Highly Precise Measurement of Kinetic Isotope Effects Using 1H-Detected 2D [13C,1H]-HSQC NMR Spectroscopy

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• 作者：Kathryn A. Manning ; Bharathwaj Sathyamoorthy ; Alexander Eletsky ; Thomas Szyperski ; Andrew S. Murkin
• 刊名：The Journal of the American Chemical Society
• 出版年：2012
• 出版时间：December 26, 2012
• 年：2012
• 卷：134
• 期：51
• 页码：20589-20592
• 全文大小：252K
• 年卷期：v.134,no.51(December 26, 2012)
• ISSN：1520-5126

文摘

A new method is presented for measuring kinetic isotope effects (KIEs) by ¹H-detected 2D [¹³C,¹H]-heteronuclear single quantum coherence (HSQC) NMR spectroscopy. The high accuracy of this approach was exemplified for the reaction catalyzed by glucose-6-phosphate dehydrogenase by comparing the 1-¹³C KIE with the published value obtained using isotope ratio mass spectrometry. High precision was demonstrated for the reaction catalyzed by 1-deoxy-d-xylulose-5-phosphate reductoisomerase from Mycobacterium tuberculosis. 2-, 3-, and 4-¹³C KIEs were found to be 1.0031(4), 1.0303(12), and 1.0148(2), respectively. These KIEs provide evidence for a cleanly rate-limiting retroaldol step during isomerization. The high intrinsic sensitivity and signal dispersion of 2D [¹³C,¹H]-HSQC offer new avenues to study challenging systems where low substrate concentration and/or signal overlap impedes 1D ¹³C NMR data acquisition. Moreover, this approach can take advantage of highest-field spectrometers, which are commonly equipped for ¹H detection with cryogenic probes.