Open AccessHighly Precise Measurement of Kinetic Isotope Effects Using 1H-Detected 2D [13C,1H]-HSQC NMR Spectroscopy
Author(s) -
Kathryn A. Manning,
Bharathwaj Sathyamoorthy,
Alexander Eletsky,
Thomas Szyperski,
Andrew S. Murkin
Publication year - 2012
Publication title -
journal of the american chemical society
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.115
H-Index - 612
eISSN - 1520-5126
pISSN - 0002-7863
DOI - 10.1021/ja310353c
Subject(s) - chemistry , heteronuclear single quantum coherence spectroscopy , heteronuclear molecule , kinetic isotope effect , analytical chemistry (journal) , nuclear magnetic resonance spectroscopy , mass spectrometry , spectroscopy , isotope , kinetic energy , stereochemistry , chromatography , deuterium , nuclear physics , physics , quantum mechanics
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.