A strategy for the construction of a profluorescent caged enzyme is described. An active site-directed peptide-based affinity label was designed, synthesized, and employed to covalently label a non-active site residue in the cAMP-dependent protein kinase. The modified kinase displays minimal catalytic activity and low fluorescence. Photolysis results in partial cleavage of the enzyme-bound affinity label, restoration of enzymatic activity (60 – 80%) and a strong fluorescent response (10 – 20 fold). The caged kinase displays analogous behavior in living cells, inducing a light-dependent loss of stress fibers that is characteristic of cAMP action. This strategy furnishes molecularly engineered enzymes that can be remotely controlled in time, space, and total activity
