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• W2059484946 abstract "To explore the role that surface and active center charges play in electrostatic attraction of ligands to the active center gorge of acetylcholinesterase (AChE), and the influence of charge on the reactive orientation of the ligand, we have studied the kinetics of association of cationic and neutral ligands with the active center and peripheral site of AChE. Electrostatic influences were reduced by sequential mutations of six surface anionic residues outside of the active center gorge (Glu-84, Glu-91, Asp-280, Asp-283, Glu-292, and Asp-372) and three residues within the active center gorge (Asp-74 at the rim and Glu-202 and Glu-450 at the base). The peripheral site ligand, fasciculin 2 (FAS2), a peptide of 6.5 kDa with a net charge of +4, shows a marked enhancement of rate of association with reduction in ionic strength, and this ionic strength dependence can be markedly reduced by progressive neutralization of surface and active center gorge anionic residues. By contrast, neutralization of surface residues only has a modest influence on the rate of cationicm-trimethylammoniotrifluoroacetophenone (TFK+) association with the active serine, whereas neutralization of residues in the active center gorge has a marked influence on the rate but with little change in the ionic strength dependence. Brownian dynamics calculations for approach of a small cationic ligand to the entrance of the gorge show the influence of individual charges to be in quantitative accord with that found for the surface residues. Anionic residues in the gorge may help to orient the ligand for reaction or to trap the ligand. Bound FAS2 on AChE not only reduces the rate of TFK+ reaction with the active center but inverts the ionic strength dependence for the cationic TFK+ association with AChE. Hence it appears that TFK+ must traverse an electrostatic barrier at the gorge entry imparted by the bound FAS2 with its net charge of +4. To explore the role that surface and active center charges play in electrostatic attraction of ligands to the active center gorge of acetylcholinesterase (AChE), and the influence of charge on the reactive orientation of the ligand, we have studied the kinetics of association of cationic and neutral ligands with the active center and peripheral site of AChE. Electrostatic influences were reduced by sequential mutations of six surface anionic residues outside of the active center gorge (Glu-84, Glu-91, Asp-280, Asp-283, Glu-292, and Asp-372) and three residues within the active center gorge (Asp-74 at the rim and Glu-202 and Glu-450 at the base). The peripheral site ligand, fasciculin 2 (FAS2), a peptide of 6.5 kDa with a net charge of +4, shows a marked enhancement of rate of association with reduction in ionic strength, and this ionic strength dependence can be markedly reduced by progressive neutralization of surface and active center gorge anionic residues. By contrast, neutralization of surface residues only has a modest influence on the rate of cationicm-trimethylammoniotrifluoroacetophenone (TFK+) association with the active serine, whereas neutralization of residues in the active center gorge has a marked influence on the rate but with little change in the ionic strength dependence. Brownian dynamics calculations for approach of a small cationic ligand to the entrance of the gorge show the influence of individual charges to be in quantitative accord with that found for the surface residues. Anionic residues in the gorge may help to orient the ligand for reaction or to trap the ligand. Bound FAS2 on AChE not only reduces the rate of TFK+ reaction with the active center but inverts the ionic strength dependence for the cationic TFK+ association with AChE. Hence it appears that TFK+ must traverse an electrostatic barrier at the gorge entry imparted by the bound FAS2 with its net charge of +4. The high catalytic efficiency of acetylcholinesterase (AChE, 1The abbreviations used are: AChE, acetylcholinesterase; FAS2, fasciculin 2; TFK+,m-trimethylammoniotrifluoroacetophenone; TFK0,m-tert-butyltrifluoroacetophenone; PhAc, phenylacetate; pNPhAc, p-nitrophenylacetate; ACTh, acetylthiocholine. 