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. 1992 Oct;11(10):3561–3568. doi: 10.1002/j.1460-2075.1992.tb05439.x

Substrate inhibition of acetylcholinesterase: residues affecting signal transduction from the surface to the catalytic center.

A Shafferman 1, B Velan 1, A Ordentlich 1, C Kronman 1, H Grosfeld 1, M Leitner 1, Y Flashner 1, S Cohen 1, D Barak 1, N Ariel 1
PMCID: PMC556814  PMID: 1396557

Abstract

Amino acids located within and around the 'active site gorge' of human acetylcholinesterase (AChE) were substituted. Replacement of W86 yielded inactive enzyme molecules, consistent with its proposed involvement in binding of the choline moiety in the active center. A decrease in affinity to propidium and a concomitant loss of substrate inhibition was observed in D74G, D74N, D74K and W286A mutants, supporting the idea that the site for substrate inhibition and the peripheral anionic site overlap. Mutations of amino acids neighboring the active center (E202, Y337 and F338) resulted in a decrease in the catalytic and the apparent bimolecular rate constants. A decrease in affinity to edrophonium was observed in D74, E202, Y337 and to a lesser extent in F338 and Y341 mutants. E202, Y337 and Y341 mutants were not inhibited efficiently by high substrate concentrations. We propose that binding of acetylcholine, on the surface of AChE, may trigger sequence of conformational changes extending from the peripheral anionic site through W286 to D74, at the entrance of the 'gorge', and down to the catalytic center (through Y341 to F338 and Y337). These changes, especially in Y337, could block the entrance/exit of the catalytic center and reduce the catalytic efficiency of AChE.

