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• W2059358513 abstract "The contribution of the oxyanion hole to the functional architecture and to the hydrolytic efficiency of human acetylcholinesterase (HuAChE) was investigated through single replacements of its elements, residues Gly-121, Gly-122 and the adjacent residue Gly-120, by alanine. All three substitutions resulted in about 100-fold decrease of the bimolecular rate constants for hydrolysis of acetylthiocholine; however, whereas replacements of Gly-120 and Gly-121 affected only the turnover number, mutation of residue Gly-122 had an effect also on the Michaelis constant. The differential behavior of the G121A and G122A enzymes was manifested also toward the transition state analogm-(N,N,N-trimethylammonio)trifluoroacetophenone (TMTFA), organophosphorous inhibitors, carbamates, and toward selected noncovalent active center ligands. Reactivity of both mutants toward TMTFA was 2000–11,000-fold lower than that of the wild type HuAChE; however, the G121A enzyme exhibited a rapid inhibition pattern, as opposed to the slow binding kinetics shown by the G122A enzyme. For both phosphates (diethyl phosphorofluoridate, diisopropyl phosphorofluoridate, and paraoxon) and phosphonates (sarin and soman), the decrease in inhibitory activity toward the G121A enzyme was very substantial (2000–6700-fold), irrespective of size of the alkoxy substituents on the phosphorus atom. On the other hand, for the G122A HuAChE the relative decline in reactivity toward phosphonates (500–460-fold) differed from that toward the phosphates (12–95-fold). Although formation of Michaelis complexes with substrates does not seem to involve significant interaction with the oxyanion hole, interactions with this motif are a major stabilizing element in accommodation of covalent inhibitors like organophosphates or carbamates. These observations and molecular modeling suggest that replacements of residues Gly-120 or Gly-121 by alanine alter the structure of the oxyanion hole motif, abolishing the H-bonding capacity of residue at position 121. These mutations weaken the interaction between HuAChE and the various ligands by 2.7–5.0 kcal/mol. In contrast, variations in reactivity due to replacement of residue Gly-122 seem to result from steric hindrance at the active center acyl pocket. The contribution of the oxyanion hole to the functional architecture and to the hydrolytic efficiency of human acetylcholinesterase (HuAChE) was investigated through single replacements of its elements, residues Gly-121, Gly-122 and the adjacent residue Gly-120, by alanine. All three substitutions resulted in about 100-fold decrease of the bimolecular rate constants for hydrolysis of acetylthiocholine; however, whereas replacements of Gly-120 and Gly-121 affected only the turnover number, mutation of residue Gly-122 had an effect also on the Michaelis constant. The differential behavior of the G121A and G122A enzymes was manifested also toward the transition state analogm-(N,N,N-trimethylammonio)trifluoroacetophenone (TMTFA), organophosphorous inhibitors, carbamates, and toward selected noncovalent active center ligands. Reactivity of both mutants toward TMTFA was 2000–11,000-fold lower than that of the wild type HuAChE; however, the G121A enzyme exhibited a rapid inhibition pattern, as opposed to the slow binding kinetics shown by the G122A enzyme. For both phosphates (diethyl phosphorofluoridate, diisopropyl phosphorofluoridate, and paraoxon) and phosphonates (sarin and soman), the decrease in inhibitory activity toward the G121A enzyme was very substantial (2000–6700-fold), irrespective of size of the alkoxy substituents on the