Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1990 Oct;87(19):7546–7550. doi: 10.1073/pnas.87.19.7546

Mutagenesis of essential functional residues in acetylcholinesterase.

G Gibney 1, S Camp 1, M Dionne 1, K MacPhee-Quigley 1, P Taylor 1
PMCID: PMC54784  PMID: 2217185

Abstract

The cholinesterases are serine hydrolases that show no global similarities in sequence with either the trypsin or the subtilisin family of serine proteases. The cholinesterase superfamily includes several esterases with distinct functions and other proteins devoid of the catalytic serine and known esterase activity. To identify the residues involved in catalysis and conferring specificity on the enzyme, we have expressed wild-type Torpedo acetylcholinesterase (EC 3.1.1.7) and several site-directed mutants in a heterologous system. Mutation of serine-200 to cysteine results in diminished activity, while its mutation to valine abolishes detectable activity. Two conserved histidines can be identified at positions 425 and 440 in the cholinesterase family; glutamine replacement at position 440 eliminates activity whereas the mutation at 425 reduces activity only slightly. The assignment of the catalytic histidine to position 440 defines a rank ordering of catalytic residues in cholinesterases distinct from trypsin and subtilisin and suggests a convergence of a catalytic triad to form a third, distinct family of serine hydrolases. Mutation of glutamate-199 to glutamine yields an enzyme with a higher Km and without the substrate-inhibition behavior characteristic of acetylcholinesterase. Hence, modification of the acidic amino acid adjacent to the serine influences substrate association and the capacity of a second substrate molecule to affect catalysis.

