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 mumol/min.mg) obtained for homoserine-peptide phosphorylation at 12.5 mM Mg2+ compares favorably to the velocities observed for the corresponding serine- (21 mumol/min.mg), threonine- (3.2 mumol/min.mg), and hydroxyproline-peptides (1 mumol/min.mg). However, the Km for homoserine kinase activity is modest (1.3 mM) relative to the Km associated with the phosphorylation of the serine-containing substrate (22 microM). The effect of Mg2+ concentration on the kinetic parameters kcat, Km, and kcat/Km was investigated for both serine- and homoserine-peptides. Both substrates display similar kcat/Km versus [Mg2+] profiles, with the most notable difference that the optimal Mg2+ concentration is higher for the homoserine-containing peptide. In addition, the Km for the serine-peptide was found to be independent of [Mg2+], whereas the Km 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.
Prorok
M
M
Department of Chemistry, State University of New York, Buffalo 14214.
Sukumaran
D K
DK
Lawrence
D S
DS
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D007202:Indicators and Reagents; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology*; D009842:Oligopeptides / Q000138:chemical synthesis; D010770:Phosphotransferases / Q000302:isolation & purification / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor); D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D007202:Indicators and Reagents; D009842:Oligopeptides; D010770:Phosphotransferases; D011494:Protein Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); C021370:homoserine kinase; D008274:Magnesium