Inhibition and activation of kinases by reaction products: a quick, reporter-free assay.

Kinases are widely distributed in nature and are implicated in many human diseases, thus an understanding of their activity and regulation is of fundamental importance. Several kinases are known to be inhibited by ADP, however thorough investigation of this phenomenon is hampered by the lack of a simple and effective assay for studying this inhibition. We now present a quick, general approach for measuring the effects of reaction products on kinase activity. Based on isothermal titration calorimetry, this is the first universal, reporter-free, continuous assay for probing kinase inhibition or activation by ADP. In applications to an aminoglycoside phosphotransferase (APH(3')-IIIa) and pantothenate kinases from E. coli (EcPanK) and P. aeruginosa (PaPanK), we found ADP to be an efficient inhibitor of all three kinases, with Ki values similar to or lower than the Km values of ATP. Interestingly, ADP was an activator at low concentrations and an inhibitor at high concentrations of EcPanK. This unusual effect was quantitatively modelled, and attributed to cooperative interactions between the two subunits of the dimeric enzyme. Importantly, our results suggest that at typical bacterial intracellular concentrations of ATP and ADP (approx. 1.5 mM and 180 μM respectively), all three kinases are partially inhibited by ADP, allowing enzyme activity to rapidly respond to changes in the levels of both metabolites.

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