phosphorus atom. On the other hand, for the G122A HuAChE the relative decline in reactivity toward phosphonates (500–460-fold) differed from that toward the phosphates (12–95-fold). Although formation of Michaelis complexes with substrates does not seem to involve significant interaction with the oxyanion hole, interactions with this motif are a major stabilizing element in accommodation of covalent inhibitors like organophosphates or carbamates. These observations and molecular modeling suggest that replacements of residues Gly-120 or Gly-121 by alanine alter the structure of the oxyanion hole motif, abolishing the H-bonding capacity of residue at position 121. These mutations weaken the interaction between HuAChE and the various ligands by 2.7–5.0 kcal/mol. In contrast, variations in reactivity due to replacement of residue Gly-122 seem to result from steric hindrance at the active center acyl pocket. The catalytic efficiency of acetylcholinesterase (AChE, 1The abbreviations used are: AChE, acetylcholinesterase; HuAChE, human acetylcholinesterase; TcAChE,Torpedo californica acetylcholinesterase; ATC, acetylthiocholine; TB, 3,3-dimethylbutyl thioacetate; DFP, diisopropyl phosphorofluoridate; DEFP, diethyl phosphorofluoridate; paraoxon,p-nitrophenyl diethyl phosphate; TMTFA,m-(N,N,N-trimethylammonio)trifluoro-acetophenone. EC 3.1.1.7) and its high reactivity toward a variety of covalent and noncovalent inhibitors seem to originate from the unique architecture of the active center, currently investigated by x-ray crystallography (1Sussman J.L. Harel M. Frolow F. Oefner C. Goldman A. Toker L. Silman I. Science. 1991; 253: 872-879Crossref PubMed Scopus (2440) Google Scholar, 2Harel M. Schalk I. Ehret-Sabatier 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, 3Harel M. Quinn D.M. Nair H.K. Silman I. Sussman J.L. J. Am. Chem. Soc. 1996; 118: 2340-2346Crossref Scopus (344) Google Scholar, 4Raves 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) and site-directed mutagenesis (5Shafferman A. Kronman C. Flashner Y. Leitner M. Grosfeld H. Ordentlich A. Gozes Y. Cohen S. Ariel N. Barak D. Harel M. Silman I. Sussman J.L. Velan B. J. Biol. Chem. 1992; 267: 17640-17648Abstract Full Text PDF PubMed Google Scholar, 6Ordentlich A. Barak D. Kronman C. Flashner Y. Leitner M. Segall Y. Ariel N. Cohen S. Velan B. Shafferman A. J. Biol. Chem. 1993; 268: 17083-17095Abstract Full Text PDF PubMed Google Scholar, 7Vellom D.C. Radic Z. Li Y. Pickering S.N. Camp A. Taylor P. Biochemistry. 1993; 32: 12-17Crossref PubMed Scopus (260) Google Scholar, 8Barak D. Kronman C. Ordentlich A. Ariel N. Bromberg A. Marcus D. Lazar A. Velan B. Shafferman A. J. Biol. Chem. 1994; 269: 6296-6305Abstract Full Text PDF PubMed Google Scholar, 9Taylor P. Radic Z. Annu. Rev. Pharmacol. Toxicol. 1994; 34: 281-320Crossref PubMed Scopus (609) Google Scholar, 10Ordentlich A. Barak D. Kronman C. Ariel N. Segall Y. Velan B. Shafferman A. J. Biol. Chem. 1996; 271: 11953-11962Abstract Full Text PDF PubMed Scopus (106) Google Scholar). The x-ray structures of AChE are characterized by a deep and narrow “gorge,” which penetrates halfway into the enzyme and contains the catalytic site at about 4 Å from its base (1Sussman J.L. Harel M. Frolow F. Oefner C. Goldman A. Toker L. Silman I. Science. 1991; 253: 872-879Crossref PubMed Scopus (2440) Google Scholar). Several functional subsites in the active center gorge were identified, including the catalytic triad (Ser-203(200), 2Amino acids and numbers refer to HuAChE, and the numbers in parentheses refer to the positions of analogous residues in TcAChE according to the recommended nomenclature (55Massoulie J. Sussman J.L. Doctor B.P. Soreq H. Velan B. Cygler M. Rotundo R. Shafferman A. Silman I. Taylor P. Shafferman A. Velan B. Multidisciplinary Approaches to Cholinesterase Functions. Plenum Publishing Corp., New York1992: 285-288Crossref Google Scholar). His-447(440), and Glu-334(327)) (1Sussman J.L. Harel M. Frolow F. Oefner C. Goldman A. Toker L. Silman I. Science. 