on [S] for unmodified AChE was fitted to Equation 1, the Haldane equation for substrate inhibition (30Haldane J.B.S. Enzymes. Longmans, Green, New York1930: 84Google Scholar), by weighted nonlinear regression analysis (assuming constant percent error in v) with Fig.P (BioSoft, version 6.0). v=Vmax[S][S]1+[S]KSS+Kapp(Eq. 1) In Equation 1, V max = k cat[E]tot, where k cat is the maximal substrate turnover rate, [E]tot is the total concentration of AChE active sites, K SS is the substrate inhibition constant, and K app is the apparent Michaelis constant. [E]tot values for both wild type and unmodified H287C AChE were calculated by assuming 450 units/nmol (13De Ferrari G.V. Mallender W.D. Inestrosa N.C. Rosenberry T.L. J. Biol. Chem. 2001; 276: 23282-23287Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 15Mallender W.D. Szegletes T. Rosenberry T.L. Biochemistry. 2000; 39: 7753-7763Crossref PubMed Scopus (125) Google Scholar), 3One unit of AChE activity corresponds to 1 μmol of acetylthiocholine hydrolyzed/min under standard pH-stat assay conditions at pH 8 (13De Ferrari G.V. Mallender W.D. Inestrosa N.C. Rosenberry T.L. J. Biol. Chem. 2001; 276: 23282-23287Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 20Rosenberry T.L. Scoggin D.M. J. Biol. Chem. 1984; 259: 5643-5652Abstract Full Text PDF PubMed Google Scholar). Our conventional spectrophotometric assay at 412 nm is conducted in pH 7 buffer. With wild type AChE and 0.5 mm acetylthiocholine, this assay results in 4.8 ΔA 412nm/min with 1 nm AChE or about 76% of the pH-stat assay standard. and for radiomethylated AChE these [E]tot values were converted to dpm/nmol ratios that were also applied to the modified AChEs. Some residual unmodified H287C AChE remained after treatment with the MTS reagents, and fractionation of the reaction mixtures by affinity chromatography did not completely resolve the two enzyme populations. Because substrate hydrolysis by these two AChEs was characterized by different kinetic parameters, Equation 1 was extended to Equation 2. v=Vmax1[S][S]1+[S]KSS+Kapp+Vmax2[S][S]1+[S]KSS2+Kapp2(Eq. 2) Hydrolysis rates in Equation 2 are the sum of two terms from the Haldane equation. V max1 = k cat[E]1 and V max2 = k cat2[E]2. V max1, [E]1, k cat, K SS, and K app refer to the unmodified AChE population and V max2, [E]2, k cat2, K SS2, and K app2, to the modified. When fitting data for these AChE mixtures, k cat, K SS, and K app were fixed at the values obtained for unmodified H287C AChE; [E]tot was fixed at values calculated from dpm/nmol ratios; [E]1 was set to [E]tot - [E]2; and k cat2, [E]2, K SS2, and K app2 were allowed to vary. Inhibition Constants for Reversible Inhibitors—At low concentrations of substrate S with a homogeneous AChE, hydrolysis rates are determined by the second order hydrolysis rate constants z. In the absence of inhibitor, z is denoted z I=0 and corresponds to V max/K app. Measured z at various fixed [I] were fitted according to Equation 3 by weighted nonlinear regression analyses to obtain the inhibition constant K I and the experimental parameter α (13De Ferrari G.V. Mallender W.D. Inestrosa N.C. Rosenberry T.L. J. Biol. Chem. 2001; 276: 23282-23287Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 16Szegletes T. Mallender W.D. Rosenberry T.L. Biochemistry. 1998; 37: 4206-4216Crossref PubMed Google Scholar). vI=0v≈zI=0z=1+[I]KI1+α[I]KI(Eq. 3) In Equation 3, values of z and z I = 0 either were determined as pseudo first-order rate constants ([S] = [S]initial e -zt (14Szegletes T. Mallender W.D. Thomas P.J. Rosenberry T.L. Biochemistry. 1999; 38: 122-133Crossref PubMed Scopus (152) Google Scholar)) or approximated by single rate measurements v and v I=0, in all cases at low substrate concentration ([S] < 0.2K app). K I is the equilibrium dissociation constant for I with E, and the constant α is the ratio of the second order rate constant with saturating I to that in the absence of I. With acetylthiocholine, 0 < α < 0.1 for P-site inhibitors (16Szegletes T. Mallender W.D. Rosenberry T.L. Biochemistry. 1998; 37: 4206-4216Crossref PubMed Google Scholar) and α ≅ 0 for A-site inhibitors. With two distinct populations of AChE composed of H287C AChE partially modified by MTS reagents, Equation 3 may be extended to Equation 4. vI=0v≈zI=0z=B(1-R2)1+α[I]KI1+[I]KI+R21+α2[I]KI21+[I]KI2-1(Eq. 4) In Equation 4, K I and α refer to the unmodified AChE population, K I2 and α2 refer to the modified enzyme, and R 2 is the fraction of total activity contributed by the second population in the absence of inhibitor. Data were fitted to Equation 4 by weighted nonlinear regression analysis with K I and α fixed at the values obtained for unmodified H287C AChE and α2 assigned equal to α. The B term reduced the emphasis on the v I = 0 point and was 0.99 ± 0.06 for all analyses. Molecular Modeling—Molecular modeling was performed with InsightII software (Accelrys, Inc.) on a Silicon Graphics Indigo II workstation. The crystal structure of Torpedo californica AChE complexed with tacrine (1ACJ) was taken as the initial structure, and incompletely resolved