Inhibitors of Different Structure Induce Distinguishing Conformations in the Omega Loop, Cys69–Cys96, of Mouse Acetylcholinesterase
2002; Elsevier BV; Volume: 277; Issue: 45 Linguagem: Inglês
10.1074/jbc.m204391200
ISSN1083-351X
AutoresJianxin Shi, Zoran Radić, Palmer Taylor,
Tópico(s)Computational Drug Discovery Methods
ResumoWe have shown previously that association of reversible active site ligands induces a conformational change in an omega loop (Ω loop), Cys69–Cys96, of acetylcholinesterase. The fluorophore acrylodan, site-specifically incorporated at positions 76, 81, and 84, on the external portion of the loop not lining the active site gorge, shows changes in its fluorescence spectrum that reflect the fluorescent side chain moving from a hydrophobic environment to become more solvent-exposed. This appears to result from a movement of the Ω loop accompanying ligand binding. We show here that the loop is indeed flexible and responds to conformational changes induced by both active center and peripheral site inhibitors (gallamine and fasciculin). Moreover, phosphorylation and carbamoylation of the active center serine shows distinctive changes in acrylodan fluorescence spectra at the Ω loop sites, depending on the chirality and steric dimensions of the covalently conjugated ligand. Capping of the gorge with fasciculin, although it does not displace the bound ligand, dominates in inducing a conformational change in the loop. Hence, the ligand-induced conformational changes are distinctive and suggest multiple loop conformations accompany conjugation at the active center serine. The fluorescence changes induced by the modified enzyme may prove useful in the detection of organophosphates or exposure to cholinesterase inhibitors. We have shown previously that association of reversible active site ligands induces a conformational change in an omega loop (Ω loop), Cys69–Cys96, of acetylcholinesterase. The fluorophore acrylodan, site-specifically incorporated at positions 76, 81, and 84, on the external portion of the loop not lining the active site gorge, shows changes in its fluorescence spectrum that reflect the fluorescent side chain moving from a hydrophobic environment to become more solvent-exposed. This appears to result from a movement of the Ω loop accompanying ligand binding. We show here that the loop is indeed flexible and responds to conformational changes induced by both active center and peripheral site inhibitors (gallamine and fasciculin). Moreover, phosphorylation and carbamoylation of the active center serine shows distinctive changes in acrylodan fluorescence spectra at the Ω loop sites, depending on the chirality and steric dimensions of the covalently conjugated ligand. Capping of the gorge with fasciculin, although it does not displace the bound ligand, dominates in inducing a conformational change in the loop. Hence, the ligand-induced conformational changes are distinctive and suggest multiple loop conformations accompany conjugation at the active center serine. The fluorescence changes induced by the modified enzyme may prove useful in the detection of organophosphates or exposure to cholinesterase inhibitors. acetylcholinesterase mouse acetylcholinesterase 7-[[(methylethoxy)phosphinyl]-oxyl]-1-methylquinolinium iodide O,O-dimethylO-(2,2-dichlorovinyl)phosphate diisopropyl fluorophosphate 3,3-dimethylbutyl methylphosphonyl thiocholine 6-acryloyl-2-dimethylaminonaphthalene N,N-dimethylcarbamoylN-methyl-7-hydroxyquinolinium Acetylcholinesterase (AChE)1 plays a pivotal role in neurotransmission by terminating the action of neurotransmitter, acetylcholine, at neuromuscular junction and other cholinergic synapses (1Rosenberry T.L. Adv. Enzymol. Relat. Areas Mol. Biol. 1975; 43: 103-218PubMed Google Scholar, 2Taylor P. Hardman J.G. Limbird L.E. Goodman & Gilman's The Pharmacological Basis of Therapeutics. 10th Ed. McGraw-Hill Medical Publishing Division, New York2001: 161-1176Google Scholar, 3Quinn D.M. Chem. Rev. 1987; 87: 955-979Crossref Scopus (955) Google Scholar). AChE is one of most efficient enzymes known with hydrolysis of its natural substrate reaching diffusion-controlled limits. Inhibitors of AChE target two sites in the active site gorge: an active center at the base of a narrow gorge 20 Å in depth and a peripheral site at the gorge rim (4Taylor P. Lappi S. Biochemistry. 