NMR studies of the mechanism of cyclic AMP-dependent protein kinase

NMR has been used to study the role of the divalent cation, the conformations, arrangement, and exchange rates of the enzyme-bound metal-ATP and peptide substrates, the mechanism of the phosphoryl transfer, and the structure and role of the regulatory subunit on type II cyclic AMP (cAMP)-dependent protein kinase from bovine heart. The active complex consists of an enzyme-ATP-metal bridge in which the metal is beta, gamma coordinated, with delta chirality at P beta, and a torsional angle at the adenine-ribose bond in the high-anti range (x approximately 80 degrees). The bound heptapeptide substrate Leu-Arg-Arg-Ala-Ser-Leu-Gly is extended in conformation, forming either a coil or, less likely, a beta turn but not an alpha helix or beta sheet. The distance from the gamma-P of bound ATP analogs to the Ser-OH of the bound peptide (5.3 +/- 0.7 A) would permit a metaphosphate or an elongated phosphorane intermediate or transition state. The regulatory subunit (R2) blocks the peptide- or protein-binding site of the catalytic subunit. The 31P chemical shift of cAMP is not greatly altered on binding to R2, but the resonance is broadened to approximately 32 Hz, which indicates no chemical change but marked immobilization of bound cAMP. A narrower (approximately 7 Hz) 31P resonance at 4.44 ppm is assigned to P-serine-95 of R2 because it disappears with catalytic subunit, Mg2+, and an ADP-generating system.