1991; 253: 872-879Crossref PubMed Scopus (2440) Google Scholar, 5Shafferman A. Kronman C. Flashner Y. Leitner M. Grosfeld H. Ordentlich A. Gozes Y. Cohen S. Ariel N. Barak D. Harel M. Silman I. Sussman J.L. Velan B. J. Biol. Chem. 1992; 267: 17640-17648Abstract Full Text PDF PubMed Google Scholar, 11Gibney G. Camp S. Dionne M. MacPhee-Quigley K. Taylor P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7546-7550Crossref PubMed Scopus (90) Google Scholar, 12Shafferman A. Velan B. Ordentlich A. Kronman C. Grosfeld H. Leitner M. Flashner Y. Cohen S. Barak D. Ariel N. EMBO J. 1992; 11: 3561-3568Crossref PubMed Scopus (200) Google Scholar), the acyl pocket (Phe-295 (288) and Phe-297(290)) (6Ordentlich A. Barak D. Kronman C. Flashner Y. Leitner M. Segall Y. Ariel N. Cohen S. Velan B. Shafferman A. J. Biol. Chem. 1993; 268: 17083-17095Abstract Full Text PDF PubMed Google Scholar, 7Vellom D.C. Radic Z. Li Y. Pickering S.N. Camp A. Taylor P. Biochemistry. 1993; 32: 12-17Crossref PubMed Scopus (260) Google Scholar, 9Taylor P. Radic Z. Annu. Rev. Pharmacol. Toxicol. 1994; 34: 281-320Crossref PubMed Scopus (609) Google Scholar), and the “hydrophobic subsite.” The latter accommodates the alcohol portion of the covalent adduct (tetrahedral intermediate) and may include residues Trp-86(84), Tyr-133(130), Tyr-337(330), and Phe-338(331), which operate through nonpolar and/or stacking interactions, depending on the substrate (6Ordentlich A. Barak D. Kronman C. Flashner Y. Leitner M. Segall Y. Ariel N. Cohen S. Velan B. Shafferman A. J. Biol. Chem. 1993; 268: 17083-17095Abstract Full Text PDF PubMed Google Scholar, 10Ordentlich A. Barak D. Kronman C. Ariel N. Segall Y. Velan B. Shafferman A. J. Biol. Chem. 1996; 271: 11953-11962Abstract Full Text PDF PubMed Scopus (106) Google Scholar, 13Ordentlich A. Barak D. Kronman C. Ariel N. Segall Y. Velan B. Shafferman A. J. Biol. Chem. 1995; 270: 2082-2091Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Stabilization of the charged moieties of substrates and other ligands at the active center is mediated by cation-π interactions with the residue at position 86 rather than through true ionic interactions (1Sussman J.L. Harel M. Frolow F. Oefner C. Goldman A. Toker L. Silman I. Science. 1991; 253: 872-879Crossref PubMed Scopus (2440) Google Scholar, 2Harel M. Schalk I. Ehret-Sabatier 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, 6Ordentlich A. Barak D. Kronman C. Flashner Y. Leitner M. Segall Y. Ariel N. Cohen S. Velan B. Shafferman A. J. Biol. Chem. 1993; 268: 17083-17095Abstract Full Text PDF PubMed Google Scholar, 12Shafferman A. Velan B. Ordentlich A. Kronman C. Grosfeld H. Leitner M. Flashner Y. Cohen S. Barak D. Ariel N. EMBO J. 1992; 11: 3561-3568Crossref PubMed Scopus (200) Google Scholar,13Ordentlich A. Barak D. Kronman C. Ariel N. Segall Y. Velan B. Shafferman A. J. Biol. Chem. 1995; 270: 2082-2091Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Another important component of the AChE active center functional architecture is an arrangement of hydrogen bond donors that can stabilize the tetrahedral transition enzyme-substrate complex through accommodation of the negatively charged carbonyl oxygen (14Kraut J. Annu. Rev. Biochem. 1977; 46: 331-358Crossref PubMed Scopus (1079) Google Scholar). Structural and modeling studies (1Sussman J.L. Harel M. Frolow F. Oefner C. Goldman A. Toker L. Silman I. Science. 