'Royal College of Obstetricians & Gynaecologists (RCOG)'
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