1The abbreviations used are: AChE, acetylcholinesterase; FAS2, fasciculin 2; TFK+,m-trimethylammoniotrifluoroacetophenone; TFK0,m-tert-butyltrifluoroacetophenone; PhAc, phenylacetate; pNPhAc, p-nitrophenylacetate; ACTh, acetylthiocholine. EC 3.1.1.7) as well as the rapid rates of reaction of selective AChE inhibitors are primarily addressed with cationic ligands. The physiological and most rapidly hydrolyzed substrate of AChE, acetylcholine, as well as its highest affinity inhibitors,m-trimethylammoniotrifluoroacetophenone (TFK+) and fasciculin 2 (FAS2), carry one or more positive charges. Inhibition of AChE by small cationic reversible inhibitors likeN-methylacridinium appears diffusion limited (1Nolte H.-J. Rosenberry T.L. Neumann E. Biochemistry. 1980; 19: 3705-3711Crossref PubMed Scopus (157) Google Scholar) as is conjugation of the active serine by cationic TFK+ (2Quinn D.M. Seravalli J. Nair H.K. Medhekar R. Husseini B. Radic̀ Z. Vellom D.C. Pickering N. Taylor P. Quinn D.M. Balasubramanian A.S. Doctor B.P. Taylor P. Enzymes of the Cholinesterase Family. Plenum Publishing Corp., New York1995: 203-207Crossref Google Scholar). Initial rates of acetylcholine hydrolysis by Electrophorus electricus AChE also appear limited by the initial diffusion-controlled association of reactants (3Rosenberry T.L. Adv. Enzymol. Relat. Areas Mol. Biol. 1975; 43: 103-218PubMed Google Scholar, 4Quinn D.M. Chem. Rev. 1987; 87: 955-979Crossref Scopus (922) Google Scholar). Early kinetic studies suggested a net negative charge at the active center, a finding borne out from an overall analysis of ionizable groups (5Antosiewicz J. Briggs J.M. Elcock A.H. Gilson M.K. McCammon J.A. J. Comput. Chem. 1996; 17: 1633-1644Crossref Scopus (135) Google Scholar, 6Antosiewicz J. McCammon J.A. Gilson M.K. Biochemistry. 1996; 35: 7819-7833Crossref PubMed Scopus (414) Google Scholar) in the three-dimensional structure of Torpedo californica AChE (7Sussman J.L. Harel M. Frolow F. Oefner C. Goldman A. Toker L. Silman I. Science. 1991; 253: 872-879Crossref PubMed Scopus (2389) Google Scholar) and mouse AChE (8Bourne Y. Taylor P. Marchot P. Cell. 1995; 83: 503-512Abstract Full Text PDF PubMed Scopus (319) Google Scholar). In addition, the net negative charges on the enzyme appear to be strategically distributed for rapid catalysis. Theoretical calculations based on the crystal structure ofTorpedo AChE (9Tan R.C. Truong T.N. McCammon J.A. Sussman J.L. Biochemistry. 1993; 32: 401-403Crossref PubMed Scopus (134) Google Scholar, 10Ripoll D.R. Faerman C.H. Axelsen P.H. Silman I. Sussman J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5128-5132Crossref PubMed Scopus (249) Google Scholar) suggest the presence of a strong electrostatic field that directs cations into the active center gorge of the enzyme. The existence of such a field has been recently supported experimentally by analysis of electrooptical properties of snake AChE in strong, external electric fields (11Porschke D. Creminon D. Cousin X. Bon C. Sussman J. Silman I. Biophys. J. 1996; 70: 1603-1608Abstract Full Text PDF PubMed Scopus (44) Google Scholar). A comprehensive mutagenesis study of human