A direct NMR method for the measurement of competitive kinetic isotope effects
A direct NMR method for the measurement of competitive kinetic isotope effects
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DOI：
10.1038/nchembio.352
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发表时间：
2010-06-01
影响因子：
14.8
通讯作者：
Bennet, Andrew J.
中科院分区：
文献类型：
--
作者：
Chan, Jefferson;Lewis, Andrew R.;Bennet, Andrew J.
我们提出了一种技术，使用（13）C NMR光谱测量动力学同位素对酶催化反应的二级速率常数（k（cat）/K（m））的影响。仅使用毫克量的同位素标记的底物，精确的竞争性KIE可以确定，同时直接在NMR光谱仪中进行反应。我们的研究结果为霍乱弧菌唾液酸酶催化水解天然底物类似物支持这些反应的协调酶促过渡态。
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.