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 sialidaseĝ€"catalyzed hydrolysis of natural substrate analogs support a concerted enzymatic transition state for these reactions.
|Original language||English (US)|
|Number of pages||3|
|Journal||Nature chemical biology|
|State||Published - Jun 2010|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology