Characterization of Phosphorylated and Native cGMP‐Dependent Protein Kinase

Pure preparations of cGMP‐dependent protein kinase are autophosphorylated in the presence of cAMP and cGMP. The stoichiometry and functional significance of this reaction has been studied. Pure preparations of cGMP‐dependent protein kinase contained 1.4 ± 0.1 (7) mol phosphate/mol subunit as determined by chemical phosphate analysis. The protein‐bound phosphate was hydrolyzed by 1 M NaOH, but was stable in 0.1 M HCl or 0.8 M NH 4 OH or hot 16% trichloracetic acid. In the presence of cAMP and [γ‐ 32 P]ATP. Mg another 2.5 ± 0.2 (5) mol phosphate/mol subunit were incorporated into the enzyme yielding a total of 4.2 ± 0.06 (5) mol phosphate/mol subunit. cGMP also stimulated the phosphorylation of the enzyme although to a lower extent than cAMP. The autophosphorylation reaction was half‐maximally stimulated at 0.12 μM and 7.0 μM cGMP and cAMP, respectively. cAMP‐dependent autophosphorylation of the native enzyme did not change the apparent K A for cGMP in a phosphotransferase assay ( K A = 0.15 μM) but decreased the K A for cAMP from 19.6 μM to 1.75 μM. Phosphorylation did not affect the apparent K m for the substrate peptide but doubled the apparent V of the phosphotransferase reaction determined in the presence of saturating concentrations of cGMP or cAMP. Phosphorylation did not change the affinity of the enzyme for cGMP, when cGMP binding was determined in the absence of ATP (apparent K d = 18 nM). ATP · Mg decreased the affinity of the native enzyme for cGMP about threefold to an apparent K d of 54 nM; in contrast, ATP · Mg had only a minimal effect on the binding kinetics of the phosphorylated enzyme. These results support the notion that autophosphorylation of cGMP‐dependent protein kinase considerably influences the kinetic parameter of the enzyme.