residues were fully defined using InsightII. Hydrogens were added at pH 7.0, and the resulting structure was energy-minimized for several days to eliminate steric overlap. The histidine at position 287 was mutated in silico to a cysteine, and reagents in Table I were attached to H287C through a disulfide linkage. Energy minimizations were initiated from three different placements of the tethered ligand: extending away from the enzyme, into the P-site, and into the A-site. For tethers too short to reach the A-site, the carbon-carbon bonds were stretched until the quaternary amine group was within contact distance of Trp-86. Five computation cycles were performed, each involving energy minimization with the Discover 3 force fields in InsightII followed by a 250-fs interval of molecular dynamics at elevated temperatures (1000-2000 K). This prevented entrapment of the structure in a local energy minimum. The total potential energy function contained a van der Waals contact term (which included hydrophobic and aromatic interaction energies) and an electrostatic term. The peptide backbone and AChE side chains were fixed during energy minimizations with two exceptions: For minimizations in which tacrine or acridinium had to traverse a constriction between the P-site and the A-site (V and VI starting outside the A-site and IV starting in the A-site), Phe-337 and Tyr-72 were allowed to move. Without this flexibility the ligands were trapped and could not traverse the energy barrier at the constriction. Nevertheless, by the final cycle of energy minimization, Phe-337 and Tyr-72 had returned to their starting positions. When III-V were initially placed outside the active site, local energy minima were reached with the quaternary amine equidistant from Asp-74 and Asp-292. This location was an artifact, apparently resulting from the absence of explicit waters in the minimizations and the inappropriate extension of electrostatic interactions through the vacuum. To obtain the structures in Fig. 4 and the corresponding potential energy values in Table IV, Asp-292 was mutated to alanine to remove these local minima and allow significantly lower minima to be attained with the quaternary amine close to Asp-74.Table IVMinimized potential energies and tethered ligand locations in H287C AChE modified by MTS reagentsH287C modificationTotal potential energyaThe total potential energy minima were obtained as outlined under “Experimental Procedures.”Distance from Asp-74bThe distance indicated is measured between the Oδ of Asp-74 and the quaternary ammonium nitrogen. Values are means ± S.E. determined from the three initial ligand placements.kcal mol −1 Å −1ÅI−53 ± 510.4 ± 0.6II−85 ± 138.7 ± 0.2III−147 ± 54.9 ± 0.1IV−163 ± 13.7 ± 0.6V−209 ± 110.1 ± 0.3a The total potential energy minima were obtained as outlined under “Experimental Procedures.”b The distance indicated is measured between the Oδ of Asp-74 and the quaternary ammonium nitrogen. Values are means ± S.E. determined from the three initial ligand placements. Open table in a new tab Radiomethylation of H287C AChE and Modification with MTS Reagents—H287C AChE was covalently modified with the MTS reagents indicated by I-VI in Table I. The reagents lost methanesulfinic acid as they became tethered through a disulfide bond to the sulfhydryl group at residue Cys-287. Preliminary experiments indicated that tethered I-III with relatively short cationic groups had minimal effects on enzyme catalytic activity, whereas the larger cationic groups in IV-VI gave a pronounced reduction of activity. In fact, these larger groups decreased the catalytic activity to such an extent that residual unmodified AChE obscured the catalytic properties of the modified AChEs. Two procedures were implemented to circumvent this problem. First, stock H287C AChE was reductively radiomethylated to provide a sensitive radioassay of the AChE concentration in subsequent modification and purification steps. In addition, small acridinium affinity columns were introduced to separate H287C AChE treated with MTS reagents into three fractions (Fig. 1). The small amount of AChE inactivated by the labeling procedures was not retained by the column at all and was eluted in the initial buffer wash. H287C AChEs modified with larger cationic groups were bound to the resin with relatively low affinity. Merely increasing the ionic strength of the buffer solution to 0.5 m NaCl released these modified enzymes from the acridinium resin. AChEs modified with IV, V, and VI were in this category (Fig. 1, C and D). Finally, unmodified AChE and AChEs modified with the smaller cationic groups in I, II, and III bound with high affinity to the resin and were only eluted with high concentrations" @default.
• W2045542952 created "2016-06-24" @default.
• W2045542952 creator A5002695228 @default.
• W2045542952 creator A5019258325 @default.
• W2045542952 creator A5040934873 @default.
• W2045542952 creator A5062474476 @default.
• W2045542952 creator A5064362404 @default.
• W2045542952 creator A5071319098 @default.
• W2045542952 creator A5076803336 @default.