AChE was undertaken to analyze the kinetic contributions of seven surface anionic charges influencing the electric field of AChE (12Shafferman A. Ordentlich A. Barak D. Kronman C. Ber R. Bino T. Ariel N. Osman R. Velan B. EMBO J. 1994; 13: 3448-3455Crossref PubMed Scopus (74) Google Scholar). Those surface residues outside of the active center gorge had only a small influence on catalytic efficiency for both cationic and neutral substrates. Recent theoretical calculations predict that anionic residues peripheral to the active center gorge exhibit only a minor influence on catalysis rates since the directing field is the result of a large number of contributions from the protein (13Antosiewicz J. McCammon J.A. Wlodek S.T. Gilson M.K. Biochemistry. 1995; 34: 4211-4219Crossref PubMed Scopus (55) Google Scholar,14Antosiewicz J. Wlodek S.T. McCammon J.A. Biopolymers. 1996; 39: 85-94Crossref PubMed Google Scholar). Inhibition of AChE is achieved by competing ligands binding to the active center region of AChE, which is located in the center of the subunit at the base of a narrow gorge some 18–20 Å in depth. A separate set of ligands, which includes the organic cation, propidium (15Taylor P. Lappi S. Biochemistry. 1975; 14: 1989-1997Crossref PubMed Scopus (326) Google Scholar), and the peptide FAS2 (16Radić 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), binds to a site peripheral to the active center gorge. Inhibition of this site results from the ligand impeding substrate entry to the active center and exerting an allosteric influence on the conformation of the enzyme (15Taylor P. Lappi S. Biochemistry. 1975; 14: 1989-1997Crossref PubMed Scopus (326) Google Scholar, 16Radić 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, 17Epstein D.J. Berman H.A. Taylor P. Biochemistry. 1979; 18: 4749-4754Crossref PubMed Scopus (35) Google Scholar, 18Barak D. Ordentlich A. Bromberg A. Kronman C. Marcus D. Lazar A. Ariel N. Velan B. Shafferman A. Biochemistry. 1995; 34: 15444-15452Crossref PubMed Scopus (80) Google Scholar). In this study, we have neutralized a series of anionic side chains through site-specific mutagenesis to distinguish the influence of electrostatics on the kinetics of inhibitor and substrate binding at the active center from that at the peripheral site. We also investigated the influence of bound FAS2 on the kinetics of entry of cationic and neutral ligands into the active center gorge. Mutations of mouse AChE were generated from a cDNA inserted into Bluescript II SK(+) (Stratagene, San Diego, CA) or directly in expression vectors pRC/CMV or pCDNA3 (Invitrogen, San Diego, CA) using M13K07 helper phage (New England Biolabs, Beverly, MA) to obtain single-stranded DNA. Oligonucleotides were synthesized (Life Technologies, Inc.; Genosys, Woodlands, TX) to encode the mutation of interest and produce restriction sites that enabled screening of mutants. Multiple mutants were generated by subcloning several cDNA fragments containing single site mutations into the construct. Fragments were selected by the positions of the suitable restriction sites. Constructs were finally sequenced in the expression vector to confirm the mutation. Wild-type and mutant mouse AChEs were expressed in HEK-293 cells following transfection of the cells with the encoding cDNA as described previously (19Radić Z. Pickering N.A. Vellom D.C. Camp S. Taylor P. Biochemistry. 1993; 32: 12074-12084Crossref PubMed Scopus (411) Google Scholar). AChE was concentrated from the serum-free medium in which the expressing cells were grown. Purified and lyophilized FAS2 was kindly provided by Dr. Carlos Cerveñansky, Instituto de Investigaciones Biologicas, Montevideo, Uruguay, and Dr. Pascale Marchot, CNRS, University of Marseille, France. Concentrations of FAS2 stock solutions were determined by absorbance (ε276 = 4900m−1 cm−1) (20Karlsson E. Mbugua P.M. Rodriguez-Ithurralde D. J. Physiol. ( Paris ). 