Significance Mutations, deletions, or gene fusions in protein kinases have been associated with the development of many diseases in humans and have led to the emergence of the protein kinase family as an important therapeutic drug target. In the cell, kinase activity is often turned on and off allosterically by intramolecular regulatory domains, flexible linkers, or other interacting proteins. Here, we use single-molecule optical tweezers to investigate the mechanism of allosteric regulation of the cAMP-dependent PKA. This approach allowed us to determine the thermodynamic driving forces that enable PKA to transduce cAMP-binding signals to turn on its kinase activity.