Studies on the interactions between the cyclic nucleotide-binding sites of cGMP-dependent protein kinase.
The rate and equilibrium kinetics of [3H]cGMP binding to the two rapidly exchanging and two slowly exchanging sites of dimeric cGMP-dependent protein kinase from bovine lung were studied. As observed by McCune and Gill (McCune, R. W., and Gill, G. N. (1979) J. Biol. Chem. 254, 5083-5091), unlabeled cGMP retarded the dissociation of [3H]cGMP bound to the "slow" site. This effect was due to interaction of unlabeled cGMP with the "rapid" rather than the slow site. First, the potencies of unlabeled cGMP and a number of cGMP analogs correlated nearly perfectly with their affinities for the rapid site. Second, the rate of dissociation in the absence of unlabeled ligand was independent of the degree of saturation of the slow sites. Third, unlabeled ligand inhibited the rate of dissociation more (about 10-fold) than theoretically predicted (maximum 2-fold) from interaction between two similar sites in one macromolecule. A favorable free energy coupling appeared to exist between the rapid and slow sites but not between the slow sites. cGMP associated faster to the slow site than the rapid site. Mg/ATP decreased the rate of association to either site by 50% and increased about ten-fold the rate of dissociation from the slow site. The dissociation of cGMP from the slow site could be described by a single activation energy (Ea = 71 kJ X mol-1) for the whole temperature range (0-37 degrees C) tested. These data indicated that the cyclic nucleotide-binding sites of the cGMP-kinase are kinetically more homologous to those in the cAMP-dependent protein kinases than previously recognized.
D000818:Animals; D001665:Binding Sites; D002151:Calorimetry; D002417:Cattle; D006152:Cyclic GMP / Q000378:metabolism*; D007700:Kinetics; D008168:Lung / Q000201:enzymology; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D014316:Tritium
D014316:Tritium; D011494:Protein Kinases; D006152:Cyclic GMP