Multiple arginine residues contribute to the increased efficacy of peptide substrates for the cAMP-dependent protein kinase.

Efficient cAMP-dependent protein kinase substrates typically contain an arginine dyad one amino acid removed from the residue which undergoes phosphorylation (ie. Arg-Arg-X-Ser). However, several naturally occurring protein kinase inhibitors and substrates possess additional basic residues that are proximal to the arginine dyad, implying the presence of either an extended or an additional acidic subsite on the enzyme. In this study, we investigated the substrate efficacy of several multiple arginine-bearing peptides. The most efficient substrate studied, Arg-Arg-Leu-Arg-Arg-Ala-Ser-Leu-Gly, exhibits a nearly eleven-fold increase in kcat/Km relative to Leu-Arg-Arg-Ala-Ser-Leu-Gly. The enhanced kcat/Km is primarily a consequence of a reduced Km. These results suggest that a double arginine dyad, separated by a single amino acid, represents the optimal sequence for basic residues on cAMP-dependent protein kinase substrates.