DISSECTING THE COOPERATIVE REASSOCIATION OF THE REGULATORY AND CATALYTIC SUBUNITS OF CAMP-DEPENDENT PROTEIN-KINASE - ROLE OF TRP-196 IN THECATALYTIC SUBUNIT

The catalytic (C) subunit of cAMP-dependent protein kinase requires tw o distinct surfaces to form a stable complex with its physiological in hibitors, the regulatory (R) subunits and the heat-stable protein kina se inhibitors. In addition to a substrate-like segment that is common to both inhibitors, R requires a peripheral recognition site, PRS2. Th is surface is comprised of the essential phosphorylation site, Thr-197 , His-87, Trp-196, and several surrounding basic residues. To probe th e role of Trp-196 in the recognition of R, Trp-196 was replaced with A rg and Ala. Although both rC(W196A) and rC(W196R) were inhibited readi ly with cAMP-free R, they failed to form an inhibited holoenzyme compl ex with native R under conditions in which wild-type holoenzyme formed readily. Pairing rC(W196R) with mutant forms of R lacking domain B or having defects in cAMP binding sites A or B highlighted the importanc e of the conformation of R, and, in particular, the accessibility of s ite A. One of these mutants, rR(R333K), having a defect in cAMP bindin g site B formed a stable complex with rC(W196R) in the absence of cAMP . However, unlike wild-type holoenzyme, this complex was active.