ACTIVATION BY CYCLIC-GMP BINDING CAUSES AN APPARENT CONFORMATIONAL CHANGE IN CGMP-DEPENDENT PROTEIN-KINASE

Cyclic nucleotide binding activates cyclic nucleotide-dependent protei n kinases, but the molecular mechanism is unknown. In the present stud ies, cGMP binding to type I alpha or type I beta cGMP-dependent protei n kinase (PKG) caused (i) a large electronegative charge shift of each enzyme on ion exchange chromatography, (ii) an increase in the Stokes radius (>3 Angstrom) of each enzyme, and (iii) a decreased mobility o f type I beta PKG on native gel electrophoresis, These physical change s were not detected in the monomeric form of type I beta PKG upon acti vation by cGMP, However, the results of partial proteolysis of type I alpha PKG revealed some degree of cGMP-induced conformational change w ithin the PKG-monomer, since cGMP binding protects the PKG-monomer aga inst chymotryptic cleavage, The altered sensitivity to proteolysis occ urs at Met-200, which is located between the B and C alpha-helices in the high affinity site (site A), and implies that the cGMP-induced str uctural perturbations in this region may participate in activation of dimeric PKG, The cGMP-induced conformational effects observed using th e physical separation methods are likely to reflect altered interactio ns within the dimeric PKG that are caused by structural alterations wi thin the subunits.