Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Brief Communication
  • Published:

A direct NMR method for the measurement of competitive kinetic isotope effects

Nature Chemical Biology volume 6, pages 405–407 (2010)Cite this article

Subjects

Abstract

We present a technique that uses 13C NMR spectroscopy to measure kinetic isotope effects on the second-order rate constant (kcat/Km) for enzyme-catalyzed reactions. Using only milligram quantities of isotopically labeled substrates, precise competitive KIEs can be determined while following the ongoing reaction directly in a NMR spectrometer. Our results for the Vibrio cholerae sialidase–catalyzed hydrolysis of natural substrate analogs support a concerted enzymatic transition state for these reactions.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Direct NMR spectroscopic measurement of kinetic isotope effects.
Figure 2: Substrate structures and reaction time-courses for KIE measurements.

Similar content being viewed by others

References

  1. Kohen, A. & Limbach, H.-H. Isotope Effects in Chemistry and Biology (Taylor & Francis, Boca Raton, 2006).

  2. Schramm, V.L. J. Biol. Chem. 282, 28297–28300 (2007).

    Article  CAS  Google Scholar 

  3. Taylor, E.A. et al. J. Am. Chem. Soc. 129, 6984–6985 (2007).

    Article  CAS  Google Scholar 

  4. Melander, L.C.S. & Saunders, W.H.J. Reaction Rates of Isotopic Molecules (Wiley, New York, 1980).

  5. Berti, P.J., Blanke, S.R. & Schramm, V.L. J. Am. Chem. Soc. 119, 12079–12088 (1997).

    Article  CAS  Google Scholar 

  6. Singleton, D.A. & Thomas, A.A. J. Am. Chem. Soc. 117, 9357–9358 (1995).

    Article  CAS  Google Scholar 

  7. Lee, J.K., Bain, A.D. & Berti, P.J. J. Am. Chem. Soc. 126, 3769–3776 (2004).

    Article  CAS  Google Scholar 

  8. Schimerlik, M.I., Rife, J.E. & Cleland, W.W. Biochemistry 14, 5347–5354 (1975).

    Article  CAS  Google Scholar 

  9. Guo, X. & Sinnott, M.L. Biochem. J. 294, 653–656 (1993).

    Article  CAS  Google Scholar 

  10. Berger, S. & Braun, S. 200 and More NMR Experiments: A Practical Course 128 (Wiley-VCH, Weinheim, 2004).

  11. Berger, S. & Braun, S. 200 and More NMR Experiments: A Practical Course 160 (Wiley-VCH, Weinheim, 2004).

  12. Wilson, J.C., Angus, D.I. & von Itzstein, M. J. Am. Chem. Soc. 117, 4214–4217 (1995).

    Article  CAS  Google Scholar 

  13. Davies, G., Sinnott, M.L. & Withers, S.G. Glycosyl transfer. in Comprehensive Biological Catalysis (ed. Sinnott, M.L.) 119–209 (Academic, San Diego, 1998).

  14. Friebolin, H., Supp, M., Brossmer, R., Keilich, G. & Ziegler, D. Angew. Chem. Int. Ed. Engl. 19, 208–209 (1980).

    Article  Google Scholar 

  15. Kim, M.J., Hennen, W.J., Sweers, H.M. & Wong, C.H. J. Am. Chem. Soc. 110, 6481–6486 (1988).

    Article  CAS  Google Scholar 

  16. Corfield, A.P., Higa, H., Paulson, J.C. & Schauer, R. Biochim. Biophys. Acta 744, 121–126 (1983).

    Article  CAS  Google Scholar 

  17. Watson, J.N., Dookhun, V., Borgford, T.J. & Bennet, A.J. Biochemistry 42, 12682–12690 (2003).

    Article  CAS  Google Scholar 

  18. Watts, A.G., Oppezzo, P., Withers, S.G., Alzari, P.M. & Buschiazzo, A. J. Biol. Chem. 281, 4149–4155 (2006).

    Article  CAS  Google Scholar 

  19. Davies, G. & Henrissat, B. Structure 3, 853–859 (1995).

    Article  CAS  Google Scholar 

  20. Zechel, D.L. & Withers, S.G. Acc. Chem. Res. 33, 11–18 (2000).

    Article  CAS  Google Scholar 

  21. Huang, X.C., Tanaka, K.S.E. & Bennet, A.J. J. Am. Chem. Soc. 119, 11147–11154 (1997).

    Article  CAS  Google Scholar 

  22. Tanaka, Y., Tao, W., Blanchard, J.S. & Hehre, E.J. J. Biol. Chem. 269, 32306–32312 (1994).

    CAS  PubMed  Google Scholar 

  23. Indurugalla, D. & Bennet, A.J. J. Am. Chem. Soc. 123, 10889–10898 (2001).

    Article  CAS  Google Scholar 

  24. Guo, X., Laver, W.G., Vimr, E. & Sinnott, M.L. J. Am. Chem. Soc. 116, 5572–5578 (1994).

    Article  CAS  Google Scholar 

  25. Vocadlo, D.J. & Davies, G.J. Curr. Opin. Chem. Biol. 12, 539–555 (2008).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors thank W. Wakarchuk and M. Schur (Institute for Biological Sciences, National Research Council Canada, Ottawa) for providing the labeled 2,3-sialosides. This work was financially supported by the Natural Sciences and Engineering Research Council of Canada.

Author information

Authors and Affiliations

  1. Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, Canada

    Jefferson Chan, Andrew R Lewis & Andrew J Bennet

  2. Institute for Biological Sciences, National Research Council Canada, Ottawa, Ontario, Canada

    Michel Gilbert & Marie-France Karwaski

Authors
  1. Jefferson Chan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Andrew R Lewis
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Michel Gilbert
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Marie-France Karwaski
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Andrew J Bennet
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.C., all labeled 2,6-sialoside syntheses and NMR measurements; A.R.L., NMR expertise; M.G., supervisor of enzyme production; M.-F.K., expression and purification of sialyltransferases; A.J.B., project planning and design.

Corresponding author

Correspondence to Andrew J Bennet.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Methods, Supplementary Results, Supplementary Figures 1–4, Supplementary Schemes 1–2 and Supplementary Tables 1–4 (PDF 513 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chan, J., Lewis, A., Gilbert, M. et al. A direct NMR method for the measurement of competitive kinetic isotope effects. Nat Chem Biol 6, 405–407 (2010). https://doi.org/10.1038/nchembio.352

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.352

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing