Highly Precise Measurement of Kinetic Isotope Effects Using 1H-Detected 2D [13C,1H]-HSQC NMR Spectroscopy
Highly Precise Measurement of Kinetic Isotope Effects Using 1H-Detected 2D [13C,1H]-HSQC NMR Spectroscopy
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DOI：
10.1021/ja310353c
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发表时间：
2012-12-26
影响因子：
15
通讯作者：
Murkin, Andrew S.
中科院分区：
文献类型：
--
作者：
Manning, Kathryn A.;Sathyamoorthy, Bharathwaj;Murkin, Andrew S.
本文提出了一种用H-1探测的二维[C-13，H-1]-杂原子单量子相干（HSQC）NMR谱测量动力学同位素效应（KIE）的新方法。通过将1-C-13 KIE与使用同位素比质谱法获得的公开值进行比较，该方法的高准确度被证明用于葡萄糖-6-磷酸脱氢酶催化的反应。结核分枝杆菌1-脱氧-D-木酮糖-5-磷酸还原异构酶催化的反应具有较高的精密度。发现2-、3-和4-C-13 KIE分别为1.0031（4）、1.0303（12）和1.0148（2）。这些KIE提供了异构化过程中的清洁的限速逆醛醇步骤的证据。2D [C-13，H-1]-HSQC的高固有灵敏度和信号分散为研究具有挑战性的系统提供了新的途径，其中低底物浓度和/或信号重叠阻碍了ID C-13 NMR数据采集。此外，这种方法可以利用最高场的光谱仪，这是通常配备的H-1检测与低温探头。
A new method is presented for measuring kinetic isotope effects (KIEs) by H-1 detected 2D [C-13,H-1]-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-C-13 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-C-13 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 [C-13,H-1]-HSQC offer new avenues to study challenging systems where low substrate concentration and/or signal overlap impedes ID C-13 NMR data acquisition. Moreover, this approach can take advantage of highest-field spectrometers, which are commonly equipped for H-1 detection with cryogenic probes.