Biochemical Genetics

, Volume 31, Issue 7–8, pp 259–278 | Cite as

Effects of the residue adjacent to the reactive serine on the substrate interactions ofDrosophila esterase 6

  • Mark A. Myers
  • Marion J. Healy
  • John G. Oakeshott
Article

Abstract

Esterase 6 fromDrosophila melanogaster is a carboxylesterase that belongs to the serine esterase multigene family. It has a basic histidine (His) at residue 187, adjacent to the reactive serine (Ser) at residue 188, whereas most other characterized members of the family have an acidic glutamate (Glu) in the equivalent position. We have used site-directedin vitro mutagenesis to replace the His codon of the esterase 6 gene with either Gln or Glu codons. The enzymes encoded by these active-site mutants and a wild-type control have been expressed, purified, and characterized. Substitution of Gln for His at position 187 has little effect on the biochemical properties of esterase 6, but the presence of Glu at this position is associated with three major differences. First, the pH optimum is increased from 7 to 9. Second, the mutant enzyme shows decreased activity for β-naphthyl esters andp-nitrophenyl acetate but has gained the ability to hydrolyze acetylthiocholine. Finally, the Gibb’s free energy of activation for the enzyme is increased. These results suggest that residue 187 interacts directly with the substrate alkyl group and that this interaction is fully realized in the transition state. We further propose that the presence of His rather than Glu at position 187 in esterase 6 contributes significantly to its functional divergence from the cholinesterases and that this divergence is due to different interactions between residue 187 and the substrate alkyl group.

