Rapid radiochemical filter paper assay for determination of hexokinase activity and affinity for glucose-6-phosphate

Glucose phosphorylation by hexokinase (HK) is a rate-limiting step in glucose metabolism. Regulation of HK includes feedback inhibition by its product glucose-6-phosphate (G6P) and mitochondria binding. HK affinity for G6P is difficult to measure because its natural product (G6P) inhibits enzyme activity. HK phosphorylates several hexoses, and we have taken advantage of the fact that 2-deoxyglucose (2-DG)-6-phosphate does not inhibit HK activity. By this, we have developed a new method for rapid radiochemical analysis of HK activity with 2-DG as a substrate, which allows control of the concentrations of G6P to investigate HK affinity for inhibition by G6P. We verified that 2-DG serves as a substrate for the HK reaction with linear time and concentration dependency as well as expected maximal velocity and K_M. This is the first simple assay that evaluates feedback inhibition of HK by its product G6P and provides a unique technique for future research evaluating the regulation of glucose phosphorylation under various physiological conditions.NEW & NOTEWORTHY Traditionally, hexokinase activity has been analyzed spectrophotometrically in which the product formation of glucose-6-phosphate (G6P) is analyzed by an indirect reaction coupled to NADPH formation during conversion of G6P to 6-P gluconolactone. By nature, this assay prevents measurements of hexokinase (HK) affinity for inhibition by G6P. We have developed a rapid radiochemical filter paper assay to study HK affinity for G6P by use of radiolabeled 2-deoxyglucose as substrate to study physiological regulation of HK affinity for G6P-induced inhibition.