title: A direct NMR method for the measurement of competitive kinetic isotope effects.
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.
authors.affiliation: Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, Canada.
authors.forename: Jefferson
authors.initials: J
authors.lastname: Chan
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Lewis
authors.forename: Michel
authors.initials: M
authors.lastname: Gilbert
authors.forename: Marie-France
authors.initials: MF
authors.lastname: Karwaski
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Bennet
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D002247:Carbon Isotopes / Q000378:metabolism; D002384:Catalysis; D004798:Enzymes / Q000378:metabolism; D006868:Hydrolysis; D007553:Isotope Labeling; D007554:Isotopes / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008958:Models, Molecular; D019158:N-Acetylneuraminic Acid / Q000096:biosynthesis / Q000737:chemistry; D009439:Neuraminidase / Q000378:metabolism; D011684:Purine Nucleosides / Q000138:chemical synthesis; D011744:Pyrimidinones / Q000138:chemical synthesis; D014734:Vibrio cholerae / Q000201:enzymology
chemical_list: D001426:Bacterial Proteins; D002247:Carbon Isotopes; D004798:Enzymes; D007554:Isotopes; D011684:Purine Nucleosides; D011744:Pyrimidinones; C113101:forodesine; D009439:Neuraminidase; D019158:N-Acetylneuraminic Acid