1984; 79: 232-240PubMed Google Scholar).m-tert-Butyltrifluoroacetophenone (TFK0) andm-trimethylammoniotrifluoroacetophenone (TFK+) were synthesized as described earlier (21Nair H.K. Quinn D.M. Bioorg. & Med. Chem. Lett. 1993; 3: 2619-2622Crossref Scopus (14) Google Scholar). Hydrolysis of ATCh, phenylacetate, andp-nitrophenylacetate was measured spectrophotometrically at 412 nm for thiocholine (22Ellman G.L. Courtney K.D. Andres Jr., V. Featherstone R.M. Biochem. Pharmacol. 1961; 7: 88-95Crossref PubMed Scopus (20669) Google Scholar) or by measuring phenol orp-nitrophenol release at 270 and 405 nm, respectively. Kinetic constants for hydrolysis of the above substrates by wild-type and mutant AChEs were determined as described previously (23Radić 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 (83) Google Scholar). The second-order rate constants for FAS and trifluoroacetophenone association and the first-order constants of dissociation were determined, by monitoring the time course of reaction, from Equation 1 (cf. Ref. 16Radić 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). f=kon[L]kon[L]+koff(1−e−(kon[L]÷koff)t)Equation 1 where f is a fraction of enzyme inhibition at timet and ligand concentration [L]. The second-order rate constants for formation of the inhibitory complex (k on), the first-order rate constants for its dissociation (k off), and substrate catalytic constants were plotted as a function of ionic strength of the reaction medium using the Debye-Huckel limiting law implemented within the transition state theory by Glasstone et al. (24,cf. Ref. 2Quinn D.M. Seravalli J. Nair H.K. Medhekar R. Husseini B. Radic̀ Z. Vellom D.C. Pickering N. Taylor P. Quinn D.M. Balasubramanian A.S. Doctor B.P. Taylor P. Enzymes of the Cholinesterase Family. Plenum Publishing Corp., New York1995: 203-207Crossref Google Scholar); see Equation 2. kon=(kon0−konH)10−1.18‖zEzI‖·I+konHEquation 2 where k on,k on0, andk onH are second-order association rate constants at the specified ionic strength I, zero ionic strength, and infinite ionic strength, respectively.z E and z I are the charges of enzyme and inhibitor involved in the interaction. Enzyme activities in catalysis and inhibition were measured in media of varying ionic strength, generated by varying the concentration of phosphate buffer, pH 7.0, between 2 and 300 mm. To conduct the Brownian dynamics simulations, the structure for the mouse AChE·FAS2 complex was obtained from the Brookhaven Protein Data Bank (8Bourne Y. Taylor P. Marchot P. Cell. 1995; 83: 503-512Abstract Full Text PDF PubMed Scopus (319) Google Scholar). Coordinates for the missing residues Glu-1, Gly-2, Arg-3, Pro-258, Pro-259, Gly-260, Gly-261, Ala-262, Gly-263, and Gly-264 were built in using QUANTA (25Molecular Simulations Inc. (1996) San Diego, CA 92121–4777.Google Scholar). CHARMM22 with its all-atom parameter set was used to build in the protons and missing side chains (26MacKerell A.D. Wiorkiewicz-Kuczera J. Karplus M. CHARMM 22 Parameter Set. Harvard University, Cambridge, MA1995Google Scholar). Coordinates for the protons and residues 1–3 and 258–264 were relaxed using 500 steps of Adopted-Basis Newton Raphston minimization. Ionization states of the residues were modeled to represent physiological conditions. Arg and Lys residues were modeled as protonated with an overall charge of +1. Asp and Glu residues were modeled as deprotonated with an overall charge of −1. The protonation