Adenosine 3′,5′-monophosphate-dependent protein kinase: Interaction with guanidinium compounds

Abstract

Adenosine 3′,5′-monophosphate (cAMP)-dependent protein kinase consists of two dissimilar subunits which form inactive holoenzyme with the stoichiometry R 2C 2 (R represents the regulatory and C represents the catalytic subunit, respectively). A ubiquitous protein kinase inhibitor protein (PKI) also interacts with C and inhibits its activity. Using ionexchange chromatography to resolve C from C·PKI complex, we find that Mg·ATP enhances complex formation between C and PKI. We also find that guanethidine and other guanidinium compounds inhibit complex formation. Using ion-exchange resolution, we also find that guanethidine retards and MgATP enhances the rate of formation of type II holoenzyme from its components. Guanidinobenzimidazole and guanethidine are competitive inhibitors of enzyme activity with respect to protein or peptide substrates with K i values of 3 and 4 m m, respectively. Results from steady-state kinetic analysis are consistent with the idea that inhibition is based on competition between the guanidinium compound and the arginyl group of the peptide or protein substrate. We therefore propose the idea that the protein kinase inhibitor and the regulatory subunit interact with the domain of the catalytic subunit complementary to the protein substrate. Occupancy of this domain prevents the C subunit from interacting with protein substrates and thereby inhibits enzyme activity.