Skip to main content
SpringerLink
Log in

Redesigning enzyme structure by site-directed mutagenesis: tyrosyl tRNA synthetase and ATP binding

  • Letter
  • Published:

From Nature

View current issue Submit your manuscript

Abstract

We describe here a general method for systematically replacing amino acids in an enzyme. This allows analysis of their molecular roles in substrate binding or catalysis and could eventually lead to the engineering of new enzymatic activities. The gene encoding the enzyme is first cloned into a vector from which the enzyme is expressed and is then mutated in vitro to change a particular nucleotide and hence the amino acid sequence of the enzyme. We have cloned the gene for the tyrosyl tRNA synthetase of Bacillus stearothermophilus into a vector derived from the single-stranded bacteriophage M13 to facilitate mutagenesis with mismatched synthetic oligodeoxynucleotide primers. From the recombinant M13 clone we have obtained high levels of the enzyme (∼50% of soluble protein) expressed in the Escherichia coli host and have converted cysteine (Cys35) at the enzyme's active site to serine. This leads to a reduction in enzymatic activity that is largely attributable to a lower Km for ATP.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Loftfield, R. B. Prog. Nucleic Acids Res. 12, 87–128 (1972).

    Article  CAS  Google Scholar 

  2. Fersht, A. R. & Jakes, R. Biochemistry 14, 3350–3356 (1975).

    Article  CAS  Google Scholar 

  3. Winter, G., Hartley, B. S., Koch, G. L. E. & Barker, D. G. (in preparation).

  4. Barker, G. Eur. J. Biochem. 125, 357–360 (1982).

    Article  CAS  Google Scholar 

  5. Bhat, T. N., Blow, D. M., Brick, P. & Nyborg, J. J. molec. Biol. 158, 699–709 (1982).

    Article  CAS  Google Scholar 

  6. Monteilhet, C. & Blow, D. M. J. molec. Biol. 122, 407–417 (1978).

    Article  CAS  Google Scholar 

  7. Rubin, J. & Blow, D. M. J. molec. Biol. 145, 489–500 (1981).

    Article  CAS  Google Scholar 

  8. Winter, G. P., Koch, G. L. E., Dell, A. & Hartley, B. S. in Transfer RNA: Structure Properties and Recognition (eds Schimmel, P. R., Soll, D. & Abelson, J. N.) 255–265 (Cold Spring Harbor Laboratory, New York, 1979).

    Google Scholar 

  9. Barker, D. G. & Winter, G. FEBS Lett. 145, 191–193 (1982).

    Article  CAS  Google Scholar 

  10. Zoller, M. & Smith, M. Meth. Enzym. (in the press).

  11. Hutchison, C. A. III et al. J. biol. Chem. 253, 6551–6560 (1978).

    CAS  PubMed  Google Scholar 

  12. Smith, M. & Gillam, S. in Genetic Engineering Vol. 3 (eds Setlow, J. K. & Hollaender, A.) 1 (Plenum, New York, 1981).

    Book  Google Scholar 

  13. Wasylyk, B. et al. Proc. natn. Acad. Sci. U.S.A. 77, 7024–7028 (1980).

    Article  ADS  CAS  Google Scholar 

  14. Miyada, C. G., Soberon, X., Itakura, K. & Wilcox, G. Gene 17, 167–177 (1982).

    Article  CAS  Google Scholar 

  15. Gillam, S., Waterman, K. & Smith, M. Nucleic Acids Res. 2, 625–634 (1975).

    Article  CAS  Google Scholar 

  16. Wallace, R. B., Schold, M., Johnson, M. J., Dembek, P. & Itakura, K. Nucleic Acids Res. 9, 3647–3657 (1981).

    Article  CAS  Google Scholar 

  17. Suggs, S. V., Wallace, R. B., Hirose, T., Kawashima, E. & Itakura, K. Proc. natn. Acad. Sci. U.S.A. 78, 6613–6617 (1982).

