Mechanism of Activation of Protein Kinase I from Rabbit Skeletal Muscle. Mapping of the cAMP Site by Spin-Labeled Cyclic Nucleotides

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Abstract
Binding of cyclic[c]AMP to protein kinase (type I) from rabbit skeletal muscle was investigated using spin-labeled cAMP derivatives. Different compounds were synthesized with the spin label attached by spacer chains of different length at different positions on the adenine base. Immobilization of the spin label, determined by comparing the ESR spectra recorded in the presence of the kinase with those of the free ligand in solutions of different viscosities, gave information about the geometry of the cAMP site. Strong immobilization of the N-6 substituents up to a spacer length of 7 atoms indicates a rather deep cleft of the cAMP site. The depth of this cleft differs, however, when the spin label is attached to the different positions at the adenine (N-6, C-2 and C-8). Whereas the N-6 derivatives indicate a rather deep site, the C-2 derivatives reveal a significantly smaller depth and C-8 substituents (syn conformation) obviously occupy a very shallow surface with almost no immobilization. In addition the binding affinites of the spin-labeled cAMP derivatives were determined, together with those of a series of (diamagnetic) C-2 derivatives bearing hydrophobic alkyl chains of different length. The latter results helped to clarify the differences between the regions near to C-2 and N-6, respectively, of the cAMP site. N-6 spin-labeled derivatives were also investigated in the presence of ATP and protein kinase. These results are interpreted as indicative of a conformational change at the cAMP site upon formation of the holoenzyme, due to binding of ATP, leaving cAMP less strongly immobilized.

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