1975; 14: 1989-1997Crossref PubMed Scopus (338) Google Scholar). At the active center, a residue triad (Ser203-Glu334-His447) promotes acyl transfer and hydrolysis of the substrate, whereas Trp86 at the gorge base primarily stabilizes choline moiety of the substrate through a cation-π interaction. Active site inhibitors block substrate binding either by associating with the tryptophan in the choline binding site (tacrine and edrophonium) or by reacting irreversibly with catalytic serine (carbamates and organophosphates). Peripheral site inhibitors, such as propidium and gallamine, inhibit catalytic activity through both steric blockade and allosterically altering catalytic efficiency of the active center residues (4Taylor P. Lappi S. Biochemistry. 1975; 14: 1989-1997Crossref PubMed Scopus (338) Google Scholar, 5Changeux J.P. Mol. Pharmacol. 1966; 2: 369-392PubMed Google Scholar, 6Berman H.A. Taylor P. Biochemistry. 1978; 17: 1704-1713Crossref PubMed Scopus (35) Google Scholar, 7Szegletes T. Mallender W.D. Rosenberry T.L. Biochemistry. 1998; 37: 4206-4216Crossref PubMed Google Scholar, 8Szegletes T. Mallender W.D. Thomas P.J. Rosenberry T.L. Biochemistry. 1999; 38: 122-133Crossref PubMed Scopus (153) Google Scholar). To elucidate the conformational changes associated with mechanistically distinctive inhibitors, we developed a means for physically monitoring the conformation of purified mouse AChE by site-directed labeling with an environmentally sensitive fluorophore, acrylodan. Six single cysteine mutants were prepared for acrylodan conjugation (Fig. 1). Three were on the Cys69–Cys96 omega loop (Ω loop) flanking the active site gorge: L76C near the tip of the loop, and E81C and E84C on the outer surface not lining the gorge. Two residues on the opposing face of the gorge, H287C and Y124C, were selected, solvent exposure of which would be expected to be occluded by bound ligands that extend to the outer reaches of the gorge. A final residue, A262C, on a distal disulfide loop and whose temperature coefficient (B factor) would indicate flexible loop movement (9Bourne Y. Taylor P. Marchot P. Cell. 1995; 83: 503-512Abstract Full Text PDF PubMed Scopus (319) Google Scholar, 10Bourne 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), was selected as a control region. This residue is not anticipated to be influenced by ligand-induced changes in conformation. Our previous study showed a bathochromic emission shift of acrylodan conjugated at Ω loop residues 76, 81, and 84 upon binding of inhibitors, such as tacrine, edrophonium, huperzine A, andm-(N,N,N-trimethylammonio)trifluoromethyl acetophenone, that interact with Trp 86 in the choline binding site (11Shi J. Boyd A.E. Radic Z. Taylor P. J. Biol. Chem. 2001; 276: 42196-42204Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Acrylodan fluorescence is exquisitely sensitive to dipole moment of the surrounding solvent or macromolecular milieu (12Lakowicz J.R. Principles of Fluorescence Spectroscopy. 2nd Ed. Kluwer Academic and Plenum Publishers, New York1999Crossref Google Scholar, 13Lew 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, 14Prendergast 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). A bathochromic shift reflects exposure to solvent around the fluorophore. This pattern likely results from a concerted movement in the Cys69–Cys96 Ω loop upon binding of reversible inhibitors. Because a conformational change in the Ω loop induced by ligand is not reflected in the crystal structures of the AChEs studied to date (9Bourne Y. Taylor P. Marchot P. Cell. 1995; 83: 503-512Abstract Full Text PDF PubMed Scopus (319) Google Scholar, 10Bourne 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, 15Sussman J.L. Harel M. Frolow F. Oefner C. Goldman A. Toker L. Silman I. Science. 1991; 253: 872-879Crossref PubMed Scopus (2440) Google Scholar, 16Harel M. Schalk I. Ehretsabatier L. Bouet F. Goeldner M. Hirth C. Axelsen P.H. Silman I. Sussman J.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9031-9035Crossref PubMed Scopus (851) Google Scholar, 17Raves M.L. Harel M. Pang Y.P. Silman I. Kozikowski A.P. Sussman J.L. Nat. Struct. Biol. 1997; 4: 57-63Crossref PubMed Scopus (393) Google Scholar, 18Millard C.B. Kryger G. Ordentlich A. Greenblatt H, M Harel M Raves M.L. Segall Y. Barak D. Shafferman A. Silman I. Sussman J.L. Biochemistry. 