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 protein kinase is important for binding to the regulatory (R) subunit, acidic residues in R were sought that might contribute to R-C interaction. Using differential labeling by a water-soluble carbodiimide (Buechler, T. A., and Taylor, S. S. (1990) Biochemistry 29, 1937-1943), seven specific carboxylates in RI alpha were identified that were protected from chemical modification in the holoenzyme; each was then replaced 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(99) in RII alpha were mutated to Ala. Replacement of Glu(101) had no effect while rRII(E99A) was very defective. RI alpha and RII alpha thus differ in the molecular details of how they recognize C. Unlike wild-type 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 the hydrogen bonding network in cAMP-binding site A, thus contributes also to holoenzyme stability.