Abstract

ATP-dependent kinases play central roles in metabolism and cellular regulation, making their accurate quantification essential for biochemical and kinetic studies. Classical coupled assays used to measure ATP-dependent kinase activity suffer from technical limitations, including instability of substrates of the auxiliary enzymes, dependence on monovalent ions, and the need to detect cofactor disappearance. Here, we describe the characterization and standardization of an alternative coupled assay for the quantitative and continuous measurement of ATP-dependent kinase activity, based on an ADP-dependent glucokinase from Thermococcus litoralis (TlGK) coupled to glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides (LmG6PDH). The assay relies on the auxiliary enzymes TlGK and LmG6PDH, both of which were produced recombinantly and kinetically characterized at pH 7.0 and 25 degrees C. In this system, ADP generated by the kinase reaction is used to produce glucose-6-phosphate, which is subsequently oxidized to form NADH, allowing direct spectrophotometric monitoring at 340 nm. The effect of the Mg-ATP complex on assay performance was systematically analyzed, identifying the kinetic mechanism of inhibition, which is relevant for the experimental design of the coupled assay. Using a kinetic model for sequentially coupled reactions, we developed a general mathematical framework to estimate the minimum amounts of auxiliary enzymes required to ensure steady-state conditions and maintain the analytical validity of the assay. The method was experimentally validated using the ATP-dependent kinase ThiD, demonstrating that the measured initial velocity is independent of auxiliary enzyme concentrations and directly proportional to the kinase activity. Together, these results establish a kinetic framework and provide a versatile alternative assay for the quantitative measurement of ATP-dependent kinase activity.