Kinetic isotope effects of nucleoside hydrolase from Escherichia coli

Advances in Kinetic Isotope Effect Measurement Techniques for Enzyme Mechanism Study

“…The KIE of [1- 15 N]uridine determined by LC-MS is almost the same as the KIE of [1- 15 N]inosine measured with scintillation counting via radioactive labeled nucleosides reported by Schramm and coauthors [41]. The KIEs of [1′- 13 C]inosine and [1′- 13 C]uridine for E. coli nucleoside hydrolase are same (1.021) as measured by GC-MS [40] and LC-MS [42], respectively. These values indicate that the C1′-N1 bond is broken in the transition state.…”

“…Lee and coauthors [40] also reported the KIEs of [1′- 13 C]inosine and [1′- 2 H]inosine catalyzed by nucleoside hydrolase from C. fasciculate determined by GC-MS under selected ion monitoring (SIM) condition at three ion pairs (158/159, 187/188, and 217/218) were 1.021 ± 0.006 and 1.113 ± 0.008, respectively and the transition state is consistent with that reported by Schramm and coauthors [41] using radioactive substrates which means GC-MS is reliable to determine KIEs (Figure 1A). Kline and coauthors [42] determined KIEs of [1′- 13 C], [1- 15 N], [1′- 2 H], [2′- 2 H] and [5′- 2 H 2 ]uridine by nucleoside hydrolase from E. coli with LC-MS under the SIM conditions analyzing the 173/174 and 113/114 ion pairs for the ribose and uracil moiety, respectively and the elucidated transition state is consistent with a bond-energy bond-order vibrational analysis (Figure 1B). The KIE of [1- 15 N]uridine determined by LC-MS is almost the same as the KIE of [1- 15 N]inosine measured with scintillation counting via radioactive labeled nucleosides reported by Schramm and coauthors [41].…”

“…Observed KIEs of inosine ( A ) and uridine ( B ) for the enzymatic catalysis determined with GC-MS, LC-MS and radioactive-labeled methods [40,41,42]. …”

Measuring specificity in multi-substrate/product systems as a tool to investigate selectivity in vivo

“…Thus, mass spectrometry (MS) is a common analytical technology that is utilized for these types of studies [32–35]. The coupling of LC or gas chromatography to MS provides separation of multiple substrates, and therefore more accurate quantification, for multiple target analysis [36, 37]. Furthermore, tandem MS (MS/MS) can be used to acquire more spatial or structural information of analytes.…”

Transition state analysis of adenosine nucleosidase from yellow lupin (Lupinus luteus)

“…Heavy atom kinetic isotope effects were measured using a modification of the competitive method developed for stable isotopes (Parkin, 1991;Hunt et al, 2005). A reaction mixture consisting of 50% specifically labeled and 50% unlabeled adenosine 1 (1 mM total concentration) in 10 mM Tris, pH 7.2, was divided into two portions.…”