Full text

PDF
7546

Images in this article

7547Image
on p.7547

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bazan J. F., Fletterick R. J. Viral cysteine proteases are homologous to the trypsin-like family of serine proteases: structural and functional implications. Proc Natl Acad Sci U S A. 1988 Nov;85(21):7872–7876. doi: 10.1073/pnas.85.21.7872. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bomblies L., Biegelmann E., Döring V., Gerisch G., Krafft-Czepa H., Noegel A. A., Schleicher M., Humbel B. M. Membrane-enclosed crystals in Dictyostelium discoideum cells, consisting of developmentally regulated proteins with sequence similarities to known esterases. J Cell Biol. 1990 Mar;110(3):669–679. doi: 10.1083/jcb.110.3.669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chatonnet A., Lockridge O. Comparison of butyrylcholinesterase and acetylcholinesterase. Biochem J. 1989 Jun 15;260(3):625–634. doi: 10.1042/bj2600625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Claudio T., Green W. N., Hartman D. S., Hayden D., Paulson H. L., Sigworth F. J., Sine S. M., Swedlund A. Genetic reconstitution of functional acetylcholine receptor channels in mouse fibroblasts. Science. 1987 Dec 18;238(4834):1688–1694. doi: 10.1126/science.3686008. [DOI] [PubMed] [Google Scholar]
  5. Craik C. S., Roczniak S., Largman C., Rutter W. J. The catalytic role of the active site aspartic acid in serine proteases. Science. 1987 Aug 21;237(4817):909–913. doi: 10.1126/science.3303334. [DOI] [PubMed] [Google Scholar]
  6. Doctor B. P., Chapman T. C., Christner C. E., Deal C. D., De La Hoz D. M., Gentry M. K., Ogert R. A., Rush R. S., Smyth K. K., Wolfe A. D. Complete amino acid sequence of fetal bovine serum acetylcholinesterase and its comparison in various regions with other cholinesterases. FEBS Lett. 1990 Jun 18;266(1-2):123–127. doi: 10.1016/0014-5793(90)81522-p. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Epstein D. J., Berman H. A., Taylor P. Ligand-induced conformational changes in acetylcholinesterase investigated with fluorescent phosphonates. Biochemistry. 1979 Oct 16;18(21):4749–4754. doi: 10.1021/bi00588a040. [DOI] [PubMed] [Google Scholar]
  9. Froede H. C., Wilson I. B. Direct determination of acetyl-enzyme intermediate in the acetylcholinesterase-catalyzed hydrolysis of acetylcholine and acetylthiocholine. J Biol Chem. 1984 Sep 10;259(17):11010–11013. [PubMed] [Google Scholar]
  10. Gibney G., Taylor P. Biosynthesis of Torpedo acetylcholinesterase in mammalian cells. Functional expression and mutagenesis of the glycophospholipid-anchored form. J Biol Chem. 1990 Jul 25;265(21):12576–12583. [PubMed] [Google Scholar]
  11. Hall L. M., Spierer P. The Ace locus of Drosophila melanogaster: structural gene for acetylcholinesterase with an unusual 5' leader. EMBO J. 1986 Nov;5(11):2949–2954. doi: 10.1002/j.1460-2075.1986.tb04591.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Han J. H., Stratowa C., Rutter W. J. Isolation of full-length putative rat lysophospholipase cDNA using improved methods for mRNA isolation and cDNA cloning. Biochemistry. 1987 Mar 24;26(6):1617–1625. doi: 10.1021/bi00380a020. [DOI] [PubMed] [Google Scholar]
  13. Higaki J. N., Evnin L. B., Craik C. S. Introduction of a cysteine protease active site into trypsin. Biochemistry. 1989 Nov 28;28(24):9256–9263. doi: 10.1021/bi00450a004. [DOI] [PubMed] [Google Scholar]
  14. Korza G., Ozols J. Complete covalent structure of 60-kDa esterase isolated from 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced rabbit liver microsomes. J Biol Chem. 1988 Mar 5;263(7):3486–3495. [PubMed] [Google Scholar]
  15. Krupka R. M. Hydrolysis of neutral substrates by acetylcholinesterase. Biochemistry. 1966 Jun;5(6):1983–1988. doi: 10.1021/bi00870a028. [DOI] [PubMed] [Google Scholar]
  16. Lockridge O., Bartels C. F., Vaughan T. A., Wong C. K., Norton S. E., Johnson L. L. Complete amino acid sequence of human serum cholinesterase. J Biol Chem. 1987 Jan 15;262(2):549–557. [PubMed] [Google Scholar]
  17. Long R. M., Satoh H., Martin B. M., Kimura S., Gonzalez F. J., Pohl L. R. Rat liver carboxylesterase: cDNA cloning, sequencing, and evidence for a multigene family. Biochem Biophys Res Commun. 1988 Oct 31;156(2):866–873. doi: 10.1016/s0006-291x(88)80924-0. [DOI] [PubMed] [Google Scholar]
  18. MacPhee-Quigley K., Vedvick T. S., Taylor P., Taylor S. S. Profile of the disulfide bonds in acetylcholinesterase. J Biol Chem. 1986 Oct 15;261(29):13565–13570. [PubMed] [Google Scholar]
  19. Majumdar R., Balasubramanian A. S. Chemical modification of acetylcholinesterase from eel and basal ganglia: effect on the acetylcholinesterase and aryl acylamidase activities. Biochemistry. 1984 Aug 28;23(18):4088–4093. doi: 10.1021/bi00313a012. [DOI] [PubMed] [Google Scholar]
  20. Matthews D. A., Alden R. A., Birktoft J. J., Freer T., Kraut J. Re-examination of the charge relay system in subtilisin comparison with other serine proteases. J Biol Chem. 1977 Dec 25;252(24):8875–8883. [PubMed] [Google Scholar]
  21. Maulet Y., Camp S., Gibney G., Rachinsky T. L., Ekström T. J., Taylor P. Single gene encodes glycophospholipid-anchored and asymmetric acetylcholinesterase forms: alternative coding exons contain inverted repeat sequences. Neuron. 1990 Feb;4(2):289–301. doi: 10.1016/0896-6273(90)90103-m. [DOI] [PubMed] [Google Scholar]
  22. McGrath M. E., Wilke M. E., Higaki J. N., Craik C. S., Fletterick R. J. Crystal structures of two engineered thiol trypsins. Biochemistry. 1989 Nov 28;28(24):9264–9270. doi: 10.1021/bi00450a005. [DOI] [PubMed] [Google Scholar]
  23. Mercken L., Simons M. J., Swillens S., Massaer M., Vassart G. Primary structure of bovine thyroglobulin deduced from the sequence of its 8,431-base complementary DNA. Nature. 1985 Aug 15;316(6029):647–651. doi: 10.1038/316647a0. [DOI] [PubMed] [Google Scholar]
  24. Myers M., Richmond R. C., Oakeshott J. G. On the origins of esterases. Mol Biol Evol. 1988 Mar;5(2):113–119. doi: 10.1093/oxfordjournals.molbev.a040485. [DOI] [PubMed] [Google Scholar]
  25. Neet K. E., Koshland D. E., Jr The conversion of serine at the active site of subtilisin to cysteine: a "chemical mutation". Proc Natl Acad Sci U S A. 1966 Nov;56(5):1606–1611. doi: 10.1073/pnas.56.5.1606. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Okayama H., Berg P. A cDNA cloning vector that permits expression of cDNA inserts in mammalian cells. Mol Cell Biol. 1983 Feb;3(2):280–289. doi: 10.1128/mcb.3.2.280. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Polgar L., Bender M. L. The reactivity of thiol-subtilisin, an enzyme containing a synthetic functional group. Biochemistry. 1967 Feb;6(2):610–620. doi: 10.1021/bi00854a032. [DOI] [PubMed] [Google Scholar]
  28. Rosenberry T. L. Acetylcholinesterase. Adv Enzymol Relat Areas Mol Biol. 1975;43:103–218. doi: 10.1002/9780470122884.ch3. [DOI] [PubMed] [Google Scholar]
  29. Roskoski R., Jr Choline acetyltransferase and acetylcholinesterase: evidence for essential histidine residues. Biochemistry. 1974 Dec 3;13(25):5141–5144. doi: 10.1021/bi00722a014. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Takagi Y., Morohashi K., Kawabata S., Go M., Omura T. Molecular cloning and nucleotide sequence of cDNA of microsomal carboxyesterase E1 of rat liver. J Biochem. 1988 Nov;104(5):801–806. doi: 10.1093/oxfordjournals.jbchem.a122553. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES