A real-time 31P-NMR-based approach for the assessment of glycerol kinase catalyzed monophosphorylations

Abstract Phosphorous-NMR is scarcely employed to evaluate enzyme kinetics of kinase driven monophosphorylations, despite of being a powerful and reliable tool to undoubtedly detect the actual phosphoryl transfer to the targeted substrate. Another advantage is that an external supplementation source of the NMR active isotope is not required, since 31P is highly abundant in nature. Glycerol kinase (GlpK) from E. coli is an exemplary ATP-dependent kinase/phosphotransferase model to illustrate the value and usefulness of a 31P-NMR-based approach to assess the enzymatically driven monophosphorylation of glycerol. Moreover, the described approach offers an alternative to the indirect coupled glycerol kinase enzyme assays. Herein, we provided a real time 31P-NMR-based method customized for the direct assessment of the glycerol kinase enzyme activity. • Real-time detection for phosphoryl group dynamics in the GlpK driven reaction • Direct assessment of product formation (glycerol-monophosphate) • Parallel determination of cosubstrate (ATP) consumption and coproduct (ADP) generation • Method validation was performed via 31P-NMR for each phosphorylated molecule involved in the reaction in order to assist in the molecular assignments