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

14 March 2025, Version 1
This content is a preprint and has not undergone peer review at the time of posting.

Abstract

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.

Keywords

enzymatic catalysis
ultrafast spectroscopy
phosphorylation
2D-IR spectroscopy
Kinetics

Supplementary materials

Title
Description
Actions
Title
Supporting Information
Description
Experimental Details Supplementary Figures: - FTIR of ADP and Glc-6P - 2D-IR spectra of ATP for different waiting times
Actions

Comments

Comments are not moderated before they are posted, but they can be removed by the site moderators if they are found to be in contravention of our Commenting Policy [opens in a new tab] - please read this policy before you post. Comments should be used for scholarly discussion of the content in question. You can find more information about how to use the commenting feature here [opens in a new tab] .
This site is protected by reCAPTCHA and the Google Privacy Policy [opens in a new tab] and Terms of Service [opens in a new tab] apply.