The positive cooperativity of the Kemptide substrate or the ATP molecule with the PICA catalytic subunit has been studied by dynamics simulations and free energy calculations on a series of binary and ternary models. The results revealed that the first ATP binding to the PKA catalytic subunit is energetically favorable for the successive Kemptide binding, confirming the positive cooperativity. The key residues Thr51, Glu170, and Phe187 in PICA contributing to the positive cooperativity have been found. The binding of ATP to PICA induces the positive cooperativity through one direct allosteric communication network in PICA from the ATP binding sites in the catalytic loop of the large lobe to the Kemptide binding sites in the activation segment of the large lobe, two indirect ones from those in the glycine-rich loop and the beta 3 strand of the small lobe, and from those in the catalytic loop to those in the activation segment via the alpha F helix media. The Tyr204Ala mutation in the activation segment of PICA causes both the decoupling of the cooperativity and the disruption of the corresponding allosteric network through the alpha F helix media.