Measurement of Kinetic Isotope Effects by Continuously Monitoring Isotopologue Ratios Using NMR Spectroscopy

Glycoside hydrolase stabilization of transition state charge: new directions for inhibitor design

“…Aer checking that inclusion of ethanol-d 6 (10% v/v) into the buffer had no effect on the activity of TmGalA, we measured the 18 O-KIE at 50 C-an increase in temperature from 37 C to improve the solubility of 4-by following by following standard procedures. [50][51][52] We note that the expected effect of the higher temperature on the measured 18 O-KIE is smaller than the experimental error in the value (Table 1). Also, as the two enzyme-catalyzed chemical steps (k inact and k react , Scheme 1) have drastically different rstorder rate constants for the reaction of 4 with TmGalA, 38 we were able to measure the a-secondary deuterium KIE on the turnover of 4 by measuring individual rate constants for k cat , or turnover, of 4 and (1-2 H)-4.…”

“…Nonlinear least squares tting of the corrected R and F values to eqn (1) gave values for two of the three competitive KIEs (Table 1) resulting from isotopic substitution at the pseudo-anomeric center. 45,49,50 To measure the leaving group 18 O-KIE we introduced a 13 C probe nucleus at the pseudoanomeric center. However, because 13 C is a less sensitive probe nucleus than 19 F, the concentration of covalent inhibitor was increased for NMR spectroscopic analysis.…”

Fundamental Insight into Glycoside Hydrolase-Catalyzed Hydrolysis of the Universal Koshland Substrates–Glycopyranosyl Fluorides

“…We used the same isotopologue panel to measure the KIEs for the A. niger glucoamylase (Table ), and for these experiments, we used 19 F NMR spectroscopy to measure competitive KIEs by monitoring the ratios of α- d -glucopyranosyl fluoride isotopologues as the reaction progressed . We integrated individual 19 F NMR signals to obtain relative ratios ( R ) for each α- d -glucopyranosyl fluoride isotopologue as a function of the fraction of reactions ( F 1 ) for the unlabeled starting material using an inert internal standard [3,5-difluorophenyl β- d -(1- 2 H)­galactopyranoside].…”

Heavy-Atom Kinetic Isotope Effects: Primary Interest or Zero Point?

“…Other methods, e.g., neutron absorption ( 10 B/ 11 B), gas chromatography ( 12 C/ 13 C), or optical rotation ( 16 O/ 18 O), were employed, but rarely NMR spectroscopy . However, the pioneering work of Singleton dramatically changed this. ,, Unlike (IR)­MS, NMR allows isotope ratios to be measured non-destructively, without physical separation of isotopologues. As discussed below, measurements of NMR-active nuclei can be made directly, with site-specificity (e.g., 12 C/ 13 C KIEs), while carefully deployed isotope shifts ( n ΔX­(Y)) and scalar couplings ( n J XY ) allow indirect detection of any heavy atoms, affording great flexibility in experimental design.…”

“…As discussed below, measurements of NMR-active nuclei can be made directly, with site-specificity (e.g., 12 C/ 13 C KIEs), while carefully deployed isotope shifts ( n ΔX­(Y)) and scalar couplings ( n J XY ) allow indirect detection of any heavy atoms, affording great flexibility in experimental design. Moreover, with widespread access to high-field NMR spectrometers, and the development of cryoprobes, in situ heavy-atom KIE analysis using isotopically enriched samples is also feasible, Scheme . ,− …”