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

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.

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

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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.

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Authors and Affiliations

Authors

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.

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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)

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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

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