Adenosine Cyclic 3′,5′-Monophosphate Dependent Protein Kinase: Kinetic Mechanism for the Bovine Skeletal Muscle Catalytic Subunit

The kinetic mechanism for adenosine cyclic 3′,5′-monophosphate dependent protein kinase was determined from initial velocity studies in the absence and presence of the product MgADP and dead-end inhibitors. Data are consistent with random addition of MgATP and Ser-peptide and ordered release of phospho-Ser-peptide and MgADP with a dead-end E-MgADP-Ser-peptide complex. In addition to the metal required for the nucleotide, we also characterized the binding of Mg²⁺ to a second site. Increasing the Mg²⁺ results in a 5-6-fold decrease in V_max in the presence or absence of 0.1 M KCl. There is a 5-fold increase in V/K_MgATP in the absence of KCl and a 13-fold increase in V/K_MgATP at 0.1 M KCl. The effect of increasing free Mg²⁺ on V_max and V/K was also obtained with MgITP (20% the V_max with MgATP) and MgGTP (10% the V_max with MgATP) as substrates. The dissociation constant for Mg²⁺ from E-Ser-peptide-Mg²⁺ and central complexes is 2–3 mM. At low concentrations of free Mg²⁺ and no added KCl, competitive inhibitors of MgATP (K_i = 160 µM) decrease in the order adenosine = MgADP (K_i ≃ 40 µM) > AMP (K_i ≃ 8 mM). At saturating free Mg²⁺, K_i values are 10 µM (MgATP), 10 µM (MgADP), 40 µM (adenosine), and 850 µM (AMP). In addition, guanosine (1 mM) and MgPPP_i (10 mM) failed to inhibit at low or high free Mg²⁺. A mechanism for nucleotide interaction is proposed on the basis of a hydrophobic site or negatively charged group in the region of the α-phosphate in the nucleotide binding site and the presence or absence of a negative charge on the α-phosphate. We also made use of a spectrophotometric assay for protein kinase that couples the production of MgADP to the pyruvate kinase and lactate dehydrogenase reactions. On the basis of determinations of the amount of phosphorylatable Ser-peptide by spectrophotometric or radioisotopic determinations, we find that commercial samples contain 25–50% nonpeptide material by weight.