A Sensitive Coupled Enzyme Assay for Measuring Kinase and ATPase Kinetics Using ADP-Specific Hexokinase

Kinases and ATPases perform essential biological functions in metabolism and regulation. Activity of these enzymes is commonly measured by coupling ATP consumption to the synthesis of a detectable product. For most assay systems the ATP concentration during the reaction is unknown, compromising the precision of the assay. Using the ADP-specific hexokinase (ADP-HK) from the thermophilic archaeon Thermococcus litoralis the protocol outlined here allows real time coupling of ATP consumption to downstream signal change enabling accurate kinetic measurements. ADP-HK phosphorylates glucose that is then used by glucose-6-phosphate dehydrogenase to reduce NAD⁺ to NADH which can be measured at 340 nm. We have shown this assay to be sensitive to the detection of micromole quantities of ADP with no detectable background from ATP.