1999; 38: 7032-7039Crossref PubMed Scopus (260) Google Scholar), and steady state catalysis by Ω loop mutant AChEs yielded minimal evidence for the loop being involved in the catalytic cycle (19Velan B. Barak D. Ariel N. Leitner M. Bino T. Ordentlich A. Shafferman A. FEBS Lett. 1996; 395: 22-28Crossref PubMed Scopus (29) Google Scholar, 20Faerman 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), we developed a means to measure directly conformation and solvent exposure in and around the active center gorge. In this study, we investigate the conformational changes reflected in acrylodan fluorescence for peripheral site inhibitors and for a congeneric series of carbamates and organophosphates that react covalently with the active center serine. Fluorescence measurements, combined with kinetics of inhibitor association, reveal a linkage between inhibition and a conformational change in the Ω loop. Ligand conjugation at the active center and association at the peripheral site induce distinctive conformational changes in the loop. Because the character of the spectral changes is dependent on chirality and dimensions of the ligand as well as its site of association, the Ω loop exhibits considerable flexibility in the solution conformations of AChE. Acetylthiocholine iodide, 5,5′-dithiobis(2-nitrobenzoic acid), DFP, dithiothreitol, physostigmine, gallamine, neostigmine, and paraoxon were purchased from Sigma-Aldrich. Acrylodan was obtained from Molecular Probes (Eugene, OR), echothiophate was obtained from Ayerst Laboratories (Philadelphia, PA), and DDVP was obtained from Bayer Inc. (West Haven, CT). Rivastigmine was obtained as the commercial product (Exelon) from Novartis. Fasciculin 2 (purified from the venom of Dendroaspis angusticeps) was a gift of Dr. Pascale Marchot (University of Marseille, Marseille, France). Drs. Yacov Ashani and Bhupendra P. Doctor (Walter Reed Army Research Center, Washington, DC) kindly provided 7-[[(methylethoxy)phosphinyl]oxyl]-1-methylquinolinium iodide (MEPQ) and procainamide-linked Sepharose CL-4B resin. The chiral organophosphonate enantiomers, (S p)-dimethylbutyl methylphosphonothiocholine ((S p)-DMBMP-TCh), (S p)-cycloheptyl methylphosphonothiocholine, (S p)-isopropyl methylphosphonothiocholine, and (R p)-dimethylbutyl methylphosphonothiocholine ((R p)-DMBMP-TCh) were kindly provided by Dr. Harvey Berman (State University of New York, Buffalo, NY). 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 human embryonic kidney (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 (21Boyd 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 pCDNA3 (21Boyd 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. Affinity chromatography permitted one-step purification of AChE. From 4–6 liters of media, mutant and wild type enzyme were purified in quantities ranging between 5 and 25 mg, as previously described (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 specific activity determination. The cysteine-substituted enzymes show kinetics of acetylcholine hydrolysis similar to wild type enzyme (11Shi J. Boyd A.E. Radic Z. Taylor P. J. Biol. Chem. 2001; 276: 42196-42204Abstract Full Text Full Text PDF PubMed Scopus (41) 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-HCl, 100 mm NaCl, 40 mm MgCl2, pH 8.0. Conditions for acrylodan labeling and stoichiometry estimates have been described previously (11Shi J. Boyd A.E. Radic Z. Taylor P. J. Biol. Chem. 2001; 276: 42196-42204Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). 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, 0.7–1.0; E81C, 0.77–1.0; E84C, 0.77–1.0; Y124C, 0.78–1.0; A262C, 0.69–0.92; and H287C, 0.78–1.0. 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: L76C, 72–85%; E81C, 81–92%; E84C, 85–93%; Y124C, 83–92%; A262C, 77–93%; H287C, 70–82%. Picomolar concentrations of enzyme in 0.01% bovine serum albumin and 0.1 m sodium phosphate buffer, pH 7.0, were reacted with covalent inhibitor in the absence of substrate at 25 °C. Typically, four inhibitor concentrations were used. Inhibition was monitored by measuring residual enzyme activity by removal of aliquots during the course of the reaction. Bimolecular rate constants (ki) were determined by the plot of pseudo first order rate constant (k obs) against inhibitor concentration (25Radic Z. Gibney G. Kawamoto S. MacPhee-Quigley K. Bongiorno C. Taylor P. Biochemistry. 