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

Wendy Escobedo-Hinojosa; Julian L Wissner; Bernhard Hauer

doi:10.1016/j.mex.2021.101285

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

MethodsX. 2021 Feb 19:8:101285. doi: 10.1016/j.mex.2021.101285. eCollection 2021.

Authors

Wendy Escobedo-Hinojosa¹, Julian L Wissner¹, Bernhard Hauer¹

Affiliation

¹ Department of Technical Biochemistry, Institute of Biochemistry and Technical Biochemistry, University of Stuttgart, Stuttgart, Germany.

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.

Keywords: 31P-NMR; Enzymatic reaction; Glycerol kinase; Monophosphorylations; Real-time assessment.