1991; 253: 872-879Crossref PubMed Scopus (2440) Google Scholar, 6Ordentlich A. Barak D. Kronman C. Flashner Y. Leitner M. Segall Y. Ariel N. Cohen S. Velan B. Shafferman A. J. Biol. Chem. 1993; 268: 17083-17095Abstract Full Text PDF PubMed Google Scholar, 15Barak D. Ariel N. Velan B. Shafferman A. Shafferman A. Velan B. Multidisciplinary Approaches to Cholinesterase Functions. Plenum Publishing Corp., New York1992: 195-199Crossref Google Scholar) and, in particular, the recent solution of the x-ray structure of the transition state analog TMTFA complexed with TcAChE (3Harel M. Quinn D.M. Nair H.K. Silman I. Sussman J.L. J. Am. Chem. Soc. 1996; 118: 2340-2346Crossref Scopus (344) Google Scholar) revealed a three-pronged oxyanion hole formed by peptidic NH groups Gly-121(118), Gly-122(119), and Ala-204(201), in contrast to the two-pronged oxyanion holes in most of serine and cysteine proteases (16Ménard R. Storer A. Biol. Chem. Hoppe-Seyler. 1992; 373: 393-400Crossref PubMed Scopus (92) Google Scholar). The contribution of the amide nitrogen of Ala-204, rather than that of the catalytic Ser-203, to the oxyanion hole is consistent with the reverse handedness of the catalytic triads in AChE compared with serine proteases (1Sussman J.L. Harel M. Frolow F. Oefner C. Goldman A. Toker L. Silman I. Science. 1991; 253: 872-879Crossref PubMed Scopus (2440) Google Scholar). Residues Gly-121, Gly-122, and Gly-120 are part of a flexible “glycine loop” which constitutes one of the gorge walls adjacent to the catalytic serine and, due to the narrow dimensions of the gorge bottom, should be in contact with most of the AChE noncovalent ligands (1Sussman J.L. Harel M. Frolow F. Oefner C. Goldman A. Toker L. Silman I. Science. 1991; 253: 872-879Crossref PubMed Scopus (2440) Google Scholar, 2Harel M. Schalk I. Ehret-Sabatier 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, 4Raves 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). The notion that, apart from its role in accommodating the oxyanion, this loop is one of the important determinants of the active center geometry was recently supported by the x-ray structure of the huperzine A-TcAChE complex (4Raves 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). In this structure the conformation of Gly-121 is different from that observed in other TcAChE-ligand complexes, demonstrating the flexibility of the loop and the extent of its interaction with the ligand. Such conformational mobility of the glycine loop implies that its function may be modified by single replacements of Gly-120(117), Gly-121(118), and Gly-122(119). It was reported (17Millard C.B. Lockridge O. Broomfield C.A. Biochemistry. 1995; 34: 15925-15933Crossref PubMed Scopus (104) Google Scholar, 18Loewenstein-Lichtenstein Y. Glick D. Gluzman N. Sternfeld M. Zakut H. Soreq H. Mol. Pharmacol. 1996; 50: 1423-1431PubMed Google Scholar) that replacements of some of the analogous glycine residues in butyrylcholinesterase resulted in enzyme (G115(117)A) exhibiting diminished affinity toward tacrine, but not toward BW284C51, or in enzymes (G117(119)E and the G117(119)H) retaining a nearly wild type catalytic activity toward butyrylthiocholine (BTC). In HuAChE, our past attempts to introduce large residues (e.g. histidine, glutamate, and serine) at position 122 yielded no protein, underscoring the different active center void volumes of the two enzymes. Here we describe the construction of HuAChE enzymes with modified oxyanion hole, through substitution of Gly-121 and Gly-122 by alanine, and examine their reactivity toward substrates and a variety of covalent and noncovalent active