Substrate Inhibition of Acetylcholinesterase: Residues Affecting Signal Transduction from the Surface to the Catalytic Center

Overview

Journal EMBO J

Specialties Biotechnology
Molecular Biology

Date 1992 Oct 1

PMID 1396557

Citations 41

Authors

A Shafferman

B Velan

A Ordentlich

C Kronman

H Grosfeld

M Leitner

Y Flashner

S Cohen

D Barak

N Ariel

Affiliations

Soon will be listed here.

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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References

McTiernan C, Adkins S, Chatonnet A, Vaughan T, Bartels C, Kott M . Brain cDNA clone for human cholinesterase. Proc Natl Acad Sci U S A. 1987; 84(19):6682-6. PMC: 299147. DOI: 10.1073/pnas.84.19.6682. View

Dennis M, Giraudat J, Goeldner M, Hirth C, Chang J, Lazure C . Amino acids of the Torpedo marmorata acetylcholine receptor alpha subunit labeled by a photoaffinity ligand for the acetylcholine binding site. Biochemistry. 1988; 27(7):2346-57. DOI: 10.1021/bi00407a016. View

Schumacher M, Camp S, Maulet Y, Newton M, Taylor S, Friedmann T . Primary structure of Torpedo californica acetylcholinesterase deduced from its cDNA sequence. Nature. 1986; 319(6052):407-9. DOI: 10.1038/319407a0. View

KRUPKA R . Hydrolysis of neutral substrates by acetylcholinesterase. Biochemistry. 1966; 5(6):1983-8. DOI: 10.1021/bi00870a028. View

Ellman G, COURTNEY K, ANDRES Jr V, FEATHER-STONE R . A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol. 1961; 7:88-95. DOI: 10.1016/0006-2952(61)90145-9. View

Gorman C, Moffat L, Howard B . Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982; 2(9):1044-51. PMC: 369897. DOI: 10.1128/mcb.2.9.1044-1051.1982. View

Changeux J . Responses of acetylcholinesterase from Torpedo marmorata to salts and curarizing drugs. Mol Pharmacol. 1966; 2(5):369-92. View

Berman H, Yguerabide J, Taylor P . Fluorescence energy transfer on acetylcholinesterase: spatial relationship between peripheral site and active center. Biochemistry. 1980; 19(10):2226-35. DOI: 10.1021/bi00551a036. View

Rosenberry T . Acetylcholinesterase. Adv Enzymol Relat Areas Mol Biol. 1975; 43:103-218. DOI: 10.1002/9780470122884.ch3. View

10.

Wigler M, Silverstein S, Lee L, Pellicer A, Cheng Y, Axel R . Transfer of purified herpes virus thymidine kinase gene to cultured mouse cells. Cell. 1977; 11(1):223-32. DOI: 10.1016/0092-8674(77)90333-6. View

11.

Taylor P, Lappi S . Interaction of fluorescence probes with acetylcholinesterase. The site and specificity of propidium binding. Biochemistry. 1975; 14(9):1989-97. DOI: 10.1021/bi00680a029. View

12.

Sussman J, Harel M, Frolow F, Oefner C, Goldman A, Toker L . Atomic structure of acetylcholinesterase from Torpedo californica: a prototypic acetylcholine-binding protein. Science. 1991; 253(5022):872-9. DOI: 10.1126/science.1678899. View

13.

Velan B, Grosfeld H, Kronman C, Leitner M, Gozes Y, Lazar 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; 266(35):23977-84. View

14.

Velan B, Kronman C, Grosfeld H, Leitner M, Gozes Y, Flashner Y . Recombinant human acetylcholinesterase is secreted from transiently transfected 293 cells as a soluble globular enzyme. Cell Mol Neurobiol. 1991; 11(1):143-56. PMC: 11567368. DOI: 10.1007/BF00712806. View

15.

Krejci E, Duval N, Chatonnet A, Vincens P, Massoulie 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; 88(15):6647-51. PMC: 52145. DOI: 10.1073/pnas.88.15.6647. View

16.

Radic Z, Reiner E, Taylor P . Role of the peripheral anionic site on acetylcholinesterase: inhibition by substrates and coumarin derivatives. Mol Pharmacol. 1991; 39(1):98-104. View

17.

Kreienkamp H, 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; 88(14):6117-21. PMC: 52033. DOI: 10.1073/pnas.88.14.6117. View

18.

Gibney G, Camp S, Dionne M, Taylor P . Mutagenesis of essential functional residues in acetylcholinesterase. Proc Natl Acad Sci U S A. 1990; 87(19):7546-50. PMC: 54784. DOI: 10.1073/pnas.87.19.7546. View

19.

Neville L, Gnatt A, Padan R, Seidman S, Soreq H . Anionic site interactions in human butyrylcholinesterase disrupted by two single point mutations. J Biol Chem. 1990; 265(34):20735-8. View

20.

Soreq H, Ben-Aziz R, Prody C, Seidman S, Gnatt A, Neville L . 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; 87(24):9688-92. PMC: 55238. DOI: 10.1073/pnas.87.24.9688. View