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Manning KA, Sathyamoorthy B, Eletsky A, et al. Highly precise measurement of kinetic isotope effects using 1H-detected 2D [13C,1H]-HSQC NMR spectroscopy. J Am Chem Soc. 2012;134(51):20589-92doi: 10.1021/ja310353c.
Manning, K. A., Sathyamoorthy, B., Eletsky, A., Szyperski, T., & Murkin, A. S. (2012). Highly precise measurement of kinetic isotope effects using 1H-detected 2D [13C,1H]-HSQC NMR spectroscopy. Journal of the American Chemical Society, 134(51), 20589-92. https://doi.org/10.1021/ja310353c
Manning, Kathryn A, et al. "Highly precise measurement of kinetic isotope effects using 1H-detected 2D [13C,1H]-HSQC NMR spectroscopy." Journal of the American Chemical Society vol. 134,51 (2012): 20589-92. doi: https://doi.org/10.1021/ja310353c
Manning KA, Sathyamoorthy B, Eletsky A, Szyperski T, Murkin AS. Highly precise measurement of kinetic isotope effects using 1H-detected 2D [13C,1H]-HSQC NMR spectroscopy. J Am Chem Soc. 2012 Dec 26;134(51):20589-92. doi: 10.1021/ja310353c. Epub 2012 Dec 11. PMID: 23215000.
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J Am Chem Soc. 2012 Dec 26;134(51):20589-92. doi: 10.1021/ja310353c. Epub 2012 Dec 11.

Highly precise measurement of kinetic isotope effects using 1H-detected 2D [13C,1H]-HSQC NMR spectroscopy.

Journal of the American Chemical Society

Kathryn A Manning, Bharathwaj Sathyamoorthy, Alexander Eletsky, Thomas Szyperski, Andrew S Murkin

Affiliations

  1. Department of Chemistry, University at Buffalo, The State University of New York, Buffalo, New York 14260-3000, USA.

PMID: 23215000 DOI: 10.1021/ja310353c

Abstract

A new method is presented for measuring kinetic isotope effects (KIEs) by (1)H-detected 2D [(13)C,(1)H]-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-(13)C 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-(13)C 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 [(13)C,(1)H]-HSQC offer new avenues to study challenging systems where low substrate concentration and/or signal overlap impedes 1D (13)C NMR data acquisition. Moreover, this approach can take advantage of highest-field spectrometers, which are commonly equipped for (1)H detection with cryogenic probes.

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