title: Highly precise measurement of kinetic isotope effects using 1H-detected 2D [13C,1H]-HSQC NMR spectroscopy.
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.
authors.affiliation: Department of Chemistry, University at Buffalo, The State University of New York, Buffalo, New York 14260-3000, USA.
authors.forename: Kathryn A
authors.initials: KA
authors.lastname: Manning
authors.forename: Bharathwaj
authors.initials: B
authors.lastname: Sathyamoorthy
authors.forename: Alexander
authors.initials: A
authors.lastname: Eletsky
authors.forename: Thomas
authors.initials: T
authors.lastname: Szyperski
authors.forename: Andrew S
authors.initials: AS
authors.lastname: Murkin