Role of multiple basic residues in determining the substrate specificity of cyclic AMP-dependent protein kinase.

Studies on the substrate specificity of the catalytic subunit of bovine skeletal muscle adenosine 3’:5’-monophosphate-dependent protein kinase (EC 2.7.1.37,ATP:protein phosphotransferase) have been made using the synthetic peptide, Leu-Arg-Arg-Ala-Ser-Leu-Gly, corresponding to part of the local phosphorylation site sequence reported for porcine liver pyruvate kinase. The V,,,,, and apparent K,,, obtained with this peptide as substrate were 20 pmol min-’ mg-’ and 16 FM, respectively. Substitution of either of the 2 arginine residues with alanine increased the apparent K,,, more than loo-fold. Both arginine residues appeared to be essential since peptide analogs containing lysine, histidine, or homoarginine in place of one of the arginines had higher apparent K,,, values than the parent peptide. Provided at least 1 arginine was present, the V,,, was relatively insensitive to the above substitutions. These findings support the idea that multiple basic residues, in particular arginine, on the NH,-terminal side of the phosphorylated serine act as important substrate specificity determinants for the protein kinase. Replacement of the serine with threonirie resulted in a 37-fold higher apparent K,,,. Deletion of either the NH,-terminal leucine or the COOHterminal glycine had little effect on the kinetics of phosphorylation. Studies using a series of synthetic peptide analogs of the phosphorylase phosphorylation site sequence (residues 10 to 18) revealed several important differences in the specificity requirements of the protein kinase and phosphorylase kinase.