states of the His residues were determined by inspection of the availability of hydrogen bonds. All His residues were modeled as HSD (protonated at the ND1 site) except for residue 432 which was modeled as HSE (protonated at the NE2 site) and residue 447 which was modeled as HSP (doubly protonated at the ND1 and NE2 sites). The charge on the unliganded, wild-type AChE used for the simulations was −8.0. A reactive surface was placed at the gorge entrance. To do this the center of the mouth of the gorge and the gorge axis were first defined. In similar fashion to previous work with T. californica AChE (13Antosiewicz J. McCammon J.A. Wlodek S.T. Gilson M.K. Biochemistry. 1995; 34: 4211-4219Crossref PubMed Scopus (55) Google Scholar, 27Gilson M.K. Straatsma T.P. McCammon J.A. Ripoll D.R. Faerman C.H. Axelsen P.H. Silman I. Sussman J.L. Science. 1994; 263: 1276-1278Crossref PubMed Scopus (239) Google Scholar, 28Antosiewicz J. Gilson M.K. McCammon J.A. Isr. J. Chem. 1994; 34: 151-158Crossref Scopus (21) Google Scholar, 29Antosiewicz J. Gilson M.K. Lee I.H. McCammon J.A. Biophys. J. 1995; 68: 62-68Abstract Full Text PDF PubMed Scopus (46) Google Scholar), the center of the mouth of the gorge was defined by determining the geometric center of four selected atoms from residues near the gorge entrance: Tyr-72:OH, Asp-74:OD1, Phe-297:CE1, and Tyr-341:CD2. The gorge axis was then defined as the line running from Ser-203:C to the geometric center. For the reactive surface at the gorge entrance, a 12-Å reactive sphere was centered on the gorge axis 2 Å above the center of the mouth of the gorge to completely cap the entrance. Atomic charges of anionic side chains mutated to the corresponding amides were set to 0 in the AChE model to generate the corresponding mutants. Neither steric nor conformational changes potentially arising from the mutations were considered. All calculations involved in the Brownian dynamics simulations were carried out using the University of Houston Brownian Dynamics software package UHBD (30Madura J.D. Davis M.E. Gilson M.K. Wade R.C. Luty B.A. McCammon J.A. Rev. Comput. Chem. 1994; 5: 229-267Google Scholar). The simulations were conducted using a 110 × 110 × 110 grid with 1-Å spacing centered on the Ser203:C atom. Hydrodynamic radii of 35.0 and 3.5 Å were used for AChE and diffusing cationic ligand, respectively. An excluded radius of 2.0 Å was used for the ligand. Fig. 1 illustrates the reactive surface available to the diffusing ligand and several other parameters used in the simulations. The protein and solvent were assigned dielectric values of 4.0 and 78.0, respectively, with a Stern layer radius of 2.0 Å. A probe-accessible surface was used with a 1.4-Å probe radius and 300 surface points per atom. The boundary potential was determined using a single Debye-Huckel sphere of 35 Å radius and molecular charge of −8 for the AChE molecule. A step size of 0.05 ps was used for diffusion between 0 and 100 Å, 1 ps between 100 and 175 Å, and 5 ps between 175 and 300 Å from the grid center. The rate constants of enzyme-ligand encounter were calculated from large numbers of Brownian trajectories of the substrate in the neighborhood of the enzyme (30Madura J.D. Davis M.E. Gilson M.K. Wade R.C. Luty B.A. McCammon J.A. Rev. Comput. Chem. 1994; 5: 229-267Google Scholar, 31Ermak D.L. McCammon J.A. J. Chem. Phys. 1978; 69: 1352-1360Crossref Scopus (2005) Google Scholar, 32McCammon J.A. Northrup S.H. Allison S.A. J. Phys. Chem. 1986; 90: 3901-3905Crossref Scopus (63) Google Scholar, 33Madura J.D. Briggs J.M. Wade R.C. Davis M.E. Luty B.A. Ilin A. Antosiewicz J. Gilson M.K. Bagheri B. Scott L.R. McCammon J.A. Comp. Phys. Commun. 