center inhibitors. We show that reactivity of the mutants is indeed affected mainly by steric and conformational changes in the glycine loop. Furthermore, the similar changes in catalytic activity due to replacement of the adjacent residue Gly-120, which is not part of the oxyanion hole, may be also attributed to conformational mobility of the glycine loop. These results further define the functional architecture of HuAChE-active center and its role in the enzyme reactivity. Mutagenesis of AChE was performed by DNA cassette replacement into a HuAChE sequence variant (Ew4) which conserves the wild type (22Soreq H. Ben-Aziz R. Prody C.A. Seidman S. Gnatt A. Neville A. Lieman- Hurwitz J. Lev-Lehman E. Ginzberg D. Seidman S. Lapidot-Lifson Y. Zakut H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9688-9692Crossref PubMed Scopus (182) Google Scholar) coding specificity but carries new unique restriction sites (5Shafferman A. Kronman C. Flashner Y. Leitner M. Grosfeld H. Ordentlich A. Gozes Y. Cohen S. Ariel N. Barak D. Harel M. Silman I. Sussman J.L. Velan B. J. Biol. Chem. 1992; 267: 17640-17648Abstract Full Text PDF PubMed Google Scholar). Substitution of residues G120A and G121A and G122A was performed by replacement of an EspI-NarI DNA fragment with synthetic DNA duplexes carrying codon GCC(Ala) at the corresponding mutated positions. All the synthetic DNA oligodeoxynucleotides were prepared using the automatic Applied Biosystems DNA synthesizer. The sequences of all new clones were verified by the dideoxy sequencing method (U. S. Biochemical Corp. Sequenase kit). The recombinant HuAChE mutants were expressed in tripartite vectors which allow expression of the catreporter gene and the neo selection marker (5Shafferman A. Kronman C. Flashner Y. Leitner M. Grosfeld H. Ordentlich A. Gozes Y. Cohen S. Ariel N. Barak D. Harel M. Silman I. Sussman J.L. Velan B. J. Biol. Chem. 1992; 267: 17640-17648Abstract Full Text PDF PubMed Google Scholar, 23Kronman C. Velan B. Gozes Y. Leitner M. Flashner Y. Lazar A. Marcus D. Sery T. Papier A. Grosfeld H. Cohen S. Shafferman A. Gene ( Amst .). 1992; 121: 295-304Crossref PubMed Scopus (73) Google Scholar). Recombinant HuAChE and its mutants were expressed in HEK 293 cells as described previously (24Velan B. Grosfeld H. Kronman C. Leitner M. Gozes Y. Lazar A. Flashner Y. Marcus D. Cohen S. Shafferman A. J. Biol. Chem. 1991; 266: 23977-23984Abstract Full Text PDF PubMed Google Scholar) using stable recombinant cell clones expressing high levels of each of the mutants (23Kronman C. Velan B. Gozes Y. Leitner M. Flashner Y. Lazar A. Marcus D. Sery T. Papier A. Grosfeld H. Cohen S. Shafferman A. Gene ( Amst .). 1992; 121: 295-304Crossref PubMed Scopus (73) Google Scholar). Structures of the various AChE ligands are shown in Fig. 1. Acetylthiocholine iodide (ATC), ethyl(m-hydroxyphenyl)dimethylammonium chloride (edrophonium), di(p-allyl-N-methylaminophenyl)pentane-3-one (BW284C51), diisopropyl phosphorofluoridate (DFP),p-nitrophenyl diethylphosphate (paraoxon), physostygmine, and pyridostigmine were purchased from Sigma. S-3,3-Dimethylbutyl thioacetate (TB) was synthesized as described previously (6Ordentlich A. Barak D. Kronman C. Flashner Y. Leitner M. Segall Y. Ariel N. Cohen S. Velan B. Shafferman A. J. Biol. Chem. 1993; 268: 17083-17095Abstract Full Text PDF PubMed Google Scholar). Diethyl phosphorofluoridate (DEFP) was prepared according to the procedure by Saunders and Stacy (19Saunders B.C. Stacy G.J. J. Chem. Soc. (Lond.). 