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

Nat Chem Biol. 2010 Jun;6(6):405-7. doi: 10.1038/nchembio.352. Epub 2010 Apr 25.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / metabolism
  • Carbon Isotopes / metabolism
  • Catalysis
  • Enzymes / metabolism
  • Hydrolysis
  • Isotope Labeling
  • Isotopes / metabolism*
  • Kinetics
  • Magnetic Resonance Spectroscopy / methods*
  • Models, Molecular
  • N-Acetylneuraminic Acid / biosynthesis
  • N-Acetylneuraminic Acid / chemistry
  • Neuraminidase / metabolism
  • Purine Nucleosides / chemical synthesis
  • Pyrimidinones / chemical synthesis
  • Vibrio cholerae / enzymology

Substances

  • Bacterial Proteins
  • Carbon Isotopes
  • Enzymes
  • Isotopes
  • Purine Nucleosides
  • Pyrimidinones
  • forodesine
  • Neuraminidase
  • N-Acetylneuraminic Acid