1992; 31: 9760-9767Crossref PubMed Scopus (132) 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. Spectral changes in the presence of irreversible inhibitors were determined by allowing the reaction of the acrylodan-labeled enzymes to proceed until ≥99% inhibition was achieved. In the case of inhibitors with chromogenic leaving groups, the inhibited enzyme was passed through a G-50 Sephadex spin column (Roche Molecular Biochemicals) to remove the leaving group. Quantum yield changes in presence of MEPQ and paraoxon were determined by measuring the concentration of labeled enzyme by tryptophan emission and area of acrylodan fluorescence emission curve before and after organophosphate conjugation. Association of echothiophate and neostigmine with acrylodan-labeled E81C and E84C was assessed from the kinetics of change in fluorescence at 470 and 477 nm, respectively, following addition of a stoichiometric excess of inhibitor at several concentrations. Data were fitted to a single exponential approach to equilibrium. Bimolecular rate constants (ki) were determined by the plot of pseudo first order rate constant (k obs) against inhibitor concentration (25Radic Z. Gibney G. Kawamoto S. MacPhee-Quigley K. Bongiorno C. Taylor P. Biochemistry. 1992; 31: 9760-9767Crossref PubMed Scopus (132) Google Scholar). Association of rivastigmine with acrylodan-labeled E81C was monitored from the kinetics of the increase in fluorescence at 460 nm. To ensure the observed change in fluorescence upon rivastigmine association was caused by carbamoylation and not reversible binding of rivastigmine, the enzyme was reacted with the fluorescent carbamoylating agent, M7C, to ascertain the concentration of residual reactive serines (26Radic Z. Taylor P. J. Biol. Chem. 2001; 276: 4622-4633Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). We determined the bimolecular rate constants (ki) for echothiophate, neostigmine, and rivastigmine with unmodified and modified mAChE (Table I). For the wild type, E81C, and E84C mutant enzymes, the constants (ki) were obtained from measurements of enzyme activity, whereas changes in fluorescent signal were used to monitor reaction with the acrylodan-modified enzyme. A typical example of monitoring of the fluorescence change is shown in Fig.2. The data in Table I show that substitution of cysteine at the 81-position does not affect the carbamoylation and phosphorylation rates, whereas the modification at the 84-position causes a 3–4-fold reduction in rate. Upon modification of the introduced cysteine with acrylodan, reaction rates are reduced 3–4-fold compared with wild type following conjugation at the 81-position, whereas the reduction is 40–50-fold upon conjugation at the 84-position. It is important to note that the magnitude of these reductions in carbamoylation or phosphorylation rates by mutation and conjugation at each position is nearly the same, despite the inhibitors differing in their reactivity by a few orders of magnitude. In the case of rivastigmine, we measured the reaction rates by competition with excess M7C (26Radic Z. Taylor P. J. Biol. Chem. 2001; 276: 4622-4633Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) and achieved similar kinetics of inhibition. This indicates that the spectral shift produced by rivastigmine likely reflects a conformational change induced by progressive carbamoylation rather than formation of a reversible complex.Table IBimolecular rate constants for reaction of wild type and mutated AChE with echothiophate, neostigmine, and rivastigmine in the presence and absence of fluorescent (acrylodan) labelingEnzymeEchothiophateNeostigmineRivastigminek ik i,WT/k i, mutantk ik i,WT/k i, mutantk ik i,WT/k i, mutant104m−1min−1104m−1min−1104m−1min−1Wild typeaKinetic constants derived from measurements of inhibition of acetylthiocholine catalysis.235 ± 17571 ± 7372 ± 8E81CaKinetic constants derived from measurements of inhibition of acetylthiocholine catalysis.199 ± 141.2566 ± 81372 ± 21E81C-acrylodanbKinetic constants derived from intensity of the fluorescence signals.71 ± 103.3134 ± 194.398 ± 83.8E84CaKinetic constants derived from measurements of inhibition of acetylthiocholine catalysis.86 ± 42.7175 ± 33.395 ± 63.9E84C-acrylodanbKinetic constants derived from intensity of the fluorescence signals.4.8 ± 0.25015 ± 0.4399 ± 0.441Data shown as means ± standard deviation typically from three experiments. WT, wild type.a Kinetic constants derived from measurements of inhibition of acetylthiocholine catalysis.b Kinetic constants derived from intensity of the fluorescence signals. Open table in a new tab Data shown as means ± standard deviation typically from three experiments. WT, wild type. Organophosphates readily phosphorylate the active site serine (27Wilson I.B. Boyer P.D. Lardy H. Myrback K. 2nd Ed. The Enzymes. 