cGMP‐dependent protein kinase

Abstract

CGMP-dependent protein kinase 4 mol cGMP/mol enzyme to 2 different sites. Binding to site 1 (apparent Kd 17 nM) shows positive cooperatively and is inhibited by Mg.cntdot.ATP, whereas binding to site 2 (apparent Kd 100-150 nM) is non-cooperative and not affected by Mg.cntdot.ATP. Autophosphorylation of the enzyme abolishes the cooperative binding to site 1 and the inhibitory effect of Mg.cntdot.ATP. The association (K1) and dissociation (K-1) rate constant for site 2 and K1 for site 1 are not affected significantly by Mg.cntdot.ATP or autophosphorylation. The dissociation rate from site 1 measured in the presence of 1 mM unlabeled cGMP is decreased 3-fold and over 10-fold by Mg.cntdot.ATP and autophosphorylation, respectively. In contrast, the dissociation rate from site 1 measured after a 500-fold dilution of the enzyme-ligand complex is 100-fold faster than that determined in the presence of 1 mM cGMP and is only slightly influenced by Mg.cntdot.ATP or autophosphorylation. Only Kd values calculated with the latter K-1 values are similar to the Kd values obtained by equilibrium binding. Autophosphorylation of cGMP-dependent protein kinase probably affects mainly the binding characteristics of site 1.