Key words

serine esterase substrate interactions Drosophila acetylcholine 

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References

  1. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein dye binding.Anal. Biochem. 22248.CrossRefGoogle Scholar
  2. Brady, P., Richmond, R. C., and Oakeshott, J. G. (1990). Cloning of the esterase 5 locus fromDrosophila pseudoobscura and comparison with its homologue inD. melanogaster.Mol. Biol. Evol. 7525.PubMedGoogle Scholar
  3. Brot, F. E., and Bender, M. L. (1969). Use of the specificity constant of α-chymotrypsin.J. Am. Chem. Soc. 917187.CrossRefGoogle Scholar
  4. Collet, C., Nielson, K. M., Russell, R. J., Karl, M., Oakeshott, J. G., and Richmond, R. C. (1990). Molecular analysis of duplicated esterase genes inDrosophila melanogaster.Mol. Biol. Evol. 79.PubMedGoogle Scholar
  5. Cooke, P. H. (1989).Electrophoretic and Nucleotide Polymorphism at the Esterase 6 Locus of D. melanogaster Ph.D. thesis, Australian National University, Canberra.Google Scholar
  6. Craik, C. S., Roczniak, S., Largman, C., and Rutter, W. J. (1987). The catalytic role of the active site aspartic acid in serine proteases.Science 237909.PubMedGoogle Scholar
  7. Dixon, M., and Webb, E. C. (1964).Enzymes 2nd ed., Academic Press New York.Google Scholar
  8. Doctor, B. P., Chapman, T. C., Christener, C. E., Ded, C. D., De la Hoz, D. M., Gentry, M. K., Ogert, R. A., Rush, R. S., Smyth, K. K., and Wolfe, A. D. (1990). Complete amino acid sequence of fetal bovine serum acetylcholinesterase and its comparison in various regions with other cholinesterases.FEBS Lett. 266123.CrossRefPubMedGoogle Scholar
  9. Ellman, G. L., Courtney, K. D., Andres, V., Jr., and Featherstone, R. M. (1961). A new and rapid colourimetric determination of acetylcholinesterase activity.Biochem. Pharmacol. 788.CrossRefPubMedGoogle Scholar
  10. Fersht, A. R., and Leatherbarrow, R. J. (1987). Structure and activity of the tyrosyl-tRNA synthetase. In Oxender, D. L., and Fox, F. C. (eds.),Protein Engineering Alan R. Liss, New York, pp. 267–278.Google Scholar
  11. Fest, C., and Schmidt, K. J. (1973).The Chemistry of Organophosphorus Pesticides Springer, New York.Google Scholar
  12. Gibney, G., Camp, S., Dionne, M., Macphee-Quigley, K., and Taylor, P. (1990). Mutagenesis of essential functional residues in acetylcholinesterase.Proc. Natl. Acad. Sci. USA 877546.PubMedGoogle Scholar
  13. Gnagey, A. L., Forte, M., and Rosenberry, T. L. (1987). Isolation and characterisation of acetylcholinesterase from Drosophila.J. Biol. Chem. 26213290.PubMedGoogle Scholar
  14. Graf, L., Jancso, L., Szilagyi, L., Hegyi, G., Pinter, K., Naray-Szako, G., Hepp, J., Medzihradszky, K., and Rutter, W. J. (1988). Electrostatic complementarity within the substrate-binding pocket of trypsin.Proc. Natl. Acad. Sci. USA 854961.PubMedGoogle Scholar
  15. Hammock, B. D., and Roe, R. H. (1985). Analysis of juvenile hormone esterase activity. In Law, J. H., and Rilling, H. C. (eds.),Methods in Enzymology Academic Press, Orlando, FL, pp. 487–494.Google Scholar
  16. Hanzlik, T. N., Abdel-Aal, Y. A. I., Harshman, L. G., and Hammock, B. D. (1989). Isolation and sequencing of cDNA clones coding for JH esterase from Heliothis virescens.J. Biol. Chem. 26412419.PubMedGoogle Scholar
  17. Hata, Y., Matsuura, Y., Tanaka, N., Kakudo, M., Sugihara, A., Iwai, M., and Tsujisaka, Y. (1979). Low resolution crystal structure of lipase fromGeotrichum candidum (ATCC34614).J. Biochem. 861821.PubMedGoogle Scholar
  18. Kieffer, B., Goeldner, M., Hirth, C., Aebersold, R., and Chang, J. Y. (1986). Sequence determination of a peptide fragment from the electric eel acetylcholinesterase, involved in the binding of quaternary ammonium.FEBS Lett. 20291.CrossRefGoogle Scholar
  19. Krejci, E., Duval, N., Chatonnet, A., Vincens, P., and Massoulie, J. (1991). Cholinesterase-like domains in enzymes and structural proteins: Functional and evolutionary relationships and identification of a catalytically essential aspartic acid.Proc. Natl. Acad. Sci. USA 886647.PubMedGoogle Scholar
  20. Kyger, E. M., Wiegand, R. C., and Lange, L. G. (1989). Cloning of the bovine pancreatic cholesterol esterase/lysophospholipase.Biochem. Biophys. Res. Comm. 1641302.CrossRefPubMedGoogle Scholar
  21. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 227680.CrossRefPubMedGoogle Scholar
  22. Mane, S. D., Tepper, C. S., and Richmond, R. C. (1983). Studies of esterase-6 inDrosophila melanogaster. XIII. Purification and characterisation of the two major isozymes.Biochem. Genet. 211019.CrossRefPubMedGoogle Scholar
  23. Mastrapaolo, W., and Yourno, J. (1981). An ultra-violet spectrophotometric assay for α-naphthylacetate and α-naphthylbutyrate esterases.Anal. Biochem. 115188.CrossRefGoogle Scholar