4. Academic Press, New York1960: 501-520Google Scholar), presumably generating a pentavalent trigonal bipyramidal intermediate before dissociation of leaving group. The resulting phosphorylated complex resembles the tetrahedral transition states of acylation and deacylation of the trigonal esters. The diethylphosphoryl conjugate at the active site serine formed by reaction with echothiophate produces very little perturbation at positions 76, 262, and 287, consistent with their positions being well removed from the phosphorylation site (Table II). A bathochromic emission shift is observed at position 84, although of smaller magnitude when compared with the shift induced by other ligands (TablesTable II, Table III, Table IV, Table V). Interestingly, large hypsochromic shifts and enhancements of quantum yield are observed at positions 81 and 124. This pattern appears to be unusual, because the reversible active center ligands studied previously (11Shi J. Boyd A.E. Radic Z. Taylor P. J. Biol. Chem. 2001; 276: 42196-42204Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) and the dimethylphosphoryl conjugate formed from DDVP, which yields a phosphoryl serine with one methylene group shorter than echothiophate, confer little shift at position 124 and a large bathochromic shift at position 81.Table IIEffect of organophosphorates on fluorescence emission parameters of mouse AChE mutants labeled with acrylodanData are shown as mean values of at least three determinations. Relative quantum yields were determined by comparison of areas of the fluorescence emission curves between control and nonaged phosphorylated AChE. Positive chromic shifts denote bathochromic shifts, whereas negative chromic shifts denote hypsochromic shifts. Open table in a new tab Table VFluorescence emission parameters of mouse AChE mutants labeled with acrylodanView Large Image Figure ViewerDownload (PPT)Data are shown as mean values of at least three determinations. Chromic shifts were determined by comparison of fluorescence emission maximum between control and covalently modified AChE. Open table in a new tab Table IVEffect of carbamates on fluorescence emission parameters of mouse AChE mutants labeled with acrylodanView Large Image Figure ViewerDownload (PPT)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. Open table in a new tab Table IIIEffect of organophosphonates on fluorescence emission parameters of mouse AChE mutants labeled with acrylodanView Large Image Figure ViewerDownload (PPT)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 between control and nonaged phosphonylated AChE. The fluorescence emission spectrum of (R p)-DMBMP conjugate with E81C is shown in Fig. 3. 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 between control and nonaged phosphorylated AChE. Positive chromic shifts denote bathochromic shifts, whereas negative chromic shifts denote hypsochromic shifts. Data are shown as mean values of at least three determinations. Chromic shifts were determined by comparison of fluorescence emission maximum between control and covalently modified AChE. 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 are shown as mean values of at least three determinations. Relative quantum yields were determined by comparison of areas of the fluorescence emission curves between control and nonaged phosphonylated AChE. The fluorescence emission spectrum of (R p)-DMBMP conjugate with E81C is shown in Fig. 3. To confirm that the chromic shift is a result of conjugation of diethylphosphoryl group, not the retention of the thiocholine leaving group in the gorge, we examined the effect of paraoxon conjugation on chromic shift. Following reaction with the active site serine, paraoxon produces the same diethylphosphoryl conjugate as echothiophate. However, its leaving group is a neutral aromatic moiety rather than the cationic moiety of echothiophate. Because a similar spectral shift follows paraoxon conjugation, the conformational change is induced by the conjugated phosphorate, rather than being influenced by binding of residual leaving group. If we extend additional methylene units to the diisopropyl phosphoryl conjugate formed by DFP, we observe a chromic shifts at the 81- and 84-positions similar to the diethylphosphoryl conjugate. However, the hypsochromic shift at the 124-position becomes slightly smaller for the diisopropyl phosphoryl conjugate. Measurements were made immediately after reaction to preclude aging (i.e. spontaneous loss of an alkoxy moiety rendering an anionic conjugate) of the diisopropyl phosphoryl moiety (28Aldridge W.N. Reiner E. Enzyme Inhibitors as Substrates: Interactions of Esterases with Esters of Organophosphorus and Carbamic Acids. North-Holland Publishing Co., Amsterdam1972Google Scholar). To compare phosphoryl and phosphonyl conjugates of similar dimensions, racemic MEPQ was used to generate an ethyl methylphosphonyl conjugate. Kinetic studies show an enantiomeric preference of MEPQ, where presumably the S penantiomer reacts ∼10-fold faster than the R penantiomer. 