Phosphorylation of cGMP-dependent protein kinase increases the affinity for cyclic AMP

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Abstract
Cyclic-GMP-dependent protein kinase contains two binding sites for cGMP, which have different affinities for cGMP. Autophosphorylation of the enzyme affects mainly the binding of cGMP to the ''high''-affinity site (site 1). The enzyme binds cAMP and cAMP stimulates the phosphotransferase activity of the native enzyme half-maximally at 44 .mu.M. Autophosphorylation of the enzyme decreases the apparent Ka value to 7 .mu.M. Autophosphorylation does not affect the catalytic rate of the enzyme if measured at a saturating concentration of ATP. Tritiated cAMP apparently binds at 4.degree. C to one site with a Kd value of 3 .mu.M. Binding to the second site is not measurable. Autophosphorylation of the enzyme increases the affinity of the high-affinity site for cAMP sixfold (Kd 0.46 .mu.M) and allows the detection of a second site. In accordance with these data the dissociation rate of [3H]cAMP from the high-affinity site is decreased from 4.5 min-1 to 1.2 min-1 by autophosphorylation. Experiments in which unlabeled cAMP competes with [3H] cGMP for the two binding sites confirmed these results. Recalculation of the competition curves by a computer program for two binding sites indicated that autophosphorylation decreases the Kd value for binding of cAMP to the high-affinity site from 1.9 .mu.M to 0.17 .mu.M. Autophosphorylation does not affect significantly the affinity for the second site. Kd values for site 2 varied from 17 .mu.M to 40 .mu.M. These results suggest that autophosphorylation of cGMP-dependent protein kinase increases the affinity of the enzyme for the cAMP by affecting mainly the properties of binding site 1.