cGMP-dependent protein kinase: autophosphorylation changes the characteristics of binding site 1

• Published in: European journal of biochemistry Vol. 147; no. 2; pp. 361 - 365
• Main Authors: HOFMANN, F, GENSHEIMER, H.-P, GÖBEL, C
• Format: Journal Article
• Language: English
• Published: Oxford Blackwell 01.03.1985
• Subjects: Adenosine Triphosphate - metabolism; Allosteric Site; Analytical, structural and metabolic biochemistry; Binding, Competitive; Biological and medical sciences; Cyclic GMP - metabolism; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Kinetics; Phosphorylation; Protein Binding; protein kinase; Protein Kinases - metabolism; Transferases; Phosphorylation; Enzyme; Protein kinase; Transferase; Animal; Dependence; Cyclic nucleotide; 3',5'-GMP; Molecular interaction; Binding site
• ISSN: 0014-2956; 1432-1033
• DOI: 10.1111/j.1432-1033.1985.tb08758.x

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.