2Z. Radić, unpublished observation. Hence, reaction with a stoichiometric excess of MEPQ should ensure one enantiomer covalently reacts preferentially with the enzyme. No discernable emission changes are observed at residues 262 and 287. Similar to DDVP, a bathochromic shift is observed for acrylodan at both positions 81 and 84. A moderate hypsochromic shift is observed at the 124-position, and very small change at the 76-position. Table III also shows the changes in acrylodan emission for a series ofS p methylphosphonates with increasing alkoxy substituent dimensions. Because the absolute stereochemistry of the methylphosphonates is known (29Berman H.A. Leonard K. J. Biol. Chem. 1989; 264: 3942-3950Abstract Full Text PDF PubMed Google Scholar), the chiral S pmethylphosphonates will direct their phosphonyl oxygen toward the oxyanion hole, the small methylphosphonyl moiety will be directed to the acyl pocket, and the more bulky alkoxy group directed to choline binding site (30Hosea N.A. Berman H.A. Taylor P. Biochemistry. 1995; 34: 11528-11536Crossref PubMed Scopus (101) Google Scholar). For the three S p enantiomers, very little or no change in emission maxima for acrylodan at positions 124, 262, and 287 is discerned. Similar to reversible active site ligands that interact with choline binding site (11Shi J. Boyd A.E. Radic Z. Taylor P. J. Biol. Chem. 2001; 276: 42196-42204Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), bathochromic shifts are observed at the Ω loop positions with the largest shift at E84C, an intermediate value at E81C, and only small change at L76C. The ethyl methylphosphonyl conjugate, which contains the smallest alkoxy moiety among S p conjugates, induces the smallest bathochromic shift at the 81- and 84-positions. R p alkyl methylphosphonates react far more slowly with the enzyme than the S p enantiomers (30Hosea N.A. Berman H.A. Taylor P. Biochemistry. 1995; 34: 11528-11536Crossref PubMed Scopus (101) Google Scholar), and we use formation of the (R p)-3,3-dimethylbutyl methylphosphonyl enzyme as an example. Formation of initial reversible complex can be detected by an immediate reduction in quantum yield of acrylodan at 81 with little change in emission maximum (Fig.3). This is followed by a progressive hypsochromic shift that reflects the covalent reaction with the active center serine. The isoemissive point at 510 nm, evident through the course of the slow reaction, likely reflects the presence of two species (i.e. the reversible DMBMP-TCh … AChE complex and the conjugated DMBMP-AChE being the dominant species in the progressive reaction). The resulting hypsochromic shift of conjugated acrylodan to 459 nm markedly contrasts with theS p enantiomer with its bathochromic shift in emission spectrum. These two enantiomers provide the critical clue for linking fluorescence emission maxima at the 81-position to the characteristics of structural perturbations of the Ω loop. Formation of a carbamoyl serine conjugate of AChE affords an alternative means for forming a relatively stable modified enzyme conjugate (27Wilson I.B. Boyer P.D. Lardy H. Myrback K. 2nd Ed. The Enzymes. 4. Academic Press, New York1960: 501-520Google Scholar). Kinetic studies and crystallographic evidence show the carbamoyl oxygen of the covalent conjugate directed toward the oxyanion hole, and the alkyl carbamoyl group pointing toward the acyl pocket (25Radic Z. Gibney G. Kawamoto S. MacPhee-Quigley K. Bongiorno C. Taylor P. Biochemistry. 1992; 31: 9760-9767Crossref PubMed Scopus (132) Google Scholar, 31Ariel N. Ordentlich A. Barak D. Bino T. Velan B. Shafferman A. Biochem. J. 1998; 335: 95-102Crossref PubMed Scopus (79) Google Scholar, 32Bar-On P. Millard C.B. Harel M. Dvir H. Enz A. Sussman J.L. Silman I. Biochemistry. 2002; 41: 3555-3564Cros
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