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Selected References

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  1. Abramson S. N., Li Y., Culver P., Taylor P. An analog of lophotoxin reacts covalently with Tyr190 in the alpha-subunit of the nicotinic acetylcholine receptor. J Biol Chem. 1989 Jul 25;264(21):12666–12672. [PubMed] [Google Scholar]
  2. Berman H. A., Yguerabide J., Taylor P. Fluorescence energy transfer on acetylcholinesterase: spatial relationship between peripheral site and active center. Biochemistry. 1980 May 13;19(10):2226–2235. doi: 10.1021/bi00551a036. [DOI] [PubMed] [Google Scholar]
  3. Changeux J. P. Responses of acetylcholinesterase from Torpedo marmorata to salts and curarizing drugs. Mol Pharmacol. 1966 Sep;2(5):369–392. [PubMed] [Google Scholar]
  4. Dennis M., Giraudat J., Kotzyba-Hibert F., Goeldner M., Hirth C., Chang J. Y., Lazure C., Chrétien M., Changeux J. P. Amino acids of the Torpedo marmorata acetylcholine receptor alpha subunit labeled by a photoaffinity ligand for the acetylcholine binding site. Biochemistry. 1988 Apr 5;27(7):2346–2357. doi: 10.1021/bi00407a016. [DOI] [PubMed] [Google Scholar]
  5. Dougherty D. A., Stauffer D. A. Acetylcholine binding by a synthetic receptor: implications for biological recognition. Science. 1990 Dec 14;250(4987):1558–1560. doi: 10.1126/science.2274786. [DOI] [PubMed] [Google Scholar]
  6. ELLMAN G. L., COURTNEY K. D., ANDRES V., Jr, FEATHER-STONE R. M. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol. 1961 Jul;7:88–95. doi: 10.1016/0006-2952(61)90145-9. [DOI] [PubMed] [Google Scholar]
  7. Froede H. C., Wilson I. B., Kaufman H. Acetylcholinesterase: theory of noncompetitive inhibition. Arch Biochem Biophys. 1986 Jun;247(2):420–423. doi: 10.1016/0003-9861(86)90601-6. [DOI] [PubMed] [Google Scholar]
  8. Galzi J. L., Revah F., Black D., Goeldner M., Hirth C., Changeux J. P. Identification of a novel amino acid alpha-tyrosine 93 within the cholinergic ligands-binding sites of the acetylcholine receptor by photoaffinity labeling. Additional evidence for a three-loop model of the cholinergic ligands-binding sites. J Biol Chem. 1990 Jun 25;265(18):10430–10437. [PubMed] [Google Scholar]
  9. Gibney G., Camp S., Dionne M., MacPhee-Quigley K., Taylor P. Mutagenesis of essential functional residues in acetylcholinesterase. Proc Natl Acad Sci U S A. 1990 Oct;87(19):7546–7550. doi: 10.1073/pnas.87.19.7546. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kreienkamp H. J., Weise C., Raba R., Aaviksaar A., Hucho F. Anionic subsites of the catalytic center of acetylcholinesterase from Torpedo and from cobra venom. Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):6117–6121. doi: 10.1073/pnas.88.14.6117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Krejci E., Duval N., Chatonnet A., Vincens P., Massoulié J. Cholinesterase-like domains in enzymes and structural proteins: functional and evolutionary relationships and identification of a catalytically essential aspartic acid. Proc Natl Acad Sci U S A. 1991 Aug 1;88(15):6647–6651. doi: 10.1073/pnas.88.15.6647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Krupka R. M. Hydrolysis of neutral substrates by acetylcholinesterase. Biochemistry. 1966 Jun;5(6):1983–1988. doi: 10.1021/bi00870a028. [DOI] [PubMed] [Google Scholar]
  14. McGuire M. C., Nogueira C. P., Bartels C. F., Lightstone H., Hajra A., Van der Spek A. F., Lockridge O., La Du B. N. Identification of the structural mutation responsible for the dibucaine-resistant (atypical) variant form of human serum cholinesterase. Proc Natl Acad Sci U S A. 1989 Feb;86(3):953–957. doi: 10.1073/pnas.86.3.953. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. McTiernan C., Adkins S., Chatonnet A., Vaughan T. A., Bartels C. F., Kott M., Rosenberry T. L., La Du B. N., Lockridge O. Brain cDNA clone for human cholinesterase. Proc Natl Acad Sci U S A. 1987 Oct;84(19):6682–6686. doi: 10.1073/pnas.84.19.6682. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Neville L. F., Gnatt A., Padan R., Seidman S., Soreq H. Anionic site interactions in human butyrylcholinesterase disrupted by two single point mutations. J Biol Chem. 1990 Dec 5;265(34):20735–20738. [PubMed] [Google Scholar]
  17. Prody C. A., Zevin-Sonkin D., Gnatt A., Goldberg O., Soreq H. Isolation and characterization of full-length cDNA clones coding for cholinesterase from fetal human tissues. Proc Natl Acad Sci U S A. 1987 Jun;84(11):3555–3559. doi: 10.1073/pnas.84.11.3555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Radić Z., Reiner E., Taylor P. Role of the peripheral anionic site on acetylcholinesterase: inhibition by substrates and coumarin derivatives. Mol Pharmacol. 1991 Jan;39(1):98–104. [PubMed] [Google Scholar]
  19. Rosenberry T. L. Acetylcholinesterase. Adv Enzymol Relat Areas Mol Biol. 1975;43:103–218. doi: 10.1002/9780470122884.ch3. [DOI] [PubMed] [Google Scholar]
  20. Schumacher M., Camp S., Maulet Y., Newton M., MacPhee-Quigley K., Taylor S. S., Friedmann T., Taylor P. Primary structure of Torpedo californica acetylcholinesterase deduced from its cDNA sequence. 1986 Jan 30-Feb 5Nature. 319(6052):407–409. doi: 10.1038/319407a0. [DOI] [PubMed] [Google Scholar]
  21. Shafferman A., Velan B., Cohen S., Leitner M., Grosfeld H. Specific residues within an amino-terminal domain of 35 residues of interferon alpha are responsible for recognition of the human interferon alpha cell receptor and for triggering biological effects. J Biol Chem. 1987 May 5;262(13):6227–6237. [PubMed] [Google Scholar]
  22. Soreq H., Ben-Aziz R., Prody C. A., Seidman S., Gnatt A., Neville L., Lieman-Hurwitz J., Lev-Lehman E., Ginzberg D., Lipidot-Lifson Y. Molecular cloning and construction of the coding region for human acetylcholinesterase reveals a G + C-rich attenuating structure. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9688–9692. doi: 10.1073/pnas.87.24.9688. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sussman J. L., Harel M., Frolow F., Oefner C., Goldman A., Toker L., Silman I. Atomic structure of acetylcholinesterase from Torpedo californica: a prototypic acetylcholine-binding protein. Science. 1991 Aug 23;253(5022):872–879. doi: 10.1126/science.1678899. [DOI] [PubMed] [Google Scholar]
  24. Taylor P., Lappi S. Interaction of fluorescence probes with acetylcholinesterase. The site and specificity of propidium binding. Biochemistry. 1975 May 6;14(9):1989–1997. doi: 10.1021/bi00680a029. [DOI] [PubMed] [Google Scholar]
  25. Velan B., Grosfeld H., Kronman C., Leitner M., Gozes Y., Lazar A., Flashner Y., Marcus D., Cohen S., Shafferman A. The effect of elimination of intersubunit disulfide bonds on the activity, assembly, and secretion of recombinant human acetylcholinesterase. Expression of acetylcholinesterase Cys-580----Ala mutant. J Biol Chem. 1991 Dec 15;266(35):23977–23984. [PubMed] [Google Scholar]
  26. Velan B., Kronman C., Grosfeld H., Leitner M., Gozes Y., Flashner Y., Sery T., Cohen S., Ben-Aziz R., Seidman S. Recombinant human acetylcholinesterase is secreted from transiently transfected 293 cells as a soluble globular enzyme. Cell Mol Neurobiol. 1991 Feb;11(1):143–156. doi: 10.1007/BF00712806. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Wigler M., Silverstein S., Lee L. S., Pellicer A., Cheng Y. c., Axel R. Transfer of purified herpes virus thymidine kinase gene to cultured mouse cells. Cell. 1977 May;11(1):223–232. doi: 10.1016/0092-8674(77)90333-6. [DOI] [PubMed] [Google Scholar]

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