  24. McGuire, M. C., Nogueira, C. P., Bartels, C. F., Lightstone, H., van der Spek, A. F. L., Lockridge, O., and La Du, B. N. (1989). Identification of the structural mutation responsible for the dibucaine-resistant (atypical) variant form of human serum cholinesterase.Proc. Natl. Acad. Sci. USA 86953.PubMedGoogle Scholar
  25. Mouches, C., Pauplin, Y., Agarwal, M., Lemieux, L., Herzog, M., Abadon, M., Beyssat-Arnaouty, V., Hyrien, O., de Saint Vincent, B. R., Georghiou, G. P., and Pasteur, N. (1990).Proc. Natl. Acad. Sci USA 872574.PubMedGoogle Scholar
  26. Myers, M. A. (1990).Structure Function Relationships of Esterase 6 from Drosophila melanogaster, Ph.D. thesis, Australian National University, Canberra.Google Scholar
  27. Neville, L. F., Gnatt, A., Padan, R., Seidman, S., and Soreq, H. (1990). Anionic site interactions in human butyrylcholinesterase disrupted by two single point mutations.J. Biol. Chem. 26520735.PubMedGoogle Scholar
  28. Oakeshott, J. G., Collet, C., Phillis, R. W., Nielson, K. M., Russell, R. J., Chambers, G. K., Ross, V., and Richmond, R. C. (1987). Molecular cloning and characterisation of esterase-6, a serine hydrolase of Drosophila.Proc. Natl. Acad. Sci. USA 843359.PubMedGoogle Scholar
  29. Price, N. C., and Stevens, L. (1982).Fundamentals of Enzymology Oxford University Press, Oxford.Google Scholar
  30. Quinn, D. M. (1987). Acetylcholinesterase: Enzyme structure, reaction dynamics and virtual transition state.Chem. Rev. 87955.CrossRefGoogle Scholar
  31. Robertson, H. M., Preston, C. R., Phillis, R. W., Johnson-Sohlez, D. M., Benz, W. K., and Engels, W. R. (1988). A stable genomic source of P-element transposase inD. melanogaster.Genetics 118461.PubMedGoogle Scholar
  32. Rosenberry, T. L. (1975). Acetylcholinesterase.Adv. Enzymol. 43103.PubMedGoogle Scholar
  33. Rubin, G. M., and Spradling, A. C. (1982). Genetic transformation of Drosophila with transposable element vectors.Science 218348.PubMedGoogle Scholar
  34. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989).Molecular Cloning: A Laboratory Manual Cold Spring Harbor, Cold Spring Harbor, NY.Google Scholar
  35. Sanger, F., Nicklen, S., and Coulson, A. R. (1977). DNA sequencing with chain terminating inhibitors.Proc. Natl. Acad. Sci. USA 745463.PubMedGoogle Scholar
  36. Schrag, J. D., Simid, M. F., Morgan, D. G., Phillips, G. N., Jr., Chiu, W., and Tang, L. (1988). Crystallisation and preliminary X-ray diffraction analysis of 11s acetylcholinesterase.J. Mol. Biol. 2639795.Google Scholar
  37. Schrag, J. D., Yunge, L., Wu, S., and Cygler, M. (1991). Ser-His-Glu triad forms the catalytic site of the lipase fromGeotrichum candidum.Nature 351761.CrossRefPubMedGoogle Scholar
  38. Sergeev, P. V., Castilo, J. E., Peunova, N. I., and Yenikolopov, G. N. (1990). Primary structure of the esterase S gene ofDrosophila virilis.Bioorgan. Khim. 15839.Google Scholar
  39. Shafferman, 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., and Velan, B. (1992). Mutagenesis of human acetylcholinesterase.J. Biol. Chem. 26717640.PubMedGoogle Scholar
  40. Sheehan, K., Richmond, R. C., and Cochrane, B. J. (1979). Studies of esterase-6 in Drosophila melanogaster. III. The developmental pattern and tissue distribution.Insect Biochem. 9443.CrossRefGoogle Scholar
  41. Shimada, Y., Sugihara, A., Iizumi, T., and Tominaga, Y. (1990). cDNA cloning and characterisation ofGeotrichum candidum lipase II.J. Biochem. 107703.PubMedGoogle Scholar
  42. Sussman, J. L., Harel, M., Frolow, F., Oefner, C., Goldman, A., Toker, L., and Silman, I. (1991). Atomic structure of acetylcholinesterase fromTorpedo californica: A prototypic acetylcholine-binding protein.Science 253872.PubMedGoogle Scholar
  43. Taylor, J. W., Ott, J., and Eckstein, F. (1985). In vitro mutagenesis.Nucl. Acids. Res. 138764.Google Scholar
  44. Vernick, K. D., Collins, F. H., Seeley, D. C., Gwadz, R. W., and Miller, L. H. (1988). The genetics and expression of an esterase locus inAnopheles gambiae.Biochem. Genet. 26367.CrossRefPubMedGoogle Scholar
  45. White, M. W., Mane, S. D., and Richmond, R. C. (1988). Studies of esterase-6 inDrosophila melanogaster. XVIII. Biochemical differences between slow and fast allozymes.Mol. Biol. Evol. 541.PubMedGoogle Scholar
  46. Wray, W., Boulikas, T., Wray, V. P., and Hancock, R. (1981). Silver staining of proteins in polyacrylamide gels.Anal. Biochem. 118197.CrossRefPubMedGoogle Scholar

Copyright information

© Plenum Publishing Corporation 1993

Authors and Affiliations

  • Mark A. Myers
    • 1
    • 2
  • Marion J. Healy
    • 1
  • John G. Oakeshott
    • 1
  1. 1.CSIRO Division of EntomologyCanberraAustralia
  2. 2.Botany DepartmentAustralian National UniversityCanberraAustralia

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