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

被引：55

作者：

Chan, Jefferson ^{[1
]}

Lewis, Andrew R. ^{[1
]}

Gilbert, Michel ^{[2
]}

Karwaski, Marie-France ^{[2
]}

Bennet, Andrew J. ^{[1
]}

机构：

[1] Simon Fraser Univ, Dept Chem, Burnaby, BC V5A 1S6, Canada

[2] Natl Res Council Canada, Inst Biol Sci, Ottawa, ON K1A 0R6, Canada

来源：

NATURE CHEMICAL BIOLOGY | 2010年 / 6卷 / 06期

基金：

加拿大自然科学与工程研究理事会;

关键词：

TRANSITION-STATE; SALMONELLA-TYPHIMURIUM; NATURAL-ABUNDANCE; HYDROLYSIS; SIALIDASE; MECHANISMS; CATALYSIS; ENZYMES; ACID;

D O I：

10.1038/nchembio.352

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

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.

引用

页码：405 / 407

页数：3

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