Phosphorylation of hydroxyproline in a synthetic peptide catalyzed by cyclic AMP-dependent protein kinase.
The cyclic AMP-dependent protein kinase catalyzes the phosphorylation of hydroxyproline present in the heptapeptide, Leu-Arg-Arg-Ala-Hyp-Leu-Gly. The Km value for the reaction with this substrate was high (approximately 18 mM) compared to the Km values reported for the analogous threonine and serine-containing peptides, which were 0.59 mM and 0.016 mM, respectively (Kemp, B.E., Graves, D.J., Benjamini, E., and Krebs, E.G. (1977) J. Biol. Chem. 252, 4888-4894). The Vmax value with the hydroxyproline-containing peptide was 1 mumol . min-1 mg-1 in contrast to Vmax values of 6 mumol . min-1 mg-1 and 20 mumol . min-1 mg-1 for the threonine- and serine-containing peptides, respectively. Phosphate esterified to hydroxyproline present in the peptide was relatively stable in hot alkali, only 10% being released as Pi within 30 min in 0.1 N NaOH at 100 degrees C, whereas all of the phosphate was released from the phosphoserine peptide analogue under these conditions. Phosphohydroxyproline in the peptide was also more stable to acid (5.7 N HCl, 110 degrees C) than phosphoserine, the time for 50% release as Pi being 15 h in contrast to 6 h for the latter.
D000818:Animals; D002417:Cattle; D000242:Cyclic AMP / Q000494:pharmacology; D004789:Enzyme Activation; D006909:Hydroxyproline; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D009842:Oligopeptides; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D013379:Substrate Specificity
D009842:Oligopeptides; D000242:Cyclic AMP; D011494:Protein Kinases; D006909:Hydroxyproline