1948; : 695-699Crossref PubMed Google Scholar). Preparation of 2-propyl methylphosphonofluoridate (sarin) and 1,2,2-trimethylpropyl methylphosphonofluoridate (soman) followed an accepted synthetic procedure using methylphosphonodifluoride (20Monard C. Quinchon J. Bull. Soc. Chim. Fr. 1961; : 1084-1085Google Scholar) and the appropriate alcohol. m-(N,N,N-Trimethylammonio)trifluoroacetophenone (TMTFA) was prepared according to the procedure described by Nairet al. (21Nair H.K. Lee K. Quinn D.M. J. Am. Chem. Soc. 1993; 115: 9939-9941Crossref Scopus (52) Google Scholar). AChE activity was assayed according to Ellman et al. (25Ellman G.L. Courtney K.D. Andres V. Featherstone R.M. Biochem. Pharmacol. 1961; 7: 88-95Crossref PubMed Scopus (21719) Google Scholar) (in the presence of 0.1 mg/ml bovine serum albumin, 0.3 mm5,5′-dithiobis(2-nitrobenzoic acid), 50 mm sodium-phosphate buffer, pH 8.0, and various concentrations of ATC), carried out at 27 °C, and monitored by a Thermomax microplate reader (Molecular Devices). Values of inhibition constants (Ki) for the noncovalent inhibitors edrophonium, tacrine, and huperzine A were determined from the effects of various concentrations of the inhibitor on Km and Vmax of the enzyme-catalyzed hydrolysis of ATC. All the HuAChE enzymes examined formed rapid equilibria with the tested inhibitors, allowing for an immediate addition of increasing amounts of enzyme to the ATC/inhibitor mixture (preincubation of the enzymes with huperzine A for 10 min, before addition of the substrate, or simultaneous mixing yielded the same results). The values of Ki were computed from the secondary plots of the values ofKm/Vmax (determined from slopes of 1/V versus 1/[S]) versusconcentrations of the respective inhibitors as described previously (26Ashani Y. Grunewald J. Kronman C. Velan B. Shafferman A. Mol. Pharmacol. 1994; 45: 555-560PubMed Google Scholar). The rate constants of progression of the carbamylation reactions (see Scheme 1) were estimated for at least four different concentrations (and at least 10-fold in ligand concentration, around the estimated value of Kd) of carbamate (CX), by adding substrate at various time intervals, and measuring the enzyme residual activity (E). The apparent bimolecular carbamylation rate constants (ki), at different carbamate concentrations, were computed from the plot of slopes of ln(E) versus time. Only the initial slopes were considered in order to minimize the errors due to reactivation of the carbamylated enzymes. Double-reciprocal plot of kiversus [CX] were used to compute k2and Kd from the intercept and from the ratio of the slope and the intercept, respectively, according to the following equations: 1/k = 1/k2 + 1/ki[CX]; Kd =k2/ki (27Aldrich W.N. Reiner E. Enzyme Inhibitors as Substrates. Elsevier/North-Holland Biomedical Press, Amsterdam1972: 245Google Scholar). Note that whenk−1 ≫k2,Kd approaches the value of dissociation constant for the corresponding Michaelis complex. Determination of the apparent bimolecular rate constants (ki) for the irreversible inhibition of HuAChE enzymes by organophosphates and organophosphonates as well as estimation of the dissociation constants Kd and the first-order phosphorylation rate constants (k2) for paraoxon and DFP were carried out as described before (10Ordentlich A. Barak D. Kronman C. Ariel N. Segall Y. Velan B. Shafferman A. J. Biol. Chem. 1996; 271: 11953-11962Abstract Full Text PDF PubMed Scopus (106) Google Scholar, 13Ordentlich A. Barak D. Kronman C. Ariel N. Segall Y. Velan B. Shafferman A. J. Biol. Chem. 1995; 270: 2082-2091Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). The apparent first-order rate constants for the time-dependent inhibition of the wild type and the G122A HuAChE enzymes by TMTFA were determined by periodically measuring the initial rate of substrate hydrolysis of