1995; 91: 57-95Crossref Scopus (586) Google Scholar). The substrate moves under the influence of the electrostatic field of the enzyme and the random bombardment of solvent molecules. Trajectories are initiated on the surface of a sphere of radius b, the b surface, around the center of coordinates of the enzyme. This sphere is made sufficiently large so that the electrostatic forces between the ligand and the enzyme are approximately centrosymmetric for r> b. Each trajectory is continued until the substrate satisfies a predefined encounter criterion or reaches an outer spherical surface of radius q, the quit-surface. The fraction of trajectories that finish with encounters is corrected to include the additional encounters that would have occurred if the trajectories had not been truncated at the quit-surface and is then multiplied by the rate constant for the encounter of ligand with theb surface to yield the bimolecular diffusion-controlled rate constant, k on (34Davis M.E. Madura J.D. Luty B.A. McCammon J.A. Comput. Phys. Commun. 1991; 62: 187-197Crossref Scopus (467) Google Scholar). For this study, Brownian dynamics trajectories were started randomly on a b surface 55.0 Å from Ser203:C atom. A trajectory was terminated when one of three criteria were met. 1) The ligand made contact with the reactive surface. 2) The diffusing ligand reached the quit-surface, a distance greater than 300 Å from the Ser203:C atom. 3) The ligand made more than 1 × 107 steps. For the wild-type and each of the nine mutants, 3000 trajectories were conducted at 0 and 670 mm ionic strengths. Catalytic parameters for substrates, second-order association rate constants, and first-order dissociation rate constants for cationic and neutral ligands with wild-type AChE, measured as a function of ionic strength of reaction medium, are shown in Fig. 2. The cationic inhibitors TFK+ and FAS2 associate with AChE significantly faster in buffers of low than high ionic strength (Table I). The increase in rates over this ionic strength range approaches 1 order of magnitude for the TFK+monocation and exceeds 2 orders of magnitude for FAS2, a 6.5-kDa peptide that bears net charge of +4 at pH 7.0. The association rate constants for FAS2 at low ionic strength approach the values for TFK+. Both values at a low ionic strength are within the range of rate constants predicted for diffusion controlled reactions. For the cationic substrate ATCh, the ratiok cat/K m increased only 2-fold at low ionic strength indicating that the diffusion and chemical steps in catalysis by mouse AChE may be of similar magnitude. The rates of association of neutral ligands do not increase with a reduction in ionic strength. Hydrolysis rates of neutral substrates, PhAc andpNPhAc, being 1 and 2 orders of magnitude slower than ATCh hydrolysis, also were not influenced by the change in ionic strength. The association rate constants for the neutral inhibitor TFK0, which are about 2 orders of magnitude slower than TFK+, exhibit a modest decrease in rate with a reduction in ionic strength (Table I). This decrease was assumed to be consequence of indirect electrostatic interaction as in the case of thek cat versus I dependence for ATCh and PhAc; only the composite parameter (z I z E) was therefore determined, instead of individual charges. Rates of dissociation of both the charged and neutral TFKs and fasciculin were largely independent of the ionic strength.Table IRate constants for association and dissociation of inhibitors and for turnover of substrates with wild-type mouse AChEs in media of varying ionic strengthLigandk on0k onHz Iz Ek off109m−1min−1109m−1min−110−3 min−1TFK+9801-aCorrected for TFK+ and TFK0 hydration (cf. 35). ± 601301-aCorrected for TFK+ and TFK0 hydration (cf. 35).