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
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on SpringerLink
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Kohen, A. & Limbach, H.-H. Isotope Effects in Chemistry and Biology (Taylor & Francis, Boca Raton, 2006).
Schramm, V.L. J. Biol. Chem. 282, 28297–28300 (2007).
Taylor, E.A. et al. J. Am. Chem. Soc. 129, 6984–6985 (2007).
Melander, L.C.S. & Saunders, W.H.J. Reaction Rates of Isotopic Molecules (Wiley, New York, 1980).
Berti, P.J., Blanke, S.R. & Schramm, V.L. J. Am. Chem. Soc. 119, 12079–12088 (1997).
Singleton, D.A. & Thomas, A.A. J. Am. Chem. Soc. 117, 9357–9358 (1995).
Lee, J.K., Bain, A.D. & Berti, P.J. J. Am. Chem. Soc. 126, 3769–3776 (2004).
Schimerlik, M.I., Rife, J.E. & Cleland, W.W. Biochemistry 14, 5347–5354 (1975).
Guo, X. & Sinnott, M.L. Biochem. J. 294, 653–656 (1993).
Berger, S. & Braun, S. 200 and More NMR Experiments: A Practical Course 128 (Wiley-VCH, Weinheim, 2004).
Berger, S. & Braun, S. 200 and More NMR Experiments: A Practical Course 160 (Wiley-VCH, Weinheim, 2004).
Wilson, J.C., Angus, D.I. & von Itzstein, M. J. Am. Chem. Soc. 117, 4214–4217 (1995).
Davies, G., Sinnott, M.L. & Withers, S.G. Glycosyl transfer. in Comprehensive Biological Catalysis (ed. Sinnott, M.L.) 119–209 (Academic, San Diego, 1998).
Friebolin, H., Supp, M., Brossmer, R., Keilich, G. & Ziegler, D. Angew. Chem. Int. Ed. Engl. 19, 208–209 (1980).
Kim, M.J., Hennen, W.J., Sweers, H.M. & Wong, C.H. J. Am. Chem. Soc. 110, 6481–6486 (1988).
Corfield, A.P., Higa, H., Paulson, J.C. & Schauer, R. Biochim. Biophys. Acta 744, 121–126 (1983).
Watson, J.N., Dookhun, V., Borgford, T.J. & Bennet, A.J. Biochemistry 42, 12682–12690 (2003).
Watts, A.G., Oppezzo, P., Withers, S.G., Alzari, P.M. & Buschiazzo, A. J. Biol. Chem. 281, 4149–4155 (2006).
Davies, G. & Henrissat, B. Structure 3, 853–859 (1995).
Zechel, D.L. & Withers, S.G. Acc. Chem. Res. 33, 11–18 (2000).
Huang, X.C., Tanaka, K.S.E. & Bennet, A.J. J. Am. Chem. Soc. 119, 11147–11154 (1997).
Tanaka, Y., Tao, W., Blanchard, J.S. & Hehre, E.J. J. Biol. Chem. 269, 32306–32312 (1994).
Indurugalla, D. & Bennet, A.J. J. Am. Chem. Soc. 123, 10889–10898 (2001).
Guo, X., Laver, W.G., Vimr, E. & Sinnott, M.L. J. Am. Chem. Soc. 116, 5572–5578 (1994).
Vocadlo, D.J. & Davies, G.J. Curr. Opin. Chem. Biol. 12, 539–555 (2008).
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
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
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
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nchembio.352
This article is cited by
-
Dissecting the mechanisms of a class of chemical glycosylation using primary 13C kinetic isotope effects
Nature Chemistry (2012)
-
Mechanistic evidence for a front-side, SNi-type reaction in a retaining glycosyltransferase
Nature Chemical Biology (2011)