    Article  ADS  Google Scholar 

  18. Sanger, F., Nicklen, S. & Coulson, A. R. Proc. natn. Acad. Sci. U.S.A. 74, 5463–5467 (1977).

    Article  ADS  CAS  Google Scholar 

  19. Clewell, D. B. & Helinski, D. R. J. Bact. 110, 1135–1146 (1972).

    CAS  PubMed  Google Scholar 

  20. Hohn, B., Lechner, H. & Marvin, D. A. J. molec. Biol. 56, 143–154 (1971).

    Article  CAS  Google Scholar 

  21. Barker, D. G., Bruton, C. J. & Winter, G. (in preparation).

  22. McElroy, W. D., De Luca, M. & Travis, J. Science 157, 151–160 (1967).

    ADS  Google Scholar 

  23. Fersht, A. R. Biochemistry 14, 5–12 (1975).

    Article  CAS  Google Scholar 

  24. Kerr, A. K., Ashmore, J. P. & Koetzle, T. F. Acta crystallogr. B31, 2022–2026 (1975).

    Article  Google Scholar 

  25. Kistenmacher, T. J., Rand, G. A. & Marsh, R. E. Acta Crystallogr. B30, 2573–2578 (1974).

    Article  Google Scholar 

  26. Crampton, M. R. in Chemistry of the −SH group (ed. Patai, S.) 379–415 (Wiley-Inter-science, New York, 1974).

    Google Scholar 

  27. Paul, I. C. in Chemistry of the −SH group (ed. Patai, S.) 111–149 (Wiley-Interecience, New York, 1974).

    Google Scholar 

  28. Fersht, A. R. & Dingwall, C. Biochemistry 18, 1245–1249 (1979).

    Article  CAS  Google Scholar 

  29. Heinrikson, R. L. & Hartley, B. S. Biochem. J. 105, 17–24 (1967).

    Article  CAS  Google Scholar 

  30. Sutcliffe, J. G. Cold Spring Harb. Symp. quant. Biol. 43, 77–90 (1978).

    Article  Google Scholar 

  31. Hong, G. F. Biosci. Rep. 1, 243–252 (1981).

    Article  CAS  Google Scholar 

  32. Messing, J. Recombinant DNA Tech. Bull. 2, 43–48 (1979).

    CAS  Google Scholar 

  33. Cohen, S. N., Chang, A. C. Y. & Hsu, L. Proc. natn. Acad. Sci. U.S.A. 69, 2110–2114 (1972).

    Article  ADS  CAS  Google Scholar 

  34. Winter, G. & Fields, S. Nucleic Acids Res. 8, 1965–1974 (1980).

    Article  CAS  Google Scholar 

  35. Grunstein, M. & Hogness, D. S. Proc. natn. Acad. Sci. U.S.A. 72, 3961–3965 (1975).

    Article  ADS  CAS  Google Scholar 

  36. Denhardt, D. T. Biochem. biophys. Res. Commun. 23, 641–646 (1966).

    Article  CAS  Google Scholar 

  37. Laemmli, U. K. Nature 227, 680–683 (1970).

    Article  ADS  CAS  Google Scholar 

  38. Atkinson, A. et al. J. appl. Biochem. 1, 247–258 (1979).

    CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK

    Greg Winter

  2. Department of Chemistry, Imperial College of Science and Technology, London, SW7 2AY, UK

    Alan R. Fersht & Anthony J. Wilkinson

  3. Department of Biochemistry, Faculty of Medicine, University of British Columbia, 2146 Health Sciences Mall, Vancouver, British Columbia, Canada, V6T 1W5

    Mark Zoller & Michael Smith

Authors
  1. Greg Winter
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Alan R. Fersht
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Anthony J. Wilkinson
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Mark Zoller
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Michael Smith
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Winter, G., Fersht, A., Wilkinson, A. et al. Redesigning enzyme structure by site-directed mutagenesis: tyrosyl tRNA synthetase and ATP binding. Nature 299, 756–758 (1982). https://doi.org/10.1038/299756a0

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/299756a0

  • Springer Nature Limited

This article is cited by

Search

Navigation