Tracking the enzymatic activity of Hexokinase with 2D-IR spectroscopy

Hexokinase catalyzes the first step of glycolysis, transferring a phosphate group from adenosine triphosphate (ATP) to glucose (Glc) to produce glucose-6-phosphate (Glc-6P) and adenosine diphosphate (ADP). This enzymatic reaction is crucial in biochemistry and medical diagnostics, such as Glc level assessments using the hexokinase/glucose-6-phosphate dehydrogenase method. In this study, we employ two-dimensional infrared (2DIR) spectroscopy to monitor hexokinase activity in real time by observing the asymmetric stretching vibrations of the (PO2)−-groups in ATP and ADP. Compared to Fouriertransform infrared (FTIR), 2D-IR offers enhanced structural resolution and reduced solvent interference. Our results reveal distinct cross-peaks in the ATP spectrum absent in ADP, attributed to intramolecular coupling of (PO2)−- groups. This work highlights the advantages of 2D-IR spectroscopy for studying enzymatic processes.