+1−2.3 ± 0.21.1 ± 0.3TFK02.21-aCorrected for TFK+ and TFK0 hydration (cf. 35). ± 0.34.81-aCorrected for TFK+ and TFK0 hydration (cf. 35).ND1-bDue to nonlinearity of the k on versus I dependence, only a composite parameter (z I z E) = 1.1 ± 0.2 was determined, instead of individual charges.ND1-bDue to nonlinearity of the k on versus I dependence, only a composite parameter (z I z E) = 1.1 ± 0.2 was determined, instead of individual charges.15 ± 1FAS249 ± 30.071+4−1.2 ± 0.14.4 ± 2.6Substrate(k cat/K m)0(k cat/K m)Hz Iz E109m−1min−1109m−1 min−1ATCh2.5 ± 0.21.5+1−1.1 ± 0.4PhAc0.12 ± 0.030.120—PNPAc0.0075 ± 0.00110.00750—k on0 and k onH are second-order association rate constants for inhibitor at zero and 670 mm ionic strengths, respectively. (k cat/K m)0 and (k cat/K m)H are second-order rate constants for substrate turnover at zero and 670 mm ionic strengths, respectively. z E andz I are charges of the enzyme and ligand, respectively, involved in the interaction. Constantsk on0 for inhibitors, (k cat/K m)0 for substrates and z E were obtained by nonlinear regression analysis of Equation 2 using fixed values ofk onH, (k cat/K m)H andz I, from experimental data presented in Fig 2. Dashes denote indeterminant parameters. The first-order dissociation rate constants, k off, appeared independent of ionic strength and were calculated as a mean of values obtained at different ionic strengths.1-a Corrected for TFK+ and TFK0 hydration (cf. 35).1-b Due to nonlinearity of the k on versus I dependence, only a composite parameter (z I z E) = 1.1 ± 0.2 was determined, instead of individual charges. Open table in a new tab k on0 and k onH are second-order association rate constants for inhibitor at zero and 670 mm ionic strengths, respectively. (k cat/K m)0 and (k cat/K m)H are second-order rate constants for substrate turnover at zero and 670 mm ionic strengths, respectively. z E andz I are charges of the enzyme and ligand, respectively, involved in the interaction. Constantsk on0 for inhibitors, (k cat/K m)0 for substrates and z E were obtained by nonlinear regression analysis of Equation 2 using fixed values ofk onH, (k cat/K m)H andz I, from experimental data presented in Fig 2. Dashes denote indeterminant parameters. The first-order dissociation rate constants, k off, appeared independent of ionic strength and were calculated as a mean of values obtained at different ionic strengths. The catalytic constants for hydrolysis of ATCh by wild-type and mutant mouse AChEs determined in 100 mm phosphate buffer, pH 7.0, are listed in Table II. Of the 17 mutants studied, mutations of aspartyl and glutamyl residues located on the enzyme surface to their corresponding amidated residues produced less than 5-fold variations in kinetic constants. In all cases, K mincreases. By contrast, the active center mutations, except for E202Q, produced substantial changes in catalytic parameters including increases in K m of up to nearly 3 orders of magnitude and decreases in k cat of more than an order of magnitude. The ionic strength dependences of catalytic parameters for ATCh with wild-type and seven mutants are presented in Fig. 3.Table IICatalytic parameters for hydrolysis of ATCh by wild-type and mutant mouse AChEs in 0.1 m phosphate buffer, pH 7.0EnzymeK mK ssk catbk cat/K mμmmm105min−1109m−1min−1Wild type2-aData of Radić et al. (19).46 ± 315 ± 21.4 ± 0.10.23 ± 0.013.0Surface mutantsE84Q120 ± 3112 ± 71.8 ± 0.30.23 ± 0.031.5E91Q69 ± 311 ± 21.5 ± 0.10.24 ± 0.052.2D280V73 ± 410 ± 11.2 ± 0.20.23 ± 0.031.6D283N84 ± 214 ± 61.8 ± 0.50.17 ± 0.012.1E292Q85 ± 5014 ± 91.6 ± 0.30.33 ± 0.111.9D372N62 ± 109.4 ± 0.31.2 ± 