INTERACTION OF THE REGULATORY AND CATALYTIC SUBUNITS OF CAMP-DEPENDENT PROTEIN-KINASE - ELECTROSTATIC SITES ON THE TYPE I-ALPHA REGULATORY SUBUNIT

Since a basic surface on the catalytic (C) subunit of cAMP-dependent p rotein kinase is important for binding to the regulatory (R) subunit, acidic residues in R were sought that might contribute to R-C interact ion. Using differential labeling by a water-soluble carbodiimide (Buec hler, T. A., and Taylor, S. S. (1990) Biochemistry 29, 1937-1943), sev en specific carboxylates in RI alpha were identified that were protect ed from chemical modification in the holoenzyme; each was then replace d with Ala. Of these, rRI(E15A/E106A/D107A)), rRI(E105A), rRI(D140A), rRI(E143A), and rRI(D258A) all were defective in holoenzyme formation and define negative electrostatic surfaces on RI alpha. An additional conserved car boxylate, Glu(101) in RI alpha and the equivalent, Glu(9 9) in RII alpha were mutated to Ala. Replacement of Glu(101) had no ef fect while rRII(E99A) was very defective. RI alpha and RII alpha thus differ in the molecular details of how they recognize C. Unlike wild-t ype RI, two additional mutants, rRI(D170A) and rRI(K242A), inhibited C -subunit stoichiometrically in the presence of cAMP and show increases in both on- and off-rates. Asp(170), which contributes directly to th e hydrogen bonding network in cAMP-binding site A, thus contributes al so to holoenzyme stability.