title: cGMP-dependent protein kinase. Autophosphorylation changes the characteristics of binding site 1.
abstract: cGMP-dependent protein kinase binds 4 mol cGMP/mol enzyme to two different sites. Binding to site 1 (apparent Kd 17 nM) shows positive cooperativity and is inhibited by Mg . ATP, whereas binding to site 2 (apparent Kd 100-150 nM) is non-cooperative and not affected by Mg . ATP. Autophosphorylation of the enzyme abolishes the cooperative binding to site 1 and the inhibitory effect of Mg . ATP. The association (K1) and dissociation (K-1) rate constant for site 2 and K1 for site 1 are not affected significantly by Mg . ATP or autophosphorylation. The dissociation rate from site 1 measured in the presence of 1 mM unlabelled cGMP is decreased threefold and over tenfold by Mg . 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 . ATP or autophosphorylation. Only Kd values calculated with the latter K-1 values are similar to the Kd values obtained by equilibrium binding. These results suggest that autophosphorylation of cGMP-dependent protein kinase affects mainly the binding characteristics of site 1.
authors.forename: F
authors.initials: F
authors.lastname: Hofmann
authors.forename: H P
authors.initials: HP
authors.lastname: Gensheimer
authors.forename: C
authors.initials: C
authors.lastname: Göbel
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000495:Allosteric Site; D001667:Binding, Competitive; D006152:Cyclic GMP / Q000378:metabolism; D007700:Kinetics; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*
chemical_list: D000255:Adenosine Triphosphate; D011494:Protein Kinases; D006152:Cyclic GMP