• W2045542952 creator A5080773870 @default.
• W2045542952 date "2003-10-01" @default.
• W2045542952 modified "2023-09-27" @default.
• W2045542952 title "Inhibitors Tethered Near the Acetylcholinesterase Active Site Serve as Molecular Rulers of the Peripheral and Acylation Sites" @default.
• W2045542952 cites W1563813749 @default.
• W2045542952 cites W1587234113 @default.
• W2045542952 cites W1931662328 @default.
• W2045542952 cites W1965894713 @default.
• W2045542952 cites W1971048984 @default.
• W2045542952 cites W1972719099 @default.
• W2045542952 cites W1978861766 @default.
• W2045542952 cites W2018164924 @default.
• W2045542952 cites W2021704864 @default.
• W2045542952 cites W2029531895 @default.
• W2045542952 cites W2030128867 @default.
• W2045542952 cites W2035043090 @default.
• W2045542952 cites W2037992892 @default.
• W2045542952 cites W2041706446 @default.
• W2045542952 cites W2050302468 @default.
• W2045542952 cites W2059120868 @default.
• W2045542952 cites W2064340111 @default.
• W2045542952 cites W2067045168 @default.
• W2045542952 cites W2073417483 @default.
• W2045542952 cites W2084897312 @default.
• W2045542952 cites W2085958374 @default.
• W2045542952 cites W2086758152 @default.
• W2045542952 cites W2104847646 @default.
• W2045542952 cites W2109490221 @default.
• W2045542952 cites W2139644513 @default.
• W2045542952 cites W2149196013 @default.
• W2045542952 cites W2150996942 @default.
• W2045542952 cites W2325615512 @default.
• W2045542952 cites W2413286905 @default.
• W2045542952 cites W2949704785 @default.
• W2045542952 cites W4250975437 @default.
• W2045542952 cites W4317707744 @default.
• W2045542952 cites W988479680 @default.
• W2045542952 doi "https://doi.org/10.1074/jbc.m304797200" @default.
• W2045542952 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12851386" @default.
• W2045542952 hasPublicationYear "2003" @default.
• W2045542952 type Work @default.
• W2045542952 sameAs 2045542952 @default.
• W2045542952 citedByCount "44" @default.
• W2045542952 countsByYear W20455429522012 @default.
• W2045542952 countsByYear W20455429522013 @default.
• W2045542952 countsByYear W20455429522014 @default.
• W2045542952 countsByYear W20455429522015 @default.
• W2045542952 countsByYear W20455429522016 @default.
• W2045542952 countsByYear W20455429522017 @default.
• W2045542952 countsByYear W20455429522019 @default.
• W2045542952 countsByYear W20455429522020 @default.
• W2045542952 countsByYear W20455429522021 @default.
• W2045542952 countsByYear W20455429522022 @default.
• W2045542952 countsByYear W20455429522023 @default.
• W2045542952 crossrefType "journal-article" @default.
• W2045542952 hasAuthorship W2045542952A5002695228 @default.
• W2045542952 hasAuthorship W2045542952A5019258325 @default.
• W2045542952 hasAuthorship W2045542952A5040934873 @default.
• W2045542952 hasAuthorship W2045542952A5062474476 @default.
• W2045542952 hasAuthorship W2045542952A5064362404 @default.
• W2045542952 hasAuthorship W2045542952A5071319098 @default.
• W2045542952 hasAuthorship W2045542952A5076803336 @default.
• W2045542952 hasAuthorship W2045542952A5080773870 @default.
• W2045542952 hasBestOaLocation W20455429521 @default.
• W2045542952 hasConcept C126322002 @default.
• W2045542952 hasConcept C161790260 @default.
• W2045542952 hasConcept C181199279 @default.
• W2045542952 hasConcept C185592680 @default.
• W2045542952 hasConcept C193557335 @default.
• W2045542952 hasConcept C2778816929 @default.
• W2045542952 hasConcept C41183919 @default.
• W2045542952 hasConcept C46762472 @default.
• W2045542952 hasConcept C55493867 @default.
• W2045542952 hasConcept C71240020 @default.
• W2045542952 hasConcept C71924100 @default.
• W2045542952 hasConceptScore W2045542952C126322002 @default.
• W2045542952 hasConceptScore W2045542952C161790260 @default.
• W2045542952 hasConceptScore W2045542952C181199279 @default.
• W2045542952 hasConceptScore W2045542952C185592680 @default.
• W2045542952 hasConceptScore W2045542952C193557335 @default.
• W2045542952 hasConceptScore W2045542952C2778816929 @default.
• W2045542952 hasConceptScore W2045542952C41183919 @default.
• W2045542952 hasConceptScore W2045542952C46762472 @default.
• W2045542952 hasConceptScore W2045542952C55493867 @default.
• W2045542952 hasConceptScore W2045542952C71240020 @default.
• W2045542952 hasConceptScore W2045542952C71924100 @default.
• W2045542952 hasIssue "40" @default.
• W2045542952 hasLocation W20455429521 @default.
• W2045542952 hasOpenAccess W2045542952 @default.

• next