aliquots of the reaction mixture. The inhibitor concentrations used were 7.5–75 nmfor the wild type and 5.0–50 μm for the G122A HuAChEs. Following the kinetic treatment of Nair et al. (21Nair H.K. Lee K. Quinn D.M. J. Am. Chem. Soc. 1993; 115: 9939-9941Crossref Scopus (52) Google Scholar) and assuming a two-state inhibition mechanism (Scheme 2), the values ofkon and koff could be estimated from the linear plots of kobs versus inhibitor concentration according to Equation1. kobs=kon′[TMTFA]+koffEquation 1 E+TMTFAket⇌koffkonE­TMTFAScheme 2 Since in aqueous solution TMTFA is a mixture of the free ketone (TMTFAket) and the ketone hydrate (TMTFAhyd), corrected values of the association rate constants were obtained from kon = k′on (1 + [TMTFAhyd]/[TMTFAket]), using the ratio of hydrated and ketone forms of TMTFA (62,500), as determined by19F NMR (21Nair H.K. Lee K. Quinn D.M. J. Am. Chem. Soc. 1993; 115: 9939-9941Crossref Scopus (52) Google Scholar). To measure directly the values of koff, the enzyme was inhibited by excess TMTFA (over 90% inhibition), and the mixture was filtered rapidly through a column (Ultrafree-Biomax30k, Millipore) to remove free inhibitor. Regeneration of enzymatic activity for the wild type and the G122A enzymes followed first-order kinetics, yielding values of the dissociation rate constants (koff). For adduct of the G121A HuAChE, activity was completely restored within processing time required for removal of the free inhibitor. TMTFA behaved toward this enzyme as a rapid reversible inhibitor, and the corresponding inhibition constant (Ki) could be determined as described above for noncovalent inhibitors. Models of the tetrahedral adducts of wild type, G120A, G121A, and G122A HuAChEs with ATC and TMTFA were performed on an Indigo 2 workstation using SYBYL modeling software (Tripos Inc.). Initial models of the substrate adducts were constructed as described before (6Ordentlich A. Barak D. Kronman C. Flashner Y. Leitner M. Segall Y. Ariel N. Cohen S. Velan B. Shafferman A. J. Biol. Chem. 1993; 268: 17083-17095Abstract Full Text PDF PubMed Google Scholar), and those of TMTFA were built in analogy to the x-ray structure of the TcAChE-TMTFA conjugate (3Harel M. Quinn D.M. Nair H.K. Silman I. Sussman J.L. J. Am. Chem. Soc. 1996; 118: 2340-2346Crossref Scopus (344) Google Scholar). The resulting structures were optimized by molecular mechanics using the AMBER and the MAXMIN force fields (with AMBER charge parameters for the enzyme). For most of the starting geometries, for adducts of the G120A and G121A enzymes, a conformational flip of residue at position 121 occurred during the optimization process. Replacement of residue Gly-121 by alanine resulted in an enzyme with a 100-fold lower value of the turnover number (kcat) for both ATC and its noncharged analog TB (Table I; for structures see Fig. 1), as compared with the wild type HuAChE. On the other hand, this substitution had only a limited effect on the Km values for both substrates. Past studies with these isosteric substrates demonstrated that in their respective Michaelis complexes with HuAChE the alkoxy substituents are accommodated in a different manner (6Ordentlich A. Barak D. Kronman C. Flashner Y. Leitner M. Segall Y. Ariel N. Cohen S. Velan B. Shafferman A. J. Biol. Chem. 1993; 268: 17083-17095Abstract Full Text PDF PubMed Google Scholar). Thus, the lack of significant effect on the values of Km for either substrate may suggest that during the formation of Michaelis complexes there is no significant stabilization due to interaction of the substrate carbonyl moieties with the oxyanion hole. Replacement of the second oxyanion hole element Gly-122 by alanine also