Mechanism of Activation of Protein Kinase I from Rabbit Skeletal Muscle. The Equilibrium Parameters of Ligand Interaction and Protein Dissociation

Abstract

Protein kinase I (EC 2.7.1.37) from rabbit skeletal muscle binds c[cyclic]AMP and ATP with high affinity; cAMP promotes and ATP retards dissociation (activation) of the tetrameric enzyme. The interrelationship of ligand interaction with protein dissociation was probed by quantitative ligand binding, using the filter assay technique, and by computer simulation of binding curves in terms of Scatchard plots. A comparison of the experimental and computed binding data strongly confirms the supposition that the dimeric regulatory subunit (R2) binds cAMP cooperatively. ATP binding suggests that the interaction of the 2 catalytic subunits with R2 is also strongly cooperative, whereas the binding of ATP to the holoenzyme is non-cooperative. The underlying random model, possessing low-affinity and high-affinity sites not only for ATP but also for cAMP, allows the determination of a minimal set of equilibrium parameters required for description and also an estimation of their magnitude. It further provides a basis for an explanation of the different behavior of protein kinases I and II reported in the literature.