0.10.26 ± 0.031.9E84Q/E91Q131 ± 2016 ± 21.2 ± 0.10.17 ± 0.050.92D280V/D283N60 ± 918 ± 21.4 ± 0.20.23 ± 0.112.3E84Q/E91Q/D280V/D283N240 ± 5211 ± 41.1 ± 0.10.23 ± 0.040.46E84Q/E91Q/D280V/D283N/D372N162 ± 617 ± 70.82 ± 0.360.24 ± 0.090.51E84Q/E91Q/D280V/D283N/E292Q/D372N230 ± 3214 ± 70.54 ± 0.060.38 ± 0.000.23Active center mutantsD74N2-aData of Radić et al. (19).1,300 ± 140530 ± 1700.84 ± 0.1100.065E202Q200 ± 40140 ± 120.85 ± 0.0600.43E450Q2-bData of Hosea et al. (37).140 ± 1059 ± 220.034 ± 0.0041.8 ± 0.10.024D74N/E202Q700 ± 2918 ± 20.24 ± 0.034.9 ± 2.20.034D74N/E202Q/E450Q18,000 ± 2,5000.04 ± 0.010.00022Active center and surface mutantsD74N/D280V/D283N1,600 ± 320390 ± 1310.36 ± 0.1000.023Constants were obtained by nonlinear regression analysis of the following equation: v=(1+bS/Kss)(1+S/Kss)·V(1+Km/S)where S denotes the substrate concentration,K m and K ss Michaelis-Menten and substrate inhibition constants, and b the productivity ratio of the ternary SES complex to the ES complex (cf., Radić et al. (19Radić Z. Pickering N.A. Vellom D.C. Camp S. Taylor P. Biochemistry. 1993; 32: 12074-12084Crossref PubMed Scopus (411) Google Scholar)). Values are means of 2–5 separate measurements.2-a Data of Radić et al. (19Radić Z. Pickering N.A. Vellom D.C. Camp S. Taylor P. Biochemistry. 1993; 32: 12074-12084Crossref PubMed Scopus (411) Google Scholar).2-b Data of Hosea et al. (37Hosea N.A. Radić Z. Tsigelny I. Berman H.A. Quinn D.M. Taylor P. Biochemistry. 1996; 35: 10995-11004Crossref PubMed Scopus (70) Google Scholar). Open table in a new tab Constants were obtained by nonlinear regression analysis of the following equation: v=(1+bS/Kss)(1+S/Kss)·V(1+Km/S)where S denotes the substrate concentration,K m and K ss Michaelis-Menten and substrate inhibition constants, and b the productivity ratio of the ternary SES complex to the ES complex (cf., Radić et al. (19Radić Z. Pickering N.A. Vellom D.C. Camp S. Taylor P. Biochemistry. 1993; 32: 12074-12084Crossref PubMed Scopus (411) Google Scholar)). Values are means of 2–5 separate measurements. The Michaelis constant was found to increase, with ionic strength for wild-type and all mutants resulting in a decrease ink cat/K m. A modest increase ink cat for all the surface mutants and wild-type AChE was evident, whereas k cat for active center mutants was independent of ionic strength. Catalytic parameters for hydrolysis of neutral substrates (data not shown) did not show a dependence on ionic strength, with exception of K mand k cat for PhAc which show a slight increase at high ionic strength for wild-type AChE. The catalytic constants for active center mutants were similar to the constants for wild-type, with the exception of k cat which was reduced about an order of magnitude in the mutants. Of the three substrates, ATCh and PhAc have similark cat values that are significantly higher thank cat for pNPhAc, indicating a common rate-limiting step in the chemical step of catalysis, presumably deacylation. The acylation step appears slower than deacylation forpNPhAc (3Rosenberry T.L. Adv. Enzymol. Relat. Areas Mol. Biol. 1975; 43: 103-218PubMed Google Scholar). Substitution of active center anionic residues reduces k cat for ATCh and PhAc to the levels ofpNPhAc indicating a shift in the rate-limiting step to the acylation step for these substrates, with a concomitant loss of the ionic strength dependence. This suggests that the increases ofk cat for ATCh and PhAc at higher ionic strengths are due to an enhanced deacylation step of hydrolysis. Rates of deacetylation of mouse AChE thus depend on solvent-accessible charged moieties in the AChE active cent" @default.
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