resulted in similar decreases in the values of kcat for ATC and TB (18- and 15-fold, respectively). However, for this enzyme theKm value for ATC was also affected (6-fold), whereas practically no effect was observed on the corresponding value for TB (Table I). As in the case of the G121A enzyme the nearly equivalent decrease in the turnover numbers for both substrates indicates that the structural modification of the oxyanion hole affects mainly interactions with the substrate acyl moiety. Replacement of Gly-120 by alanine was carried out assuming that although this residue is not a constituent of the oxyanion hole, its substitution may affect the conformation of the glycine loop. Indeed, the effects on the catalytic parameters for ATC are similar to those observed for the G121A enzyme (see Table I), suggesting that the structure of the loop may be similarly affected by the two replacements. However, since the poorly expressed G120A enzyme could be obtained only in extremely low quantities and due to the limited solubility of TB in water, kinetic studies with the noncharged substrate could not be carried out.Table IKinetic constants for ATC and TB hydrolysis by HuAChE and its derivativesAChE typeKmkcatkappaThe apparent bimolecular rate constant (kapp) was calculated from the ratiokcat/Km.ΔΔG bΔΔG # represents the change in free energy relative to the wild type enzyme required to reach the transition state (ES#), evaluated from the effect of mutation on the value of kapp.ATCTBATCTBATCTBATCTBmm×10−5 min−1× 10−8 m−1 min−1kcal/molWT0.10.334.00.3400.9G120A0.05—cLow availability of the G121A enzyme and limitation of TB aqueous solubility precluded examination of this reaction.0.06—cLow availability of the G121A enzyme and limitation of TB aqueous solubility precluded examination of this reaction.1.2—cLow availability of the G121A enzyme and limitation of TB aqueous solubility precluded examination of this reaction.2.09—cLow availability of the G121A enzyme and limitation of TB aqueous solubility precluded examination of this reaction.G121A0.10.90.040.0030.40.0032.753.4G122A0.570.440.220.020.40.052.751.72Values represent mean of triplicate determinations with standard deviation not exceeding 20%.a The apparent bimolecular rate constant (kapp) was calculated from the ratiokcat/Km.b ΔΔG # represents the change in free energy relative to the wild type enzyme required to reach the transition state (ES#), evaluated from the effect of mutation on the value of kapp.c Low availability of the G121A enzyme and limitation of TB aqueous solubility precluded examination of this reaction. Open table in a new tab Values represent mean of triplicate determinations with standard deviation not exceeding 20%. The kinetics of AChE inhibition by TMTFA has been studied extensively since the tetrahedral covalent adduct is believed to mimic the transition state of the acylation process (21Nair H.K. Lee K. Quinn D.M. J. Am. Chem. Soc. 1993; 115: 9939-9941Crossref Scopus (52) Google Scholar, 28Brodbeck U. Schweikert K. Gentinetta R. Rottenberg M. Biochim. Biophys. Acta. 1979; 567: 357-369Crossref PubMed Scopus (77) Google Scholar, 29Nair H.K. Seravalli J. Arbuckle T. Quinn D.M. Biochemistry. 1994; 33: 8566-8576Crossref PubMed Scopus (80) Google Scholar). TMTFA was shown to behave as a tight binding time-dependent inhibitor, a behavior characteristic of other trifluoroketone inhibitors of serine proteases (30Brady K. Abeles R.H. Biochemistry." @default.
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• W2059358513 title "Functional Characteristics of the Oxyanion Hole in Human Acetylcholinesterase" @default.
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