The cyclic AMP-dependent protein kinase from bovine cardiac muscle is a homoserine kinase

The catalytic subunit of the cAMP-dependent protein kinase from bovine cardiac muscle phosphorylates homoserine in the synthetic peptide Leu-Arg-Arg-Ala-Hse-Leu-Gly. Phosphorylation of the primary alcohol of the homoserine residue was established via NMR spectroscopy. Two-dimensional correlated and nuclear Overhauser effect spectroscopies provided the sequence-specific chemical shift assignments of the substrate peptide and its phosphorylated counterpart. Coupled and decoupled ³¹P NMR experiments established the presence of phosphate on the homoserine residue. The maximal velocity (6.4 μmol/min · mg) obtained for homoserine-peptide phosphorylation at 12.5 mM Mg²⁺ compares favorably to the velocities observed for the corresponding serine- (21 μmol/min · mg), threonine- (3.2 μmol/min · mg), and hydroxyproline-peptides (1 μmol/min · mg). However, the K(m) for homoserine kinase activity is modest (1.3 mM) relative to the K(m) associated with the phosphorylation of the serine-containing substrate (22 μM). The effect of Mg²⁺ concentration on the kinetic parameters k(cat), K(m), and k(cat)/K(m) was investigated for both serine- and homeserine-peptides. Both substrates display similar k(cat)/K(m) versus [Mg²⁺] profiles, with the most notable difference that the optimal Mg²⁺ concentration is higher for the homoserine-containing peptide. In addition, the K(m) for the serine-peptide was found to be independent of [Mg²⁺], whereas the K(m) for the homoserine-peptide was observed to be dependent upon [Mg²⁺]. These results suggest that the long homoserine side chain may induce an unusually large off rate for the peptide and/or may misalign the hydroxyl moiety in the active site.