A Dithio-Coupled Kinase and ATPase Assay

Abstract

Kinases and ATPases produce adenosine diphosphate (ADP) as a common product, so an assay that detects ADP would provide a universal means for activity-based screening of enzymes in these families. Because it is known that most kinases accept ATPβS (sulfur on the β-phosphorous) as a substrate in place of adenosine triphosphate (ATP), the authors have developed a continuous assay using this substrate, with detection of the ADPβS product using dithio reagents. Such an assay is possible because dithio groups react selectively with ADPβS and not with ATPβS. Thiol detection was done using both Ellman’s reagent (DTNB) and a recently developed fluorescent dithio reagent, DSSA. Therefore, the assay can be run in both absorbance and fluorescence detection modes. The assay was used to perform steady-state kinetic analyses of both hexokinase and myosin ATPase. It was also used to demonstrate the diastereoselectivity of hexokinase (R) and myosin ATPase (S) for the isomers of ATPβS, consistent with previous results. When run in fluorescence mode using a plate reader, an average Z′ value of 0.54 was obtained, suggesting the assay is appropriate for high-throughput screening.