A REQUIREMENT OF HYDROPHOBIC AND BASIC-AMINO-ACID RESIDUES FOR SUBSTRATE RECOGNITION BY CA2+ CALMODULIN-DEPENDENT PROTEIN-KINASE IA

The substrate recognition determinants of Ca2+/calmodulin-dependent protein kinase Ia were investigated by using peptide analogues based on the amino acid sequence around Ser-9 of synapsin I. The K-m and V-max for the synthetic peptide Leu-Arg-Arg-Arg-Leu-Ser-Asp-Ala-Asn-Phe are 3.9 mu M and 18.5 mu mol/(min.mg), respectively. Deletion of Leu at the -5 position lowers the V-max/K-m by 470-fold. The requirement for a hydrophobic residue at -5 was confirmed by the 90- to 2400-fold reduction in V-max/K-m produced by Arg, Ala, or Asp substitutions, but only 2.6-fold decrease after Phe substitution at this position. A hydrophobic residue is similarly required at the +4 position. Deletion of Phe at this position produces a 67-fold reduction, and substitution of Ala for Phe a 43-fold reduction in V-max/K-m. In contrast, substitution with Leu increases V-max/K-m by 1.8-fold. Arg at -3 is also required for recognition as shown by an approximate to 240-fold decrease in V-max/K-m after Ala substitution at this position. Positions -2, -4, and +1 appear to play secondary roles in substrate recognition. Arg at -2 and -4 are positive determinants, since Ala substitution at these positions decreases V-max/K-m by 4.7- and 11-fold, respectively. Asp at +1 is a negative influence, since Ala and Leu substitutions at this position increase V-max/K-m by 2.3- and 6.3-fold, respectively. Substitution of Ala for Leu at -1 or Thr for Ser at the 0 position has little effect on phosphorylation kinetics. Thus Ca2+/calmodulin-dependent protein kinase Ia has the minimal substrate recognition motif of Hyd-Xaa-Arg-Xaa-Xaa-(Ser*/Thr*)-Xaa-Xaa-Xaa-Hyd, where Hyd represents a hydrophobic amino acid residue.