We present a technique that uses (13)C NMR spectroscopy to measure kinetic isotope effects on the second-order rate constant (k(cat)/K(m)) 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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| http://dx.doi.org/10.1038/nchembio.352 | DOI Listing |
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