Time-resolved Monitoring of Enzyme Activity with Ultrafast Hyper-CEST Spectroscopy
Abstract
We propose a method to dynamically monitor the progress of an enzymatic reaction using NMR of hyperpolarized ^(129)Xe in a host-guest system. It is based on a displacement assay originally designed for fluorescence experiments that exploits the competitive binding of the enzymatic product on the one hand and a reporter dye on the other hand to a supramolecular host. Recently, this assay has been successfully transferred to NMR, using xenon as a reporter, cucurbit[6]uril as supramolecular host, and Hyper-CEST as detection technique. Its advantage is that the enzyme acts on the unmodified substrate and only the product is detected through immediate inclusion into the host. We here apply a method that drastically accelerates the acquisition of Hyper-CEST spectra in vitro using magnetic field gradients. This allows monitoring the dynamic progress of the conversion of lysine to cadaverine with a temporal resolution of ~30 s. Moreover, the method only requires to sample the very early onset of the reaction (<0.5 % of substrate conversion where the host itself is required only at μM concentrations) at comparatively low reaction rates, thus saving enzyme material and reducing NMR acquisition time. The obtained value for the specific activity agrees well with previously published results from fluorescence assays. We furthermore outline how the Hyper-CEST results correlate with xenon T_2 measurements performed during the enzymatic reaction. This suggests that ultrafast Hyper-CEST spectroscopy can be used for dynamically monitoring enzymatic activity with NMR.
Additional Information
© 2017 John Wiley & Sons. Issue Online 04 June 2018; Version of Record online: 18 January 2018; Accepted manuscript online: 23 December 2017; Manuscript accepted: 10 December 2017; Manuscript revised: 07 December 2017; Manuscript received: 10 October 2017. Nils Bogdanoff is gratefully acknowledged for assisting with data processing. This work was supported by the European Research Council under the European Community's Seventh Framework Programme (FP7/2007-2013)/ERC grant agreement no. 242710 and funding SCHR 995/5-1 from the German Research Foundation to L.S. as well as funding HE 5967/4-1 from the German Research Foundation to A.H.Attached Files
Accepted Version - D-pfert_et_al-2017-Magnetic_Resonance_in_Chemistry.pdf
Supplemental Material - mrc4702-sup-0001-Data_S1.pdf
Files
Name | Size | Download all |
---|---|---|
md5:cb8f87685a0ebce39b45f25c5b3aa486
|
975.3 kB | Preview Download |
md5:44e189eaf3059ae2e749034efdf32046
|
605.0 kB | Preview Download |
Additional details
- Eprint ID
- 84034
- Resolver ID
- CaltechAUTHORS:20180102-135848866
- 242710
- European Research Council (ERC)
- SCHR 995/5-1
- Deutsche Forschungsgemeinschaft (DFG)
- HE 5967/4-1
- Deutsche Forschungsgemeinschaft (DFG)
- Created
-
2018-01-02Created from EPrint's datestamp field
- Updated
-
2021-11-15Created from EPrint's last_modified field